Targets of Activity

Representational difference analysis (RDA) was used to identify Drosophila genes repressed by HP1. In this technique, RNA samples from two different sources are compared to identify specific RNAs that are overrepresented in one sample relative to the other. RNA from third instar larvae lacking functional HP1 (HP1-/-) were compared to RNA from wild-type larvae (HP1+/+) to look for RNAs overexpressed in the HP1-/- sample. Four prominent bands were obtained after three rounds of subtractive hybridization. After cloning and sequencing of DNA fragments from these four bands, two euchromatic genes, Ser4 and CG13135, were identified whose expression levels were anticipated to be higher in HP1(-/-) larvae than in wild type. Subsequently, it was confirmed by Northern blot analysis that the expression of these two genes is repressed 2.5- to 3-fold in wild-type compared with HP1(-/-) larvae (Hwang, 2001).

To ensure that the effects of HP1 mutations on the expression of Ser4 and CG13135 are caused by HP1 dosage and not by specific alleles or linked modifiers, larvae that carried different HP1 mutations were generated by crossing Su(var)2-504/+ to Su(var)2-505/+ or Su(var)2-504/+ to Su(var)2-5149/+. Su(var)2-504 and Su(var)2-5149 encode truncated HP1 proteins, whereas Su(var)2-505 encodes a frame-shift mutation at codon 10. Each allele was isolated in a separate mutational screen in a different genetic background. Expression of Ser4 and CG13135 was elevated in all HP1-mutant larvae, whereas their expression in sibs decreased in proportion to the number of functional copies of the HP1 gene present. In larvae with one functional dose of HP1, expression of both genes was increased 1.5- to 2-fold, whereas in HP1-null larvae, the average expression levels of both genes were 2.5- to 3-fold of those observed in wild type. These results show that loss of Ser4 and CG13135 repression is caused by reduced functional HP1 gene dosage and not by genetic background (Hwang, 2001).

To test whether CG13135 and Ser4 are repressed further by HP1 overexpression, flies were generated with three or four copies of functional HP1 by using the Dp(2;2)P90 chromosome, which carries a tandem duplication including the HP1 gene. The expression of CG13135 and Ser4 decreases progressively with the increased HP1 gene dosage across the entire range of HP1 dosage examined. The repressive effect of HP1 on the expression of CG13135 (13-fold from 0-4 doses) is greater than on Ser4 (6-fold from 0-4 doses). HP1 transcripts increase in HP1(+/+/+) and (+/+/+/+) larvae, as expected (Hwang, 2001).

Several other modifiers of PEV have been reported, including Su(var)2-1, 2-4, 3-6, and 3-9. The aggregate histone 4 acetylation level is increased in Su(var)2-1 mutants and this mutation displays a lethal interaction with the histone deacetylase inhibitor N-butyrate. Human SUV39H1 and murine Suv39H1 (mammalian orthologs of Drosophila Su(var)3-9) encode histone H3-specific methyltransferases that selectively methylate Lys-9 of the amino terminus of histone H3 in vitro. Methylated Lys-9 on histone H3 creates a binding site for HP1 proteins in yeast and mammals. Su(var)2-4 and Su(var)3-6 are strong dominant suppressors of PEV, although the mechanisms of these suppressors are unknown. Su(var)3-6 encodes a protein phosphatase that might be essential for modification of chromosomal proteins such as Su(var)3-7. The effects of these modifiers on the expression of CG13135 and Ser4 were investigated. Most of the PEV-modifier mutations tested significantly elevate the levels of Ser4 and CG13135 expression. In particular, the increased expression of HP1-regulated genes caused by Su(var)3-9 mutation parallels the effect of HP1 mutations, which is consistent with recent findings that histone H3 methylation promotes HP1 binding (Hwang, 2001).

CG13135 and Ser4 are localized in cytological regions 31A and 25B of chromosome 2L, respectively. Cytological region 31 is a prominent euchromatic site of HP1 binding on the left arm of chromosome 2. Two other genes (CG4791 and CG4897) were chosen in cytological region 31; these are positioned, respectively, within 100 kb proximal and distal to CG13135. Tested were performed to see whether these two genes also are regulated by HP1. Like CG13135 and Ser4, the expression level of CG4791 is progressively decreased in response to increasing HP1 copy number. CG4897 expression also is decreased, although it seems to be less sensitive to HP1 dosage. The expression profiles of CG4791 and CG4897 in PEV-suppressor mutations differ in magnitude from those of CG13135. Importantly, however, the Su(var)3-9 and Su(var)2-1 mutations significantly enhance the expression of all three genes. Thus, three genes in the euchromatic region 31 are similarly repressed by HP1, and this repression depends on the dosage of histone modifiers (Hwang, 2001).

These results provide evidence that HP1 represses genes at their endogenous euchromatic locations. All previous examples of HP1-dependent repression involve artificial repression of normally active euchromatic genes when they are translocated close to heterochromatin by chromosome rearrangements. Hints at connections between HP1 and euchromatic gene regulation have come from yeast 2-hybrid protein screens and coimmunoprecipitation assays in which HP1-family proteins were found to associate with transcription corepressors. Although HP1 has long been known to bind at several euchromatic sites by chromosome immunostaining, this study provides evidence that euchromatic HP1-binding sites represent domains of HP1-dependent gene repression. Although HP1 could be acting indirectly by regulating the regulators of euchromatic genes, the linear inverse response to HP1 from 0-4 doses (across two levels of underexpression and two levels of overexpression) and the observable binding of HP1 to the chromosomal interval containing HP1 target genes suggest that HP1 repression is direct. Furthermore, the Su(var)3-9 and Su(var)2-1 proteins are required for the HP1-dependent repression of euchromatic genes. These in vivo data implicate specific covalent modifications of histones as prerequisites for higher-order euchromatin structure organized by HP1 and, further, suggest that the mechanism of HP1-mediated repression in euchromatin shares features with HP1-dependent heterochromatin-mediated silencing in PEV (Hwang, 2001).

Three of the HP1-repressed genes map to region 31, which is one of the most prominent HP1-binding euchromatic regions in the Drosophila genome. Is region 31 of Drosophila chromosome 2 a domain of intercalary heterochromatin? Region 31 is a well banded interval in polytene nuclei, lacking the disorganized, attenuated appearance of pericentric heterochromatin. Although a 2-fold under-replication of the interval cannot be ruled out, region 31 is neither dramatically under-replicated nor significantly late replicating in polytene chromosomes, nor does it contain easily broken regions (weak points) or ectopic pairing sites characteristic of intercalary heterochromatin. Meiotic recombination is not suppressed significantly across region 31 (28) in contrast to the pericentric heterochromatin, where recombination is absent. Sequence analysis reveals no significant homology to any of the major satellite DNA sequences characteristic of pericentric heterochromatin or any significant amount of repetitious DNA sequences in tandem. The density of ORFs in region 31 (128 ORFs) is not significantly lower than the adjacent numbered euchromatic segments (108 ORFs in region 30; 106 ORFs in region 32). In contrast, gene density is thought to be much lower per unit length of DNA in heterochromatin than in euchromatin. Taken together, these observations strongly suggest that region 31 is not simply an island of heterochromatin in a sea of euchromatin. Instead, it is thought that region 31 represents a euchromatin domain subject to repression by HP1 (Hwang, 2001).

Silencing of euchromatic genes by PEV results in mosaic expression of such genes in tissues where the genes are normally expressed uniformly. It is not known whether HP1-mediated repression of region 31 genes is similarly mosaic or whether HP1 reduces expression of target genes uniformly in all cells. Additionally, it is not known whether HP1 is targeted directly to down-regulated region 31 genes or whether such targeting is general or tissue specific (Hwang, 2001).

Heterochromatin protein 1 (HP1) is a major component of heterochromatin. It has been reported to bind to a large number of genes and to many, but not all, transposable elements (TEs). The genomic signals responsible for targeting of HP1 have remained elusive. Whole-genome and computational approaches have been used to identify genomic features that are predictive of HP1 binding in Drosophila melanogaster. Genes in repeat-dense regions are more likely to be bound by HP1, particularly in pericentric chromosomal regions. TEs are bound by HP1 only if they are flanked by other repeats, suggesting a cooperative mechanism of binding. Genome-wide DamID mapping of HP1 in larvae and adult flies reveals that repeat-flanked genes typically bind HP1 throughout development, whereas repeat-free genes display developmentally dynamic HP1 association. Furthermore, computational analysis shows that HP1 preferentially binds to transcribed regions of long genes. Finally, low but significant amounts of HP1 were detected along the entire X chromosome in male, but not female, flies, suggesting a link between HP1 and the dosage compensation complex. These results provide insights into the mechanisms of HP1 targeting in the natural genomic context (de Wit, 2005).

HP1 and gene silencing in heterochromatin

Line HS-2 of Drosophila, carrying a silenced transgene in the pericentric heterochromatin, was used to investigate in detail the chromatin structure imposed by this environment. Digestion of the chromatin with micrococcal nuclease (MNase) shows a nucleosome array with extensive long-range order, indicating regular spacing, and with well-defined MNase cleavage fragments, indicating a smaller MNase target in the linker region. The repeating unit is about 10 bp larger than that observed for bulk Drosophila chromatin. The silenced transgene shows both a loss of DNase I-hypersensitive sites and decreased sensitivity to DNase I digestion within an array of nucleosomes lacking such sites; within such an array, sensitivity to digestion by MNase is unchanged. The ordered nucleosome array extends across the regulatory region of the transgene, a shift that could explain the loss of transgene expression in heterochromatin. Highly regular nucleosome arrays are observed over several endogenous heterochromatic sequences, indicating that this is a general feature of heterochromatin. However, genes normally active within heterochromatin (rolled and light) do not show this pattern, suggesting that the altered chromatin structure observed is associated with regions that are silent, rather than being a property of the domain as a whole. The results indicate that long-range nucleosomal ordering is linked with the heterochromatic packaging that imposes gene silencing (Sun, 2001).

Over 30 genetic functions reside within D. melanogaster heterochromatin: those characterized require a heterochromatic environment for their proper expression, exhibiting a variegating phenotype or reduced expression when rearrangements place them adjacent to a breakpoint in euchromatin. Particularly striking is the observation that expression of the heterochromatic genes rolled and light in their endogenous heterochromatic position is reduced in larvae mutant for HP1, suggesting that proper maintenance of heterochromatin structure is required for expression of these genes. Thus, it was somewhat surprising to observe that light and rolled have nucleosome arrays similar to those observed for the euchromatic transgene, rather than the heterochromatic transgene. This suggests that the regulation of these heterochromatic genes by HP1 may not be based on the impact of HP1 on heterochromatin structure in general (which is correlated with silencing of transgenes such as hsp70-white) but may be the consequence of a context-dependent (positive or negative) activity, similar to that displayed by RAP1 in S. cerevisiae. Alternatively, the impact of HP1 on a heterochromatic gene may reflect packaging of the surrounding area, rather than the transcribed region. A more detailed analysis of the chromatin structure encompassing these genes and their regulatory regions will be required to resolve this question (Sun, 2001)

HP1 promotes chromosome looping

A transgene inserted in euchromatin exhibits mosaic expression when targeted by a fusion protein made of the DNA-binding domain of GAL4 and the heterochromatin-associated protein HP1. The silencing responds to the loss of a dose of the dominant modifiers of position-effect variegation Su(var)3-7 and Su(var)2-5, the locus encoding HP1. The genomic environs of the insertion site at 87C1 comprise the dispersed repetitive elements micropia and alpha gamma. In the presence of the GAL4-HP1 chimera, the polytene chromosomes of this line form loops between the insertion site of the transgene and six other sections of chromosome 3R, as well as, rarely, with pericentric and telomeric heterochromatin. In contrast to the insertion site of the transgene at 87C, the six loop-forming sites in the euchromatic arm have each been described as intercalary heterochromatin. Moreover, GAL4-HP1 tethering on one homolog trans-inactivates the reporter on the other. HP1, probably together with other partners, could thus facilitate the coalescence of dispersed middle repetitive sequences, and organize the heterochromatic structure responsible for the variegated silencing of nearby euchromatic genes (Seum, 2001).

Looping, as seen here, and the presumed higher availability of complex-forming proteins at blocks of heterochromatin explain both long-distance effects and expansion of repression observed in variegating rearrangements. These experiments directly test and visualize some of these predictions. (1) It has been found that variegation of a euchromatic insertion of a transgene seems to require two conditions: proximity of middle repetitive DNA, and local presence of HP1, a heterochromatin-associated protein and modifier of position-effect variegation. Indeed, overexpression of wild-type HP1 does not promote variegated silencing at 87C in the absence of HP1 at the site. HP1 must be targeted there by the GAL4 DNA-binding domain. (2) It is observed that the region forms loops with sites of intercalary heterochromatin and with telomeric and pericentric heterochromatin. In contrast, in a non-variegating line, induction of GAL4-HP1 does not promote loops. Pairing and silencing appear correlated. (3) HP1 targeting on one homolog trans-inactivates the reporter on the other. The chromosome pairing and looping promoted by HP1 result in trans-inactivation and variegation of a transgene. These observations place HP1 in a pivotal role. It interacts with Su(var)3-7 and recruits it at ectopic sites. Su(var)3-7 is itself a protein found to interact with repetitive DNAs. Targeted HP1 may induce the pairing observed with domains of intercalary heterochromatin by recruiting Su(var)3-7 bound to middle repetitive elements near its 87C insertion site and at sites of intercalary, telomeric or pericentric heterochromatin. In a general model, position-effect variegation could result from expansion of heterochromatin blocks, but could also develop discontinuously by the attachment of Su(var)3-7, HP1 and other partners at dispersed middle repetitive sequences. The visible consequence is ectopic pairing within and among chromosomes (Seum, 2001).

It is also speculated that genes in proximity to the anchoring sites of loops variegate when GAL4-HP1 is expressed. This has been tested for one reporter, but other genes should be tested, whether at 87C or at the sites of intercalary heterochromatin. As an example, the bithorax complex of homeotic genes lies at 89E, a region of intercalary heterochromatin able to loop with Wink-A7 (a reporter containing three binding sites for the yeast GAL4 transcriptional activator) in the presence of GAL4-HP1. In preliminary experiments, no homeotic phenotypes were detected in the presence of GAL4-HP1; however, this needs a more comprehensive examination (Seum, 2001).

The Drosophila homolog of the human AF10 is an HP1-interacting suppressor of position effect variegation

In chromosomal rearrangements of acute myeloid leukaemia patients the mixed lineage leukaemia (MLL) gene, a human homolog of the Drosophila gene trithorax, is frequently fused to AF10. The identification and a functional characterization is described of the Drosophila homolog dAF10 (Alhambra). dAF10 functions in heterochromatin-dependent genomic silencing of position effect variegation, a phenomenon associated with chromosomal rearrangements that cause mosaic expression of euchromatic genes when relocated next to heterochromatin. dAF10 can associate with the heterochromatin protein 1 (HP1) in vitro and in vivo. The results indicate that dAF10 is an HP1-interacting component of the heterochromatin-dependent gene silencing pathway, which either contributes to the stability of the heterochromatin complex or to its function (Linder, 2001).

Cloning of the Drosophila homolog dAF10, was initiated by a database search. This search found the corresponding annotated transcription unit (CG1070), which maps into region 84C1-2 on the right arm of chromosome 3. Sequence analysis of both the genomic DNA and various EST clones confirmed the chromosomal location and revealed that dAF10 encodes different splicing variants of which the two major forms could be assigned unambiguously (Linder, 2001).

In order to establish the temporal pattern of the dAF10 expression, developmental Northern blot analysis was performed. dAF10 codes for four different transcripts with distinct temporal expression profiles during the Drosophila life cycle. One of two major transcripts, ~5 kb in length, is constitutively expressed with high levels during embryogenesis and in adult females. The second major transcript of ~3 kb is restricted to embryogenesis and females. In addition, two splicing variants of the transcript (6 and 8 kb) were found from later stages of embryogenesis onwards. Whole-mount in situ hybridization to oocytes and embryos revealed that the 5 and 3 kb transcripts are expressed maternally. Both splicing variants were found in nurse cells and later equally distributed in oocytes. They are maintained in fertilized eggs and decease gradually until the preblastoderm stage. Zygotic dAF10 expression is initiated during syncytial blastoderm (stage 4) and develops a stripe pattern similar to pair-rule segmentation genes. The 3 and 5 kb transcripts were recovered in two corresponding full-size cDNA clones. The 5 kb transcript encodes a 4131 bp open reading frame (ORF) corresponding to a 1377 amino acids polypeptide, which contains a PHD-finger next to an extended PHD-finger domain and a leucine zipper motif as observed with human AF10. The 3 kb transcript starts with an alternative exon and codes for an 826 amino acid protein, which lacks the N-terminal PHD-finger/extended PHD-finger domains. Thus, only the larger of the two major transcripts codes for the human AF10 homolog. However, both variants contain the leucine zipper motif and a PLVVL pentamer motif found in a subset of proteins that interact with HP1 in vitro. This observation suggests that dAF10 may bind to HP1 and function in an HP1-dependent manner (Linder, 2001).

A possible dAF10::HP1 interaction was examined via pull-down assays involving a GST-HP1 fusion protein and in vitro translated, labelled dAF10. HP1 binds via its chromo shadow domain to full-size dAF10 and to the subfragment that contains the PLVVL motif. These findings suggest that dAF10 may function in an HP1-dependent gene silencing pathway (Linder, 2001).

The results establish that dAF10 and HP1 can associate in vitro and that both components act in heterochromatin-induced gene silencing. It has been proposed that this type of silencing results from a co-operative assembly of heterochromatin as multimeric complexes proceeding from a pre-existing block of heterochromatin. HP1 action involves the recognition of an epigenetic methylation mark at lysine 9 of the N-terminus of histone and is likely to cause chromatin remodelling involving homo- and/or hetero-philic protein-protein interactions. Furthermore, human HP1 was found to be associated with the lamin B receptor, a component of the inner nuclear membrane. These observations are consistent with the argument that HP1 is a constitutive component of heterochromatin, which initiates silencing at distinct sites and may also function in the subnuclear localization of heterochromatin (Linder, 2001).

dAF10 exerts a spatiotemporally restricted expression profile. This finding, the in vitro association between HP1 and dAF10 and their genetic interaction in suppression of PEV suggest that dAF10 is a component of a distinct heterochromatin-dependent silencing process. dAF10 may either contribute to the stability of the heterochromatin complex or serve as an attachment site for other proteins to join the silencing complex. Interestingly, the human AF10 is frequently fused with the trithorax homolog MLL of AML patients. It is therefore tempting to speculate that combining MLL with AF10 in a chimeric MLL-AF10 fusion protein causes a switch in cellular memory. The protein may attach to HP1 and cause gene silencing instead of maintaining target gene expression (Linder, 2001).

HP1 nucleates the formation of silent chromatin at non-centric locations

HP1 is a conserved non-histone chromosomal protein enriched in heterochromatin. On Drosophila polytene chromosomes, HP1 localizes to centric and telomeric regions, along the fourth chromosome, and to specific sites within euchromatin. HP1 associates with centric regions through an interaction with methylated lysine nine of histone H3, a modification generated by the histone methyltransferase SU(VAR)3-9. This association correlates with a closed chromatin configuration and silencing of euchromatic genes positioned near heterochromatin. To determine whether HP1 is sufficient to nucleate the formation of silent chromatin at non-centric locations, HP1 was tethered to sites within euchromatic regions of Drosophila chromosomes by fusing HP1 to a heterologous DNA-binding domain. At 25 out of 26 sites tested, tethered HP1 caused silencing of a nearby reporter gene. The site that did not support silencing was upstream of an active gene, suggesting that the local chromatin environment did not support the formation of silent chromatin. Silencing correlates with the formation of ectopic fibers between the site of tethered HP1 and other chromosomal sites, some containing HP1. The ability of HP1 to bring distant chromosomal sites into proximity with each other suggests a mechanism for chromatin packaging. Silencing was not dependent on SU(VAR)3-9 dosage, suggesting a bypass of the requirement for histone methylation (Li, 2003).

Distinct HP1 and Su(var)3-9 complexes bind to sets of developmentally coexpressed genes depending on chromosomal location

Heterochromatin proteins are thought to play key roles in chromatin structure and gene regulation, yet very few genes have been identified that are regulated by these proteins. Large-scale mapping and analysis was performed of in vivo target loci of the proteins HP1, HP1c, and Su(var)3-9 in Drosophila Kc cells, which are of embryonic origin. For each protein, ~100-200 target genes were identified among >6000 probed loci. HP1 and Su(var)3-9 bind together to transposable elements and genes that are predominantly pericentric. In addition, Su(var)3-9 binds without HP1 to a distinct set of nonpericentric genes. On chromosome 4, HP1 binds to many genes, mostly independent of Su(var)3-9. The binding pattern of HP1c is largely different from that of HP1 and Su(var)3-9. Target genes of HP1 and Su(var)3-9 show lower expression levels in Kc cells than do nontarget genes, but not if they are located in pericentric regions. Strikingly, in pericentric regions, target genes of Su(var)3-9 and HP1 are predominantly embryo-specific genes, whereas on the chromosome arms Su(var)3-9 is preferentially associated with a set of male-specific genes. These results demonstrate that, depending on chromosomal location, the HP1 and Su(var)3-9 proteins form different complexes that associate with specific sets of developmentally coexpressed genes (Greil, 2003).

The DamID chromatin profiling technique was used to map in vivo target genes of HP1 and Su(var)3-9 in cultured D. melanogaster Kc167 cells. In brief, this technique involves in vivo expression of a trace amount of a chromatin protein of interest fused to Escherichia coli DNA adenine methyltransferase (Dam). As a result, DNA in the target loci of the chromatin protein is preferentially methylated by the tethered Dam. Subsequently, methylated DNA fragments are isolated, labeled with a fluorescent dye, and hybridized to a microarray. To correct for nonspecific binding of Dam and local differences in DNA accessibility, methylated DNA fragments of control cells transfected with Dam alone are labeled with a different fluorescent dye and cohybridized. The obtained ratio of fluorescent dyes reflects the extent of protein binding to the probed gene. The protocol was modified by replacing the purification of methylated fragments with a PCR-based selective amplification of methylated fragments. Control experiments confirm that the relative abundance of methylated sequences was conserved in the PCR amplification step. This new protocol is much more efficient and requires considerably smaller amounts (>20-fold reduction) of genomic DNA compared with the original protocol (Greil, 2003).

Based on the well documented roles of HP1 and Su(var)3-9 in the silencing of reporter genes, it was expected that the genes identified as targets would be mostly inactive. IndeedSu(var)3-9 and HP1 preferentially associate with genes of low expression levels. This preference is more prominent for Su(var)3-9 than for HP1; in fact, genes that are bound by Su(var)3-9 without HP1 are more often inactive than genes that are bound by both proteins. Formally, binding of Su(var)3-9 may be either the cause or the consequence of gene silencing. In the latter case, Su(var)3-9 complexes would mark genes that are already inactive due to other silencing mechanisms. However, if Su(var)3-9 is indeed actively involved in the silencing of its target genes, then Su(var)3-9 complexes may be more potent silencers if they lack HP1 (Greil, 2003).

Although HP1 and Su(var)3-9 generally display a preference for genes of low activity, a considerable fraction of their target genes are expressed, sometimes even at high levels. Many of these active target genes are located in pericentric regions and on chromosome 4. Earlier findings already demonstrated that the pericentric genes light and rolled are active, and it was confirmed that these genes are also bound by HP1 and Su(var)3-9. Association of HP1 was reported recently with ecdysone- and heat shock-induced puffs on polytene chromosomes. The expression of lt, rl, and hsp70 genes is reduced in HP1-deficient larvae, suggesting that HP1 may facilitate rather than suppress transcription of certain genes. Attempts were made to extend these observations in Kc cells by microarray mRNA expression profiling after RNA interference of HP1 and Su(var)3-9. No changes in expression of the HP1 or Su(var)3-9 target genes were detected after knockdown of either of the two proteins. According to these results, HP1 and Su(var)3-9 may have only redundant roles in gene regulation in Kc cells. However, it should be noted that the dsRNA-induced reduction of HP1 and Su(var)3-9, although substantial, may have been insufficient or not long enough to cause detectable alterations in gene regulation. In addition, the previously reported changes in expression of the lt and rl genes in HP1-deficient larvae were only ~2.5-fold. Such modest changes in gene expression may have been missed in a microarray-based assay. Finally, HP1 and Su(var)3-9 complexes may not be essential for gene regulation in Kc cells. Heterochromatin-mediated silencing of a reporter gene is not fully developed until late embryogenesis. Kc cells appear to be embryonic, so it is possible that the regulatory functions of HP1 and Su(var)3-9 initiate only later in development. Furthermore, the lack of a visible phenotype of Su(var)3-9 null mutants suggests that a role of Su(var)3-9 in gene regulation may be redundant (Greil, 2003).

Among the target loci of HP1 and Su(var)3-9, two conspicuous groups of developmentally coregulated genes were identified. In the first group, many of the nonpericentric genes that are exclusively bound by Su(var)3-9 in Kc cells are highly expressed in adult males, but much less in females, embryos, larvae, and Kc cells. Extrapolating these data to the entire genome [comparison of Su(var)3-9 binding and developmental expression patterns was only possible for ~2700 genes], it is anticipated that 50-100 male-specific genes are bound by Su(var)3-9 in Kc cells. This could be an underestimate, because testis-specific genes may be underrepresented in the cDNA libraries present on the microarray (Greil, 2003).

Su(var)3-9 may contribute to repression of these male-specific genes in early stages of development and in adult females. Kc cells are female, as judged from the absence of a Y chromosome and expression of the female-specific but not the male-specific splicing variant of doublesex. Therefore binding of Su(var)3-9 to these genes may reflect either the female or the embryonic origin of the Kc cells. Alternatively, aberrant expression of these genes in embryos, larvae, and female adults may not lead to a detectable phenotype under laboratory conditions (Greil, 2003).

The second group of developmentally coregulated target genes is formed by a set of embryo-specific genes. Strikingly, these embryo-specific target genes are strongly concentrated in pericentric regions, and are typically bound by both HP1 and Su(var)3-9. This suggests a specialized role for pericentric HP1 and Su(var)3-9 in the embryonic gene expression program. In Kc cells, these pericentric target genes are generally not repressed, consistent with the embryonic origin of these cells. Both HP1 and Su(var)3-9 are present in embryos, suggesting that the lack of repression of pericentric target genes cannot be attributed to the absence of either HP1 or Su(var)3-9 during this developmental stage. Rather, these proteins may facilitate gene expression in the embryo, or perhaps mark the embryonic pericentric genes for silencing later in development. The clustering of these genes in the pericentric region may play a role in their coordinated regulation (Greil, 2003).

It is likely that the genomic binding pattern of the proteins studied here depends at least in part on the cell type or developmental stage. Evidence that the target specificity of heterochromatin proteins is dynamic comes from recent observations that HP1 binds to induced but not to uninduced heat-shock and ecdysone-responsive genes. The HP1 binding map obtained in Kc cells shows only limited overlap with the banding pattern of HP1 staining on polytene chromosome arms in salivary glands. Of 91 nonpericentric target loci identified, only nine coincide with HP1 bands in polytene chromosomes. Although this comparison should be interpreted with caution because of the different methodology and a >10-fold difference in mapping resolution (the median size of the stained polytene chromosome regions is 74 kb, whereas the median size of the genomic regions probed by the microarray is 3.7 kb), it suggests that many target loci may be cell type-specific. An example of this is region 31, a broad (~0.5 Mb) region on chromosome 2 that is bound by HP1 in polytene chromosomes in salivary gland tissue. In Kc cells, three out of 50 probed loci in this region are associated with HP1, which is unlikely to account for the extensive HP1 staining of region 31 in polytene chromosomes. This suggests that much of the binding of HP1 to this region in salivary gland cells is cell type-specific (Greil, 2003).

The heterogeneity of the HP1 and Su(var)3-9 complexes, in terms of both protein composition and target gene expression status, further complicates the matter of defining heterochromatin. By morphological criteria, heterochromatin in Drosophila chromosomes is concentrated in pericentric regions. However, most pericentric genes, although bound by HP1 and Su(var)3-9, are transcriptionally active, contrary to the repressive role that is generally attributed to heterochromatin. On the 'euchromatic' chromosome arms, genes bound by Su(var)3-9 are often repressed, yet these genes typically lack the classical heterochromatin marker protein HP1. Over the long term, it may be more useful to define different types of chromatin according to their protein composition, including posttranslational modifications and histone variants. This will require a much more sophisticated nomenclature than 'euchromatin' and 'heterochromatin'. The transcriptional status of a gene may be expected to be controlled by the combinatorial action of the proteins that are associated with it. Global approaches to study chromatin composition on a gene-by-gene basis, such as described here, will be essential to catalog the different chromatin types and to understand their role in gene regulation (Greil, 2003).

Heterochromatin protein 1 is associated with induced gene expression in Drosophila euchromatin

Heterochromatin protein 1 (HP1) is a conserved nonhistone chromosomal protein, which is involved in heterochromatin formation and gene silencing in many organisms. In addition, it has been shown that HP1 is also involved in telomere capping in Drosophila. This study shows novel striking features of this protein demonstrating its involvement in the activation of several euchromatic genes in Drosophila. By immunostaining experiments using an HP1 antibody, it was found that HP1 is associated with developmental and heat shock-induced puffs on polytene chromosomes. Because the puffs are the cytological phenotype of intense gene activity, a detailed analysis was performed of the heat shock-induced expression of the HSP70 encoding gene in larvae with different doses of HP1. It was found that HP1 is positively involved in Hsp70 gene activity. These data significantly broaden the current views of the roles of HP1 in vivo by demonstrating that this protein has multifunctional roles (Piacentini, 2003).

In contrast to the most commonly cited role of HP1 in heterochromatin formation, the present data show a clear association of HP1 with induced gene expression in euchromatin. Association takes place whether the induction occurs as a result of the developmental stage (as with the ecdysone regulated puffs), a heat shock-induced response, or induced ectopic expression (as with the GAL4/UAS transgene). In addition, the recruitment of HP1 to transgenic, developmental, and heat shock-induced puffs suggests that the association of HP1 with gene expression depends on the induction per se and not on a specific type of induction, specific promoter, or specific transcript (Piacentini, 2003).

These analyses of gene expression have failed to detect a difference in heat shock-induced puffs between individuals with or without a functional HP1 gene. Although, the puff formation is not visibly affected, a quantitative Northern analysis reveals that genotypes with different doses of the HP1-encoding gene differ in the amount of Hsp70 transcripts. During the first hours after heat shock, the amount of Hsp70 transcripts in mutant larvae lacking HP1 and in transgenic larvae carrying four doses of the HP1-encoding gene is, respectively, lower and higher compared with the transcript level in wild-type larvae, thus, showing that HP1 affects heat shock RNA, either its expression or stability. The results of formaldehyde cross-linked chromatin immunoprecipitation (X-ChIP) assay show that, after heat shock induction, HP1 accumulates on the coding regions and not on the promoter region. This is consistent with a role of this protein on transcription rates, transcript elongation, transcript processing, or transcript stability rather than a role in gene induction. This role seems to be corroborated by observations suggesting that HP1 accumulation depends on the presence of Hsp70 transcripts and by the integrity of its chromo domain. Because it has been shown that the chromo domain could be a module of interaction with RNA, it is proposed that HP1 may directly bind the Hsp70 transcripts. However, whatever the mechanism, it is clear that these results suggest a new role for HP1 in its association with induced, actively transcribed genes in euchromatin, and predict also its biochemical association with factors compatible with gene expression. Given that the physiological and heat shock-induced genes show accumulation of this protein, the network of interacting proteins may include mediators of the induction itself, such as hormone receptors and HSF. An interesting point in this regard, is that the accumulation of HP1 on heat shock-induced puffs seems coincident with its removal from many other sites including the developmental puffs. This opens the possibility that HP1 could be involved, at least in part, in the well-known extensive silencing of the genome after heat shock (Piacentini, 2003).

The positive versus negative effects of HP1 are thought to be determined by its interacting proteins. Whether the positive and negative effects will map to the same interacting protein domains of HP1 will be interesting to determine. The activator and repressor activities require distinct protein domains for different DNA-protein, RNA-protein, or protein-protein interactions. HP1 has different domains that it shares with other PEV modifier proteins or transcriptional regulators that should confer to it the necessary structural flexibility required for multiple functional roles. Further studies will reveal whether HP1 has multiple separate, nonoverlapping functions acting as either positive or negative transcriptional regulator also in euchromatin, depending on chromosomal context (Piacentini, 2003).

Drosophila atm/telomere fusion is required for telomeric localization of HP1 and telomere position effect

Terminal deletions of Drosophila chromosomes can be stably protected from end-to-end fusion despite the absence of all telomere-associated sequences. The sequence-independent protection of these telomeres suggests that recognition of chromosome ends might contribute to the epigenetic protection of telomeres. In mammals, Ataxia Telangiectasia Mutated (ATM) is activated by DNA damage and acts through an unknown, telomerase-independent mechanism to regulate telomere length and protection. The Drosophila homolog of ATM is encoded by the telomere fusion (common alternative name: ATM) gene. In the absence of ATM, telomere fusions occur even though telomere-specific Het-A sequences are still present. High levels of spontaneous apoptosis are observed in ATM-deficient tissues, indicating that telomere dysfunction induces apoptosis in Drosophila. Suppression of this apoptosis by p53 mutations suggests that loss of ATM activates apoptosis through a DNA damage-response mechanism. Loss of ATM reduces the levels of heterochromatin protein 1 (HP1) at telomeres and suppresses telomere position effect. It is proposed that recognition of chromosome ends by ATM prevents telomere fusion and apoptosis by recruiting chromatin-modifying complexes to telomeres (Oikemus, 2004).

Drosophila atm is required to protect telomeres from fusion. HP1 and HOAP localize to the telomeres of polytene chromosomes, as well as other sites, and are required for telomere protection in mitotic cells. Immunostaining was used to examine the distribution of HP1 and HOAP on wild-type and atm- polytene chromosomes. HP1 staining at the chromocenter, fourth chromosome, and several euchromatic sites is unaffected by loss of atm, whereas HP1 staining is reduced at most atm- telomeres. At the tip of chromosome 2R, similar levels of HP1 staining at an internal site (cytological position 60F) can be observed in wild-type and mutant chromosomes, whereas HP1 is specifically reduced at the telomere of the mutant chromosome. The normal levels of HP1 at sites other than the telomere indicate that the lack of telomere staining at atm- chromosomes is not due to differences in chromosome preparations or to global changes in chromatin structure in atm- cells. Rather, atm is specifically required to recruit or maintain HP1 to chromosome ends (Oikemus, 2004).

Immunostaining of the same chromosomes for HOAP reveal reduced staining at the telomeres of most atm- chromosomes compared with wild type. Similar decreases in HP1 and HOAP localization at telomeres are seen in atmDelta356/ Df(3R)PG4 and atmtefu/Df(3R)PG4 animals, indicating that this phenotype is not allele specific. Quantification of the fluorescence intensity associated with HOAP and HP1 staining further demonstrates that there is a reproducible reduction at atm- telomeres compared with wild type. In contrast, HP1 staining at an internal chromosomal site (60F) is not reduced (Oikemus, 2004).

HP1 promotes heterochromatin formation, in part, by recruiting histone-modifying enzymes. To probe whether atm mutations alter chromatin at the telomeres of mitotic cells, telomere position effect (TPE) at three telomeres was examined. When a white reporter gene is placed adjacent to telomere-associated sequences (TAS), gene expression is silenced. At each site tested, TPE is partially suppressed by mutations in atm. In transgenes inserted at nontelomeric genomic positions, placement of the TAS from the telomere of chromosome arm 2L next to the white reporter gene is sufficient to silence white expression. Unlike TAS in their normal location adjacent to telomeres, silencing by a nontelomeric TAS is not affected by atm mutations. These results indicate that the suppression of TPE is due to the specific action of atm on gene expression near telomeres (Oikemus, 2004).

In other organisms, loss of telomere protection can be due to the attrition or degradation of telomere repeat sequences. In Drosophila, it is possible to recover terminal deletions that remove all telomere-specific sequences. However, these observations do not rule out the possibility that telomere-specific sequences contribute to telomere protection or TPE at normal Drosophila telomeres. In fact, the number of telomere repeats has been shown to influence some forms of TPE. To test whether the telomere defects in atm- animals could be due to loss of telomere sequences, fluorescent in situ hybridization was performed using a probe to the Het-A retrotransposon, which is specific to telomere DNA. Hybridization was performed with wild-type and atm- diploid and polytene chromosomes. In mitotic chromosomes from diploid neuroblast cells, the levels of Het-A hybridization are variable, but not significantly different between wild-type and atm mutant cells. In polytene chromosomes, HeT-A sequences are strongly detected at two telomeres of both wild-type and atm- chromosomes. Previous analysis of HP1 mutants demonstrated that telomere-specific sequences were still present at chromosome fusion sites (Fanti, 1998b). In atm mutant cells, Het-A hybridization is also detected at sites of chromosome fusion. These results indicate that the reduction of telomeric HP1-HOAP and the fusion of telomeres in atm- cells is not a direct or indirect result of telomere sequence loss (Oikemus, 2004).

Both wild-type and terminally deleted Drosophila chromosomes are protected from telomere fusion and are capped with the telomere-protection proteins HP1 and HOAP (Biessmann, 1988; Fanti, 1998; Cenci, 2003). These results indicate that sequence-independent mechanisms can recruit and maintain telomere protection complexes to chromosome ends. This study has demonstrated that Drosophila atm/tefu is required to prevent chromosome end fusions, to regulate levels of HP1 and HOAP at telomeres, and to promote telomere-position effect. It is also found that atm is required for induction of apoptosis by ionizing radiation. Given the conserved role of ATM family proteins in recognizing DNA breaks, it is suggested that Drosophila ATM protects telomeres by recognizing chromosome ends and recruiting chromatin-modifying proteins to those ends (Oikemus, 2004).

To date ATM protein has not been directly detected at Drosophila telomeres. However, on the basis of results in mammalian cells, it may be necessary to develop antibodies specific for activated forms of ATM to probe ATM activity at telomeres (Bakkenist, 2003). However, several observations presented here indicate that Drosophila ATM acts at telomeres to prevent chromosome fusions. (1) The chromosome rearrangements observed are consistent with a defect in telomere protection rather than translocations due to defective DNA repair or replication. Most chromosome fusions occur near the ends of chromosome arms, and this study demonstrates that the fused chromosomes still contain telomeric DNA sequences. (2) A high frequency of acentric chromosome fragments is not observed during metaphase. In animals mutant for other damage-signaling genes such as the Drosophila homologs of ATR and ATRIP, acentric chromosome fragments are often observed during metaphase, suggesting that these mutations cause a defect in DNA repair or replication that is not observed in atm- animals. (3) Circular chromosomes do not undergo rearrangements in atmtefu mutant animals, strongly indicating that chromosome fusions are due to fusion of existing chromosome ends rather than the creation of new chromosome breaks. (4) ATM is specifically required for localization of HP1 to telomeres but not centromeric or euchromatic sites. (5) Loss of atm suppresses silencing by telomere-associated sequences when they are adjacent to telomeres, but not when they are at euchromatic sites (Oikemus, 2004).

The telomere fusion defect seen in atm- animals is consistent with a partial defect in telomere protection. Whereas ~80% of atm- metaphases contain a chromosome fusion, >95% of metaphases from animals lacking HP1 or HOAP contain a fusion (Fanti, 1998; Cenci, 2003). Furthermore, in some cells lacking HP1 or HOAP, nearly all telomeres appear to be fused. This extreme phenotype has not been observed in atm mutant nuclei. Consistent with a partial defect in telomere protection, the levels of HP1 and HOAP at polytene telomeres are found to be reduced, but not eliminated, in atm- animals. In mitotic cells, formation of repressive chromatin is also partially disrupted. The interpretation of these results is that reduced and variable levels of HP1 at the telomeres of atm- animals are sufficient to protect some, but not all telomeres from fusion. The results also indicate that another pathway, possibly involving other DNA damage-response proteins, must contribute to HP1 and HOAP localization, TPE, and telomere protection (Oikemus, 2004).

The direct target of ATM at telomeres is unclear. The decrease in HP1 and HOAP levels at atm- telomeres is not due to a loss of telomere sequences; wild-type and atm- chromosomes exhibit similar levels of a telomere-specific retrotransposon sequence as assayed by FISH, and even sites of fusion retain this sequence. This result is consistent with previous demonstrations that the sequences at chromosome ends are not required for telomere protection or for telomeric localization of HP1 and HOAP. Instead, ATM is likely to affect the interaction of HP1 and HOAP with telomeres by regulating the formation of the HP1-HOAP complex or by modification of telomeric chromatin. Other proteins in the DNA damage-response pathway may act with ATM to maintain telomere protection. Although Chk1, Chk2, and p53 are targets of mammalian ATM during the DNA damage response, Drosophila homologs of these proteins do not appear to be required for telomere protection; animals lacking one or more of these genes do not exhibit the high levels of apoptosis associated with loss of ATM. Mutations in homologs of other ATM targets such as NBS1 or SMC1 have not been described in Drosophila (Oikemus, 2004 and references therein).

Recruitment of HP1 and HOAP by ATM is likely to alter chromatin structure at telomeres. HP1 plays a conserved role in heterochromatin formation, histone modification, and gene silencing . In Drosophila, both HP1 and HOAP are required for gene silencing at pericentric heterochromatin. In addition, HP1 is required for gene silencing near fourth chromosome and terminally deleted telomeres, and for repression of P-element transposition by subtelomeric P-element insertions. HP1 homologs are also associated with telomere function in other eukaryotes. In mammals, all three HP1 homologs are found at telomeres, and loss of histone H3 methylases leads to reduced levels of HP1 homologs at telomeres as well as elongated telomeres. In contrast, overexpression of mammalian HP1 homologs is associated with decreased telomere length (Sharma, 2003). The fission yeast homolog of HP1 is not required for telomere protection, but does regulate telomere length, telomere clustering, and telomeric gene silencing. Interestingly, as in Drosophila telomere protection, some aspects of telomere function in fission yeast are controlled by an epigenetic mechanism. Together, these observations indicate that a requirement for HP1 in telomere function and chromatin structure is conserved, but that its precise role at the telomere may differ among organisms (Oikemus, 2004 and references therein).

Regulation of telomere chromatin structure is also a conserved function of ATM-like kinases. Fission yeast Rad3 and budding yeast Mec1 are required for gene silencing at telomeres and mutations in human ATM are associated with altered nucleosomal periodicity at telomeres. The conserved role of ATM-kinases in telomere protection and telomeric chromatin structure suggests that these functions might be linked. The finding that Drosophila ATM is required for TPE and HP1-HOAP localization to telomeres demonstrates one mechanism by which ATM can influence telomere chromatin. It is possible that in organisms that utilize sequence-specific binding proteins such as TRF2 (see Drosophila TRF2) to protect telomeres, regulation of telomeric heterochromatin by ATM and HP1 plays a minor role in protection of normal telomeres, but a more important role at short telomeres that cannot recruit sufficient levels of TRF2. Such a model might explain the synergistic telomere defects seen in cells lacking both telomerase and ATM. The lack of an obvious TRF2 homolog may explain why ATM and HP1 play such striking roles in the protection of Drosophila telomeres (Oikemus, 2004 and references therein).

In addition to preventing chromosome end fusion by DNA repair enzymes, telomere protection is required to prevent activation of DNA damage responses, including the induction of p53-dependent apoptosis and senescence. This analysis of the cellular effects of ATM loss indicates that induction of p53-dependent apoptosis is a conserved consequence of unprotected telomeres in metazoans. Because these unprotected telomeres lead to anaphase bridges and chromosome breaks, p53 may be directly activated by unprotected telomeres or may be activated by subsequent chromosome breaks. Drosophila ATM is required for the induction of apoptosis following IR. Because the spontaneous apoptosis in atm- animals is, by definition, ATM independent, a different pathway must be able to activate Drosophila p53 in response to unprotected telomeres. Similarly, loss of mammalian ATM reduces, but does not eliminate p53-dependent apoptosis in response to unprotected telomeres. Other DNA damage-response pathways may activate Drosophila p53 in the absence of ATM (Oikemus, 2004 and references therein).

In yeast, insects, and mammals, ATM-kinases are required to activate cellular responses to DNA ends generated by exogenous DNA damage, but also to suppress activation of these pathways by telomeres. Specific recognition of telomere sequences by telomere repeat-binding proteins provides one means to distinguish telomeric DNA ends from damage-induced DNA breaks. However, this mechanism is not sufficient to explain the epigenetic regulation of telomere protection in Drosophila. The requirement of ATM to recruit HP1 and HOAP to Drosophila telomeres suggests that recognition of chromosome ends contributes to chromatin-mediated telomere protection. This model may help explain how terminally deleted chromosomes can be stably inherited without any telomere-specific sequences. Future studies should reveal which other damage response proteins help ATM protect telomeres, what their targets are at telomeres, and how these proteins distinguish between damage-induced DNA ends and the natural ends of chromosomes (Oikemus, 2004).

Mechanisms of HP1-mediated gene silencing in Drosophila: HP1-mediated silencing can operate in a SU(VAR)3-9-independent and -dependent manner

HP1 is a structural component of silent chromatin at telomeres and centromeres. Euchromatic genes repositioned near heterochromatin by chromosomal rearrangements are typically silenced in an HP1-dependent manner. Silencing is thought to involve the spreading of heterochromatin proteins over the rearranged genes. HP1 associates with centric heterochromatin through an interaction with methylated lysine 9 of histone H3, a modification generated by SU(VAR)3-9. The current model for spreading of silent chromatin involves HP1-dependent recruitment of SU(VAR)3-9, resulting in the methylation of adjacent nucleosomes and association of HP1 along the chromatin fiber. To address mechanisms of silent chromatin formation and spreading, HP1 was fused to the DNA-binding domain of the E. coli lacI repressor and expressed in Drosophila melanogaster stocks carrying heat shock reporter genes positioned 1.9 and 3.7 kb downstream of lac operator repeats. Association of lacI-HP1 with the repeats results in silencing of both reporter genes and correlates with a closed chromatin structure consisting of regularly spaced nucleosomes, similar to that observed in centric heterochromatin. Chromatin immunoprecipitation experiments have demonstrated that HP1 spreads bi-directionally from the tethering site and associates with the silenced reporter transgenes. To examine mechanisms of spreading, the effects of a mutation in Su(var)3-9 were investigated. Silencing is minimally affected at 1.9 kb, but eliminated at 3.7 kb, suggesting that HP1-mediated silencing can operate in a SU(VAR)3-9-independent and -dependent manner (Danzer, 2004).

Upon daily production of the lacI-HP1 fusion protein (produced by heat shock), silencing of the reporter genes is observed at ectopic locations, even within regions of robust transcriptional activity. By contrast, a single pulse of lacI-HP1 in the embryo results in at best, partial silencing at the larval stage. This lack of mitotic stability is reminiscent of results obtained using tethered Polycomb (Pc), a protein required for the stable silencing of homeotic loci. Faithfully inherited silencing was observed with a single pulse of Gal4-Pc only when the transgene included a PRE (Polycomb Response Element), thought to stabilize the silencing complex. To date, HP1-mediated gene silencing has been shown to be relatively independent of DNA sequences; therefore, the continued presence of HP1 appears to be required for heritability of the silenced state (Danzer, 2004 and references therein).

Upon association of HP1 at these ectopic locations, changes were observed in gene expression and chromatin structure at least 3.7 kb from the lac repeat array. Sequences adjacent to the tethering site are relatively inaccessible to nuclease digestion and packaged into regular nucleosome arrays, mimicking a heterochromatic state. Such chromatin features are similar to those that form over euchromatic genes when placed into juxtaposition with heterochromatin. HP1 might cause chromatin reorganization through the recruitment of chromatin remodeling factors. An interaction between HP1 and chromatin remodeling machines has been documented in mammalian systems. Chromatin reorganization might also occur through the spread of HP1 along the chromosome. The data clearly demonstrate HP1 association within the promoter regions of silenced reporter genes up to 3.7 kb from the tethering site (Danzer, 2004).

In contrast to the silencing over several kb shown here, an HP1 tethering system using a stably integrated reporter gene in mammalian cell culture has demonstrated only short range effects over a few hundred base pairs. In this case, HP1Hsalpha was recruited to a reporter transgene through an interaction with tethered KRAB/KAP1 interaction partners. Silencing and a less accessible chromatin structure were apparent at 0.28 kb from the tethering site, but not at 2.78 kb. One possible explanation for the difference between these two tethering studies might be that human HP1Hsalpha and Drosophila HP1 have distinctly different silencing mechanisms. This is thought unlikely since human HP1Hsalpha localizes appropriately and rescues the lethality of Su(var)2-5 mutants when expressed in Drosophila. A second possibility is that lacI-HP1 overexpression enhances spreading, whereas the KRAB/KAP1 system operates under endogenous levels of HP1. A third explanation to account for the different results might be the manner in which the HP1 proteins are recruited to the reporter gene. Using the lacI-HP1 tethering system, recruitment occurs through a heterologous DNA-binding domain fused to the N terminus of HP1, thus leaving the CSD available for homodimerization and/or interaction with other partners. In the KRAB/KAP1 tethering system, recruitment occurs through an interaction between the HP1Hsalpha CSD and the transcriptional co-repressor KAP1, which may limit its availability for interactions with partners that are required for long distance spreading (Danzer, 2004 and references therein).

Transcriptional repressors can regulate gene expression over both short and long distances. Short-range repressors such as Giant and Krüppel operate at distances of less than 100 bp. These repressors frequently bind to sites within the promoter region and recruit histone deacetylases that locally deacetylate histone tails. By contrast, long-range silencing is hypothesized to involve the spread of silencing factors along the chromatin fiber, deacetylation of histone tails and generation of the MeH9K3 modification throughout the region. In experiments described here, silencing was observed 3.7 kb from the HP1 tethering site, implying that HP1 acts as a long-range silencer. Evidence of HP1 spreading is demonstrated by chromatin immunoprecipitation experiments that place HP1 near the promoter region of the silenced reporter genes. As the distance from the tethering site increases, the amount of HP1 association decreases, supporting a linear spreading model. However, these data do not exclude the possibility that HP1 association and silencing occur through a looping mechanism that is mediated by the `stickiness' of silencing proteins (Danzer, 2004 and references therein).

One proposed linear spreading model involves the association of HP1, subsequent recruitment of SU(VAR)3-9, and methylation of adjacent histones, forming new HP1-binding sites. This model was tested by examining the effects of HP1 tethering in a Su(var)3-9 mutant background. In the absence of SU(VAR)3-9, HP1 induced silencing of the hsp26 reporter persisted at 1.9 kb from the tethering site. Consistent with this finding Su(var)3-906 also had virtually no effect on silencing of a mini-white transgene positioned 0.5 kb from the HP1 tethering site. Taken together, these data suggest that silencing up to 1.9 kb is not heavily dependent upon SU(VAR)3-9 activity. It is speculated that HP1 might self-propagate for a limited distance along the chromosome, perhaps by multimerization through the CSD or by MeK9H3-independent interactions with histones. The introduction of HP1 mutants that abolish homodimerization into the tethering system will shed light on this issue (Danzer, 2004).

In contrast to the persistence of silencing at 1.9 kb in the Su(var)3-9 mutant, a substantial loss of silencing was observed at 3.7 kb. Heat shock-induced expression of hsp70 during HP1 tethering in a Su(var)3-906 mutant background was equal to expression levels observed in the non-tethering and GFP-tethering conditions. Several explanations could account for the different SU(VAR)3-9 requirements observed for silencing the hsp26 and hsp70 reporters. (1) The hsp70 transgene promoter might be stronger than the hsp26 transgene promoter -- this is thought unlikely since the hsp26 transgene appears to show greater fold induction than hsp70 at all five of the genomic insertion sites tested here under non-tethering conditions. (2) The two heat shock genes could have different mechanisms of transcriptional activation. This idea is inconsistent with years of research demonstrating that the regulatory elements and trans-activators for these two genes are nearly identical. (3) Alternatively, the differences observed might be due to multiple mechanisms of HP1-mediated silencing. Silencing at long distances (between 1.9 and 3.7 kb) may require SU(VAR)3-9, as current models for HP1 spreading would predict. By contrast, silencing at short distances (less than 1.9 kb) is relatively independent of SU(VAR)3-9, and would suggest alternate mechanisms of HP1 spreading that might involve self-propagation. This model is favored since several recent reports demonstrate that HP1 can be found independently of SU(VAR)3-9 and MeK9H3 on chromosomes. In particular, others have demonstrated that several genes silenced in Drosophila Kc cells were associated with HP1, but not SU(VAR)3-9. Thus, understanding of the role of HP1 in gene regulation will depend upon knowledge about the method of localization and the interaction partners at a given genomic site (Danzer, 2004).

Telomeric trans-silencing: an epigenetic repression combining RNA silencing and heterochromatin formation

The study of P-element repression in Drosophila led to the discovery of the telomeric Trans-Silencing Effect (TSE), a repression mechanism by which a transposon or a transgene inserted in subtelomeric heterochromatin (Telomeric Associated Sequence or TAS) has the capacity to repress in trans in the female germline, a homologous transposon, or transgene located in euchromatin. TSE shows variegation among egg chambers in ovaries when silencing is incomplete. This study reports that TSE displays an epigenetic transmission through meiosis, which involves an extrachromosomal maternally transmitted factor. This silencing is highly sensitive to mutations affecting both heterochromatin formation (Su(var)205 encoding Heterochromatin Protein 1 and Su(var)3-7) and the repeat-associated small interfering RNA (or rasiRNA) silencing pathway (aubergine, homeless, armitage, and piwi). In contrast, TSE is not sensitive to mutations affecting r2d2, which is involved in the small interfering RNA (or siRNA) silencing pathway, nor is it sensitive to a mutation in loquacious, which is involved in the micro RNA (or miRNA) silencing pathway. These results, taken together with the recent discovery of TAS homologous small RNAs associated to PIWI proteins, support the proposition that TSE involves a repeat-associated small interfering RNA pathway linked to heterochromatin formation, which was co-opted by the P element to establish repression of its own transposition after its recent invasion of the D. melanogaster genome. Therefore, the study of TSE provides insight into the genetic properties of a germline-specific small RNA silencing pathway (Josse, 2007; full text of article).

Repression of transposable elements (TEs) involves complex mechanisms that can be linked to either small RNA silencing pathways or chromatin structure modifications depending on the species and/or the TE family. Drosophila species are particularly relevant to the study of these repression mechanisms since some families of TEs are recent invaders, allowing genetic analysis to be carried out on strains with or without these TEs. In some cases, crossing these two types of strains induces hybrid dysgenesis, a syndrome of genetic abnormalities resulting from TE mobility. In D. virilis, repression of hybrid dysgenesis has been correlated to RNA silencing since small RNAs of the retroelement Penelope, responsible for dysgenesis, were detected in nondysgenic embryos but not in dysgenic embryos. In D. melanogaster, repression of retrotransposons can be established by noncoding fragments of the corresponding element (I factor, ZAM, and Idefix) and can be in some cases (gypsy, mdg1, copia, Het-A, TART, and ZAM, Idefix) sensitive to mutations in genes from the Argonaute family involved in small RNA silencing pathways. In the same species, strong repression of the DNA P TE, by a cellular state that has been called 'P cytotype', can be established by one or two telomeric P elements inserted in heterochromatic 'Telomeric Associated Sequences' (TAS) at the 1A cytological site corresponding to the left end of the X chromosome. This includes repression of dysgenic sterility resulting from P transposition. This P cytotype is sensitive to mutations affecting both Heterochromatin Protein 1 (HP1) (Ronsseray, 1996) and the Argonaute family member AUBERGINE (Reiss, 2004). P repression corresponds to a new picture of TE repression shown, using an assay directly linked to transposition, to be affected by heterochromatin and small RNA silencing mutants (Josse, 2007).

In the course of the study of P cytotype, a new silencing phenomenon has been discovered. Indeed, a P-lacZ transgene or a single defective P element inserted in TAS can repress expression of euchromatic P-lacZ insertions in the female germline in trans, if a certain length of homology exists between telomeric and euchromatic insertions. This homology-dependent silencing phenomenon has been termed Trans-Silencing Effect (TSE) (Roche, 1998). Telomeric transgenes, but not centromeric transgenes, can be silencers and all euchromatic P-lacZ insertions tested can be targets. TSE is restricted to the female germline and has a maternal effect since repression occurs only when the telomeric transgene is maternally inherited (Ronsseray, 2001). Further, when TSE is not complete, variegating germline lacZ repression is observed from one egg chamber to another, suggesting a chromatin-based mechanism of repression. Recently, an extensive analysis of small RNAs complexed with PIWI family proteins (AUBERGINE, PIWI, and AGO3) was performed in the Drosophila female germline. The latter study showed that most of the RNA sequences associated to these proteins derive from TEs. TSE corresponds likely to such a situation (Josse, 2007).

This study analyzed the genetic properties of TSE and shows that it has an epigenetic transmission through meiosis, which involves an extrachromosomal maternally transmitted stimulating component. Further, in order to investigate the mechanism behind TSE, a candidate gene analysis was performed to identify genes whose mutations impair TSE. It was found that TSE is strongly affected both by mutations in genes involved in heterochromatin formation and in the recently discovered small RNA silencing pathway called 'repeat-associated small interfering RNAs' (rasiRNA) pathway. In contrast, this study shows that TSE is not sensitive to genes specific to the classical RNA interference pathway linked to small interfering RNAs (siRNA) or to the micro RNA (miRNA) pathway. This suggests thus that TSE involves a rasiRNA pathway linked to heterochromatin formation and that such a mechanism, working in the germline, may underlie epigenetic transmission of repression through meiosis (Josse, 2007).

Developmental control of late replication and S phase length

Fast, early embryonic cell cycles have correspondingly fast S phases. In early Drosophila embryos, forks starting from closely spaced origins replicate the whole genome in 3.4 min, ten times faster than in embryonic cycle 14 and a hundred times faster than in a wing disc. It is not known how S phase duration is regulated. This study examined prolongation of embryonic S phases, its coupling to development, and its relationship to the appearance of heterochromatin. Imaging of fluorescent nucleotide incorporation and GFP-PCNA gave exquisite time resolution of S phase events. In the early S phases, satellite sequences replicated rapidly despite a compact chromatin structure. In S phases 11-13, a delay in satellite replication emerged in sync with modest and progressive prolongation of S phase. In S phase 14, major and distinct delays ordered the replication of satellites into a sequence that occupied much of S phase. This onset of late replication required transcription. Satellites only accumulated abundant heterochromatin protein 1 (HP1) after replicating in S phase 14. By cycle 15, satellites clustered in a compact HP1-positive mass, but replication occurred at decondensed foci at the surface of this mass. It is concluded that the slowing of S phase is an active process, not a titration of maternal replication machinery. Most sequences continue to replicate rapidly in successive cycles, but increasing delays in the replication of satellite sequences extend S phase. Although called constitutively heterochromatic, satellites acquire the distinctive features of heterochromatin, compaction, late replication, HP1 binding, and aggregation at the chromocenter, in successive steps coordinated with developmental progress (Shermoen, 2010).

This work shows that heterochromatin, long recognized as a key factor in the developmental programming of gene expression, also plays an integral role in the timing of the early embryonic cell cycles. Satellite sequences successively acquire features of heterochromatin, becoming late replicating by cycle 14, which prolongs S phase. This prolongation of S phase slows the early cell cycles and allows the progression to MBT (Shermoen, 2010).

This description of the successive introduction of the features of heterochromatin reveals a lack of interdependency of these features. For example, because it occurs earlier, the compaction of the satellite sequences is independent of late replication and of HP1 binding. It was also possible to visualize the events of late replication with unprecedented spatial and temporal resolution that gives insights into the replication of compacted HP1-bound chromatin (Shermoen, 2010).

Origin spacing could contribute to S phase length. However, electron microscopy studies show that origin spacing changes only slightly, from 7.9 to 10.6 kb, from preblastoderm embryos to cycle 14. Because forks are thought to converge at a rate of 3 kb/min, the additional separation would extend S phase by about 1 min, a minor contribution to the change from a 3.4 to a 50 min S phase (Shermoen, 2010).

S phase duration would also increase if all of the replicons did not replicate at the same time. However, asynchrony in replicon firing can occur in two ways, organized and unorganized. Unorganized asynchrony means that origins fire at different times without regard to their position in the genome. In this case, early- and late-firing origins can be juxtaposed. When replication from an early-firing origin reaches an adjacent later-firing origin just before it fires, one fork, rather than two, replicates the interorigin distance, doubling replication time. Greater unorganized asynchrony will result in passive replication of later origins and reduce the number of origins. Thus, given the known origin spacing, unorganized asynchrony is unlikely to make a very major contribution to the more than 10-fold increase in S phase length between preblastoderm cycles and cycle 14 (Shermoen, 2010).

If replication asynchrony is organized so that large regions of the genome (replication units) have many similarly behaving replicons, early-initiated forks invading a late region from the outside will not have time to replicate a significant portion of the large domain. The insulation resulting from distance can greatly magnify the impact of asynchrony on S phase duration. Organization of genomes into large replication units is widespread but poorly understood. This study shows that the satellite sequences are replication units and that embryonic changes in S phase duration result from change in the schedules of their replication. In preblastoderm cycles, satellite sequences replicate early, finishing in synch with general replication. Subsequently, satellite replication is increasingly delayed in parallel to S phase prolongation. Importantly, when satellite replication is late, it is deferred, not slow. For example, a 3.4 Mb Y-chromosomal repeat of the simple sequence AATAC begins to replicate 18 min into S phase 14. Each type of satellite sequence exhibits distinct replication delays. The 11 Mb X-chromosomal repeat of 359 bases, termed the 359 sequence has almost no delay in S phase 14, whereas AATAT and AATAACATAG finish replicating after 359 but before AATAC. The stereotyped schedules suggest that each replication unit has a characteristic 'lateness' parameter. This lateness parameter appears to be continuously variable in that there are many replication units, each with its own schedule of replication (Shermoen, 2010).

Though its replication is delayed, a unit such as AATAC replicates quickly once initiated (10 min). Although the 359 satellite is more slowly replicating (~15 min), it is suggested that it may be composed of separately and asynchronously replicating subdomains that were sometimes resolved. It is concluded that the dynamics of replication within a replication unit change only modestly during the early cycles (from 3.4 to roughly 10 min) (Shermoen, 2010).

In summary, the results argue that by S phase 14, the genome is replicated as a series of units, each of which replicates relatively quickly, but that a temporal program of sequential replication of these units creates a long S phase. This replication program resembles a consensus view of replication in slowly replicating cells of mammals and plants. It is concluded that progression from coincident replication of all of the replication units in a rapid S phase to sequential replication in a prolonged S phase 14 underlies prolongation of early embryonic S phases in Drosophila (Shermoen, 2010).

In widely divergent species and biological settings, heterochromatin has common characteristics including compaction, transcriptional quiescence, late replication, 'repressive' histone modifications, and association of specific heterochromatin proteins. This intimate association suggests mechanistic coupling of these features. If this were so, the various heterochromatin characteristics would emerge coordinately at the same moment during development. Instead, the current observations show temporal uncoupling during early Drosophila embryogenesis (Shermoen, 2010).

Although it was suggested that heterochromatin appears at cycle 14, both the cytological and biochemical manifestations of heterochromatin develop progressively. Foci of compacted chromatin that align with satellite sequences appeared in preblastoderm embryos prior to, and hence independently of, late replication and HP1 recruitment. Furthermore, HP1 binding to satellite sequences occurred late in cycle 14, after the onset of late replication. Because HP1 would have to decorate the satellite sequences at the onset of cycle 14 if it were required to suppress early replication and promote late replication, it is concluded that the late replication of satellite sequences is specified independently of the HP1 binding. Finally, satellite sequences reorganize; the previously independent foci of satellites aggregate into a large coherent HP1-positive region at the very beginning of interphase 15. This intimate association of satellites, which makes the chromocenter more coherent, is downstream of cycle 14 events and the MBT (Shermoen, 2010).

Together, these findings show that satellite sequences acquire the features of heterochromatin progressively. Compaction is present early, late replication is introduced subsequently, and recruitment of HP1 and then chromocenter maturation follow. Onset of position-effect variegation suggests that heterochromatic suppression of transcription begins in G2 of cycle 14 and mounts subsequently. Thus, heterochromatin does not form in a single step, and it acquires increasing influence during critical developmental events surrounding the MBT and gastrulation (Shermoen, 2010).

If compaction of chromatin prevents replication, decompaction might accompany or provoke replication. Real-time observations of PCNA and HP1 in cycle 15 show replication adjacent to, but not overlapping, HP1-bright foci of compacted chromatin. A more diffuse HP1 region appears adjacent to bright HP1 foci; PCNA overlies these fainter partner foci. Each partner focus appears and disappears as the PCNA signal rises and declines. It is concluded from this that replication does not occur in the compacted domain and that the sequences in the compacted HP1-bright focus unfurl during replication (Shermoen, 2010).

The persistence of in situ foci for 359 and AATAC shows that the satellites are not fully decondensed during replication. The size of partner HP1 foci also argues for limited decompaction. If an entire focus of compacted HP1-bright chromatin were to disperse, it would expand in volume, but the partner focus is about the same size as the brighter parent focus. Thus, it is suggested that a partner focus represents decompaction of a portion of the sequences harbored in the adjacent HP1-bright focus (Shermoen, 2010).

Following replication, heterochromatic sequences rapidly recompact. After an initial expansion, the partner HP1 focus does not grow throughout replication, and it shrinks and disappears as replication declines. When pulsed with fluorescent nucleotides for less than the replication time of the satellite, fluorescence overlies the compacted satellite sequence. Thus, it is suggested that DNA is “spooled” out of compacted foci, replicated, and returned to compacted foci shortly after replication. It the duration of replication-associated decompaction in embryonic cycle 15 is roughly estimated as 1 min. The dynamics, which are not easily consistent with decompaction of large topological domains, suggest that active replication forks drive local unfolding of chromatin structure, but the possibility cannot be excluded that transient decompaction might promote replication (Shermoen, 2010).

Mechanisms that couple the changing cell-cycle behavior with development are of great interest. Previous work suggested that the gradual prolongation of early cycles is secondary to gradual prolongation of S phase. A model in which the exponentially increasing amounts of DNA titrate replication components to prolong S phase is attractive but not presently supported (Shermoen, 2010).

The current results show that if a titration mechanism governs S phase duration, it is indirect. Injection of α-amanitin in cycle 13 prevented onset of late replication, accelerated S phase 14, and caused an early synchronous mitosis. Thus, activity of at least one of the DNA-dependent RNA polymerases is required to slow S phase, and the replication “hardware” needed for a rapid S phase is not limiting. Accordingly, if a titration mechanism were involved, the titrated component would regulate an upstream process. For example, transcription is restricted prior to cycle 14, and titration of a repressor might derepress transcription in late cycle 13, indirectly triggering onset of late replication (Shermoen, 2010).

Three findings suggest an abrupt switch to late replication at the beginning of cycle 14: the dramatic increase in S phase length, the accompanying switch of satellite sequences to delayed replication, and the requirement for transcription in cycle 13 for this transition. However, the late replication program of cycle 14 is anticipated by slight delays in replication of satellite sequences in cycles 12 and 13. These early changes suggest a more progressive process. It is proposed that early slight changes in replication timing and transcription initiate a positive feedback process that precipitates an abrupt change at the MBT. Rapid cell cycles suppress transcription and limit the time available to modify newly replicated chromatin, but, once the cycle begins to slow, transcription and heterochromatin modifications would accelerate to create conditions permissive for late replication, which would further slow the cycle (Shermoen, 2010).

Cooperative and antagonistic contributions of two heterochromatin proteins to transcriptional regulation of the Drosophila sex determination decision

Eukaryotic nuclei contain regions of differentially staining chromatin (heterochromatin), which remain condensed throughout the cell cycle and are largely transcriptionally silent. RNAi knockdown of the highly conserved heterochromatin protein HP1 in Drosophila was previously shown to preferentially reduce male viability. This study reports a similar phenotype for the telomeric partner of HP1, HOAP (Caravaggio), and roles for both proteins in regulating the Drosophila sex determination pathway. Specifically, these proteins regulate the critical decision in this pathway, firing of the establishment promoter of the masterswitch gene, Sex-lethal (Sxl). Female-specific activation of this promoter, SxlPe, is essential to females, as it provides SXL protein to initiate the productive female-specific splicing of later Sxl transcripts, which are transcribed from the maintenance promoter (SxlPm) in both sexes. HOAP mutants show inappropriate SxlPe firing in males and the concomitant inappropriate splicing of SxlPm-derived transcripts, while females show premature firing of SxlPe. HP1 mutants, by contrast, display SxlPm splicing defects in both sexes. Chromatin immunoprecipitation assays show both proteins are associated with SxlPe sequences. In embryos from HP1 mutant mothers and Sxl mutant fathers, female viability and RNA polymerase II recruitment to SxlPe are severely compromised. These genetic and biochemical assays indicate a repressing activity for HOAP and both activating and repressing roles for HP1 at SxlPe (Li, 2011).

The canonical heterochromatin protein HP1 is most commonly associated with constitutive heterochromatin and gene repression. This study reports a critical role for it in regulating one of the earliest decisions in metazoan development, whether to embark on a female or male path of sexual differentiation and dosage compensation. The role of heterochromatin in mammalian dosage compensation has been recognized from early work on the mouse. Although Drosophila utilizes a different mechanism to equalize X-linked gene dose, through hyper-activation of the single male X chromosome via chromatin modification, this study provides the first evidence of a role for heterochromatin proteins in the early events of Drosophila sex determination. HP1, together with its telomere partner HOAP, influence the critical decision in sex determination - activation of SxlPe, the Sxl establishment promoter (Li, 2011).

Reductions in HOAP preferentially compromise male viability. This was observed for two different cav mutant alleles and by reducing HOAP through RNAi. The presence of SxlPm-derived transcripts that have been spliced in the female mode in cav mutant males suggested inappropriate Sxl activation to be responsible for this reduced viability. In situ data indicating inappropriate firing of SxlPe in male embryos from cav2248 heterozygous parents support this view, as does the rescue of the cav2248 male viability defect by Sxl loss of function mutations. The more pronounced male lethality observed from reducing HOAP by RNAi expression driven by maternal, versus paternal, contribution of Actin5C GAL4 is consistent with such an early requirement for HOAP for male viability (Li, 2011).

Previous reports have shown that reducing HP1 by RNAi similarly reduces male viability preferentially. RT-PCR assays of SxlPm transcripts in HP1 mutants, however, suggested a more complex scenario as incorrect sex specific transcripts were observed in both sexes. This pointed to an activation, as well as repressor, role for HP1. Consistent with an activation role, reduction of maternal HP1 severely compromised female viability when the dose of Sxl was also reduced in the progeny, and ChIP assays of embryos from this cross showed recruitment of RNAP II to SxlPe to be impaired. This effect of reducing HP1 on female viability was strictly maternal, as was the antagonizing effect of simultaneously reducing maternal HOAP. Moreover, the partial rescue of the Su(var)205 maternal effect by the C-terminally truncated cav1 allele, which produces a protein that is compromised for HP1-binding, points to an involvement of HP1 in the antagonizing activity of HOAP. Finally, ChIP assays show a dependence of HP1 on HOAP for its association with SxlPe. Combined, these data indicate both antagonistic and cooperative roles for these heterochromatin proteins in regulating SxlPe, whereby HOAP acts as a repressor and HP1 acts as both an activator and repressor. The reliance of HP1 on HOAP for recruitment to the promoter would suggest HOAP may also have a role in the activation function of HP1 at the promoter, although this was not readily apparent in the assays used in this study (Li, 2011).

Although the data clearly show maternal roles for HOAP and HP1 in regulating the activity of SxlPe, for both HOAP and HP1, RNAi knockdown data indicate a substantial zygotic component in their effects on male viability. These zygotic effects, observed only in progeny carrying both an interference RNA transgene and a GAL4 driver transgene, suggest additional later sex-specific roles for both proteins. Such roles could be related to those observed for HP1 and SU(VAR)3-7 in male dosage compensation. Because the effect of reducing these proteins on the chromosomal distribution of DCC proteins is the opposite of those observed for males that are deficient for DCC proteins, as predicted to occur with inappropriate SxlPe expression, the activities of heterochromatin proteins in dosage compensation appear to be distinct from the early roles of HP1 and HOAP at SxlPe. In addition, there may be zygotic roles for heterochromatin proteins in sex-specific gene expression, as proposed for HP1 (Li, 2011).

Previous analysis of SxlPe indicated that 400 bp immediately upstream of the promoter are sufficient for sex-specific regulation, but distal sequences, extending to -1700 bp, are required for wild type levels of expression, E-box binding sites for antagonistically acting bHLH proteins, which are encoded by zygotically expressed X-linked and autosomal signal elements (XSE and ASE) and direct an X counting mechanism, are distributed throughout both regions (Li, 2011).

Both HP1 and HOAP are enriched in the region proximal to SxlPe which contains binding sites for both positive and negative E-box proteins. Within the SxlPe promoter distal region, HOAP alone is enriched in two peaks where there is a striking relationship with E-box binding sites for positive factors, but those for negative factors appear essentially devoid of HOAP. HOAP may antagonize positive factors but permit negative factors to bind in the SxlPe distal region, in an HP1-independent repressing role. Whereas loss of HOAP de-represses SxlPe in males, the strength and uniformity of expression does not approach that in wild type females. This indicates continued influence from the X counting mechanism in cav mutant males. SxlPe is also expressed prematurely in female embryos. This de-repression by reduced levels of maternal HOAP in both sexes indicates that HOAP is present at SxlPe in both sexes of wild type embryos. However, whether the proximal and distal SxlPe regions have the same or different compositions of HOAP and HP1 in the two sexes cannot be determined from ChIP assays, as the embryos are of mixed sexual identity (Li, 2011).

The interdependency of HOAP and HP1 for their binding to the SxlPe proximal region, most notably the dependence of HP1 on HOAP, also indicates both proteins are in this region in, at least, wild type female embryos. In spite of this interdependency, the genetic data show HOAP repression antagonizes HP1 activation. HOAP repression appears to also be partly HP1-dependent; the mutant HOAP protein from the cav1 allele which lacks HP1-binding also antagonizes HP1 activation. This combination of antagonistic and cooperative interactions suggests a model in which maternal HOAP and HP1 first cooperate to repress SxlPe prior to its activation. The repressive structure formed by maternal HOAP and HP1 likely serves to reduce the sensitivity of SxlPe to spurious fluctuations in zygotic XSE levels, ensuring it is only activated in females where an effective ratio of activating to repressing transcription factors exists. HP1 is retained at SxlPe during its activation in females, where it presumably switches into an activation role. In early embryos constitutive heterochromatin proteins may be more appropriate for such regulation than the Polycomb Group of facultative heterochromatin proteins, as they would not be subject to cross regulatory signals from body plan specification pathways (Li, 2011).

How HP1 switches over to transcriptional activation mode in the SxlPe proximal region is unclear. Changes in HP1 phosphorylation and/or association with other factors could alter its activity. Several XSE (X-linked element) binding sites are nearby, making them strong candidates. Presumably, this would only occur in females where the XSE dose surpasses a threshold and SxlPe is activated (Li, 2011).

This report provides the most clearly defined role for HP1 in developmental control of a euchromatic gene in a metazoan species, and the first evidence of a bifunctional regulatory role for it in such a context. Prior reports describing HP1 in transcriptional activation have focused on it in the context of transcription elongation. ChIP data at SxlPe, however, show a requirement of it for association of RNAP II with the promoter, more consistent with a role in transcription initiation. A role in initiation is also in keeping with the position of HP1 on the gene; very little HP1 is found elsewhere on the Sxl gene, even during the time of SxlPe activity. This dependence of RNAP II association on HP1 is similar to what is observed in the accumulation of noncoding RNAs at S. pombe centromeric repeats and mating type locus. Nonetheless, it is possible that the loss of RNAP II at SxlPe reflects reduced stability of all RNAP II isoforms as a consequence of an early defect in transcription elongation, rather than a defect in RNAP II recruitment to the promoter (Li, 2011).

Pausing of RNAP II in promoter proximal regions prior to activation has been observed in a high proportion of genes under developmental control in Drosophila embryos, and such pauses have also been implicated in regulation of alternative splicing. While SxlPm appears to have the features of a promoter with paused RNAP II in a genome wide RNAP II ChIP study of 0-4 hr embryos, RNAP II was absent from SxlPe. It is likely that the collection window for this study did not precisely coincide with the time of SxlPe activity. A more narrowly timed collection indicates paused RNAP II at SxlPe, suggesting that, like SxlPm, it is a pre-loaded promoter. A preloaded SxlPe also readily explains how generalized up-regulation of phosphorylation of the RNAP II CTD by the loss of Nanos, causes SxlPe activation in males with an unchanged X:A ratio (Li, 2011).

Finally, the dominant negative activity of the cav2248 allele suggests a role for the partially deleted SRY-like HMG box in HOAP association with SxlPe. ChIP data show HOAP association with the SxlPe proximal region is required for HP1 association. This proposed role for the HMG box of HOAP in SxlPe regulation is of particular interest with regards to a recent report linking HP1 and KAP-1 (TIF1β) to SRY-dependent repression of testis-specific genes in the ovary. Because mammalian sex determination is inextricably linked to gonad sex determination, SRY and HOAP each appear to constitute early decision points in their respective sex determination pathways. There are, perhaps, unexpected parallels between these divergent pathways (Li, 2011).

POF and HP1 bind expressed exons, suggesting a balancing mechanism for gene regulation

Two specific chromosome-targeting and gene regulatory systems are present in Drosophila melanogaster. The male X chromosome is targeted by the male-specific lethal complex believed to mediate the 2-fold up-regulation of the X-linked genes, and the highly heterochromatic fourth chromosome is specifically targeted by the Painting of fourth (POF) protein, which, together with heterochromatin protein 1 (HP1), modulates the expression level of genes on the fourth chromosome. This study used chromatin immunoprecipitation and tiling microarray analysis to map POF and HP1 on the fourth chromosome in S2 cells and salivary glands at high resolution. The enrichment profiles were complemented by transcript profiles to examine the link between binding and transcripts. The results show that POF specifically binds to genes, with a strong preference for exons, and the HP1 binding profile is a mirror image of POF, although HP1 displays an additional 'peak' in the promoter regions of bound genes. HP1 binding within genes is much higher than the basal HP1 enrichment on Chromosome 4. The results suggest a balancing mechanism for the regulation of the fourth chromosome where POF and HP1 competitively bind at increasing levels with increased transcriptional activity. In addition, the results contradict the idea that transposable elements are a major nucleation site for HP1 on the fourth chromosome (Johansson, 2007).

Previously studies have shown that POF and HP1 colocalize at the resolution given by polytene chromosomes and that the same set of genes is regulated by these two proteins (Johansson, 2007a). This study shows that POF and HP1 binding colocalize at the gene level, which also provides insights into the modes of action for this regulatory system. The bias in within-gene binding towards exons shown by POF was also observed for HP1. HP1, like POF, preferentially binds exons but, in contrast to POF, HP1 shows a high basal binding to the fourth chromosome and a peak in binding associated with promoters for most targeted genes. The specificity of HP1 to certain promoters at the individual target gene level has previously been reported in mammals. According to the current data the promoter peak of HP1 is a more general characteristic of HP1-bound genes on the fourth chromosome and is related to transcription. It has been proposed that the presence of H3K9me at promoters is connected to gene repression, but that H3K9me within the genes is associated with gene activity. If the HP1 profile is assumed to be linked to the presence of H3K9me this implies that a combination of these two binding profiles is linked to the transcription of genes on the fourth chromosome (Johansson, 2007).

The classical view is that HP1 is associated with gene repression. However, a number of recent reports have linked HP1 to gene activation, based on the enrichment of H3K9me and HP1 on active genes. It is well known that HP1 is enriched in pericentric regions and that genes in those regions are, therefore, connected to high HP1 levels. It has been shown that mutation in HP1 causes a reduction in the expression of a number of heterochromatin-located genes in Drosophila e.g., light and rolled, supporting the idea that some genes depend on their heterochromatic surroundings for correct expression. However, it has also been demonstrated that HP1 is associated with the transcribed regions of active genes located in euchromatic regions. In addition, HP1 has been shown to associate with developmental and heatshock-induced puffs of the polytene chromosome, which is indicative of intense gene activity. It has been shown that H3K9 methylation occurs in the transcribed region of active genes in mammalian chromatin and, in fact, increases during activation of transcription. In that case HP1 was found to be associated with the transcribed genes of several mammalian cell lines and also in primary cells. However, it is important to note that, except for the heterochromatin genes light and rolled,, it is not clear whether the binding of HP1 is associated with facilitated transcription. The current results indicate not only that HP1 binds to active genes on the fourth chromosome, but also that the genes on the fourth chromosome are up-regulated upon loss of HP1. Thus, although the genes on the fourth chromosome are bound in response to gene activity, HP1 still causes repression. It may be that, for example, heat-shock induced genes attract HP1 as a modulator that represses uncontrolled gene expression. In contrast to HP1, the loss of POF leads to a general decrease in gene expression from the fourth chromosome. The strong correlation with respect to binding between HP1 and POF and their correlation with transcription support the balancing model (Johansson, 2007a): POF stimulates and HP1 represses gene expression and the interdependent binding of these two proteins fine tunes the expression output from the fourth chromosome. It should be noted that the correlation between HP1 and POF seems to be linear, suggesting that highly expressed genes have the same POF/HP1 ratio as genes with weak expression, although they bind higher amounts of both proteins. A balancing mechanism may act as a buffering system in which the dual recruitment of a repressing and a stimulating factor makes the transcription efficiency more stable and less sensitive to fluctuations. Balancing mechanisms may be more general. For example, this may explain the proposed binding of HP1 to the male X chromosome. The facilitated transcription of X chromosomal genes by acetylation of H4K16 may need to be tempered by a repressing factor to reach the expected 2-fold increase. This repressing function might be supported by HP1, Su(var)3-7, or other unknown factors not yet linked to dosage compensation (Johansson, 2007).

The targeting of POF to the fourth chromosome shows similarities to the targeting of the MSL complex to the male X chromosome. The striking similarity between POF and the dosage-compensating MSL complex in evolutionary terms, their function as chromosome-wide regulators, and their binding profiles, as presented as presented in this study, supports a common origin. For the MSL complex, expressed genes are the main targets. In addition, in fly embryos, S2 cells, and in a cell line derived from larval imaginal discs (Clone8 cells), MSL binding is also associated with expressed genes, but does not correlate with level of expression. It has been demonstrated that, to a large extent, MSL binding is stable throughout development and that the binding reflects the expression levels in young embryos (4–5 h). It is hypothesized that a similar strong correlation between levels of transcript and binding as well as cell type differences as seen for POF, might be true also for MSL if studied at higher resolution. A correlation between binding levels and levels of transcription would be in line with the expected 2-fold increase of gene expression independendent of expression levels (Johansson, 2007).

It has been shown that transgenes inserted on the fourth chromosome are often partially silenced and that the localization of these variegated insertions in some regions of chromosome 4 is correlated to their distance from the transposable element 1360 (Sun, 2004). This, along with the fact that the 1360 element can contribute to the silencing of an adjacent reporter when close to pericentric heterochromatin, suggests that 1360 elements may serve as HP1 recruitment signals. Further support for this hypothesis is provided by the suggestion that repeat flanked genes are more likely to bind HP1. The current results show that HP1 binds Chromosome 4 genes, but no indications were found of transposable elements such as 1360 acting as nucleation sites for HP1. It should be stressed that these results do not contradict the reported correlation between transgenic silencing and distance from 1360 elements. It is possible that 1360 elements under certain conditions serve as nucleation sites for heterochromatin formation and spread, but that this does not involve HP1. It is also possible that 1360 elements act as initial nucleation sites for HP1, which are not maintained in the two cell types analyzed. Furthermore, silenced transgene insertions on Chromosome 4 are linked to regions with relatively high binding of HP1, but these regions are typically expressed. This implies that the transcriptional consequences of high HP1 levels differ between inserted transgenes and endogenous Chromosome 4 genes. It is speculated that POF is needed for expression of these Chromosome 4 genes and that inserted transgenes will be repressed by HP1 but will fail to recruit POF (Johansson, 2007).

HP1a, Su(var)3-9, SETDB1 and POF stimulate or repress gene expression depending on genomic position, gene length and expression pattern in Drosophila melanogaster

Heterochromatin protein 1a (HP1a) is a chromatin-associated protein important for the formation and maintenance of heterochromatin. In Drosophila, the two histone methyltransferases SETDB1 and Su(var)3-9 mediate H3K9 methylation marks that initiates the establishment and spreading of HP1a-enriched chromatin. Although HP1a is generally regarded as a factor that represses gene transcription, several reports have linked HP1a binding to active genes, and in some cases, it has been shown to stimulate transcriptional activity. To clarify the function of HP1a in transcription regulation and its association with Su(var)3-9, SETDB1 and the chromosome 4-specific protein Painting of fourth (POF), genome-wide expression studies were performed and the results were combined with available binding data in Drosophila melanogaster. The results suggest that HP1a, SETDB1 and Su(var)3-9 repress genes on chromosome 4, where non-ubiquitously expressed genes are preferentially targeted, and stimulate genes in pericentromeric regions. Further, it was shown that on chromosome 4, Su(var)3-9, SETDB1 and HP1a target the same genes. In addition, it was found that transposons are repressed by HP1a and Su(var)3-9 and that the binding level and expression effects of HP1a are affected by gene length. The results indicate that genes have adapted to be properly expressed in their local chromatin environment (Lundberg, 2013).

Heterochromatin protein 1 is a protein that has been well-studied in many model organisms, including Schizosaccharomyces pombe, mouse and D. melanogaster. Although D. melanogaster HP1a is best known for its role in heterochromatin formation and silencing, several reports have linked HP1a to regulation of transcriptional activity of heterochromatic and some euchromatic genes. This study asked if these conflicting results could partly be explained by a region-specific function of HP1a and the proteins involved in HP1a binding, i.e. SETDB1, Su(var)3-9 and POF. Based on polytene chromosome staining, it was clear that POF, HP1a and SETDB1 overlapped on chromosome 4 but not on the pericentromeric section or on the most distal part of the tip, which was only bound by HP1a. These POF and SETDB1 unbound regions also correspond to regions that are independent of SETDB1 for maintaining a proper H3K9me2 and me3 pattern. In line with previous studies, it was found that Su(var)3-9 binds to chromosome 4 when considering expressed genes, and more interestingly, this binding to active genes is, on average, stronger than the binding of Su(var)3-9 to active genes in the pericentromeric regions, although loss of Su(var)3-9 had minor effects on the methylation pattern of chromosome 4. The putative function of Su(var)3-9 on chromosome 4 therefore remains elusive (Lundberg, 2013).

In addition to the persistent binding of POF to chromosome 4, it is interesting to note the presence of occasional binding to region 2L:31. It is known that POF binds to HP1a binding sites where HP1a binding is dependent on SETDB1, and since it has been previously shown that binding of HP1a in region 2L:31 is dependent on SETDB1, this could partially explain the sporadic binding of POF in this region. Region 2L:31 displayed similar properties to other euchromatic regions that are unbound by SETDB1 and HP1a. Thus, the reason for the targeting of this particular region remains to be explained (Lundberg, 2013).

HP1a has long been known for its repressive function. It was initially identified as a dominant suppressor of position-effect variegation and was named Su(var)205, and it has been reported that HP1a represses gene expression on chromosome 4. However, several studies have reported an activating function of HP1a. The current study suggests that these conflicting reports can at least be partly explained by the observation that HP1a has different functions in different regions; chromosome 4 genes are, on average, repressed, whereas pericentromeric genes are stimulated. It is therefore believed that it is important to look at different groups of genes when studying the effects of HP1a. Otherwise, these opposing effects may cancel each other out on a genome-wide level (Lundberg, 2013).

Nevertheless, the conflicting results cannot be fully explained by the current findings. For example, another study found that transcription was reduced in an RNAi-mediated HP1a knock-down, in contrast to the current results. Therefore, technical differences between experiments should also be considered; in the current study, the possibility of a maternal contribution of HP1a cannot be excluded, as mutants were studied in first-instar larvae from heterozygous mothers, and it is thus likely that we have a reduction in HP1a levels rather than complete removal. It has been shown that maternal HP1a contributes to ~20% of the HP1a protein found in heterozygous mutant first-instar larvae. Previously studies have shown that the average level of gene expression of chromosome 4 is comparable with, or even higher than, that of genes on other chromosomes. At least to some extent, this is a consequence of POF-mediated stimulation of gene expression output, which counteracts the repressing nature of the 4th chromosome (Johansson, 2007; Stenberg, 2009; Johansson, 2012). It is hypothesized that due to POF and other factors, genes on the 4th chromosome have evolved to be functional in this repressive chromatin environment. A decrease in HP1a is mainly expected to cause a reduction of the low affinity binding of HP1a in the gene body, and consequently a de-repression of gene expression. However, prolonged loss or very strong depletion of HP1a will most probably have dramatic effects on the overall structure of chromosome 4 chromatin, and thus lead to a dysfunctional chromatin structure with decreased gene expression. This implies that the genes have adapted to be properly expressed in the local chromatin environment (Lundberg, 2013).

Previous studies have shown that POF is involved in stimulating expression of active genes on chromosome 4. The observed effects in the Pof mutant on genes in the pericentromeric regions and region 2L:31 are most likely explained by indirect effects when HP1a is being redistributed from chromosome 4 to other binding sites, as binding of HP1a to chromosome 4 is dependent on the presence of POF (Riddle, 2012; Figueiredo, 2012; Johansson, 2007). The increased transcriptional output of chromosome 4 genes in the Setdb1 mutant is likely due to loss of the repressive methylation marks, which in turn will reduce HP1a binding. Although it is known that HP1a binding to promoters is independent of methylation marks, it is possible that HP1a binding remains in promoters, where it exerts an activating function (Lundberg, 2013).

The increased chromosome 4 expression observed in the Su(var)3-9 mutant is surprising but could be explained by indirect effects; it is speculated that when Su(var)3-9 is lost from the pericentromeric regions, SETDB1 is redirected from chromosome 4 to sustain normal H3K9 methylation in the pericentromeric regions, thus decreasing HP1a binding to chromosome 4. This could explain why both the Setdb1 and the Su(var)3-9 mutants give such similar up-regulating effects on chromosome 4 expression. An alternative explanation for this effect is that the observed binding of Su(var)3-9 to chromosome 4 has a yet-unknown repressing function independent of the HKMT function of Su(var)3-9 (Lundberg, 2013).

The HP1a Pof double-mutant displayed weak non-significant up-regulation of chromosome 4, with marginally larger error bars than for the HP1a mutant, which supports the suggested balancing mechanism of chromosome 4, where HP1a and POF fine-tune the transcriptional output; in the absence of both components, the overall expression will not change but individual genes will start losing proper transcriptional control (Lundberg, 2013).

Although previous studies have indicated that SETDB1 and Su(var)3-9 have separate main targets, the data show that the majority of genes that are up- or down-regulated in Su(var)3-9 mutants are correspondingly up- or down-regulated in Setdb1 mutants. These results suggest that a redundancy exists between these two proteins, in which both proteins, to some extent, have the ability to be redirected to other locations when needed, as we know that Su(var)3-9 has a chromosome 4 binding capacity. Alternatively, the HP1a system might affect a number of genetic networks so that even if different regions are affected by Su(var)3-9 and Setdb1, the same genetic networks may be indirectly affected. Because Su(var)3-9 affects larger regions than Setdb1, it is likely that more HP1a will be released and redirected to other regions in the Su(var)3-9 mutant than in a Setdb1 mutant, thus causing repression of genes normally unbound by HP1a. This would explain why more genes are down-regulated in the Su(var)3-9 mutant compared with the Setdb1 mutant (Lundberg, 2013).

These results provide strong support for the suggested model in which transposons are repressed by HP1 proteins, as shown for HP1a, the HP1 homolog Rhino and also Su(var)3-9. In contrast, neither SETDB1 nor POF had any effects on transposon expression. Because SETDB1 is known to have a role in repression of chromosome 4, one could speculate that SETDB1 has a greater influence on repression of transposons located specifically on chromosome 4 than in other parts of the genome. However, due to the repetitive nature of the transposons and the methods used in this study, it was not possible to distinguish effects for transposons in specific regions (Lundberg, 2013).

The observation that chromosome 4 displays a stronger effect on non-ubiquitously expressed genes (NUEGs), both in terms of down-regulation in the Pof mutant and up-regulation in the HP1a mutant, is supported by previous findings on chromosome 4. One potential explanation for this is that NUEGs have evolved to respond to a regulatory mechanism, whereas UEGs are more robust in expression. Although weak, it is noteworthy that the effect of the HP1a mutant in the pericentromeric regions (decreased gene expression) was slightly stronger for UEGs than NUEGs; this is in line with the relatively strong binding peak found in promoters compared with the gene body in pericentromeric UEGs, as it has been proposed that HP1a in the promoter has a stimulating effect and HP1a in the gene body of chromosome 4 genes has an inhibiting effect. Note that the number of NUEGs exceeds the number of UEGs on a whole-genome level and on chromosome 4, whereas in pericentromeric regions, the UEGs are over-represented (Lundberg, 2013).

In summary, these data support a model in which HP1a binding to promoters in general has a positive function for transcriptional output, whereas HP1a binding in gene bodies has a negative function. If binding in the gene body is relatively large compared with binding to the promoter, the negative function will dominate. In contrast, if the binding to the promoter is stronger than the gene body, the stimulating effect will be larger, albeit not dominating. A reduction in HP1a levels will initially affect the low-affinity gene binding and sequentially, the loss of HP1a will also affect the promoter binding (Lundberg, 2013).

The average binding level of HP1a is constant irrespective of gene length (the HP1a binding per length unit is constant), implying that longer genes have more HP1a molecules bound in total. This finding, in combination with the suggestion that the repressive effect of HP1a is mainly observed in the gene body, could explain the greater de-repression of longer genes. Stronger binding of HP1a to long genes has also been suggested in previous studies. In addition, some chromatin marks mostly associated with active chromatin have been shown to bind differently to different gene lengths, suggesting that gene length affects the level of association with chromatin marks. Furthermore, there are indications that HP1 proteins are involved with transcription machinery; the mammalian HP1 isoform gamma and H3K9me3 regulate transcriptional activation by associating with the RNA polymerase II (RNP2), and HP1 can interact with and guide the recruitment of the histone chaperone complex FACT to active genes, which facilitates RNP2 transcription elongation. This, along with the current findings, suggests a mechanism in which HP1a is involved in transcriptional elongation. It is speculated that HP1a slows down the progression of the RNP2 through the length of the gene body. HP1a binding mechanisms could also be connected with RNA interactions, as HP1a has been shown to directly interact with RNA transcripts and heterogeneous nuclear ribonucleoproteins, and HP1a association to centric regions in Drosophila and mice is sensitive to RNase treatment (Lundberg, 2013).

Interestingly, it was discovered that a group of non-annotated short genes (<0.5 kb) were repressed by HP1a, even though the HP1a binding levels appeared to be low (which might be explained by technical aspects in determining the binding levels). The lack of annotation and lower wild-type expression level suggest that this group consists of many short genes encoding ncRNAs (Lundberg, 2013).

In the pericentromeric regions of the genome, an interesting connection was observed between the binding and stimulating effects of HP1a and the position of the gene; the closer the gene is located to the centromeric chromatin, the more strongly HP1a binds and stimulates it (Lundberg, 2013).

In conclusion, it was found that HP1a has opposite functions in different genomic regions, repressing expression on chromosome 4 and stimulating expression in pericentromeric regions. Furthermore, the targets of Su(var)3-9 and SETDB1 are considerably more redundant than previously reported, and the overlap between HP1a, Su(var)3-9 and SETDB1 on chromosome 4 genes is extensive. It is however important to note that the different effects caused by HP1a, SETDB1, Su(var)3-9 and POF could all be interrelated to create a balanced genome. Therefore, it is hard to distinguish the separate effects caused by the different proteins (Lundberg, 2013).

Protein Interactions

Heterochromatin protein 1 (HP1) is localized at heterochromatin sites where it mediates gene silencing. The chromo domain of HP1 is necessary for both targeting and transcriptional repression. In the fission yeast Schizosaccharomyces pombe, the correct localization of Swi6 (the HP1 equivalent) depends on Clr4, a homolog of the mammalian SUV39H1 histone methylase. Both Clr4 and SUV39H1 specifically methylate lysine 9 of histone H3. In this study it has been shown show that HP1 can bind with high affinity to histone H3 methylated at lysine 9 but not at lysine 4. The chromo domain of HP1 is identified as its methyl-lysine-binding domain. A point mutation in the chromo domain, which destroys the gene silencing activity of HP1 in Drosophila, abolishes methyl-lysine-binding activity. Genetic and biochemical analysis in S. pombe shows that the methylase activity of Clr4 is necessary for the correct localization of Swi6 at centromeric heterochromatin and for gene silencing. These results provide a stepwise model for the formation of a transcriptionally silent heterochromatin: SUV39H1 places a 'methyl marker' on histone H3, which is then recognized by HP1 through its chromo domain. This model may also explain the stable inheritance of the heterochromatic state (Bannister, 2001).

Recent studies show that heterochromatin-associated protein-1 (HP1) recognizes a 'histone code' involving methylated Lys9 (methyl-K9) in histone H3. Using in situ immunofluorescence, it has been demonstrated that methyl-K9 H3 and Drosophila HP1 co-localize to the heterochromatic regions of Drosophila polytene chromosomes. NMR spectra show that methyl-K9 binding of HP1 occurs via its chromo (chromosome organization modifier) domain. This interaction requires methyl-K9 to reside within the proper context of H3 sequence. NMR studies indicate that the methylated H3 tail binds in a groove of HP1 consisting of conserved residues. Using fluorescence anisotropy and isothermal titration calorimetry, it has been determined that this interaction occurs with a KD of ~100 µM, with the binding enthalpically driven. A V26M mutation in HP1, which disrupts its gene silencing function, severely destabilizes the H3-binding interface, and abolishes methyl-K9 H3 tail binding. Sequence diversity in chromo domains may lead to diverse functions in eukaryotic gene regulation. For example, the chromo domain of the yeast histone acetyltransferase Esa1 does not interact with methyl- K9 H3, but instead shows preference for unmodified H3 tail (Jacobs, 2001).

HP1 is thought to affect chromatin structure through interactions with other proteins in heterochromatin. Chromo domains located near the amino (amino chromo) and carboxy (chromo shadow) termini of HP1 may mediate such interactions, as suggested by domain swapping, in vitro binding and 3D structural studies. Several HP1-associated proteins have been reported, providing candidates that might specifically complex with the chromo domains of HP1. However, such association studies provide little mechanistic insight and explore only a limited set of potential interactions in a largely non-competitive setting. To determine how chromo domains can selectively interact with other proteins, random peptide phage display libraries were probed using chromo domains from HP1. The results demonstrate that a consensus pentapeptide is sufficient for specific interaction with the HP1 chromo shadow domain. The pentapeptide is found in the amino acid sequence of reported HP1-associated proteins, including the shadow domain itself. Peptides that bind the shadow domain also disrupt shadow domain dimers. These results suggest that HP1 dimerization, which is thought to mediate heterochromatin compaction and cohesion, occurs via pentapeptide binding. In general, chromo domains may function by avidly binding short peptides at the surface of chromatin-associated proteins (Smothers, 1999).

The available 3D structure of the chromo domain suggests how it might bind short peptides. The mouse Mod1 chromo domain contains a hydrophobic groove, formed by the triple antiparallel strands and carboxy-terminal helix of the module. This hydrophobic groove is a candidate protein-interaction site and, on the basis of sequence conservation, is expected to be present in all chromo domains (including shadow domains). The chromo shadow consensus pentapeptide derived in this study would fit into this groove. This is consistent with the hydrophobicity of the consensus, providing a biochemically avid site for the protein-peptide interactions that were observed (Smothers, 1999).

To investigate the potential biological significance of peptides selected by the chromo shadow domain, Drosophila protein sequence databases as well as reported HP1-associated protein sequences were examined. Given the short length of the selected peptides, p values from database searches are too insignificant to identify previously unknown HP1-interacting proteins with confidence. Nevertheless, using a conservative standard for similarity, the consensus sequence is found in reported HP1-associated proteins including D. melanogaster HP1 itself; Su(var)3-7, which co-immunoprecipitates and co-localizes with HP1; transcription intermediate factors (TIFs), and the p150 subunit of chromatin assembly factor-1 (CAF-1) (Smothers, 1999).

HP1 chromo shadow domains self-dimerize in vitro, possibly because the consensus is contained within the chromo shadow region of the HP1 sequence. Other studies provide genetic and cytological evidence that HP1 may dimerize through its chromo shadow domain in vivo. To ascertain whether the peptides that were identified in vitro are relevant to this interaction, it was asked whether or not shadow-specific peptides can disrupt self-dimerization. Beads saturated with chromo shadow domain protein were incubated with phage displaying either streptavidin-interacting or chromo shadow domain-interacting peptides. Chromo shadow protein freed by competitive interaction was concentrated and examined by SDS-PAGE and silver staining. All six chromo shadow domain-interacting peptides, but neither of the control peptides, were found to disrupt chromo shadow dimers; this result was confirmed using synthesized peptides. The fact that all six peptides successfully disrupt self-dimerization suggests that the consensus pentamer is critical for chromo shadow interactions in general. Furthermore, this result provides a means by which HP1 dimerization can mediate heterochromatin compaction and cohesion (Smothers, 1999).

Position-effect variegation results from mosaic silencing by chromosomal rearrangements juxtaposing euchromatin genes next to pericentric heterochromatin. An increase in the amounts of the heterochromatin-associated Su(var)3-7 and HP1 proteins augments silencing. Using the yeast two-hybrid protein interaction trap system, HP1 has been isolated using Su(var)3-7 as a bait. Three binding sites on Su(var)3-7 for HP1 have been delimited. On HP1, the C-terminal moiety, including the chromo shadow domain, is required for interaction. In vivo, both proteins co-localize not only in heterochromatin, but also in a limited set of sites in euchromatin and at telomeres. When delocalized to the sites bound by the protein Polycomb in euchromatin, HP1 recruits Su(var)3-7. In contrast with euchromatin genes, a decrease in the amounts of both proteins enhances variegation of the light gene, one of the few genetic loci mapped within pericentric heterochromatin. This body of data supports a direct link between Su(var)3-7 and HP1 in the genomic silencing of position-effect variegation (Delattre, 2000).

The HP1 chromo domain, like the Polycomb chromo domain, has chromosome binding activity, but only to distinct chromosomal sites. A chimeric HP1-Polycomb protein consisting of the chromo domain of Polycomb in the context of HP1 binds to both heterochromatin and Polycomb binding sites in polytene chromosomes. In flies expressing chimeric HP1-Polycomb protein, endogenous HP1 is mislocalized to Polycomb binding sites, and endogenous Polycomb is misdirected to the heterochromatic chromocenter, suggesting that both proteins are recruited to their distinct chromosomal binding sites through protein-protein contacts. Chimeric HP1-Polycomb protein expression in transgenic flies promotes heterochromatin-mediated gene silencing, supporting the view that the chromo domain homology reflects a common mechanistic basis for homeotic and heterochromatic silencing (Platero, 1995).

The ability of a chimeric HP1-Polycomb (PC) protein to bind both to heterochromatin and to euchromatic sites of PC protein binding was exploited to detect stable protein-protein interactions in vivo. Endogenous PC protein is recruited to ectopic heterochromatic binding sites by the chimeric protein. Posterior sex combs (PSC) protein also is recruited to heterochromatin by the chimeric protein, demonstrating that PSC protein participates in direct protein-protein interaction with PC protein or PC-associated proteins. In flies carrying temperature-sensitive alleles of Enhancer of zeste[E(z)] the general decondensation of polytene chromosomes that occurs at the restrictive temperature is associated with loss of binding of endogenous PC and chimeric HP1-Polycomb protein to euchromatin, but binding of HP1 and chimeric HP1-Polycomb protein to the heterochromatin is maintained. The E(z) mutation also results in the loss of chimera-dependent binding to heterochromatin by endogenous PC and PSC proteins at the restrictive temperature, suggesting that interaction of these proteins is mediated by E(Z) protein. A myc-tagged full-length Suppressor 2 of zeste [SU(Z)2] protein interacts poorly or not at all with ectopic Pc-G complexes, but a truncated SU(Z)2 protein is strongly recruited to all sites of chimeric protein binding. Trithorax protein is not recruited to the heterochromatin by the chimeric HP1-Polycomb protein, suggesting either that this protein does not interact directly with Pc-G complexes or that such interactions are regulated. Ectopic binding of chimeric chromosomal proteins provides a useful tool for distinguishing specific protein-protein interactions from specific protein-DNA interactions important for complex assembly in vivo (Platero, 1996).

The Su(var)3-7 locus of Drosophila melanogaster has two alternative transcripts and seven scattered xzinc fingers, each preceded by a tryptophan box. An increase in the dose of Su(var)3-7 enhances the genomic silencing of position-effect variegation caused by centromeric heterochromatin. The product of Su(var)3-7 is a nuclear protein that associates with pericentromeric heterochromatin at interphase, whether on diploid chromosomes from embryonic nuclei or on polytene chromosomes from larval salivary glands. Large amounts of protein are detected in the cytoplasm. The protein does not associate with mitotic chromosomes, but associates with the partially heterochromatic chromosome 4 during interphase. Since these phenotypes and localizations resemble those described by others for the Su(var)2-5 locus and its heterochromatin-associated protein HP1, the presumed co-operation of the two proteins was tested further. The effect of the dose of Su(var)3-7 on the silencing of a number of variegating rearrangements and insertions is strikingly similar to the effect of the dose of Su(var)2-5 reported by others. The two loci interact genetically, and the two proteins co-immunoprecipitate from nuclear extracts. These results suggest that SU(VAR)3-7 and HP1 co-operate in building the genomic silencing associated with heterochromatin (Cléard, 1997).

The heterochromatin-associated nonhistone chromosomal protein HP1 exerts dosage-dependent effects on the silencing of genes juxtaposed to pericentric heterochromatin. HP1 is multiply phosphorylated in Drosophila tissue, predominantly at serine and threonine residues. Phosphorylation is relatively rapid and phosphate is incorporated into existing protein. Maternally synthesized HP1 is underphosphorylated. The appearance of more highly phosphorylated HP1 isoforms at 1.5-2 h of development coincides with the embryonic stage at which cytologically visible heterochromatin appears (HP1 concentrates in heterochromatin). The extent of HP1 phosphorylation is lower in polytene tissue, where heterochromatin is underrepresented. These results are consistent with a role for phosphorylation of HP1, either in the assembly and maintenance of heterochromatin (Eissenberg, 1994).

Position-effect varigation, the spotty appearance of gene inactivation that accompanies placing of active euchromatic genes near heterochromatin, is accompanied by compaction of the corresponding chromosomal regions. The compaction can be continuous (bands and interbands located distal to the eu-heterochromatic junction fuse into one dense block), or discontinuous (two or more zones of compaction are separated by morphologically and functionally normal regions). In both continuous and discontinuous compaction, the blocks of dense material contain the immunochemically detectable protein HP1, characterized as specific for heterochromatin. The regions undergoing compaction do not contain HP1 when they have a normal banding pattern. Thus, it has been proposed that HP1 is one of the factors involved in compaction (Belyaeva, 1993).

The origin recognition complex (ORC) is required to initiate eukaryotic DNA replication and also engages in transcriptional silencing in S. cerevisiae. There is a striking preferential but not exclusive association of Drosophila ORC2 with heterochromatin on interphase and mitotic chromosomes. DmORC is found on chromatin at all cell cycle stages of the embryonic syncytium in a diffuse, granular pattern throughout the DNA but is highly concentrated at foci along the apical surface of the interphase nuclei, consistent with the known orientation of pericentric heterochromatin. No differences in DmORC distribution are apparent in embryos after cellularization. HP1, a heterochromatin-localized protein required for position effect variegation (PEV), colocalizes with DmORC2 at these sites. Consistent with this localization, intact DmORC and HP1 are found in physical complex. DmORC2, 5 and 6 are also found in this complex. Neither DmORC2 nor 6 show reproducible interactions with HP1. The association of Origin recognition complex subunit 1 (ORC1) with HP1 is shown biochemically to require the chromodomain and shadow domains of HP1. Amino acid residues 161-319 of DmORC1 are likely to carry multiple sites of contact with HP1. The amino terminus of DmORC1 contains a strong HP1-binding site, mirroring an interaction found independently in Xenopus by a yeast two-hybrid screen. Heterozygous DmORC2 recessive lethal mutations result in a suppression of PEV. These results indicate that ORC may play a widespread role in packaging chromosomal domains through interactions with heterochromatin-organizing factors (Pak, 1997).

The distinct structural properties of heterochromatin accommodate a diverse group of vital chromosome functions: only rudimentary molecular details of its structure are available. A powerful tool in the analysis of its structure in Drosophila has been a group of mutations that reverse the repressive effect of heterochromatin on the expression of a gene placed next to it ectopically. Several genes from this group are known to encode proteins enriched in heterochromatin. The best characterized of these is the heterochromatin-associated protein, HP1. HP1 has no known DNA-binding activity, hence its incorporation into heterochromatin is likely to be dependent on other proteins. To examine HP1 interacting proteins, three distinct oligomeric species of HP1 have been isolated from the cytoplasm of early Drosophila embryos and their compositions analyzed. The two larger oligomers share two properties with the fraction of HP1 that is most tightly associated with the chromatin of interphase nuclei: an underphosphorylated HP1 isoform profile and an association with subunits of the origin recognition complex (ORC). HP1 localization into heterochromatin is disrupted in mutants for the ORC2 subunit. These findings support a role for the ORC-containing oligomers in localizing HP1 into Drosophila heterochromatin that is strikingly similar to the role of ORC in recruiting the Sir1 protein to silencing nucleation sites in Saccharomyces cerevisiae (Huang, 1998).

The actin-related proteins have been identified by virtue of their sequence similarity to actin. While their structures are thought to be closely homologous to actin, they exhibit a far greater range of functional diversity. The Drosophila actin-related protein, Arp4, has been localized to the nucleus. It is most abundant during embryogenesis but is expressed at all developmental stages. Within the nucleus Arp4 is primarily localized to the centric heterochromatin. It is also present at much lower levels in numerous euchromatic bands, as indicated by polytene chromosome spreads. The only other protein in Drosophila reported to be primarily localized to centric heterochromatin in polytene nuclei is Heterochromatin protein 1 (HP1). Genetic evidence has linked HP1 to heterochromatin-mediated gene silencing and alterations in chromatin structure. The relationship between Arp4 and Heterochromatin protein 1 was investigated by labeling embryos and larval tissues with antibodies to Arp4 and HP1. Arp4 and HP1 exhibit almost superimposable heterochromatin localization patterns, remain associated with the heterochromatin throughout prepupal development, and exhibit similar changes in localization during the cell cycle. Polytene chromosome spreads indicate that the set of euchromatic bands labeled by each antibody overlap, but are not identical. In parallel, Arp4 and HP1 undergo several shifts in their nuclear localization patterns during embryogenesis, shifts that correlate with developmental changes in nuclear functions. The significance of their colocalization was further tested by examining nuclei that express mutant forms of HP1. In these nuclei the localization patterns of HP1 and Arp4 are altered in parallel fashion. The morphological, developmental and genetic data suggest that, like HP1, Arp4 may have a role in heterochromatin functions (Frankel, 1997).

Heterochromatin-associated protein 1 (HP1) is a nonhistone chromosomal protein with a dose-dependent effect on heterochromatin mediated position-effect silencing. It is multiply phosphorylated in vivo. Hyperphosphorylation of HP1 is correlated with heterochromatin assembly. HP1 is phosphorylated by casein kinase II in vivo at three serine residues located at the N and C termini of the protein. Alanine substitution mutations in the casein kinase II target phosphorylation sites dramatically reduce the heterochromatin binding activity of HP1, whereas glutamate substitution mutations, which mimic the charge contributions of phosphorylated serine, apparently have wild-type binding activity. It is proposed that phosphorylation of HP1 promotes protein-protein interaction between HP1 and target binding proteins in heterochromatin (Zhao, 1999).

The methods used to identify HP1 phosphorylation sites involved direct comparison of the in vivo and in vitro tryptic peptide map by high concentration PAGE, rHP1 phosphopeptide sequencing, and radioactivity detection of each amino acid derivative. For all three sites common to phosphorylated recombinate HP1 (rHP1) and HP1 derived from whole flies (dHP1), the targets are good fits to CKII consensus motifs, which, together with the sensitivity of rHP1 phosphorylation to spermine, heparin, and anti-Drosophila CKII serum, strongly suggests that HP1 is a substrate for CKII. CKII is a ubiquitous cyclic nucleotide-independent protein kinase that appears not to directly mediate known signaling pathways. CKII activity has been found to increase in response to some mitogens, and its substrates include a number of transcription factors involved in growth control. Because CKII is found both in the nucleus and the cytoplasm, and because alanine substitution has no effect on nuclear targeting, HP1 phosphorylation by CKII could occur in either compartment (Zhao, 1999 and references).

CKII consensus target sites are found at the N and/or C terminus of HP1 homologs from Drosophila virilis, Schizosaccaromyces pombe, mealybug, mouse, and human. Not all HP1 homologs have CKII targets at both ends (some have neither), but in several such cases the homologous position is occupied by glutamate. Little or nothing is known about the functional homology between Drosophila melanogaster HP1 and its structural homologs in other species, but such apparent structural conservation suggests functional conservation. Nevertheless, the data presented here showing that CKII phosphorylation is required for efficient heterochromatin targeting by the unique D. melanogaster HP1 suggest that such structural conservation is likely to be functionally significant. CKII phosphorylation could contribute to HP1 heterochromatin binding by promoting a conformational shift that permits (1) additional kinases to phosphorylate internal targets in, for example, the HP1 linker region between the chromo domains, or (2) the exposure of sites for protein-protein interactions. Either of these results could facilitate heterochromatin assembly. This interval is serine/threonine-rich and includes two consensus targets for protein kinase A and one for protein kinase C (Zhao, 1999 and references).

Phosphorylation on both the N- and the C-terminal CKII sites is required for heterochromatin binding. Although the Ser->Ala mutation on the C-terminal site does not discernibly alter the heterochromatin binding activity of the mutant fusion protein, the Ser->Ala mutation on the N-terminal site conspicuously reduces heterochromatin binding. The double Ser->Ala mutation (S15A,S202A) almost completely eliminates heterochromatin binding, although the protein can still get into the nucleus. The double mutant appears to have a generally more severe effect. However, care should be taken in interpreting quantitative differences, because levels of fusion protein expression vary from cell to cell in these assays. Although the effect of the single C-terminal substitution was not detectable by the X-gal staining method, it is possible that each mutation exerts some effect on HP1 heterochromatin binding activity because the combined mutations have the most dramatic effect on heterochromatin binding. Although there are two CKII sites at the C terminus of HP1, only the more downstream site was mutated. The upstream site is dependent on the phosphorylation of the downstream serine, so when the first serine was mutated to alanine, the second one as a CKII target was also disabled. Thus any effect attributable to the downstream serine could also reflect a requirement for phosphorylation of the upstream serine. Experiments are in progress to identify additional sites of HP1 phosphorylation and to test their role in HP1 localization and silencing activity (Zhao, 1999).

Although transformants were recovered with Ser->Glu mutations in the N or C terminus, germline transformants with Ser->Ala mutations could be recovered. The significance of this finding is unclear, but it may represent a kind of 'dominant negative' phenotype. In the absence of heat shock, basal levels of wild-type HP1·beta-galactosidase fusion protein are not toxic, although such transgenic lines are not as healthy as wild-type flies. However, mutant HP1 fusion protein may be toxic at low basal levels. A reasonable speculation is that the nonphosphorylated HP1 participates in only some HP1-dependent activities or sequesters heterochromatin factors in an inactive form (Zhao, 1999).

The most basic HP1 isoforms in vivo are phosphorylated at CKII sites. Thus, CKII phosphorylation does not directly account for the hyperphosphorylation that accompanies the appearance of heterochromatin in the early embryonic development. Indeed, it probably accounts for the maternally loaded HP1 isoforms seen in unfertilized oocytes. Nevertheless, the mutational analysis shows that CKII phosphorylation is essential for heterochromatin binding. CKII is an ubiquitous eukaryotic serine/threonine protein kinase that phosphorylates more than 100 substrates, many of which control cell division or signal transduction. These substrates include a striking number of nuclear proteins involved in DNA replication and transcription. CKII modifies protein-DNA binding and protein-protein interaction. In Drosophila, CKII is present in both the cytoplasmic and nuclear compartments. CKII phosphorylation enhances the DNA binding activity of the Engrailed protein and modulates Antennapedia activity and dorsoventral patterning. Drosophila DNA topoisomerase II is stimulated by CKII phosphorylation. Significant HP1 phosphorylation still occurs in vivo in tissues treated with sufficient cycloheximide to block all detectable nascent protein synthesis. This turnover of phosphate uncoupled from a new synthesis suggests that HP1 phosphorylation could regulate its chromatin association, an example being the dynamic dissociation and reassociation of HP1 that reportedly takes place during mitosis. Alternatively, phosphorylation-dephosphorylation may be regulated during decondensation of heterochromatin to permit DNA replication in late S phase (Zhao, 1999).

Methylation of lysine 9 in histone H3 is recognized by HP1

Specific modifications to histones are essential epigenetic markers---heritable changes in gene expression that do not affect the DNA sequence. Methylation of lysine 9 in histone H3 is recognized by heterochromatin protein 1 (HP1), which directs the binding of other proteins to control chromatin structure and gene expression. HP1 uses an induced-fit mechanism for recognition of this modification, as revealed by the structure of its chromodomain bound to a histone H3 peptide dimethylated at Nzeta of lysine 9. The binding pocket for the N-methyl groups is provided by three aromatic side chains, Tyr21, Trp42 and Phe45, which reside in two regions that become ordered on binding of the peptide. The side chain of Lys9 is almost fully extended and surrounded by residues that are conserved in many other chromodomains. The QTAR peptide sequence preceding Lys9 makes most of the additional interactions with the chromodomain, with HP1 residues Val23, Leu40, Trp42, Leu58 and Cys60 appearing to be a major determinant of specificity by binding the key buried Ala7. These findings predict which other chromodomains will bind methylated proteins and suggest a motif that they recognize (P. R. Nielsen, 2002).

Interaction of HP1 with Suppressor of variegation 3-9

The consensus peptide is found in only a subset of the proteins reported to interact with HP1. The consensus is absent from inner centromere protein (INCENP), origin recognition complex (ORC) proteins, an actin-related protein (ARP4), Suppressor of variegation 3-9, SP100 proteins and lamin B receptor (LBR). The simplest explanation for this apparent discrepancy is that these proteins engage HP1 differently from proteins that contain the consensus. For example, the amino chromo domain or hinge region of HP1 may be necessary for many of these interactions. In fact, INCENP specifically engages the hinge region of mammal HP1 orthologs, and both the amino chromo and the chromo shadow domains of HP1 are required to associate with ORC complexes. For ARP4 and Su(var)3-9, the means by which HP1 engages them is undetermined. Perhaps less easily explained are the SP100 and LBR proteins, factors that are proposed to interact with the chromo shadow domain of HP1 yet lack the consensus sequence determined from this study (Smothers, 1999 and references therein).

In Drosophila, heterochromatin protein 1 (HP1) suppresses the expression of euchromatic genes that are artificially translocated adjacent to heterochromatin by expanding heterochromatin structure into neighboring euchromatin. The purpose of this study was to determine whether HP1 functions as a transcriptional repressor in the absence of chromosome rearrangements. HP1 normally represses the expression of four euchromatic genes in a dosage-dependent manner. Three genes regulated by HP1 map to cytological region 31 of chromosome 2, which is immunostained by anti-HP1 antibodies in the salivary gland. The repressive effect of HP1 is decreased by mutation in Su(var)3-9, whose mammalian ortholog encodes a histone H3 methyltransferase and mutation in Su(var)2-1, which is correlated with histone H4 deacetylation. These data provide genetic evidence that an HP1-family protein represses the expression of euchromatic genes in a metazoan, and that histone modifiers cooperate with HP1 in euchromatic gene repression (Hwang, 2001).

Su(var)3-9 is a dominant modifier of heterochromatin-induced gene silencing. Like its mammalian and Schizosaccharomyces pombe homologs, Su(var) 3-9 encodes a histone methyltransferase (HMTase), which selectively methylates histone H3 at lysine 9 (H3-K9). In Su(var)3-9 null mutants, H3-K9 methylation at chromocenter heterochromatin is strongly reduced, indicating that Su(var)3- 9 is the major heterochromatin-specific HMTase in Drosophila. Su(var)3-9 interacts with the heterochromatin-associated HP1 protein and with another silencing factor, Su(var)3-7. Notably, interaction between Su(var)39 and Hp1 is interdependent and governs distinct localization patterns of both proteins. In Su(var)3-9 null mutants, concentration of Hp1 at the chromocenter is nearly lost without affecting Hp1 accumulation at the fourth chromosome. By contrast, in Hp1 null mutants, Su(var)3-9 is no longer restricted at heterochromatin but broadly disperses across the chromosomes. Despite this interdependence, Su(var)3-9 dominates the PEV modifier effects of Hp1 and Su(var)3-7 and is also epistatic to the Y chromosome effect on PEV. Finally, the human SUV39H1 gene is able to partially rescue Su(var)3-9 silencing defects. Together, these data indicate a central role for the SU(VAR)3- 9 HMTase in heterochromatin-induced gene silencing in Drosophila (Schotta, 2002).

In Drosophila, histone H3-K9 methylation is strongly enriched in chromocenter heterochromatin and the fourth chromosome. Immunocytological studies revealed that SU(VAR)3-9 preferentially causes H3-K9 methylation within chromocenter heterochromatin. Although these results suggest a significant role of H3-K9 methylation in altering chromatin structure and gene activity during development, further studies are required to understand how integral components of higher order chromatin complexes in heterochromatin are assembled and their function is regulated (Schotta, 2002).

Su(var)3-9 and Hp1 represent evolutionarily conserved components of heterochromatin protein complexes. Interaction between the two proteins has been suggested for the mammalian homologs. In Drosophila, the N-terminus of Su(var)3-9 and the chromo-shadow domain region of Hp1 constitute the sites where these proteins interact. Ectopic association of Su(var)3-9-EGFP along euchromatic regions in Hp1-deficient salivary gland nuclei and strongly reduced binding of Hp1- EGFP to chromocenter heterochromatin in Su(var)3-9-deficient nuclei suggest that interaction between both proteins is essential for their association with chromocenter heterochromatin. H3-K9 methylation creates chromodomain-dependent binding sites of Hp1. Strong reduction of Hp1-EGFP heterochromatin binding in Su(var)3-9 null mutants might reflect a requirement of Hp1 binding to methylated H3-K9 for heterochromatin-association of Su(var)3-9- Hp1 complexes. These results suggest a multistep control for heterochromatin association of Su(var)3-9-Hp1 complexes. After primary association of Su(var)3-9 with heterochromatin, consecutive H3-K9 methylation by Su(var)3-9 would create binding sites of Hp1, which finally results in stable association of Su(var)3-9-Hp1 complexes with heterochromatin. These processes are likely to be controlled by several other as yet unknown factors. In these processes the chromodomain as well as the SET domain of Su(var)3-9 might be directly involved. Fusion proteins deleting either the chromodomain or the SET domain only show restricted binding to heterochromatin (Schotta, 2002).

Although Su(var)3-9 associates with the fourth chromosome, H3-K9 methylation in the fourth chromosome is not changed in Su(var)3-9 null mutants, suggesting that H3-K9 methylation in this chromosome is controlled by a different HMTase activity. In contrast to Su(var)3-9 association with chromocenter heterochromatin, which depends on the chromodomain and the SET domain, for its binding to the fourth chromosome the N-terminus is sufficient. A special chromatin structure of the fourth chromosome is also indicated by identification of Painting of fourth (Pof), a chromosome four-specific protein. Different requirements of Su(var)3-9 and Hp1 association with the fourth chromosome and chromocenter heterochromatin suggest occurrence of heterochromatin protein complexes of different composition, as well as differential control of their assembly (Schotta, 2002).

Structure-function analysis with transgenic Su(var) 3- 9- EGFP protein variants reveals new aspects of their role in heterochromatin localization of Su(var)3-9. In contrast to studies with human SUV39H1, in vivo heterochromatin association of Su(var)3-9-EGFP protein variants was analyzed in nuclei deficient for the endogenous Su(var)3-9 protein. The N-terminus of Su(var)3-9 (amino acids 81-188), which contains the interaction domain to Hp1 and Su(var)3-7, is involved in heterochromatin association of the protein. However, association of the truncated protein is restricted to the fourth chromosome and the central region of chromocenter heterochromatin. Deletion of the chromodomain in Su(var)3-9 also affects its normal chromosomal distribution and reduces binding to chromocenter heterochromatin, but not with the fourth chromosome. In contrast, deletion or point mutations of the chromodomain result in ectopic distribution of human SUV39H1 in HeLa cells. These findings might indicate functional differences between the Su(var)3-9 and SUV39H1 chromodomain. In both Su(var)3-9 and SUV39H1, the N-terminus contains the interaction surface for Hp1 and Hp1ß, respectively. In human cells, overexpression of SUV39H1 results in ectopic chromosomal distribution. In contrast, even after strong overexpression of Su(var)3-9, no comparable effects were observed in Drosophila (Schotta, 2002).

Deletion of the SET domain or an exchange of the Su(var)3-9 SET domain with the SET domain of the Trx protein strongly affects heterochromatin distribution of the proteins. The proteins become concentrated within the middle of chromocenter heterochromatin, but again show normal association with the fourth chromosome. This suggests that the SET domain of Su(var)3-9 is directly involved in the control of Su(var)3-9 association with chromocenter heterochromatin. In Drosophila, aberrant heterochromatin distribution of Su(var)3-9 proteins with SET domain mutations could be causally connected with suppression of heterochromatin-induced gene silencing. Comparable results have been obtained for clr4, the Schizosaccharomyces pombe homolog of Su(var)3-9, where mutations in the SET domain show defects in silencing and mating-type switching. However, in S.pombe swi6, the homolog of Su(var)2-5 represents the main dosage-dependent component of gene silencing at the mat2/3 locus, whereas only subtle effects of clr4 are reported. These functional differences observed for Su(var)3-9 and Hp1 orthologs in fission yeast, Drosophila and mammals might reflect considerable functional and/or structural differences of the silencing complexes in these organisms (Schotta, 2002).

Aberrant heterochromatin distribution of Su(var)3-9 SET domain mutant proteins suggests involvement of other factors in a functional control of the SET domain. These factors might also affect its HMTase activity. Mutations in genes encoding these putative regulatory genes should be genetically epistatic to the triplo-dependent enhancer effect of Su(var)3-9. Proteins like the SET domain-binding factor Sbf1, which has been shown to be involved in regulation of the phosphorylation state of the SET domain, might also play a central role. Identification of PEV enhancer mutations like ptn D (see pitkin) that cause ectopic binding of Su(var)3-9 and Hp1 to many euchromatic sites indicates the existence of different positive as well as negative control mechanisms for chromosomal distribution of heterochromatin protein complexes. Further studies of modifiers of PEV mutations will contribute substantially to understanding of the complex regulatory processes involved in the control of higher order chromatin structure and heterochromatin-induced gene silencing (Schotta, 2002).

The chromodomain of the HP1 family of proteins recognizes histone tails with specifically methylated lysines. Structural, energetic, and mutational analyses are presented of the complex between the Drosophila HP1 chromodomain and the histone H3 tail with a methyllysine at residue 9, a modification associated with epigenetic silencing. The histone tail inserts as a beta strand, completing the beta-sandwich architecture of the chromodomain. The methylammonium group is caged by three aromatic side chains, whereas adjacent residues form discerning contacts with one face of the chromodomain. Comparison of dimethyl- and trimethyllysine-containing complexes suggests a role for cation-pi and van der Waals interactions, with trimethylation slightly improving the binding affinity (Jacobs, 2002).

Heterochromatin protein 2 (HP2), a partner of HP1 in Drosophila heterochromatin

Heterochromatin protein 1 (HP1), first discovered in Drosophila melanogaster, is a highly conserved chromosomal protein implicated in both heterochromatin formation and gene silencing. This study reports characterization of an HP1-interacting protein, heterochromatin protein 2 (HP2), which codistributes with HP1 in the pericentric heterochromatin. HP2 is a large protein with two major isoforms of approximately 356 and 176 kDa. The smaller isoform is produced from an alternative splicing pattern in which two exons are skipped. Both isoforms contain the domain that interacts with HP1; the larger isoform contains two AT-hook motifs. Mutations recovered in HP2 act as dominant suppressors of position effect variegation, confirming a role in heterochromatin spreading and gene silencing (Shaffer, 2002).

HP2 is a large chromosomal protein that interacts with HP1 both in a yeast two-hybrid assay and by coimmunoprecipitation. The two proteins colocalize on polytene chromosomes; HP2 is recruited to ectopic HP1 sites in vivo. HP1 both recognizes H3-mK9, a marker of heterochromatin, and interacts with the modifying methyltransferase, SU(VAR)3-9. These interactions suggest a model for the spread of this packaging form along the chromatin fiber, a property of heterochromatin inferred from the observation of PEV. The finding that mutations in HP2 can lead to suppression of PEV indicates a similar requirement for its participation in heterochromatin formation and spreading, with associated gene silencing. HP1 also has been shown to play a role in silencing at some euchromatic sites, including genes within region. HP2 is observed in that region of the genome as well, although, overall, HP2 association with euchromatic sites appears to be less than that found for HP1 (Shaffer, 2002).

The gene for HP2 produces two transcripts, generated by inclusion or omission of exons 5 and 6; two proteins are detected with the predicted sizes of 176 and 356 kDa. The larger protein contains two AT hooks, protein motifs thought to contribute to heterochromatin assembly and stability by binding to AT-rich satellite DNA through the minor groove. One of the point mutations in HP2 leading to suppression of PEV is located close to the AT hooks. Although there are no other recognizable motifs, HP2 does have a recognizable and unusual amino acid composition. Both isoforms are very rich in serine, as well as the four charged amino acids. BLAST searches of the nonredundant protein database with HP2-L find no proteins with significant similarity when compositional bias-based statistics are used. BLAST searches without compositional bias compensation find a wide variety of proteins rich in serine and the charged amino acids, including another HP1-binding protein, mouse ATRX (Shaffer, 2002).

ATRX, a transcription regulator, is localized to pericentric heterochromatin and the short arms of acrocentric chromosomes; mutations in the gene result in changes in DNA methylation patterns. The segment of the protein (amino acids 325-1176) that interacts with a mouse HP1 homologue does not possess any recognizable structure, but also is rich in serine and the charged amino acids (53.7%). This suggests that HP1 may interact with proteins such as HP2 and ATRX on the basis of a compositional motif and suggests further that HP2 might have multiple interaction sites for HP1. Although HP1 may well act as a dimer, interactions of HP1 with a very large protein such as HP2 could be important in condensation of large chromatin domains. The identification of a novel protein of this type, and demonstration that mutations in the protein result in suppression of PEV, indicates the importance of identifying and characterizing additional partners of HP1 to further exploration of the mechanisms involved in heterochromatin condensation and gene silencing (Shaffer, 2002).

Novel Drosophila Heterochromatin protein 1 (HP1)/Origin recognition complex-associated protein (HOAP) repeat motif in HP1/HOAP interactions and chromocenter associations

Association of the highly conserved heterochromatin protein, HP1, with the specialized chromatin of centromeres and telomeres requires binding to a specific histone H3 modification of methylation on lysine 9. This modification is catalyzed by the Drosophila Su(var)3-9 gene product and its homologues. Specific DNA binding activities are also likely to be required for targeting this activity along with HP1 to specific chromosomal regions. The Drosophila HOAP protein is a DNA-binding protein that was identified as a component of a multiprotein complex of HP1 containing Drosophila origin recognition complex (ORC) subunits in the early Drosophila embryo. Direct physical interactions are demonstrated between the HOAP protein and HP1 and specific ORC subunits. Two additional HP1-like proteins (HP1b and HP1c) were recently identified in Drosophila, and the unique chromosomal distribution of each isoform is determined by two independently acting HP1 domains (hinge and chromoshadow domain). Heterochromatin protein 1/origin recognition complex-associated protein (HOAP) is found to interact specifically with the originally described predominantly heterochromatic HP1a protein. Both the hinge and chromoshadow domains of HP1a are required for its interaction with HOAP, and a novel peptide repeat located in the carboxyl terminus of the HOAP protein is required for the interaction with the HP1 hinge domain. Peptides that interfere with HP1a/HOAP interactions in co-precipitation experiments also displace HP1 from the heterochromatic chromocenter of polytene chromosomes in larval salivary glands. A mutant for the HOAP protein also suppresses centric heterochromatin-induced silencing, supporting a role for HOAP in centric heterochromatin (Badugu, 2003).

The Drosophila HOAP protein is required for telomere capping

HOAP (HP1/ORC-associated protein) has been isolated from Drosophila embryos as part of a cytoplasmic complex that contains heterochromatin protein 1 (HP1) and the origin recognition complex subunit 2 (ORC2). caravaggio, a mutation in the HOAP-encoding gene, causes extensive telomere-telomere fusions in larval brain cells, indicating that HOAP is required for telomere capping. These analyses indicate that HOAP is specifically enriched at mitotic chromosome telomeres, and strongly suggest that HP1 and HOAP form a telomere-capping complex that does not contain ORC2 (Cenci, 2003).

HP1 controls telomere capping, telomere elongation, and telomere silencing by two different mechanisms in Drosophila

HP1 is a conserved chromosomal protein, first discovered in Drosophila, which is predominantly associated with the heterochromatin of many organisms. It has been shown that HP1 is required for telomere capping, telomere elongation, and transcriptional repression of telomeric sequences. Several studies have suggested a model for heterochromatin formation and epigenetic gene silencing in different species that is based on interactions among histone methyltransferases (HMTases), histone H3 methylated at lysine 9 (H3-MeK9), and the HP1 chromodomain. This model has been extended to HP1 telomeric localization by data showing that H3-MeK9 is present at all of the telomeres. This model has been tested, and it has been found that the capping function of HP1 is due to its direct binding to telomeric DNA, while the silencing of telomeric sequences and telomere elongation is due to its interaction with H3-MeK9 (Perrini, 2004).

In heterozygous HP1 mutant stocks, over time, the telomeres show a strong elongation, and the transcription of both TART and HeT-A is significantly increased. The telomeres from the strain carrying the HP1 mutation are clearly elongated. It is clear that, compared to the wild-type telomere of the second chromosome left arm, this elongation can result from either the addition of both types of telomeric transposons or from a sort of telomeric rearrangement. Telomere elongation was also found in Hp1 mutant [Su(var)2-502/Cy and Su(var)2-505/Cy] stocks (Perrini, 2004).

The transcription of the telomeric transposons were examined in heterozygous and trans-heterozygous Su(var)2-504/Su(var)2-505 and Su(var)2-502/Su(var)2-505 mutant larvae. HeT-A transcription is very low in wild-type larvae, while these transcripts are clearly present in heterozygous (mutant/wild-type) larvae and are even more abundant in trans-heterozygous mutant larvae. Similar results were found for TART. Interestingly, similar amounts of HeT-A transcripts were found in both male and female mutant larvae, thus suggesting that the HeT-A sequences being transcribed are mainly those at the telomeres rather than those on the Y chromosome (Perrini, 2004).

Using real-time RT-PCR analysis, it was found that, in Su(var)2-502/Su(var)2-505 mutants, the HeT-A transcription is 95.76 times higher than in wild-type, and similar results were also observed in Su(var)2-504/Su(var)2-505. Since, the Su(var)2-502 point mutation that disrupts the HP1 chromodomain also strongly derepresses both TART and HeT-A, it appears that the silencing of telomere transposons requires a functional HP1 chromodomain. Considering that this mutation does not affect protein localization or telomere stability, it is concluded that the HP1 chromodomain, although dispensable for telomere capping, is required for telomeric transposon silencing and, most probably, telomere elongation. Since it is known that the HP1 chromodomain binds H3-Me3K9, and that the presence of this modified histone seems to overlap HP1 in all the telomeres, it is possible that repressive telomeric chromatin is formed by the interaction of HP1 with the modified histone. To test this idea, it was asked if the methylation of H3-K9 at the telomeres could also be affected by the absence of HP1. It has been recently shown that HP1 mutations affect heterochromatic H3-K9 methylation, thus suggesting that HMTase SU(VAR)3-9 and HP1 are probably functionally interdependent in forming the pericentromeric heterochromatin mediated by the H3-K9 methylation in Drosophila . Polytene chromosomes from HP1 mutant larvae were immunostained with an H3-Me3K9-specific antibody. It was found that H3-Me3K9 is absent from telomeres of mutant larvae completely lacking HP1. Most importantly, it was found that H3-Me3K9 is also absent from telomeres in Su(var)2-502/Su(var)2-505 mutant larvae. Su(var)2-502 is a mutation in the chromodomain of HP1 that is a strong dominant suppressor of variegation and is homozygous lethal, but does not affect telomeric binding of the mutant protein. As confirmation, it was found that the imaginal disks of Su(var)2-502/Su(var)2-505 mutant larvae are similar to wild-type imaginal disks, while those lacking HP1 show extensive apoptosis. These data clearly show that the methylation of H3-K9 at the telomeres depends on the presence of the HP1 chromodomain. The HP1 chromodomain and H3-Me3K9 are not, however, required for telomere capping, but both are necessary for the control of telomeric transposon transcription and telomere elongation (Perrini, 2004).

HP1 has the ability to directly interact with DNA in vitro. This suggests that a direct interaction of HP1 with telomeric DNA may be necessary for the telomere capping function. Direct HP1-DNA binding was tested in vivo by using a crosslinking assay with cis-diamminedichloroplatinum (cis-DDP). cis-DDP is considered a useful crosslinker to identify non-histone proteins that interact directly with DNA. The most frequent site of primary binding for cis-DDP on DNA is the N7 of guanine, exposed on the surface of the major groove. To confirm the direct DNA binding of HP1 in vitro, recombinant HP1 (500 ng) and genomic DNA (12 mg) isolated from Drosophila adults were mixed in the presence of 0.1 mM cis-DDP and incubated for 90 min at 37°C. Then, the DNA-protein complexes were subjected to hydroxyapatite purification, and DNA bound proteins were eluted by 1.5 M thiourea, subjected to SDS-gel electrophoresis, and analyzed on Western blot by the C1A9 HP1 monoclonal antibody. The presence of clear immunosignals shows that HP1 crosslinks to DNA, suggesting a direct interaction between the two molecules (Perrini, 2004).

To test the DNA binding activity of HP1 in vivo, crosslinking was induced by cis-DDP in intact nuclei purified from Drosophila larvae. This approach allows the detection of DNA-protein interactions under conditions very close to those existing in vivo, since the interactions are stabilized before the disruption of the nucleus. After purification of the crosslinked complexes, it was found that HP1 is present among the crosslinked nuclear components, suggesting that HP1 also has a DNA binding activity in vivo (Perrini, 2004).

An immunoprecipitation assay (X-ChIP assay) was used to test the capacity of HP1 to bind telomeric DNA in vivo. cis-DDP crosslinked complexes from intact nuclei were purified by gel-filtration chromatography. The nuclei used came exclusively from female larvae to avoid confusion from the tandem array of telomeric HeT-A and TART-related sequences found at the centromeric region of the Y chromosome. The complexes were then immunoprecipitated with a monoclonal HP1 antibody. To examine the presence of telomeric sequences among the immunoprecipitated DNA, a PCR analysis was performed with specific pairs of primers covering fragments of the telomeric HeT-A region. The telomeric sequence is amplified only in the DNA of the immunoprecipitated sample and in the genomic DNA, but not in the DNA of the immunoprecipitated control. Thus, HP1 is directly bound to the telomeric region of HeT-A (Perrini, 2004).

HP1 is also located at the extremity of stable terminal deletions that lack both HeT-A and TART telomeric transposons. Since some terminal deletions of the X chromosome end inside the yellow gene, whether HP1 directly binds to the yellow sequences of such terminal deletions was tested by using the immunoprecipitation assay in larvae carrying the yTdl4 terminal deletion. The PCR analysis of the immunoprecipitates was performed by using pairs of primers located proximal to the original breakpoint in the terminal deletion. Amplified yellow sequences were found only in the DNA of immunoprecipitates from the yTdl4 strain. These data show that HP1 can directly bind telomeric DNA independent of specific sequences. It was also found, by using a gel shift assay, that HP1 is capable of binding both double- and single-strand telomeric DNA, but competition experiments have also suggested that it has a major affinity for single-strand DNA. Since HP1 does not have any obvious DNA binding domain, this raises the question of what part of the protein is responsible for this binding. As discussed above, the chromodomain appears to be dispensable for telomeric binding. A COOH-terminal region corresponding to the HP1 chromoshadowdomain has been found to be required for the nuclear localization of HP1, and an additional functional domain inside the hinge portion specifies HP1 heterochromatin binding. Intriguingly, this domain also permits the HP1 telomeric localization, thus suggesting a role of the hinge region in HP1 telomeric DNA binding. To test this suggestion, a gel shift assay was done on the series of HP1 fragments. Only the HP1 fragments containing the hinge regions are capable of producing a gel shift of single-strand HeT-A DNA. These results strongly suggest that the hinge region is required for the direct binding of HP1 to telomeric DNA (Perrini, 2004).

It is concluded that the involvement of HP1 in telomere capping and in controlling telomeric DNA transcription, and probably elongation, is mediated by two different types of binding to the telomeres. Telomere capping depends on the direct binding of HP1 to telomeric sequences, while the transcriptional control of such sequences depends on the interaction of the HP1 chromodomain with H3-Me3K9. Interestingly, the observation that H3-K9 methylation depends on the presence of HP1 at the telomeres suggests that this histone modification is due to a previous interaction of HP1 with a specific HMTase (Perrini, 2004).

Together these data suggest a simple model for HP1 function at the telomeres. It is suggested that HP1 first directly binds telomeric DNA and recruits a yet unknown specific HMTase. The enzyme would then methylate H3-K9, creating an additional binding site for HP1. The spreading of HP1, HMTase, and H3-Me3K9 interactions would form the telomeric repressive chromatin. Since the present data seem to exclude an involvement of RNAi, suggesting instead a preferential affinity of HP1 for single-strand HeT-A DNA, it is proposed that HP1 is probably recruited to the telomeric DNA by its specific recognition of the protruding telomeric ends. At present, it cannot be excluded that this affinity is not potentiated by yet another telomeric component. Supporting this possibility is the recent finding that the HP1-interacting HOAP protein is also required for telomere capping. It is not known yet if the telomeric functions of HP1 are evolutionarily conserved. A suggestion of this possibility comes from recent studies showing the existence of the telomeric position effect in human cells that depends on a specific higher-order organization of telomeric chromatin in which HP1 is probably involved (Perrini, 2004).

HP1 involvement in a silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin

Histone lysine methylation is a central modification to mark functionally distinct chromatin regions. In particular, H3-K9 trimethylation has emerged as a hallmark of pericentric heterochromatin in mammals. H4-K20 trimethylation is also focally enriched at pericentric heterochromatin. Intriguingly, H3-K9 trimethylation by the Suv39h HMTases is required for the induction of H4-K20 trimethylation, although the H4 Lys 20 position is not an intrinsic substrate for these enzymes. By using a candidate approach, Suv4-20h1 and Suv4-20h2 were identified as two novel SET domain HMTases that localize to pericentric heterochromatin and specifically act as nucleosomal H4-K20 trimethylating enzymes. Interaction of the Suv4-20h enzymes with HP1 isoforms suggests a sequential mechanism to establish H3-K9 and H4-K20 trimethylation at pericentric heterochromatin. Heterochromatic H4-K20 trimethylation is evolutionarily conserved, and in Drosophila, Suv4-20 is a novel position-effect variegation modifier. Together, these data indicate a function for H4-K20 trimethylation in gene silencing and further suggest H3-K9 and H4-K20 trimethylation as important components of a repressive pathway that can index pericentric heterochromatin (Schotta, 2004).

These data suggest H4-K20 trimethylation is a mark of silenced chromatin domains. Therefore whether this modification would indeed be important for gene silencing in well-described PEV models in Drosophila was investigated. A single, homozygous-viable P-element insertion (P{GT1}BG00814) into the third exon of Suv4-20 has been identified in the course of the Drosophila gene disruption project. H4-K20 trimethylation at polytene chromatin is nearly lost in homozygous mutant larvae, demonstrating that the P-element insertion (Suv4-20BG00814) represents a strong hypomorphic allele of Suv4-20. Because the Suv4-20 locus maps on the X chromosome, the classical PEV rearrangement In(1)wm4 cannot be used to analyze a potential modifier effect of Suv4-20. Therefore, another PEV rearrangement was analyzed that translocates a different marker, Stubble (Sb), close to pericentric heterochromatin (T(2;3)SbV). The dominant mutation Stubble induces short bristles, but heterochromatin-induced silencing of SbV results in wild-type (long) bristles. Homozygous Suv4-20BG00814 as well as control wild-type females were crossed to T(2;3)SbV males. In the progeny, the extent of SbV reactivation was determined as the ratio of short bristles (active SbV) to long bristles (inactive SbV). In males and females of the wild-type crosses, only 1%-2% of bristles show a Sb phenotype, indicating that SbV is largely inactivated. In contrast, SbV becomes derepressed in the progeny of Suv4-20BG00814 flies, because now ~25% of the bristles are short. This result classifies Suv4-20 as a dominant PEV modifier and further indicates a functional role for Suv4-20-dependent H4-K20 trimethylation in gene silencing (Schotta, 2004).

cis-Acting determinants of heterochromatin formation on Drosophila melanogaster chromosome four

The heterochromatic domains of Drosophila melanogaster (pericentric heterochromatin, telomeres, and the fourth chromosome) are characterized by histone hypoacetylation, high levels of histone H3 methylated on lysine 9 (H3-mK9), and association with heterochromatin protein 1 (HP1). While the specific interaction of HP1 with both H3-mK9 and histone methyltransferases suggests a mechanism for the maintenance of heterochromatin, it leaves open the question of how heterochromatin formation is targeted to specific domains. Expression characteristics of reporter transgenes inserted at different sites in the fourth chromosome define a minimum of three euchromatic and three heterochromatic domains, interspersed. A search was performed for cis-acting DNA sequence determinants that specify heterochromatic domains. Genetic screens for a switch in phenotype demonstrate that local deletions or duplications of 5 to 80 kb of DNA flanking a transposon reporter can lead to the loss or acquisition of variegation, pointing to short-range cis-acting determinants for silencing. This silencing is dependent on HP1. A switch in transgene expression correlates with a switch in chromatin structure, judged by nuclease accessibility. Mapping data implicate the 1360 transposon as a target for heterochromatin formation. It is proposed that heterochromatin formation is initiated at dispersed repetitive elements along the fourth chromosome and spreads for approximately 10 kb or until encountering competition from a euchromatic determinant (Sun, 2004).

The published DNA sequence of the fourth chromosome shows a fairly uniform distribution of genes across region 101F-102F at a normal gene density. However, this region is enriched in repetitious sequences compared to similar intervals on the other euchromatic chromosome arms. The average transposable element density is 10 to 15 per Mb in the major chromosome arms but over 82 per Mb for chromosome 4 due to an order-of-magnitude increase in remnants of long interspersed element (LINE)-like and TIR elements (elements that transpose via a DNA intermediate, flanked by short inverted repeats). The most abundant TIR in chromosome 4 is 1360, a repetitive sequence that is abundant in the chromocenter, pericentric heterochromatin, and the telomeres of the major chromosome arms. 1360 is the only transposable element found across the whole of the fourth chromosome, including the centromere and telomere (Sun, 2004).

While there appear to be some 'hot spots' for P element insertion, heterochromatic domains are also distributed across the chromosome. Variegating inserts are not restricted to juxtaposition with repetitious DNA or even to gene-free regions. In fact, most (17 of 18) of the variegating P elements lie within 2 kb of a gene, and 10 variegating P elements lie within the transcribed portion of nine different genes. Thus, the heterochromatic domains are not restricted to tandem repeat arrays; rather, the local pattern of dispersed repetitious elements, particularly 1360 in the region examined, appears to be critical for heterochromatin formation (Sun, 2004).

HP1, a consistent marker of heterochromatic domains, is prominently and extensively associated with the fourth chromosome, as shown by immunofluorescent staining of the polytene chromosomes. All of the insertion lines from this study showing a variegating phenotype that have been examined directly show a loss of silencing as a consequence of the introduction of a mutation (a hypomorph) in the gene for HP1. This group includes lines with the P element inserted into the genes BEST:CK01140, bt, and ATPsyn-beta. One can infer that a significant number of fourth-chromosome genes are packaged with HP1. While much of the Drosophila heterochromatin at centromeres and telomeres is made up of tandem repeats, classical genetics have identified several genes within the pericentric heterochromatin, and data from genome sequencing suggest that several hundred genes may reside in these regions in D. melanogaster. Many of the genes on the fourth chromosome have specific developmental functions and must have some developmental regulation superimposed on the effects of domain packaging. It will be of interest to determine how these genes function within a heterochromatic environment (Sun, 2004).

The switch in phenotype of the 2-M59A.R reporter transgene from a red-eye to a variegating phenotype resembles the classical phenomenon of PEV, in which a euchromatic white gene is brought into juxtaposition with heterochromatin by a chromosomal rearrangement. In a current model, PEV reflects the spread of heterochromatin across the rearrangement breakpoint, with the euchromatic reporter gene being packaged into heterochromatin in a stochastic process. The model assumes the presence of initiators of heterochromatin formation within each heterochromatic domain and the presence of a barrier to the spread of heterochromatin, removed by rearrangement, normally separating the domains. HP1 appears designed to play a central role in such spreading, being able to recognize both a key histone modification, methylation of lysine 9 in histone H3, and histone H3-K9 methyltransferases, including SU(VAR)3-9. This model is supported by recent findings for Schizosaccharomyces pombe showing that the spreading of heterochromatin upon removal of a putative boundary to the silent mating type region requires the yeast homologue of HP1 and the H3-K9 methyltransferase. In the analysis presented in this study, in contrast to classical PEV, the switch can be related to the loss of a relatively small fragment of DNA, pointing to local cis-acting determinants controlling heterochromatin spreading. As observed in other cases, the silencing is dependent on HP1, as shown by the loss of silencing upon the loss of HP1 in lines with small deletions (Sun, 2004).

The results shown here also reveal the reciprocal effect, the conversion of a variegating to a red-eye phenotype upon the deletion of DNA flanking the reporter transgene. The underlying mechanism for the switch in phenotype involves a shift in the chromatin structure of the transgenes, shown by changes in XbaI sensitivity of the hsp26-pt gene. This reciprocal local position effect suggests a model of competitive equilibrium between the two types of chromatin, rather than supporting the common perception that heterochromatin is a dominant form. In this model, the balance between heterochromatin and euchromatin may be determined by the presence and/or strength of nearby initiator elements for each form of chromatin, presumably acting to determine the modification state of the histone cores. The effect of euchromatin initiator elements might explain some of the observed discrepancies and will need to be taken into account in developing a detailed model of chromatin packaging. The idea of a competitive equilibrium is supported by recent experiments demonstrating that an increase in transcription factor for a variegating reporter gene can antagonize heterochromatin silencing (Sun, 2004).

The role of histone H2Av variant replacement and histone H4 acetylation in the establishment of Drosophila heterochromatin: H2Av is required for HP1 recruitment

Activation and repression of transcription in eukaryotes involve changes in the chromatin fiber that can be accomplished by covalent modification of the histone tails or the replacement of the canonical histones with other variants. The histone H2A variant of Drosophila melanogaster, Histone H2A variant (H2Av), localizes to the centromeric heterochromatin, and it is recruited to an ectopic heterochromatin site formed by a transgene array. His2Av behaves genetically as a PcG gene and mutations in His2Av suppress position effect variegation (PEV), suggesting that this histone variant is required for euchromatic silencing and heterochromatin formation. His2Av mutants show reduced acetylation of histone H4 at Lys 12, decreased methylation of histone H3 at Lys 9, and a reduction in HP1 recruitment to the centromeric region. Neither H2Av accumulation nor histone H4 Lys 12 acetylation is affected by mutations in either Su(var)3-9 or Su(var)2-5. The results suggest an ordered cascade of events leading to the establishment of heterochromatin, requiring the recruitment of the histone H2Av variant followed by H4 Lys 12 acetylation as necessary steps before H3 Lys 9 methylation and HP1 recruitment can take place (Swaminathan, 2005).

Recent results suggest that H3 trimethylated at Lys 27 facilitates Pc binding to silenced regions and this modification is carried out by the Enhacer of zeste [E(z)] protein present in the ESC-E(z) complex. Since a reduction in Pc on polytene chromosomes was observed in His2Av mutants, whether recruitment of the ESC-E(z) complex is also impaired in these mutants was examined. In wild type, E(z) can be observed at multiple sites throughout the genome. The levels and localization of E(z) do not appear to be altered in the His2Av810 mutant compared to wild type. Whether H3 Lys 27 methylation is affected by mutations in His2Av was examined. The levels and distribution of this modification appear to be the same in polytene chromosomes from wild-type and His2Av810 mutant larvae. This result was confirmed by Western analysis, which shows equal levels of H3 trimethylated at Lys 27 in wild-type and His2Av810 mutant larvae. These results suggest that H2Av is required upstream of Pc recruitment in the process of Pc-mediated silencing. Since neither recruitment of the E(z) complex nor H3 Lys 27 methylation seem to be affected in His2Av mutants, H2Av replacement might take place after H3 Lys 27 methylation and before Pc recruitment. Alternatively, Pc repression might require at least two parallel and independent pathways, one involving H2Av recruitment and a second one leading to H3 Lys 27 methylation, both of which might be required for proper Pc recruitment (Swaminathan, 2005).

Formation of heterochromatin requires deacetylation of H3 Lys 9 followed by methylation of the same residue and recruitment of HP1. The heterochromatin of Drosophila chromosomes is enriched in dimethylated and trimethylated histone H3 in the Lys 9 residue. To analyze the possible role of H2Av in heterochromatin assembly, the localization was examined of H3 dimethylated at Lys 9 in polytene chromosomes from larvae carrying a mutation in the His2Av gene. Antibodies against histone H3 dimethylated in Lys 9 stain the pericentric heterochromatin in wild-type larvae. Interestingly, polytene chromosomes from His2Av810 mutants show a decrease in the amount of methylated H3 Lys 9, whereas the presence of Su(Hw), used as a control, is the same in chromosomes from wild-type and His2Av810 mutant larvae. Since modification of this residue is important for HP1 recruitment, whether localization of HP1 in heterochromatin is also affected by mutations in His2Av was examined. In wild-type larvae, HP1 localizes preferentially to the pericentric heterochromatin of the chromocenter, but accumulation of HP1 is dramatically reduced in the His2Av810 mutant (Swaminathan, 2005).

To confirm these results, Western analyses of protein extracts obtained from wild-type and His2Av mutant larvae was carried out using antibodies against HP1 and histone H3 dimethylated in Lys 9. The results show little or no accumulation of histone H3 methylated in Lys 9, and lower levels of HP1 in the His2Av810 mutant. Methylation of histone H3 at the Lys 9 residue is carried out by the Su(var)3-9 histone methyltransferase, and HP1 is encoded by the Su(var)2-5 gene. In order to ensure that the observed effects on the levels of HP1 or the methylation of H3 Lys 9 were not caused by alterations in transcription of Su(var)3-9 or Su(var)2-5 due to the His2Av mutation, quantitative RT-PCR analyses of RNA obtained from wild-type and His2Av810 mutant third instar larvae were carried out . The results show that there are no significant changes in the levels of Su(var)3-9 or HP1 RNAs in His2Av810 mutant larvae when compared to wild type. These results and those from immunocytochemistry analyses confirm a role for H2Av in the methylation of H3 Lys 9 and subsequent recruitment of HP1 (Swaminathan, 2005).

Based on the observed effects of His2Av mutations on H3 Lys 9 methylation and HP1 recruitment, it appears that the presence of H2Av in heterochromatin might be required prior to these two events. To confirm this hypothesis, the pattern of H2Av distribution on polytene chromosomes from larvae carrying mutations was examined in the Su(var)2-5 and Su(var)3-9 genes. In both cases, H2Av localization appears normal, suggesting that the presence of H2Av is required prior to H3 Lys 9 methylation and HP1 recruitment during the establishment of heterochromatin (Swaminathan, 2005).

Regulation of HP1-chromatin binding by histone H3 methylation and phosphorylation

Tri-methylation of histone H3 lysine 9 is important for recruiting heterochromatin protein 1 (HP1) to discrete regions of the genome, thereby regulating gene expression, chromatin packaging and heterochromatin formation. HP1alpha, -beta, and -gamma are released from chromatin during the M phase of the cell cycle, even though tri-methylation levels of histone H3 lysine 9 remain unchanged. However, the additional, transient modification of histone H3 by phosphorylation of serine 10 next to the more stable methyl-lysine 9 mark is sufficient to eject HP1 proteins from their binding sites. Inhibition or depletion of the mitotic kinase Aurora B, which phosphorylates serine 10 on histone H3, causes retention of HP1 proteins on mitotic chromosomes, suggesting that H3 serine 10 phosphorylation is necessary for the dissociation of HP1 from chromatin in M phase. These findings establish a regulatory mechanism of protein-protein interactions, through a combinatorial readout of two adjacent post-translational modifications: a stable methylation and a dynamic phosphorylation mark (Fischle, 2005).

Although histone H3S10ph is widely seen as a hallmark of mitosis, the function of this modification during M phase has been enigmatic. The data suggest that phosphorylation of H3S10 by Aurora B disrupts the chromodomain-H3K9me3 interaction, which is important for HP1 recruitment to chromatin during interphase. This disruption causes a net shift in the dynamic HP1-chromatin binding equilibrium towards the unbound state. In this reaction sequence, dephosphorylation of H3S10 at the end of mitosis re-establishes the overall association of HP1 with chromatin (Fischle, 2005).

It is propose that this binary 'methyl/phos switching' permits dynamic control of the HP1-H3K9me interaction. Intriguingly, the mechanism for HP1 release from M-phase chromatin does not involve a temporary loss of H3K9me3, but instead requires a combination of this unchanging mark and the dynamic H3S10ph modification that is only transiently added to chromatin during mitosis. It is reasoned that stable transmission of the heterochromatin-defining H3K9me3 mark is needed to accurately convey, from one cell generation to the next, which regions of the genome are supposed to be permanently silenced. If removal of HP1 from M-phase chromatin were accomplished by H3K9me3-erasing demethylase activities, the epigenetic information underlying this mark- and effector-system would have to be accurately re-established at the end of every cell cycle (Fischle, 2005).

In addition to H3S10 phosphorylation, other mechanisms might be involved in the mitotic release of HP1 from chromatin. These might include further modifications of the H3-tail, HP1 proteins and/or their interaction partners. Nevertheless, inhibition, knockdown or depletion of Aurora B is sufficient to cause aberrant interaction of all HP1 isoforms with mitotic, condensed chromatin. Although the possibility cannot be excluded that HP1 proteins themselves might be in vivo targets of Aurora B kinase activity (for example, increased association of the xHP1aW57A mutant protein was observed with metaphase chromosomes assembled in DCPC extracts), it is known that the phosphorylation level of HP1b and HP1g does not increase during mitosis. Since phosphorylation of an H3K9me3 peptide is sufficient to dissociate HP1 from this site in vitro, it is concluded that Aurora B-mediated phosphorylation of H3S10 must be the central event in mitotic release of HP1 from chromatin (Fischle, 2005).

Notably, a fraction of HP1a, but not HP1b or HP1g, remains associated with the (peri-)centromeric chromosome region, where it performs important functions for centromere cohesion and kinetochore formation and might be required to identify and define this specialized area of heterochromatin throughout the cell cycle. This mitotic retention of HP1a at centromeres depends on a carboxy-terminal region of the protein, but is independent of the chromodomain8. It is therefore suggested that 'methyl/phos switching' uniformly disrupts HP1-chromatin interaction but that mechanisms other than chromodomain-H3K9me3 interaction are responsible for the lingering HP1a association with pericentromeric regions (Fischle, 2005).

What is the function of HP1 dissociation from chromatin during Mphase? It is tempting to speculate that removal of HP1 is important for allowing access by factors necessary for mediating proper chromatin condensation and faithful chromosome segregation during mitosis. Indeed, inhibition of Aurora B in vertebrate cells results in defects in chromosome alignment, segregation, chromatin-induced spindle assembly and cytokinesis. Furthermore, mutation of H3S10 causes faulty chromosome segregation in Tetrahymena and S. pombe, organisms that rely on HP1 and H3K9me3 for the establishment and maintenance of heterochromatin, but not in Saccharomyces cerevisiae, an organism that lacks this silencing system. Interestingly, most histone phosphorylation sites are rapidly phosphorylated early in M phase. It remains to be seen whether these bursts in histone phosphorylation are directly involved in the release of proteins bound to interphase chromatin, which might need to be removed to ensure faithful progression through mitosis. It is conceivable that similar 'methyl/phos switches' play critical roles in governing other histone-non-histone or even non-histone-nonhistone interactions (Fischle, 2005).

Drosophila PIWI associates with chromatin and interacts directly with HP1a

The interface between cellular systems involving small noncoding RNAs and epigenetic change remains largely unexplored in metazoans. RNA-induced silencing systems have the potential to target particular regions of the genome for epigenetic change by locating specific sequences and recruiting chromatin modifiers. Noting that several genes encoding RNA silencing components have been implicated in epigenetic regulation in Drosophila, a direct link was sought between the RNA silencing system and heterochromatin components. This study shows that Piwi, an Argonaute/Piwi protein family member that binds to Piwi-interacting RNAs (piRNAs), strongly and specifically interacts with heterochromatin protein 1a (HP1a), a central player in heterochromatic gene silencing. The HP1a dimer binds a PxVxL-type motif in the N-terminal domain of Piwi. This motif is required in fruit flies for normal silencing of transgenes embedded in heterochromatin. Piwi, like HP1a, is itself a chromatin-associated protein whose distribution in polytene chromosomes overlaps with HP1a and appears to be RNA dependent. These findings implicate a direct interaction between the Piwi-mediated small RNA mechanism and heterochromatin-forming pathways in determining the epigenetic state of the fly genome (Brower-Toland, 2007).

Thus, Drosophila PIWI interacts directly with HP1a and is distributed on chromosomes in a pattern overlapping HP1a. Association of Piwi with constitutive heterochromatin domains including pericentric heterochromatin, telomeres, and part of the banded portion of chromosome 4 is consistent with the profile of Piwi-associated small RNAs, which includes sequences homologous to telomeric and centromeric repetitive elements as well as some that are overrepresented in chromosome 4. The PIWI-HP1a interaction is mediated through binding of a PxVxL-type motif by dimerized chromoshadow domains of HP1a. Furthermore, the intact PxVxL motif is required in vivo for effective heterochromatic silencing. This is consistent with the wealth of data implicating Piwi at the interface of RNA silencing mechanisms with epigenetic phenomena, in particular heterochromatin-induced silencing. Piwi is a nuclear protein required for silencing of transposons and retroviruses. Piwi is required for effective silencing of multiple copies of dispersed transgenes, of tandem repeats of the white gene at euchromatic insertion sites, and of white reporter genes in the pericentric heterochromatin or fourth chromosome. Silencing in the latter two cases is dependent on HP1a. This study shows that Piwi interacts directly with HP1a. Some Piwi signal overlaps with HP1a in polytene chromosomes, suggesting that Piwi and HP1a together regulate the epigenetic state of diverse regions in the genome. This overlap is spatially complex and could therefore represent numerous varied roles for Piwi, HP1a, and the Piwi-HP1a interaction. Heterochromatin is first assembled early, while the Drosophila embryo is still a syncytium (nuclear division cycles 10-14). This is critical for the silencing observed in PEV, which may be compromised later in development. While the HP1a-PIWI association observed on polytene chromosomes might be similar to that inferred in the embryo, it might also represent a different function (Brower-Toland, 2007).

Recently, Piwi has been shown to bind in the germline to piRNAs, many of which are from heterochromatic regions. The colocalization of Piwi and HP1a in pericentric heterochromatin may indeed reflect a simple relationship between the piRNA pathway, histone modification, and heterochromatin formation similar to the S. pombe system wherein AGO1-mediated transcriptional gene silencing locally targets H3K9 methylation to create a binding site for the HP1 homolog Swi6. In Drosophila, these data on the specific interaction between PIWI and HP1a raise the possibility of an alternate pathway to HP1a-mediated heterochromatinization: A PIWI-piRNA complex might directly recruit HP1a to piRNA-corresponding genomic sequences, which could then recruit HMTs such as SU(VAR)3-9 to effect nucleation/spreading. This would represent an H3K9me-independent mode for initial HP1 localization, an alternative but potentially equally effective means for triggering local formation of heterochromatin. Conversely, if heterochromatin formation is targeted by a different mechanism, the presence of HP1a could allow stable binding of Piwi to heterochromatin for PTGS, a process that might be necessary to maintain silencing throughout the lifetime of the fly (Brower-Toland, 2007).

The SU(VAR)3-9/HP1 complex differentially regulates the compaction state and degree of underreplication of X chromosome pericentric heterochromatin in Drosophila melanogaster

In polytene chromosomes of Drosophila melanogaster, regions of pericentric heterochromatin coalesce to form a compact chromocenter and are highly underreplicated. Focusing on study of X chromosome heterochromatin, it was demonstrated that loss of either SU(VAR)3-9 histone methyltransferase activity or HP1 protein differentially affects the compaction of different pericentric regions. Using a set of inversions breaking X chromosome heterochromatin in the background of the Su(var)3-9 mutations, it was shown that distal heterochromatin (blocks h26-h29) is the only one within the chromocenter to form a big 'puff'-like structure. The 'puffed' heterochromatin has not only unique morphology but also very special protein composition as well: (1) it does not bind proteins specific for active chromatin and should therefore be referred to as a pseudopuff and (2) it strongly associates with heterochromatin-specific proteins SU(VAR)3-7 and SUUR, despite the fact that HP1 and HP2 are depleted particularly from this polytene structure. The pseudopuff completes replication earlier than when it is compacted as heterochromatin, and underreplication of some DNA sequences within the pseudopuff is strongly suppressed. So, it was shown that pericentric heterochromatin is heterogeneous in its requirement for SU(VAR)3-9 with respect to the establishment of the condensed state, time of replication, and DNA polytenization (Demakova, 2007).

In Su(var)2-5 and Su(var)3-7 mutants the chromocenter in salivary gland nuclei looks relatively loose and diffuse (Spierer, 2005). Loss or drastic reduction in the HMTase activity of SU(VAR)3-9 also results in strong decompaction of pericentric heterochromatin (PH) in polytene chromosomes. Most likely, only the functional complex of all these proteins can form compact pericentric heterochromatin. It was of interest to find out to what degree the compact state of heterochromatin is dependent on specific heterochromatin proteins, particularly SU(VAR)3-9. Using a set of chromosome rearrangements placing different parts of X chromosome heterochromatin adjacent to euchromatin, it was discovered that different portions of the polytene Xh are very differently affected by loss of SU(VAR)3-9: only the distal part of heterochromatin (heterochromatic blocks h26-h29 of the metaphase chromosome map becomes decondensed to a varying extent, while morphology of the heterochromatic blocks h30-h34 appears unaffected (Demakova, 2007).

The extent of heterochromatin decompaction depends on several different factors. The pseudopuff is more pronounced in males than in females. Although MSL2 protein, the core component of the dosage compensation complex (DCC), was not found in the pseudopuff, it is suggested that dosage compensation, a process equalizing X-linked gene expression in both sexes, might be responsible for this effect. It is known that DCC is distributed rather discretely, particularly, skipping over intercalary heterochromatin (IH) regions along the male X. Nevertheless, the whole X has a decondensed appearance and underreplication in IH regions is highly suppressed due to DCC function. So, it can be proposed that in males the 'puffed' portion of pericentric Xh relocated into the vicinity of euchromatin becomes dependent on dosage-compensating mechanisms, similarly to IH regions. Another possibility is that the Y chromosome, which represents an additional factor competing for the compaction proteins, might also contribute to the decompaction of Xh and to pseudopuff formation. To note, the fully heterochromatic Y chromosome comprises 40.9 Mb of DNA, while Xh contains ∼20 Mb (Demakova, 2007).

Among the inversions analyzed, wm4 produces the largest pseudopuff. A good explanation for this effect is currently missing; possibly, the chromatin environment in this eu-heterochromatin junction might contribute to Xh puffing, or some of the DNA sequences might be differentially represented in Xh in different inversions. And finally, decondensation is most strongly expressed on the background of two mutations, Su(var)3-9 and SuUR, despite the fact that SuUR mutation itself does not induce puffing of Xh. The SuUR mutation results in additional polytenization of some of the Xh regions and thus it might increase the amount of Xh material capable of forming the pseudopuff. So, it is believed that loss of both proteins, SUUR and SU(VAR)3-9, has an additive effect on the sizes of the decompacted region (Demakova, 2007).

The region of decondensed heterochromatin that can be called the pseudopuff does not demonstrate signs of true transcriptionally active puffs: the proteins characteristic for active decondensed chromatin (Z4, MSL2, JIL-1, and H3Me3K4) were not detected in the pseudopuff, with the exception of a very weak signal of PolIIo. At the same time, in the Su(var)3-9 mutants, HP1 and HP2 are weakly associated with the region 20F1-4, whereas SUUR and SU(VAR)3-7, in contrast, intensively bind the entire body of the pseudopuff. Recruitment of SU(VAR)3-7 into the pseudopuff in the absence of HP1 and SU(VAR)3-9 appears to be a very specific characteristic of pseudopuff heterochromatin since HP1 and SU(VAR)3-7 proteins cooperate closely in chromosome organization and development (Spierer, 2005). Presence of the SUUR and SU(VAR)3-7 in decompacted chromatin of the pseudopuff might indicate that they do not participate in the process of compaction of this heterochromatic material or that they act in this direction only in the complex with functional SU(VAR)3-9 (Demakova, 2007).

It is interesting to note that different parts of Xh respond differentially to the removal of this complex. The question then, is which features of organization permit proximal heterochromatin to maintain its dense packing in the absence of HP1 and SU(VAR)3-9 activities? It might be proposed that these regions are under control of protein complexes of another composition. For example, these complexes might not utilize HMTase SU(VAR)3-9. However, data on position-effect variegation contradict this idea, since gene inactivation induced by chromosome rearrangements in proximal heterochromatin also depends on the SU(VAR)3-9. It could be suggested that these complexes include some additional compacting proteins. It is known that, in contrast to the distal part of PH, its proximal part is enriched with satellite sequences that are associated with some specific proteins. For, example, the AT-hook protein D1 specifically binds to AT-rich satellites in deep Xh (Demakova, 2007).

In the course of investigating pseudopuff replication it was found that underreplication of the heterochromatic sequences was suppressed in the region of the eu-heterochromatic junction of the wm4 inversion in Su(var)3-9 mutant. Thus, full polytenization of at least a 45-kb fragment adjacent to euchromatin occurs. The pseudopuff region, in general, completes replication before the end of S-phase; in other words, it does so earlier than the bulk of PH and even some IH regions. Still, a notable fraction of X chromosome heterochromatin sequences remains underreplicated in the polytene chromocenter. It can be assumed that some Xh regions not only do not complete replication but also do not start it. Probably, these regions are separated from replicating chromatin by some barriers that prevent progression of replication forks from adjacent replicons. Therefore, this study demonstrates that Su(var)3-906 may act as a suppressor of underreplication. However, it is not known how this mutation can affect underreplication in other heterochromatic regions (Demakova, 2007).

Replication timing of the pseudopuff is notably changed in the absence of essential changes in transcriptional activity (the PolIIo painting of the pseudopuff looks no more intensive than that of the chromocenter). Moreover, the H3K4 methylation mark characteristic of active regions was not found in the pseudopuff at all. At the same time there exists a correlation between the shift to earlier replication and chromatin decompaction. This observation is interesting in relation to cause-effect relationships among replication timing, transcriptional activity, and decompaction of chromatin (Demakova, 2007).

It is interesting to note that the effect of the SuUR mutation, known as suppression of underreplication, was found not only in PH but also in all IH regions and that these regions complete replication earlier in SuUR mutants than in wild-type ones. The pseudopuff material in SuUR mutants is virtually at the same level of polytenization as in the Su(var)3-9 mutant. However, the SuUR mutation does not involve decompaction of the distal Xh. The same was noted for IH regions. Even if SuUR does induce decompaction of high-level chromatin structures, this is not detected by cytological means. Therefore, SuUR mutation affects replication timing differently compared to Su(var)3-9 (Demakova, 2007).

Summing up, it can be concluded that the reaction of pericentric heterochromatin to loss of SU(VAR)3-9 and HP1 varies in different regions of X heterochromatin. Only the distal part of it undergoes decondensation and forms a new structure called a pseudopuff, which has a specific organization, demonstrating some characteristics of active chromatin: decompaction and, concomitantly, earlier completion of replication. At the same time, the pseudopuff does not contain proteins of active chromatin but does contain several heterochromatic proteins (Demakova, 2007).

Histone H3S10 phosphorylation by the JIL-1 kinase in pericentric heterochromatin and on the fourth chromosome creates a composite H3S10phK9me2 epigenetic mark

The JIL-1 kinase mainly localizes to euchromatic interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase in Drosophila. However, recent findings raised the possibility that the binding of some H3S10ph antibodies may be occluded by the H3K9me2 mark obscuring some H3S10 phosphorylation sites. Therefore, this study has characterized an antibody to the epigenetic H3S10phK9me2 double mark as well as three commercially available H3S10ph antibodies. The results showed that for some H3S10ph antibodies their labeling indeed can be occluded by the concomitant presence of the H3K9me2 mark. Furthermore, it was demonstrated that the double H3S10phK9me2 mark is present in pericentric heterochromatin as well as on the fourth chromosome of wild-type polytene chromosomes but not in preparations from JIL-1 or Su(var)3-9 null larvae. Su(var)3-9 is a methyltransferase mediating H3K9 dimethylation. Furthermore, the H3S10phK9me2 labeling overlapped with that of the non-occluded H3S10ph antibodies as well as with H3K9me2 antibody labeling. Interestingly, when a Lac-I-Su(var)3-9 transgene is overexpressed, it upregulates H3K9me2 dimethylation on the chromosome arms creating extensive ectopic H3S10phK9me2 marks suggesting that the H3K9 dimethylation occurred at euchromatic H3S10ph sites. This is further supported by the finding that under these conditions euchromatic H3S10ph labeling by the occluded antibodies was abolished. Thus, these findings indicate a novel role for the JIL-1 kinase in epigenetic regulation of heterochromatin in the context of the chromocenter and fourth chromosome by creating a composite H3S10phK9me2 mark together with the Su(var)3-9 methyltransferase (Wang, 2014).

Umbrea, a chromo shadow domain protein in Drosophila melanogaster heterochromatin

Drosophila HP1-interacting protein (Hip) is a partner of heterochromatin protein 1 (HP1) and is involved in transcriptional epigenetic gene silencing and the formation of heterochromatin. Recently, it has been shown that HP1 interacts with the telomere capping factor HP1/ORC (origin recognition complex)-associated protein (HOAP). Telomeres, complexes of DNA and proteins at the end of linear chromosomes, have been recognized to protect chromosome ends from degradation and fusion events. Both proteins are located at telomeres and prevent telomere fusions. This study reports the identification and characterization of the Hip-interacting protein Umbrea (identical to the recently described HP6 encoding gene) (Greil. 2007). Umbrea interacts directly with Hip, HP1 and HOAP in vitro. Umbrea, Hip and HP1 are partners in a protein complex in vivo and completely co-localize in the pericentric heterochromatin and at telomeres. Using a Gal4-induced RNA interference system, it was found that after depletion of Umbrea in salivary gland polytene chromosomes, they exhibit multiple telomeric fusions. Taken together, these results suggest that Umbrea cooperates with Hip, HP1 and HOAP and plays a functional role in mediating normal telomere behaviour in Drosophila (Joppich, 2009).

This study identified and characterized the heterochromatin protein Umbrea by searching a yeast two-hybrid database for predicted interacting partners of the previously characterized HP1-interacting protein Hip. This study not only confirmed the predicted interaction of Umbrea and Hip but it was also found that Umbrea is able to interact with HP1. This direct interaction is not reported from the Drosophila interaction database (Joppich, 2009).

In contrast to the current results results, Greil (2007) performed no additional protein-protein interaction studies to verify the predicted interactions. For localization studies, Greil used epitope-tagged HP6 and HP1 in transfected Drosophila Kc cells. Whereas HP1 is enriched at the heterochromatic chromocentre, for HP6 localization they found a uniform nuclear staining. However, they did not detect a clear co-localization of HP6 and HP1 in the chromocentre. In a different experiment, Greil used the DamID large-scale mapping technique in transfected cell culture Drosophila Kc cells for co-localization studies with HP1 (Greil, 2007). In contrast, in this experiment they found binding of HP6 in pericentric regions of the major chromosomes and on the small chromosome 4. HP6 localization was only subtly affected after HP1 depletion. On the basis of this result, Greil speculate that an additional interaction might play a key role in targeting HP6 to heterochromatin. To functionally characterize HP6, Greil tested whether mutation of HP6 is a suppressor of PEV. However, the assay they used did not reveal such a function for HP6, suggesting that HP6 is not needed for heterochromatic transgene silencing (Joppich, 2009).

Both HP1 and Umbrea contain a chromo shadow domain. This domain mediates homodimerization of HP1 and this domain mediates heterodimerization between HP1 and Umbrea in vitro. This finding is supported by immunoprecipitation assays. Hip and HP1 are co-precipitated with Umbrea, suggesting that all three proteins are associated in a protein complex in vivo. It should be noted that three HP1-binding interfaces have been identfied in the Hip protein (Schwendemann, 2008). The presence of three binding interfaces in Hip implies a mode of cooperative binding suited to cross-linking of multiple chromo shadow domain-containing molecules like HP1 and Umbrea. It therefore cannot be ruled out that the in vivo interaction between Umbrea and HP1 is only indirect, mediated by the bridging protein Hip (Joppich, 2009).

In agreement with this model, it was found that Umbrea and HP1 use the same three binding modules within the Hip sequence. Both the chromo shadow domains of Umbrea and HP1 interact independently with the three binding interfaces of Hip. The interaction of the two different proteins with the same interaction modules in Hip supports the idea of a novel chromo shadow domain binding interface in Hip (Schwendemann, 2008). The Umbrea protein appears unique among other heterochromatin proteins since it is almost reduced to its chromo shadow domain. What might be the functional mechanism of a protein that consists of a single domain that is similar to the HP1 chromo shadow domain? In HP1 this domain provides the surface for the interaction with various other chromosomal proteins and displays the HP1 protein partner promiscuity. In agreement with this, Umbrea was shown to interact in the same way with at least three proteins. Binding of Umbrea could block the binding surface of an interacting partner to prevent the interaction with other proteins. It is known that HP1 is essential for heterochromatin localization of many proteins. It has recently been shown that Hip binding to heterochromatin depends on HP1 (Schwendemann, 2008). In this study it was found that HP1 also serves as a binding platform for Umbrea. For this experiment HP1-deficient third-instar larvae were used and the result is not consistent with experiments of Greil (2007). Greil used RNAi to reduce HP1 levels. They found that chromosomal localization of HP6 (identical to Umbrea) was only subtly affected by HP1 depletion. It is speculated that residual low amounts of HP1 after RNAi might be sufficient for Umbrea binding to chromatin (Joppich, 2009).

Umbrea binding along the arms of polytene chromosomes seems to be unaffected by Hip depletion. Given the interaction of Umbrea with both Hip and HP1, it is likely that the Umbrea/HP1 interaction is sufficient to target Umbrea to chromatin in the absence of Hip. In turn, this seems to be the case for HP1 and Hip. Their binding appeared to be unaffected after RNAi-induced Umbrea depletion. In contrast, Umbrea association with chromocentre heterochromatin depends on Hip. Different requirements of Hip for Umbrea association with chromocentre and chromosomal arms suggest occurrence of heterochromatin protein complexes of different composition that differentially regulate the assembly of Umbrea-containing complexes. Taking these findings together, it is speculated that HP1 is a key player for heterochromatin targeting and serves as an essential binding platform for chromatin localization of Hip and Umbrea and many other proteins (Joppich, 2009).

The Drosophila HOAP and HP1 proteins are stable components of telomeres and both proteins specifically interact with each other. Cytogenetic studies revealed that Umbrea also localizes to telomeres. However, molecular and genetic analyses provide the evidence for existence of three distinct domains in distal regions of chromosomes: cap complex, which is assembled on the terminal DNA in a sequence-independent manner; the retrotransposon array of He-T-A/TAHRE/TART elements; and the subterminal TAS repeats. Protein attachment to telomeric structures is not sufficient to establish that a protein is a component of the cap. Thus, from cytogenetic analyses it is not possible to assign Umbrea localization to one of the three domains in telomere ends of polytene chromosomes. But given the association of HP1 and HOAP with the cap region and the direct protein interaction of Umbrea with both HP1 and HOAP, it is speculated that Umbrea also localizes to the cap region (Joppich, 2009 and references therein).

Mutations in Su(var)2-5 and cav cause extensive telomere-telomere fusions, indicating that the encoded proteins are essential for telomere stability and required for telomere capping and telomere fusion protection. It was also shown that Umbrea physically interacts not only with HP1 but also with HOAP. Cytogenetic studies revealed that Umbrea is a component of all telomeres. On the basis of these results, a similar telomeric function for Umbrea was expected. However, cytological analysis of larval brain cells displayed neither end-to-end attachments of metaphase chromosomes nor abnormal metaphase configurations. For analysis of mutant brain cells different approaches were used. The lethality of the umbrea P-element mutant line did not allow cytological analysis since homozygous animals die early during embryogenesis. In another approach, progeny of an umbrea specific RNAi line under the control of UAS were examined in combination with different neuronal and ubiquitous Gal4 driver lines. Again, lethality precludes mutant characterization of metaphase chromosomes. Interestingly, the RNAi-induced depletion of Umbrea in salivary glands reveals a mutant phenotype. Frequent telomere-telomere attachments were found in polytene nuclei. Given the localization of Umbrea at telomeres and the interaction of Umbrea with the telomere-associated proteins HP1 and HOAP, this result is not really surprising at first glance. However, the mechanisms by which telomeres attach to each other in polytene nuclei are not currently understood. It is speculated that mitotic and polytene chromosomes have different mechanisms of telomere protection. In polytene chromosomes, telomere associations depend largely on the length of the retrotransposon arrays. On the other hand, in contrast to mitotic chromosomes, defects in the cap protein structure have not been shown to modify the frequencies of polytene telomere fusions. Important differences observed between the polytene and mitotically dividing cells are speculated to be due to the fact that salivary gland differentiation and transition from mitotic divisions to endocycles takes place in early embryogenesis. In this respect, maternally contributed HP1 from heterozygous Su(var)2-5 mutants is still sufficient to suppress telomeric fusions. However, a different RNAi-mediated approach was used to deplete Umbrea using the early embryonic driver line G61. Given the observed telomere-telomere fusion of polytene chromosomes, it is speculated that the fusion potential depends critically on the onset of Umbrea protein reduction (Joppich, 2009).

It is known that mutations in Su(var)2-5 cause both telomere fusion and telomere retrotransposon elongation. Ultimately, on the basis of umbrea-specific RNAi analyses, the telomere fusion cannot be attributed to defects in the protein cap structure or to the presence of excessive retrotransposon arrays. It might even be possible that Umbrea, like HP1, exhibits functions in both mechanisms. However, the results clearly indicate that umbrea elicits a phenotype similar to that observed in mutants in the HP1- and HOAP-encoding genes cav and Su(var)2-5. It is assumed that Umbrea, together with HP1 and HOAP (and perhaps numerous additional proteins), forms a telomere-capping complex and is required for telomere function (Joppich, 2009).

HP1 associates with heterochromatin, telomeres and multiple euchromatic sites. It is speculated that the different locations of HP1 are related to multiple different functions. Umbrea is located not only at telomeres but also in the pericentric heterochromatin, at regions along the euchromatic arms and, interestingly, in the nucleolus. Given these different positions, it is assumed that the function of Umbrea is not limited to telomeres. The gene umbrea is essential for normal development since both the umbrea P-element mutant and RNAi depletion of Umbrea are lethal. Further studies are required for understanding the function of the chromo shadow domain protein Umbrea and its relationship with other heterochromatin binding proteins (Joppich, 2009).

Systematic protein location mapping reveals five principal chromatin types in Drosophila cells

Chromatin is important for the regulation of transcription and other functions, yet the diversity of chromatin composition and the distribution along chromosomes are still poorly characterized. By integrative analysis of genome-wide binding maps of 53 broadly selected chromatin components in Drosophila cells, this study shows that the genome is segmented into five principal chromatin types (see Chromatin types are characterized by distinctive protein combinations and histone modifications) that are defined by unique yet overlapping combinations of proteins and form domains that can extend over > 100 kb. A repressive chromatin type was identified that covers about half of the genome and lacks classic heterochromatin markers. Furthermore, transcriptionally active euchromatin consists of two types that differ in molecular organization and H3K36 methylation and regulate distinct classes of genes. Finally, evidence is provided that the different chromatin types help to target DNA-binding factors to specific genomic regions. These results provide a global view of chromatin diversity and domain organization in a metazoan cell (Filion, 2010).

By systematic integration of 53 protein location maps this study found that the Drosophila genome is packaged into a mosaic of five principal chromatin types, each defined by a unique combination of proteins. Extensive evidence demonstrates that the five types differ in a wide range of characteristics besides protein composition, such as biochemical properties, transcriptional activity, histone modifications, replication timing, DNA binding factor (DBF) targeting, as well as sequence properties and functions of the embedded genes. This validates the classification by independent means and provides important insights into the functional properties of the five chromatin types (Filion, 2010).

Identifying five chromatin states out of the binding profiles of 53 proteins comes out as a surprisingly low number (one can form approximately 1016 subsets of 53 elements). It is emphasized that the five chromatin types should be regarded as the major types. Some may be further divided into sub-types, depending on how fine-grained one wishes the classification to be. For example, within each of the transcriptionally active chromatin types, promoters and 3' ends of genes exhibit (mostly quantitative) differences in their protein composition and thus could be regarded as distinct sub-types. However, these local differences are minor relative to the differences between the five principal types that are described in this study. It cannot be excluded that the accumulation of binding profiles of additional proteins would reveal other novel chromatin types. It is also anticipated that the pattern of chromatin types along the genome will vary between cell types. For example, many genes that are embedded in 'BLACK' chromatin (defined in Kc167 cells) are activated in some other cell types. Thus, the chromatin of these genes is likely to switch to an active type (Filion, 2010).

While the integration of data for 53 proteins provides substantial robustness to the classification of chromatin along the genome, a subset of only five marker proteins (histone H1, PC, HP1, MRG15 and BRM), which together occupy 97.6% of the genome, can recapitulate this classification with 85.5% agreement. Assuming that no unknown additional principal chromatin types exist in some cell types, DamID or ChIP of this small set of markers may thus provide an efficient means to examine the distribution of the five chromatin types in various cells and tissues, with acceptable accuracy (Filion, 2010).

Previous work on the expression of integrated reporter genes had suggested that most of the fly genome is transcriptionally repressed, contrasting with the low coverage of PcG and HP1-marked chromatin. BLACK chromatin, which consists of a previously unknown combination of proteins and covers about half of the genome, may account for these observations. Essentially all genes in BLACK chromatin exhibit extremely low expression levels, and transgenes inserted in BLACK chromatin are frequently silenced, indicating that BLACK chromatin constitutes a strongly repressive environment. Importantly, BLACK chromatin is depleted of PcG proteins, HP1, SU(VAR)3-9 and associated proteins, and is also the latest to replicate, underscoring that it is different from previously characterized types of heterochromatin (identified as BLUE and GREEN chromatin in this study) (Filion, 2010).

The proteins that mark BLACK domains provide important clues to the molecular biology of this type of chromatin. Loss of Lamin (LAM), Effete (EFF) or histone H1 causes lethality during Drosophila development. Extensive in vitro and in vivo evidence has suggested a role for H1 in gene repression, most likely through stabilization of nucleosome positions. The enrichment of LAM points to a role of the nuclear lamina in gene regulation in BLACK chromatin, consistent with the long-standing notion that peripheral chromatin is silent. Depletion of LAM causes derepression of several LAM-associated genes (Shevelyov, 2009), while artificial targeting of genes to the nuclear lamina can reduce their expression, suggesting a direct repressive contribution of the nuclear lamina in BLACK chromatin. D1 is a little-studied protein with 11 AT-hook domains. Overexpression of D1 causes ectopic pairing of intercalary heterochromatin (Smith, 2010), suggesting a role in the regulation of higher-order chromatin structure. SUUR specifically regulates late replication on polytene chromosomes (Zhimulev, 2003), which is of interest because BLACK chromatin is particularly late-replicating. EFF is highly similar to the yeast and mammalian ubiquitin ligase Ubc4 that mediates ubiquitination of histone H3, raising the possibility that nucleosomes in BLACK chromatin may carry specific ubiquitin marks. These insights suggest that BLACK chromatin is important for chromosome architecture as well as gene repression and provide important leads for further study of this previously unknown yet prevalent type of chromatin (Filion, 2010).

In RED and YELLOW chromatin most genes are active, and the overall expression levels are similar between these two chromatin types. However, RED and YELLOW chromatin differ in many respects. One of the conspicuous distinctions is the disparate levels of H3K36me3 at active transcription units. This histone mark is thought to be laid down in the course of transcription elongation and may block the activity of cryptic promoters inside the transcription unit. Why active genes in RED chromatin lack H3K36me3 remains to be elucidated (Filion, 2010).

The remarkably high protein occupancy in RED chromatin suggests that RED domains are 'hubs' of regulatory activity. This may be related to the predominantly tissue-specific expression of genes in RED chromatin, which presumably requires many regulatory proteins. It is noted that the DamID assay integrates protein binding events over nearly 24 hours, so it is likely that not all proteins bind simultaneously; some proteins may bind only during a specific stage of the cell cycle. It is highly unlikely that the high protein occupancy in RED chromatin originates from an artifact of DamID, e.g. caused by a high accessibility of RED chromatin. First, all DamID data are corrected for accessibility using parallel Dam-only measurements. Second, several proteins, such as EFF, SU(VAR)3-9 and histone H1 exhibit lower occupancies in RED than in any other chromatin type. Third, ORC also shows a specific enrichment in RED chromatin, even though it was mapped by ChIP, by another laboratory and on another detection platform. Fourth, DamID of Gal4-DBD does not show any enrichment in RED chromatin (Filion, 2010).

RED chromatin resembles DBF binding hotspots that were previously discovered in a smaller-scale study in Drosophila cells. Discrete genomic regions targeted by many DBFs have recently also been found in mouse ES cells , hence it is tempting to speculate that an equivalent of RED chromatin may also exist in mammalian cells. Housekeeping and dynamically regulated genes in budding yeast also exhibit a dichotomy in chromatin organization which may be related to the distinction between YELLOW and RED chromatin. The observations that RED chromatin is generally the earliest to replicate and strongly enriched in ORC binding, suggest that this chromatin type may be not only involved in transcriptional regulation but also in the control of DNA replication (Filion, 2010).

This analysis of DBF binding indicates that the five chromatin types together act as a guidance system to target DBFs to specific genomic regions. This system directs DBFs to certain genomic domains even though the DBF recognition motifs are more widely distributed. It is proposed that targeting specificity is at least in part achieved through interactions of DBFs with particular partner proteins that are present in some of the five chromatin types but not in others. The observation that yeast Gal4-DBD binds its motifs with nearly equal efficiency in all five chromatin types suggests that differences in compaction among the chromatin types represent overall a minor factor in the targeting of DBFs. Although additional studies will be needed to further investigate the molecular mechanisms of DBF guidance, the identification of five principal types of chromatin provides a firm basis for future dissection of the roles of chromatin organization in global gene regulation (Filion, 2010).

Protein complex of Drosophila ATRX/XNP and HP1a is required for the formation of pericentric beta-heterochromatin in vivo

ATRX belongs to the family of SWI2/SNF2-like ATP-dependent nucleosome remodeling molecular motor proteins. Mutations of the human ATRX gene result in a severe genetic disorder termed X-linked alpha-thalassemia mental retardation (ATR-X) syndrome. This study performed biochemical and genetic analyses of the Drosophila ortholog of ATRX (XNP). The loss of function allele of the Drosophila ATRX/XNP gene is semilethal. Drosophila ATRX is expressed throughout development in two isoforms, p185 and p125. ATRX185 and ATRX125 form distinct multisubunit complexes in fly embryo. The ATRX185 complex comprises p185 and heterochromatin protein HP1a. Consistently, ATRX185 but not ATRX125 is highly concentrated in pericentric beta-heterochromatin of the X chromosome in larval cells. HP1a strongly stimulates biochemical activities of ATRX185 in vitro. Conversely, ATRX185 is required for HP1a deposition in pericentric beta-heterochromatin of the X chromosome. The loss of function allele of the ATRX/XNP gene and mutant allele that does not express p185 are strong suppressors of position effect variegation. These results provide evidence for essential biological functions of Drosophila ATRX in vivo and establish ATRX as a major determinant of pericentric beta-heterochromatin identity (Emelyanov, 2010).

Mammalian and fly ATRX have previously been implicated in the function of heterochromatin, core histone modifications, regulation of DNA methylation, and interactions with heterochromatin protein HP1. However, this study demonstrates that metazoan ATRX can form a stable complex with HP1a in vivo. Although HP1a is known to physically interact with various partners, including histones, histone and DNA modification enzymes, DNA replication and repair proteins, nuclear structure proteins, and transcription factors, this work is the first demonstration that HP1 exists in a stable complex with a nucleosome remodeling factor (Emelyanov, 2010).

HP1a is known to homodimerize through interactions within its chromoshadow domain. Furthermore, at least two HP1a protomers are present in its complex with ATRX185. Considering the predicted molecular mass of the complex (∼200 kDa) and the large molecular excess of HP1a relative to ATRX/XNP in vivo, it is likely that ATRX185 binds a dimer of HP1a, and this heterotrimer constitutes the predominant native form of ATRX185-HP1a complex (Emelyanov, 2010).

HP1a apparently plays an important regulatory role in biochemical activities of XNP/ATRX. Interestingly, the basal ATPase activity of ATRX185 is somewhat inhibited in the absence of HP1a. Thus, HP1a may introduce a conformational change to the ATRX185 polypeptide that derepresses its enzymatic activity. HP1a also strongly stimulates the ability of ATRX185 to remodel nucleosomes in REA assay. In fact, ATRX185 possesses extremely little nucleosome remodeling activity in the absence of HP1a. This strong stimulation cannot be attributed solely to the enhanced ATPase activity of the enzyme. Therefore, HP1a may also promote nucleosome remodeling by other mechanisms. For instance, it may facilitate ATRX tethering to nucleosomes. Alternatively, ATRX may conversely stimulate HP1a interactions with chromatin templates, which will manifest as increased nucleosome remodeling in the REA assay (Emelyanov, 2010).

Importantly, the smaller isoform of ATRX/XNP (ATRX125) does not physically interact with HP1a and forms an alternative complex(es). In size-exclusion chromatography, recombinant ATRX125 is separated from the ATRX185-HP1a complex into a distinct peak with the molecular mass <150 kDa. This elution profile is unlike that of the native form of ATRX125, which fractionates in a peak with a predicted molecular mass of ∼500 kDa. Thus, the native ATRX125 likely forms a multisubunit complex with additional polypeptides that may be involved in regulation of biological functions of ATRX125 in vivo (Emelyanov, 2010).

Loss-of-function mutation of ATRX/XNP gene is semilethal. However, elimination of heterochromatin-specific ATRX185 isoform does not substantially affect fly viability. Therefore, ATRX125 has additional biological functions that do not depend on HP1a and are targeted toward euchromatic loci. For instance, the putative ATRX125 complex may play regulatory roles in transcription of certain euchromatic genes or regulate other chromatin functions (Emelyanov, 2010).

The xnp/atrx mutations are strong recessive suppressors of pericentric PEV in the X chromosome. They also have an effect on variegation of tandemly repeated transgene arrays and telomeric position effect. The latter observation suggests that ATRX/XNP may have a function in heterochromatin silencing that is independent of HP1a, as Su(var)2-5 alleles have little or no dominant effect of silencing of the telomeric 39C-5 insertion. Alternatively, the dose reduction of HP1a in heterozygous alleles of Su(var)2-5 may have a disproportionally weaker influence on its presence in telomeres, which would not be detected by analyzing PEV. On the other hand, the complete elimination of functional ATRX185-HP1a complex in homozygous xnp/atrx alleles may have a stronger effect on HP1a availability for both telomeric and pericentric silencing. Notably, xnp[5] is a weak dominant suppressor of pericentric PEV. This effect is not due to an antimorphic effect of expression of the truncated form of ATRX/XNP, as this truncated product is not localized to the normal pericentric site of Drosophila ATRX (Emelyanov, 2010).

In polytene chromosomes, ATRX185 specifically localizes to pericentric beta-heterochromatin of the X chromosome, where it overlaps with HP1a. This localization is unlikely to be explained by interactions with HP1a, because ATRX is largely excluded from other loci, where HP1a is abundantly present (e.g. chromocenter). Therefore, additional sequence determinants in the N terminus of ATRX185 (which are absent in ATRX125) are required for ATRX185 targeting toward the 20B-F cytogenetic region. In the future, it will be interesting to define these sequence motifs in the structure of ATRX185 (Emelyanov, 2010).

In a recent report (Schneiderman, 2009) it was shown that the pericentric focus of D. melanogaster ATRX in polytene chromosomes overlaps with a ∼50-kb satellite block of TAGA repeat. This sequence is not conserved in other Drosophila species, whereas the pericentric localization of ATRX/XNP is. The pericentric ATRX/XNP focus is a major site of replication-independent nucleosome replacement. However, the rapid histone turnover at this site appears to be sequence-dependent and does not require ATRX/XNP. It has also been speculated that the pericentric ATRX/XNP focus contributes to heterochromatic silencing throughout the nucleus, including ectopic loci, such as bwD. It was observed that certain variegated transgenes localized to heterochromatic sites outside of the ATRX/XNP focus are not responsive to ATRX. Thus, it remains likely that silencing by ATRX does require its physical localization to the cognate loci (Emelyanov, 2010).

Pericentric beta-heterochromatin is the most widely studied model of silent heterochromatin in vivo in Drosophila. It harbors heterochromatic genes, rRNA genes, repetitive sequences, and retrotransposon insertions, characteristic of 'heterochromatin.' The breakpoints of classical chromosome aberrations that exhibit PEV (such as w[m4]) and insertion sites of variegating transgenes are all positioned in beta-heterochromatin. The near elimination of HP1a from pericentric X beta-heterochromatin of xnp/atrx mutant flies reveals an important biochemical activity of ATRX in vivo. It is possible that the ATRX/XNP ATPase is required for efficient ATP-dependent deposition of HP1a into this genomic region. Alternatively, it is possible that most if not all HP1a that is normally associated with this region is present in a complex with ATRX/XNP. In either case, this result validates Drosophila ATRX as a major component and the determinant of pericentric beta-heterochromatin structure and function. The role of ATRX185 in its native complex with HP1a in establishment of beta-heterochromatin identity in the fly X chromosomes is yet another example of variable biochemical functions that SWI2/SNF2-like molecular motors can have in modification of chromatin structure in vivo (Emelyanov, 2010).

Specialization of a Drosophila capping protein essential for the protection of sperm telomeres

A critical function of telomeres is to prevent fusion of chromosome ends by the DNA repair machinery. In Drosophila somatic cells, assembly of the protecting capping complex at telomeres notably involves the recruitment of HOAP, HP1, and their recently identified partner, The hiphop gene was duplicated before the radiation of the melanogaster subgroup of species, giving birth to K81, a unique paternal effect gene specifically expressed in the male germline. This study shows that K81 specifically associates with telomeres during spermiogenesis, along with HOAP and HP1, and is retained on paternal chromosomes until zygote formation. In K81 mutant testes, capping proteins are not maintained at telomeres in differentiating spermatids, resulting in the transmission of uncapped paternal chromosomes that fail to properly divide during the first zygotic mitosis. Despite the apparent similar capping roles of K81 and HipHop in their respective domain of expression, it was demonstrated by in vivo reciprocal complementation analyses that they are not interchangeable. Strikingly, HipHop appeared to be unable to maintain capping proteins at telomeres during the global chromatin remodeling of spermatid nuclei. These data demonstrate that K81 is essential for the maintenance of capping proteins at telomeres in postmeiotic male germ cells. In species of the melanogaster subgroup, HipHop and K81 have not only acquired complementary expression domains, they have also functionally diverged following the gene duplication event. It is proposed that K81 specialized in the maintenance of telomere protection in the highly peculiar chromatin environment of differentiating male gametes (Dubruille, 2010).

K81 encodes a new telomere capping protein required for the transmission of functional paternal chromosomes to the diploid zygote. This finding elucidates the origin of the unique paternal effect lethal phenotype associated with K81. K81 is the first identified Drosophila telomere protein specifically expressed in the male germline. In fact, the structure and organization of telomeres in Drosophila male germ cells have remained largely unexplored. This study shows that during spermiogenesis, K81 accumulates in a small number of foci, where it is systematically associated with the HOAP and HP1 capping proteins. In contrast to HOAP, which is essentially a telomere-specific protein, HP1 is mainly enriched in pericentric heterochromatin in somatic nuclei. In addition, HP1 is also detected at telomeres and at numerous euchromatic sites on polytene chromosomes. In this regard, it is remarkable that HP1 is only retained at telomeric regions in spermatid nuclei, suggesting that its sole function in differentiating male germ cells is in capping telomeres. The lethality associated with cav (encoding HOAP) and Su(var)205 (encoding HP1) loss-of-function mutant alleles prevents direct testing of their respective roles during spermiogenesis. This study shows, however, that both HOAP and HP1 are lost from spermatid telomeres in K81 mutant testes. This loss of telomere capping proteins does not interfere with male gamete differentiation and maturation. Instead, the K81 mutant phenotype manifests itself only after fertilization and results in the incapacity of paternal chromosomes to segregate during the first zygotic mitosis. This initial defect leads to the formation of aneuploid embryos, which arrest development after a few abnormal nuclear divisions, or to the occasional escaping of haploid gynogenetic embryos that die shortly before hatching. The systematic and specific bridging of paternal chromatin during the first anaphase most likely results from the presence of chromosome end-to-end fusions. Although telomere fusions can be easily observed in cultured cells or in squashed preparations of larval brains, where they form chains of connected chromosomes, these defects appeared to be very difficult to observe in detail in Drosophila zygotes. Nonetheless, chromatin bridges associated with telomere dysfunction have been reported in syncytial embryos from mothers bearing hypomorphic alleles of mre11 or nbs, thus indicating that the DNA repair machinery presumably responsible for the fusion of uncapped telomeres is already active during early cleavage divisions (Dubruille, 2010).

The distribution of telomere capping protein foci in spermatid nuclei indicates that telomeres tend to associate within clusters during spermiogenesis. Interestingly, telomere clustering seems to be a conserved feature of animal spermiogenesis, such as in mammals, in which telomeres from the same chromosome are frequently associated in pairs. In Drosophila, telomere clustering is apparently the rule in late spermatids, as well as in the decondensing male pronucleus, because a single major focus of capping proteins is frequently observed in these nuclei. It is likely that this spectacular gathering of telomeres in a limited nuclear volume could favor the occurrence of paternal chromosome end-to-end fusions in K81 mutants (Dubruille, 2010).

Despite their critical role in chromosome protection, telomere proteins are rapidly evolving from yeasts to mammals. This tendency is observed in Drosophila, where important capping proteins such as HOAP, Verrocchio, Modigliani, and HipHop are encoded by fast-evolving genes. Previous work has shown that K81 is a relatively young gene that is restricted to the nine species comprising the melanogaster subgroup. K81 originated after the duplication of its paralog, hiphop (originally known as CG6874/l(3)neo26), presumably through a retroposition mechanism. The predicted K81 transcription start site is only about 100 bp from the 5' end of the Rb97D gene, which is expressed in primary spermatocytes and is required for male fertility. The selection of both hiphop and K81 genes was thus likely favored by the immediate acquisition of male germline-specific expression of the duplicated copy, after its landing close to Rb97D, followed by loss of hiphop expression in this lineage. In a less parsimonious, alternative scenario, an ancestral male germline-specific hiphop gene could have evolved a somatic and female germline expression following the duplication. However, this possibility does not fit with the expected requirement of HipHop for telomere protection in somatic cells. Interestingly, with a single exception, all Drosophila sequenced species outside the melanogaster subgroup have a single member of the hiphop/K81 gene family. For instance, D. ananassae, D. pseudoobscura, and D. persimilis have hiphop with the same conserved synteny as in melanogaster species but lack K81. In these three species, hiphop is thus expected to protect telomeres in all cells, including male germ cells. Most interestingly, phylogenetic analysis reveals the existence of a second, independent duplication of hiphop in the lineage leading to D. willistoni. Moreover, this D. willistoni hiphop duplicate presents a male-biased expression, allowing the possibility that it could be required in the male germline, like K81 in D. melanogaster. Although functional studies are not currently feasible in non-melanogaster species, developmental in situ expression analysis of members of this gene family may support these predictions (Dubruille, 2010).

In their respective cellular environments, HipHop and K81 are both specifically localized at telomeres, and they are required for the maintenance of the HOAP and HP1 capping proteins at chromosome ends. However, and despite the apparently identical molecular functions of K81 and HipHop, these experiments demonstrate that they cannot replace one another in vivo. When ectopically expressed in the male germline, GFP::HipHop is able to transiently restore the localization of HOAP and HP1 at spermatid telomeres in a K81 mutant background. In this genetic context, telomeres remained capped until the global replacement of histones with sperm-specific nuclear proteins. What actually triggers the loss of HipHop, HP1, and HOAP in these spermatids is not known. The fact that these proteins disappear concomitantly with the onset of global spermatid chromatin remodeling suggests a causal link, although this remains to be established. In mammals, although telomere integrity in male gametes is essential for zygote formation, little is known about the organization of telomeres in germ cells. However, a few studies point to the peculiar composition of telomere complexes in human sperm, suggesting that the unique organization of sperm chromatin imposes constraints on the structure and function of telomeres. Similarly, this study suggests that K81 specialized in the epigenetic maintenance of telomere identity in the highly peculiar chromatin environment of male gametes. This scenario also implies that HipHop lost its ability to protect sperm telomeres after the emergence of K81 function. Phylogenetic analysis of the hiphop and K81 coding sequences actually supports this subfunctionalization scenario. First, hiphop and K81 genes show a symmetrical acceleration of evolution in the melanogaster subgroup of species. Second, synonymous and nonsynonymous nucleotide substitution analysis of the coding sequences indicates that hiphop and K81 evolved under purifying selection. Finally, K81 expression in somatic cells does not rescue the zygotic lethality of hiphop mutants, thus confirming the functional divergence of both proteins (Dubruille, 2010).

The maternal expression of hiphop is apparently sufficient to protect telomeres during embryo development, as observed with mutations in other telomere capping genes. Accordingly, this study has shown that maternally expressed GFP::HipHop decorates both paternal and maternal telomeres as soon as the diploid zygote is formed. However, the early larval zygotic lethality of hiphop mutants prevented a more detailed in vivo phenotypic analysis using third instar larvae polytene chromosomes or neuroblast mitotic chromosomes. Although both mRFP1::K81 and GFP::K81 are fully able to associate with somatic telomeres, these experiments could only be carried out in a wild-type hiphop genetic background. It is thus not know whether K81 associates with somatic telomeres autonomously or through its association with other capping proteins, such as HOAP and/or HP1, in a HipHop-dependent manner (Dubruille, 2010).

The functional divergence of HipHop and K81 could reflect their adaptation to different chromatin environments. However, as new Drosophila telomere proteins are regularly discovered, it is also reasonable to consider the possibility that K81 and HipHop require one or more yet-unknown protein partners to function properly. For instance, K81 could not protect telomeres in somatic cells if its capping activity requires another factor only expressed in spermatids. Interestingly, the HP1-related protein Umbrea/HP6, which has been recently proposed to function in telomere protection (Joppich, 2009), is mainly expressed in the adult testis. Future studies should thus aim at determining whether other capping proteins are specialized in the protection of telomeres in germ cells, like K81 (Dubruille, 2010).

In conclusion, this study demonstrates that HipHop and K81 diverged not only in their domain of expression, but also in their ability to protect telomeres in their respective cellular environments. A challenge will be to understand the nature of the evolutionary pressure that ultimately shaped the diversification of the hiphop/K81 gene family in the genus Drosophila (Dubruille, 2010).

HipHop interacts with HOAP and HP1 to protect Drosophila telomeres in a sequence-independent manner

Telomeres prevent chromosome ends from being repaired as double-strand breaks (DSBs). Telomere identity in Drosophila is determined epigenetically with no sequence either necessary or sufficient. To better understand this sequence-independent capping mechanism, proteins were isolated that interact with the HP1/ORC-associated protein (HOAP) capping protein, and HipHop was identified as a subunit of the complex. Loss of one protein destabilizes the other and renders telomeres susceptible to fusion. Both HipHop and HOAP are enriched at telomeres, where they also interact with the conserved HP1 protein. A model telomere lacking repetitive sequences was developed to study the distribution of HipHop, HOAP and HP1 using chromatin immunoprecipitation (ChIP). It was discovered that they occupy a broad region >10 kb from the chromosome end and their binding is independent of the underlying DNA sequence. HipHop and HOAP are both rapidly evolving proteins yet their telomeric deposition is under the control of the conserved ATM and Mre11-Rad50-Nbs (MRN) proteins that modulate DNA structures at telomeres and at DSBs (Gao, 2009). This characterization of HipHop and HOAP reveals functional analogies between the Drosophila proteins and subunits of the yeast and mammalian capping complexes, implicating conservation in epigenetic capping mechanisms (Gao, 2010).

Telomeres shield the ends of linear chromosomes from DNA repair activities. This capping function is essential for genome integrity, as uncapping can lead to chromosome fusions. Telomeres also facilitate the elongation of chromosome ends, a function performed by the telomerase enzyme in most eukaryotic organisms studied. Loss of telomerase function does not impair genome stability immediately, but only does so when telomeric repeats become critically short after several generations. However, loss of the capping function can have immediate effects on genome integrity, suggesting that the presence of telomeric repeats is not sufficient for maintaining telomere identity. Furthermore, specialized yeast and plant cells can be immortalized in the absence of telomeric repeats with protected telomeres, suggesting that the presence of the repeats is also not necessary for capping. These results suggest that sequence-independent capping might serve as a backup mechanism in telomerase-maintained organisms (Gao, 2010 and references therein).

The understanding of this mechanism requires a clear picture of chromatin structure at telomeres. In lower eukaryotes, telomeric repeats are not packaged into regular nucleosomes, while the bulk of telomeric repeats in mammalian cells are packaged into nucleosome arrays. Partly due to the repetitive nature of telomeric sequences, it has been difficult to study how duplex-binding proteins are distributed over telomeric chromatin in most organisms. The Rap1 protein from budding yeast binds telomeric repeats to serve its functions in telomere elongation and capping regulation. Interestingly, Rap1 from budding and fission yeast and Taz1 from fission yeast have been localized to subtelomeric regions, suggesting that the binding of capping proteins need not be limited to the extreme end of a chromosome (Gao, 2010 and references therein).

In Drosophila, telomere identity is determined epigenetically. Although telomeres are elongated by the transposition of telomere-specific retrotransposons, these elements are neither necessary nor sufficient for capping. In particular, terminally deleted chromosomes that lack telomeric retrotransposons are stable, hence capped, for many generations. In addition, population studies uncovered frequent occurrences of such terminally deleted chromosomes in natural populations (Gao, 2010 and references therein).

Despite using a telomerase-independent mechanism for elongating chromosome ends, Drosophila use highly conserved factors to regulate capping. The ATM and ATR checkpoint kinases, along with the Mre11-Rad50-Nbs (MRN) complex and the ATRIP protein, respectively, control redundant pathways for capping regulation that are conserved in other organisms. Several other proteins serving capping function in Drosophila have homologs in other organisms: HP1, UbcD1, Woc and the H2A.Z histone variant. Epigenetic capping mechanisms that might be conserved in other organisms can be effectively studied in the unique system of Drosophila due to the natural uncoupling of the end capping function from the end elongation function (Gao, 2010).

Telomeres in yeast and mammals are capped by multi-subunit protein complexes that protect both the duplex and single-stranded regions of the telomere. In Drosophila, the structural constituents of the 'cap' remain poorly defined. The HP1/ORC-associated protein (HOAP) is cytologically present at telomeres, and loss of HOAP leads to telomere fusions. This study isolated HOAP-interacting proteins by affinity immunoprecipitation and identified the HP1-HOAP-interacting protein (HipHop) as a new component of the Drosophila capping complex. Using chromatin immunoprecipitation (ChIP) performed on a model telomere devoid of telomeric transposons, a large domain of telomeric chromatin was discovered enriched with HipHop, HOAP and HP1, suggesting that this capping complex prevents end fusion by maintaining a chromatin state that is independent of its underlying DNA sequence. Both HipHop and HOAP are fast-evolving proteins highlighting a common feature among telomeric-binding proteins in other organisms. On the basis of functional similarity and analogies in distribution patterns, it is suggested that HipHop and HOAP serve similar function as subunits of the capping complex that bind the duplex region of telomeric DNA in other organisms (Gao, 2010).

This study identified HipHop based on its ability to associate with HOAP through biochemical purification. Such an approach could be useful for future studies in Drosophila telomere biology. The biochemical approach was aided by an ability to epitope-tag the endogenous caravaggio cav locus, eliminating potential artifacts associated with the overproduction of bait proteins. With the recent development of the SIRT targeting method in Drosophila, biochemical purification using endogenous tags could be efficiently applied in the study of other biological processes in Drosophila (Gao, 2010).

Several lines of evidence suggest that HipHop and HOAP likely function as a complex. First, HipHop was abundantly present in HOAP IPs, suggesting a strong interaction between the two proteins. Second, bacteria expressed HipHop was able to interact with HOAP in fly extracts. Third, the changes of HOAP and HipHop levels showed inter-dependency. Fourth, the loading of both HipHop and HOAP to telomeres was under the same genetic controls of MRN and ATM. Finally, the two proteins had very similar distribution patterns on the model telomere and co-localized precisely in immunostaining experiments. On the basis of some of the same criteria, HP1 is likely to be a part of the complex. The Modigliani(Moi)/DTL protein was recently identified as another capping protein that is enriched at telomeres and interacts with both HOAP and HP1. No Moi/DTL peptides in were detected in HOAP IPs (Gao, 2010).

The model telomere D4ATD has allowed an unprecedented view of the chromatin landscape in the vicinity of a Drosophila telomere. HipHop, HOAP and HP1 were located essentially at the very end of a chromosome, strengthening earlier results from immunolocalization experiments. Remarkably, HipHop, HOAP and HP1 seem to bind to a much larger region than the immediate vicinity of the chromosome end. One possible mechanism is envisioned that could lead to such a binding pattern. After the initial recruitment of the HipHop-HOAP complex to the chromosome end, the complex 'spreads' internally to cover a larger region. It is tempting to speculate that this 'spreading' might be mediated by HP1, since a binding pattern of HP1 was observed essentially identical to those of HipHop and HOAP on D4ATD. However, results from ChIP experiments using HeT-A primers suggest that HP1 occupies a larger region than HipHop or HOAP on transposon-capped telomeres, which implies that the mere presence of HP1 on chromatin is not sufficient for HipHop or HOAP binding. In addition, HOAP can be localized to telomeres in su(var)205/hp1 mutants, suggesting that HP1 is not necessary for HOAP and possibly HipHop binding to telomeres. Whether HP1 affects the extent of HipHop-HOAP spreading requires ChIP localization of HipHop and HOAP on the model telomere in a su(var)205 mutant background (Gao, 2010).

It is suggested that the binding patterns of HipHop and HOAP on the model telomere is a qualitative reflection of their patterns on natural telomeres, since very similar binding intensity of HipHop on D4ATD versus its homologous telomere is observed in immunostaining experiments. Similar observations were documented for HP1 on polytene and HOAP on mitotic telomeres using TDs (Gao. 2010).

HipHop and HOAP share functional characteristics with capping proteins in other eukaryotes. First, they bind to the double-stranded region of the telomere in vivo. Second, they occupy a large domain on telomeric chromatin. Third, they are continuously present at the telomeres. Finally, the loss of these proteins leads to frequent telomere fusions. It is suggested that HipHop and HOAP behave similarly and might serve similar functions as the Rap1 protein in S. cerevisiae, Taz1 in S. pombe, and TRF2 in mammals. Further dissection of HipHop and HOAP's molecule function would be needed to confirm this suggestion (Gao, 2010).

The telomere loading of HipHop and HOAP is under the control of ATM and MRN. The same set of proteins mediate the loading of various telomeric factors including telomerase activity, and the Cdc13 capping protein in yeast. This high degree of functional conservation suggest that it is unlikely that these factors directly act on capping proteins, which are generally divergent at the sequence level. It is more likely that these proteins modulate a common DNA/chromatin structure at telomeres of eukaryotic cells. One conceivable candidate for this 'universal' structure is the terminal 3' overhang (reviewed in Lydall, 2009). The reduced occupancy of HipHop, HOAP and HP1 at the extreme end of the model telomere, suggests that Drosophila chromosomes might also terminate as a 3' overhang (Gao, 2010).

HipHop and HOAP seem to evolve faster than typical proteins. An interesting proposition is that this faster rate of evolution is driven by the fast-evolving telomeric retrotransposons (Villasante, 2008), to which the HipHop-HOAP complex binds. HOAP was implicated in binding DNA (Shareef, 2001). Whether HipHop is capable of binding DNA directly is currently under investigation. Under the limited resolution of immunostaining, no change was detected in HipHop-HOAP binding efficiency to telomeres with different levels of retrotransposons. Nor were observed any phenotypic effects of having a 'retrotransposon-free' telomere. Although TDs can be efficiently maintained under laboratory conditions, it remains undetermined whether there is any fitness cost for animals with a TD irrespective of the loss of essential genes. Therefore, further studies are required to identify the driving force for the fast evolution of HipHop and HOAP (Gao, 2010).

Interestingly, telomeric proteins from other systems are generally less conserved at the sequence level and show signs of fast evolution. Further investigation into the functional relationship between HipHop-HOAP and the telomeric retrotransposons in Drosophila might reveal the significance for this fast evolution of telomeric proteins in general (Gao, 2010).

The HP1a disordered C terminus and chromo shadow domain cooperate to select target peptide partners

Drosophila melanogaster heterochromatin protein 1a (HP1a) is essential for compacted heterochromatin structure and the associated gene silencing. Its chromo shadow domain (CSD) is well known for binding to peptides that contain a PXVXL motif. Heterochromatin protein 2 (HP2) is a non-histone chromosomal protein that associates with HP1a in the pericentric heterochromatin, telomeres, and the fourth chromosome. Using NMR spectroscopy, fluorescence polarization, and site-directed mutagenesis, this study identified an LCVKI motif in HP2 that binds to the HP1a CSD. The binding affinity of the HP2 fragment is approximately two orders of magnitude higher than that of peptides from PIWI (with a PRVKV motif), AF10 (with a PLVVL motif), or CG15356 (with LYPLL and LSIVA motifs). To delineate differential interactions of the HP1a CSD, its structure, backbone dynamics, and dimerization constant was characterized. This study found that the dimerization constant is bracketed by the affinities of HP2 and PIWI, which dock to the same HP1a homodimer surface. This suggests that HP2, but not PIWI, interaction can drive the homodimerization of HP1a. Interestingly, the integrity of the disordered C-terminal extension (CTE) of HP1a is essential for discriminatory binding, whereas swapping the PXVXL motifs does not confer specificity. Serine phosphorylation at the peptide binding surface of the CSD is thought to regulate heterochromatin assembly. Glutamic acid substitution at these sites destabilizes HP1a dimers, but improves the interaction with both binding partners. These studies underscore the importance of CSD dimerization and cooperation with the CTE in forming distinct complexes of HP1a (Mendez, 2011)

The DEK oncoprotein is a Su(var) that is essential to heterochromatin integrity

Heterochromatin integrity is crucial for genome stability and regulation of gene expression, but the factors involved in mammalian heterochromatin biology are only incompletely understood. This study identified the oncoprotein DEK, an abundant nuclear protein with a previously enigmatic in vivo function, as a Suppressor of Variegation [Su(var)] that is crucial to global heterochromatin integrity. DEK interacts directly with Heterochromatin Protein 1 alpha (HP1alpha) and markedly enhances its binding to trimethylated H3K9 (H3K9me3), which is key for maintaining heterochromatic regions. Loss of Dek in Drosophila leads to a Su(var) phenotype and global reduction in heterochromatin. Thus, these findings show that DEK is a key factor in maintaining the balance between heterochromatin and euchromatin in vivo (Kappes, 2011).

The observation that knocking down DEK expression leads to a marked diminution of the H3K9me3 heterochromatin mark suggested that, as a consequence of the decrease in HP1alpha being brought to histones, there is less efficient recruitment of KMT1 A/B [Su(var)3-9]. Thus, to determine if DEK is indeed a functional member of self-sustaining silencing loops acting through coordinated recruitment of HP1alpha and thus KMT 1 A/B to the four genes (Oct3/4, DHFR, PAX3, and MYOD) examined in HeLa cells, chromatin immunoprecipitation (ChIP) assays were performed. As no KMT 1A/B-specific antibodies were available, stable DEKkd or stable GFP-DEK overexpression was established in HEK293 cell lines that had been engineered previously to stably express low levels of hemagglutinin (HA)-tagged-KMT1 A/B. In the ChIP assays, a significantly reduced abundance of H3K9me3 was found at all genes examined in the HEK293 DEKkd cells, H3K9me3, control, and shDEKB1, confirming results obtained in HeLa cells. Furthermore, reduced gene-specific H3K9me3 levels coincided with a marked reduction in the abundance of KMT1 A/B. In strong support of the knockdown data, significantly increased H3K9me3 levels were identified in GFP-DEK-overexpressing HEK293 cells at the genes investigated, accompanied by increased occupancy of KMT1 A/B at these particular genes. Thus, both knockdown and overexpression studies demonstrate that DEK coordinates the recruitment of KMT1 A/B to specific genes, thus regulating H3K9 trimethylation (Kappes, 2011).

In summary, This study has identified DEK as a novel Su(var) factor with a conserved role in global heterochromatin integrity that functions by augmenting the binding of HP1alpha (and KMT1 A/B) to the H3K9me3 heterochromatic mark. As it has been shown previously that HP1alpha and KMT1 A/B are crucial to self-sustaining silencing loops, disruption of this mechanism can lead to significant changes in the epigenome of a given cell. Furthermore, DEK is the only oncoprotein described that directly affects heterochromatin integrity on a global level. This effect was seen in Drosophila and cell lines derived from patients with cervical cancer (HeLa S3), human embryonic kidney cells, and melanoma cells. The observation that DEK is a key factor in heterochromatin biology suggests that disruption of the balance between euchromatin and heterochromatin could play an important role in the pathogenesis of cancers in which DEK expression is altered. Most notably, the findings indicate that DEK, a nonhistone protein with no known enzymatic activity, plays a vital role in global heterochromatin integrity across species (Kappes, 2011).

HP1a targets the Drosophila KDM4A demethylase to a subset of heterochromatic genes to regulate H3K36me3 levels

Recent discoveries of histone demethylases demonstrate that histone methylation is reversible. However, mechanisms governing the targeting and regulation of histone demethylation remain elusive. A Drosophila melanogaster JmjC domain-containing protein, dKDM4A (Histone demethylase 4A), is a histone H3K36 demethylase. dKDM4A specifically demethylates H3K36me2 and me3 both in vitro and in vivo. Affinity purification and mass spectrometry analysis revealed that Heterochromatin Protein 1a (HP1a) associates with dKDMA4A. The chromoshadow domain of HP1a and a HP1-interacting motif of dKDM4A are responsible for this interaction. HP1a stimulates the histone H3K36 demethylation activity of dKDM4A and this stimulation depends on the H3K9me binding motif of HP1a. Finally, in vivo evidence is provided suggesting that HP1a and dKDM4A interact with each other and loss of HP1a leads to increased level of histone H3K36me3. Collectively, these results suggest a function of HP1a in transcription facilitating H3K36 demethylation at transcribed and/or heterochromatin regions (Lin, 2012).

This study has identified one of the JmjC domain-containing KDM4 orthologs in Drosophila, dKDM4A. The in vitro demethylation assay shows that dKDM4A demethylates histone H3K36me3 and me2 using an oxidative demethylation mechanism which requires Fe (II) and α-ketoglutarate as cofactors. Overexpression of dKDM4A in Drosophila S2 cells reduces the level of histone H3K36me3, whereas knockdown of endogenous dKDM4A increases the level of histone H3K36me3 and me2. These results together demonstrate that dKDM4A is a bona fide histone H3K36 demethylase in vivo (Lin, 2012).

Through multidimensional protein identification technology (MudPIT) analysis of the affinity-purified native dKDM4A complex, it was found that HP1a associates with dKDM4A. More importantly, it was demonstrated that HP1a stimulates the demethylation activity of dKDM4A, while the HP1a CD mutant V26M, that cannot bind methyl-K9 histone H3, fails to stimulate dKDM4 activity. In addition, dKDM4A directly binds to the HP1 CSD and this binding requires an intact HP1 CSD dimer interface. A consensus HP1 binding PxVxL motif of dKDM4A is responsible for its interaction with CSD of HP1a. Interestingly, overexpression of dKDM4A causes the spread of HP1a to euchromatin regions, presumably through this specific interaction, and dKDM4A-V423A, which does not bind to HP1a, failed to localize HP1a to euchromatin. These data suggest HP1a-dKDM4A is a euchromatic H3K36 demethylase complex (Lin, 2012).

Set2 mediated histone H3K36 methylation is an important mark on chromatin during transcription elongation . In fungi, such as S. cerevisiae, S. pombe, and N. crassa, a sole histone lysine-methyltransferase Set2 is responsible for all three methylation states of H3K36. In Drosophila, histone H3K36 methylation is catalyzed by two enzymes, dSet2 and dMes-4. Although yeast Set2 is the only histone methyltransferase that catalyzes methylation of histone H3K36, two histone H3K36 demethylases, Jhd1 and Rph1, are responsible for demethylation of histone H3K36 at different modification states in budding yeast. In Drosophila, there are three histone demethylases that govern demethylation of histone H3K36. dKDM2 has been identified as a histone H3K36me2 demethylase (Lagarou, 2008). This study demonstrates that dKDM4A is a histone H3K36me3 and me2 demethylase, and dKDM4B has demethylation activity on both histone H3K9 and K36me3/me2 in vitro. Therefore, histone H3K36 methylation in Drosophila is likely regulated by highly specific enzymes in both directions. Since both modification and de-modification enzymes possess high modification state specificity, histone H3K36 may be subjected to much more sophisticated regulation in higher eukaryotes than in yeast (Lin, 2012).

Purification of the dKDM4A complex from S2 cells revealed a specific association of HP1a with dKDM4A. Three of the HP1-like chromatin proteins (HP1a, HP1b, HP1c) in Drosophila share high amino acid sequence similarity. Both HP1a and HP1b localize to euchromatin and heterochromatin, while HP1c is found only in euchromatin. It is unclear whether these HP1-like chromatin proteins have specific or redundant functions in transcription regulation. However, this study demonstrates that dKDM4A specifically interacts with HP1a, but not HP1b and HP1c. Furthermore, HP1b and HP1c cannot stimulate dKDM4A demethylation activity in vitro. A previous study showed that the yeast homolog of KDM4, Rph1 (ScKDM4), did not stably associate with any other protein. It was speculated that the C-terminal ZF domain of Rph1, which can potentially bind to DNA, allows Rph1 to function without associated factors. Unlike other proteins in the KDM4 family which commonly contain PHD, tudor or ZF domains, dKDM4A only has JmjN and JmjC domains. This study found that HP1a stably associates with dKDM4A and stimulates its demethylation activity. Since the H3K9 binding motif is required for this stimulation, it is proposed that the CD of HP1a might contribute to target dKDM4A to specific loci, particularly to H3K9me enriched regions, to regulate gene expression (Lin, 2012).

In S. pombe, the HP1 homolog recruits a JmjC domain-containing protein Epe1 to heterochromatin loci where they function together to counteract repressive chromatin. This study shows that HP1a directly interacts with dKDM4A through a consensus binding motif PxVxL. Most importantly, the presence of HP1a stimulates histone demethylation activity of dKDM4A in vitro, and HP1a is required for maintaining normal level of H3K36me3 in vivo as well. Since Epe1 on its own seems to have no histone demethylation activity, it would be interesting to see whether a similar scenario also occurs in S. pombe, in which Swi6 may stimulate enzymatic activity of Epe1 towards other non-histone substrates (Lin, 2012).

HP1 has been reported to associate with actively transcribed euchromatin regions. Mammalian HP1γ and histone H3K9 methylation are enriched at the coding region of active genes, implying that they may play a role during transcription elongation. In yeast, histone H3K36me3 appears to be a repressive mark at coding region of actively transcribed genes. In higher eukaryotes, histone H3K9 methylation, which is absent in the budding yeast, might replace the role of K36 methylation in the coding regions of transcribed genes. However, the mechanism by which HP1 functions in active transcription is largely unknown. The current findings suggest a possible role of HP1a in recruitment of the histone H3K36me3/me2 demethylase dKDM4A to transcribed regions to remove histone H3K36 methylation. The formation of the HP1a-dKDM4A complex may help to release HP1a from heterochromatin regions, thus targeting it to specific gene loci. It is also possible that dKDM4A, which targets histone modification marks within the 3' ORF of actively transcribed genes, recruits HP1a to euchromatic regions. A model is favored in which HP1a facilitates recruitment of dKDM4A, because the HP1a CD mutant, V26M, fails to stimulate dKDM4A activity. This result suggests that HP1a binding to histone H3 is required for the enhancement of dKDM4A demethylation activity. It is speculated that HP1a-mediated histone demethylation may serve as a regulatory mechanism to control chromatin states during active transcription elongation. Alternatively, a similar mechanism might also apply to maintaining silenced states of heterochromatin (Lin, 2012).

HP1a recruitment to promoters is independent of H3K9 methylation in Drosophila melanogaster

Heterochromatin protein 1 (HP1) proteins, recognized readers of the heterochromatin mark methylation of histone H3 lysine 9 (H3K9me), are important regulators of heterochromatin-mediated gene silencing and chromosome structure. In Drosophila three histone lysine methyl transferases (HKMTs) are associated with the methylation of H3K9: Su(var)3-9, Setdb1 (Eggless), and G9a. To probe the dependence of HP1a binding on H3K9me, its dependence on these three HKMTs, and the division of labor between the HKMTs, correlations were examined between HP1a binding and H3K9me patterns in wild type and null mutants of these HKMTs. Su(var)3-9 was shown to control H3K9me-dependent binding of HP1a in pericentromeric regions, while Setdb1 controls it in cytological region 2L:31 and (together with POF) in chromosome 4. HP1a binds to the promoters and within bodies of active genes in these three regions. More importantly, however, HP1a binding at promoters of active genes is independent of H3K9me and POF. Rather, it is associated with heterochromatin protein 2 (HP2) and open chromatin. These results support a hypothesis in which HP1a nucleates with high affinity independently of H3K9me in promoters of active genes and then spreads via H3K9 methylation and transient looping contacts with those H3K9me target sites (Figueiredo, 2012).

Chromosome 4 is considered to be a repressive environment that is enriched in heterochromatin markers such as HP1a and methylated H3K9. It contains large blocks of repeated sequences and transposable elements interspersed with the genes, and transgenes inserted on the 4th chromosome often show variegated expression because of partial silencing. Despite its heterochromatic nature genes located on the 4th chromosome are expressed as strongly on average, or even more strongly, than genes on other chromosomes. Traditionally, the division of genomes into heterochromatic and euchromatic regions was based on cytological characteristics of chromatin in interphase. Today more elaborate definitions of chromatin states are available based on chromatin components, such as the five principal chromatin types defined in (Filion, 2010). According to these definitions, pericentromeric heterochromatin and the 4th chromosome is highly enriched in 'green-chromatin'. Similar definitions have been constructed by the modENCODE project, distinguishing nine chromatin states (Kharchenko, 2011), one of which (chromatin state 7) corresponds to 'green-chromatin'. HP1a and H3K9me2/me3 are the key components distinguishing green-chromatin and chromatin state 7. Maps of HP1a and H3K9me2/me3 in chromatin from dissected salivary gland tissue correlate with previously reported high-resolution ChIP-chip and DamID maps of chromatin from various cell lines, embryos and fly heads. Thus, the main regional chromosome organization into this chromatin type appears to be stable during development (Figueiredo, 2012).

The results show that the regional enrichment of HP1a depends on region-specificity of the HKMTs. The regional differences observed in whole chromosomes confirm previous results based on chromosome stainings, i.e., loss of Su(var)3-9 causes a reduction of HP1a and H3K9me in pericentromeric regions but not the 4th chromosome while loss of Setdb1 causes reductions of HP1a and H3K9me on the 4th chromosome and in region 2L:31. Loss of G9a results in no difference in H3K9me. No clear indications of redundancy were seen between the different HKMTs (Figueiredo, 2012).

The most important observations in this study are the fundamental differences between HP1a enrichment responses in gene bodies and promoters to losses of HKMT and H3K9 methylation. Upon loss of the region-specific HKMT, HP1a is strongly reduced or lost in gene bodies but the HP1a promoter peak is retained. These effects were observed in the pericentromeric region in Su(var)3-9 mutants and both the 4th chromosome and region 2L:31 in Setdb1 mutants. The observed HP1a binding in promoters is strongly indicative of H3K9me-independent nucleation sites. Interestingly, although the interaction between HP1a and methylated H3K9 is well documented, and was confirmed in these experiments, HP1 proteins have been shown to bind only weakly to reconstituted methylated nucleosomal arrays and purified native chromatin. For example, H3 peptides containing H3K9me3 bind HP1 with relatively weak (μM) affinity. This is in stark contrast to their nM affinity for unmodified histones and the stable interaction of HP1, probably to the histone fold region of H3, that occurs in S phase when DNA replication disrupts the histone octamers. It is concluded that HP1a binds to two distinct targets in chromatin: very stably and methylation-independently to promoters of active genes (probably via interactions within the nucleosomes) and less stably (but with perfect correlation) to methylated H3K9 sites (Figueiredo, 2012).

Considering the methylation-dependent and -independent binding of HP1a it is interesting to note that HP1a is essential for viability>, in contrast to the three studied HKMTs. Su(var)3-9 is not required for viability and homozygous null mutant stocks can be kept. The same is true for G9a. Setdb1 is claimed to be essential in Drosophila and is certainly required for female fertility. Nevertheless, in uncrowded conditions Setdb110.1a homozygous flies hatch although they have decreased viability, and pairwise crossings of the HKMT mutants have showed no clear effects in terms of reduced viability. The findings of H3K9me-independent HP1a binding to promoters tempt speculation that HP1a may be essential for survival due to the methylation-independent promoter binding of HP1a. However, HP1a has also been associated with non-chromatin based functions such as linkage to hnRNP particles, suggesting it may also be involved in RNA compaction, although the importance of this function remains elusive (Figueiredo, 2012).

The characterization of the HP1a bound promoter peaks led to two important findings. Firstly, promoters in the 4th chromosome and region 2L:31 have a significantly higher A/T content compared to promoters at other chromosomal locations. The presence of A/T rich motifs in general HP1a target sites have previously been reported and the results confirm that this is also true for promoter-specific HP1a targets. It has been shown that poly(dA:dT) tracts in promoters disfavour nucleosomes and modulate gene expression levels. In addition, promoters in the 4th chromosome are more DNase sensitive than promoters at other genomic locations, suggesting that the chromatin structure is more open within these promoters. In fact, it has been proposed that HP1a promotes an open chromatin structure at bound promoters. In contrast, gene bodies in the 4th chromosome are slightly less accessible to DNase, which again indicates that the HP1a binding to promoters is fundamentally different to the HP1a targeting in gene bodies. The slightly reduced DNase sensitivity in gene bodies is also consistent with the previously observed reduction of transcription elongation efficiency of genes on the 4th chromosome. Secondly, in a search for chromatin-associated factors that correlate with the HP1a binding promoter peaks, Heterochromatin protein 2 was found to show a close to perfect correlation. HP2 has previously been shown to interact with HP1a , and it has been suggested that HP2 interaction with the HP1a chromo-shadow domain drives HP1a dimerization (Mendez, 2011). The results show that the HP1a-HP2 interactions mainly occur at the HP1a bound promoters. This is consistent with previous observations that all mutations affecting HP1 dimerization abolish the interactions between HP1 and non-modified H3, since these interactions should mainly occur at promoters according to the current data (Figueiredo, 2012).

The results provide strong support for the model proposed by (Dialynas, 2006) that high affinity HP1a binding to the histone fold provides a nucleation site for HP1a targeting to chromatin. It is interesting to note that this incorporation is suggested to occur when the histone fold region of H3 becomes exposed because of active transcription, histone variant exchange or replication. A link between HP1 and replication has also been demonstrated by observed interactions between HP1 and the origin recognition complex (ORC), and the requirement of human ORC association with HP1 for correct targeting to heterochromatin. In addition, HP1a modulates replication timing in Drosophila and reduced levels of HP1a result in delayed replication of chromosome 4. It is speculated that HP1a binding to promoters avoids delay of this heterochromatic region's replication, that it provides an epigenetic nucleation mark for HP1a, and that the resulting nucleation is followed by a low affinity spreading to gene bodies. A transient looping contact model is envisioned in which the low affinity between HP1a and H3K9me provides the means for spreading of HP1a, analogous to the proposed model for the interactions of another Drosophila chromodomain protein, Polycomb (Pc). The chromo-domain of Pc interacts with H3K27me, but the nucleation sites for Pc are the Polycomb Response Elements, which have lower levels of H3K27me. Thus, the nucleation appears to be independent of H3K27me and is followed by spreading caused by transient contacts between Pc and H3K27me. Similarly to HP1a and H3K9me, the affinity of the Pc chromo-domain to H3K27me is relatively weak, with a dissociation constant in the μM range. In the case of HP1a the proposed spreading correlates with (and thus presumably depends on) at least three factors: H3K9me, active transcription and Painting of fourth (POF). The spreading appears to be generally restricted to transcribed genes, although there are two exceptions (onecut and CG1909) on the 4th chromosome. On the 4th chromosome, where POF binds to gene bodies, the HP1a enrichment is much higher than in region 2L:31. It should be stressed that HP1a and POF bindings are interdependent and POF also requires Setdb1 to target the 4th chromosome. Thus, the relationships between these factors remain elusive. Why is the gene body targeting of HP1a on the 4th chromosome Pof-dependent? This cannot be explained by expression differences, because although expression levels drop in Pof mutants the reductions are minor. It is hypothesized that POF binding to nascent RNA on active chromosome 4 genes may stabilize the interaction between HP1a and H3K9me as an adaptor system linking histone marks to nascent RNA, similar to the chromatin adaptor model for alternative splicing (Figueiredo, 2012).

The enrichment of H3K9me on the 4th chromosome mainly depends on Setdb1, but in the most proximal region of the 4th chromosome the H3K9me is Su(var)3-9 dependent. Thus, the proximal region of chromosome 4 is similar to the proximal region of other chromosome arms in this respect. Position-effect variegation studies have shown that although most variegated (partially silenced) transgenic inserts on the 4th chromosome are suppressed in Setdb1, but not Su(var)3-9 mutants, the reporter insertion 118E-10 is suppressed in Su(var)3-9 mutants. Interestingly, this transgene is inserted in the pericentric region on the 4th chromosome, i.e., the region that according to this study is dependent on Su(var)3-9 (Figueiredo, 2012).

In summary, this study reports dual binding properties of the HP1a protein: an H3K9me methylation-independent binding at promoters and a methylation-dependent binding within gene bodies suggested to occur by spreading. Like arms of other chromosomes, the proximal region of the 4th chromosome is enriched in HP1a and Su(var)3-9-dependent H3K9me. However, in contrast to other chromosome arms, the gene-rich portion of the 4th chromosome is enriched in HP1a and H3K9me, and here the enrichment within gene bodies depends on Setdb1. The methylation-independent HP1a promoter binding correlates with HP2 and with 'open' chromatin structure. It is suggested that the methylation-independent and -dependent binding of HP1a are fundamental steps in the transmission, propagation and spreading of this epigenetic mark, hence the current observations provide important insights and the basis of a novel model of gene regulation in highly heterochromatic regions (Figueiredo, 2012).

Analysis of the heterochromatin protein 1 (HP1) interactome in Drosophila

Heterochromatin protein 1 (HP1) was first described in Drosophila melanogaster as a heterochromatin associated protein required for epigenetic gene silencing. Most eukaryotes have at least three HP1 homologs that play differential roles in heterochromatin and euchromatin. However, despite the fact that the three HP1 proteins bind to different regions of the genome, several studies show that most of the interactions occur in a manner specific to HP1a. In addition, little is known about the overall interaction network of the three Drosophila HP1 homologs, HP1a, HP1b, and HP1c. This first comprehensive proteomic analysis of Drosophila HP1 homologs was performed by coupling a double-affinity purification approach with MudPIT analysis to identify interacting proteins of Drosophila HP1. This analysis found that 160-310 proteins co-eluted with HP1, including a number of novel HP1-binding partners along with the previously identified HP1 binding proteins. Finally, this study showed that slight and unique binding preferences might exist between the three HP1 proteins in Drosophila. These studies are the first to systematically analyze the interactome of HP1 paralogs and provide the basic clues as to the molecular mechanism by which HP1 might control cellular processes. Most eukaryotes have at least three HP1 homologs with similar domain structures but with differential roles in heterochromatin and euchromatin. However, little is known about the overall interactome of the three Drosophila HP1 homologs, HP1a, HP1b, and HP1c. The present study compared interacting proteins of three HP1 homologs in Drosophila. To better understand the underlying mechanisms for gene regulation of HP1, a double-affinity purification and MudPIT mass spectrometry were employed to identify differential proteins as well as common binding proteins of HP1. Therefore, this study provides not only the comparative proteomic analysis but also molecular mechanism underlying the HP1 homolog-specific function (Ryu, 2014).

Heterochromatin-associated interactions of Drosophila HP1a with dADD1, HIPP1, and repetitive RNAs

Heterochromatin protein 1 (HP1a) has conserved roles in gene silencing and heterochromatin and is also implicated in transcription, DNA replication, and repair. This study identifies chromatin-associated protein and RNA interactions of HP1a by BioTAP-XL mass spectrometry and sequencing from Drosophila S2 cells, embryos, larvae, and adults. The results reveal an extensive list of known and novel HP1a-interacting proteins, of which three were selected for validation. A strong novel interactor, dADD1 (Drosophila ADD1) (CG8290), is highly enriched in heterochromatin, harbors an ADD domain similar to human ATRX, displays selective binding to H3K9me2 and H3K9me3, and is a classic genetic suppressor of position-effect variegation. Unexpectedly, a second hit, HIPP1 (HP1 and insulator partner protein-1) (CG3680), is strongly connected to CP190-related complexes localized at putative insulator sequences throughout the genome in addition to its colocalization with HP1a in heterochromatin. A third interactor, the histone methyltransferase MES-4, is also enriched in heterochromatin. In addition to these protein-protein interactions, this study found that HP1a selectively associated with a broad set of RNAs transcribed from repetitive regions. It is proposed that this rich network of previously undiscovered interactions will define how HP1a complexes perform their diverse functions in cells and developing organisms (Alekseyenko, 2014).

A major epigenetic programming mechanism guided by piRNAs

A central enigma in epigenetics is how epigenetic factors are guided to specific genomic sites for their function. It has been reported that a Piwi-piRNA complex associates with piRNA-complementary transposon targets in the Drosophila genome and regulates their epigenetic state. This study reports that Piwi-piRNA complexes bind to numerous piRNA-complementary sequences throughout the genome, implicating piRNAs as a major mechanism that guides Piwi and Piwi-associated epigenetic factors to program the genome. To test this hypothesis, it was demonstrated that inserting piRNA-complementary sequences to an ectopic site leads to Piwi, HP1a, and Su(var)3-9 recruitment to the site as well as H3K9me2/3 enrichment and reduced RNA polymerase II association, indicating that piRNA is both necessary and sufficient to recruit Piwi and epigenetic factors to specific genomic sites. Piwi deficiency drastically changed the epigenetic landscape and polymerase II profile throughout the genome, revealing the Piwi-piRNA mechanism as a major epigenetic programming mechanism in Drosophila (Huang, 2013).

This study has systematically demonstrated the existence of the Piwi-piRNA epigenetic guidance mechanism and its function as a major mechanism of guiding epigenetic factors to their target sites in Drosophila. This mechanism provides a clear and effective answer to the long-standing question on how epigenetic factors are recruited to their specific target sites to achieve epigenetic programming throughout the genome. Given that some other Piwi proteins and piRNAs also exist in the nucleus of other organisms including mammals, this mechanism might have profound significance in diverse organisms (Huang, 2013).

Whole-genome mapping produced the high-resolution map of Piwi and piRNA binding to the genome. The perfect colocalization of Piwi and piRNA binding sites is expected given their association as molecular complexes. These Piwi-piRNA complexes directly bind to many regions in the genome, exerting epigenetic repression at most of the target sites. This may account for the diverse biological functions of Piwi in different cell types during development. In particular, many Piwi-piRNA complexes bind to transposon sequences; this may be a major mechanism that is responsible for transposon silencing by Piwi as reported in many studies (Huang, 2013).

Furthermore, this analysis has revealed, at the whole-genome scale, the dependence of HP1a and H3K9 methylation on Piwi, which suggests that HP1a and histone methylases are recruited by Piwi-piRNA complexes as a major mechanism to many sites in the genome. It is important to note that Lei and colleagues reported that, in several piRNA clusters, HP1 binding is apparently unaffected by Argonaute proteins, including Piwi (Moshkovich and Lei, 2010). This result was fully anticipated because Piwi colocalizes with HP1a at many, but not all, HP1a-containing bands on polytene chromosomes. The binding of HP1a to chromatin at Piwi-free sites must be via a different mechanism, possibly via the canonical H3K9me2/3- mediated mechanism. These data, combined with the current findings, indicate that there are at least two different ways for recruiting HP1a to the chromatin, with Piwi-piRNA mechanism as a main way of recruitment, as demonstrated in this study and suggested by previous polytene staining data (Huang, 2013).

The results demonstrate that piRNA is both necessary and sufficient to bring Piwi to specific genomic sites in a sequence-specific manner and reveal a crucial role of piRNA in guiding epigenetic factors to specific sites in the genome. This epigenetic guidance mechanism is similar to the RNAi-mediated heterochromatin formation in the fission yeast in that both are mediated by small RNAs and Piwi/Ago. However, it distinctly differs from the yeast pathway in three major aspects. First, it recruits HP1a without H3K9 methylation, which then leads to recruitment of HMT and H3K9 methylation. This is in stark contrast to the yeast RNAi pathway in which the RITS complex first recruits HMT, which then leads to the methylation of H3K9 and eventual recruitment of HP1. In addition, this is also in sharp contrast to the known H3K9 methylation-dependent mechanism of HP1a recruitment in higher eukaryotes and represents a novel H3K9 methylation-independent mechanism. The recruitment of HMT by HP1a would lead to H3K9 methylation, which would result in further recruitment of HP1a molecules to the site, thereby stabilizing the repressive state of the chromatin. Second, the Piwi-piRNA-mediated epigenetic guidance mechanism can lead to transcriptional repression or activation, depending on the genomic context Last, the Piwi-piRNA mechanism involves single-stranded piRNAs and Piwi proteins rather than double- stranded siRNAs and Ago proteins. Given the genomic complexity of the higher eukaryotes, piRNAs, mostly 24-32 nt in length, are ideal candidate molecules for conferring sequence specificity in a genome-wide context. Indeed, the extreme complexity of the identified piRNAs, with more than 20,000 piRNAs associated with Piwi alone in Drosophila and more than 58,000 piRNAs in mammals, renders the Piwi-piRNA pathway a likely major epigenetic factor guidance mechanism in Drosophila, and possibly even in mammals (Huang, 2013).

High-resolution mapping analysis suggests that piRNAs might associate with euchromatin by binding to nascent RNA transcripts of 100-800 bp yet with heterochromatin by directly binding to DNA. This is in perfect agreement with previous observation that Piwi binding to euchromatin and heterochromatin is sensitive to RNaseIII and RNaseH that selectively digest double-stranded RNA and RNA-DNA hybrid, respectively. Given the complexity of the heterochromatic context, it is not clear so far exactly how piRNA binds to heterochromatic DNA. However, it is conceivable that such direct binding might occur between piRNA and single-stranded DNA (e.g., during DNA replication or transcription) or between piRNA and DNA duplex. Future studies will resolve these hypotheses (Huang, 2013).

It is also worthy noting that the sequence specificity of piRNA binding to its targets is additive with respect to individual base pairs. Each mismatch compromises the piRNA binding efficacy by 40%, so that a piRNA carrying three point mutations retains only 10% its binding ability to target sequences. This is in contrast to siRNA targeting that requires perfect complementarity and miRNA targeting that requires a mismatch in the middle position yet requires perfect match in the base 2-7 'seed sequences'. Indeed, the graded sequence specificity of piRNA binding to its target sequences might create a mechanism of quantitative regulation that allows piRNAs to guide Piwi and epigenetic factors to even more genomic sites with graded effects as well as bestows tolerance to point mutations that frequently occur in heterochromatic and repetitive sequences (Huang, 2013).

The Hmr and Lhr hybrid incompatibility genes suppress a broad range of heterochromatic repeats

Hybrid incompatibilities (HIs) cause reproductive isolation between species and thus contribute to speciation. Several HI genes encode adaptively evolving proteins that localize to or interact with heterochromatin, suggesting that HIs may result from co-evolution with rapidly evolving heterochromatic DNA. Little is known, however, about the intraspecific function of these HI genes, the specific sequences they interact with, or the evolutionary forces that drive their divergence. The genes Hmr and Lhr genetically interact to cause hybrid lethality between Drosophila melanogaster and D. simulans, yet mutations in both genes are viable. This study reports that Hmr and Lhr encode proteins that form a heterochromatic complex with Heterochromatin Protein 1 (HP1a). Using RNA-Seq analyses it was discovered that Hmr and Lhr are required to repress transcripts from satellite DNAs and many families of transposable elements (TEs). By comparing Hmr and Lhr function between D. melanogaster and D. simulans several satellite DNAs and TEs were identified that are differentially regulated between the species. Hmr and Lhr mutations also cause massive overexpression of telomeric TEs and significant telomere lengthening. Hmr and Lhr therefore regulate three types of heterochromatic sequences that are responsible for the significant differences in genome size and structure between D. melanogaster and D. simulans and have high potential to cause genetic conflicts with host fitness. It was further found that many TEs are overexpressed in hybrids but that those specifically mis-expressed in lethal hybrids do not closely correlate with Hmr function. These results therefore argue that adaptive divergence of heterochromatin proteins in response to repetitive DNAs is an important underlying force driving the evolution of hybrid incompatibility genes, but that hybrid lethality likely results from novel epistatic genetic interactions that are distinct to the hybrid background (Satyaki, 2014).

Previous work has shown that Lhr (also known as HP3) interacts with HP1a. This study report that Hmr also interacts with Lhr, and both are present in a complex together with HP1a. Consistent with this interaction, many of the roles reported in this study for Lhr and Hmr have been described for HP1a, including localizing to heterochromatin, regulating TE and pericentric gene expression, and controlling telomere length. However, unlike mutations in Su(var)205 which enodes HP1a, mutations in Hmr and Lhr are viable. Furthermore, Hmr and Lhr do not localize to the 359 bp satellite which forms a substantial fraction of X-linked pericentric heterochromatin. These findings suggest that Hmr and Lhr are not ubiquitous heterochromatin proteins, leaving open the intriguing question of what guides their localization specificity (Satyaki, 2014).

The interaction of Hmr and Lhr with HP1a has recently been independently reported. Thomae (2013) also report other findings similar to the current observations including repressive effects of Hmr and Lhr on TEs in somatic tissues and their localization to telomeres. Several conclusions are similar between the two studies and with previously published conclusions. Thomae observe upregulation of TEs in hybrids but conclude that they are unlikely to be the direct cause of hybrid lethality, a conclusion that was reach in this study using different methods. Their conclusion that hybrids are highly sensitive to Hmr dosage is in concordance with previous studies, such as the previous observation that a ~9.7 kb Hmr+ transgene causes dosage-dependent lethality to hybrid females (Barbash, 2003). This conclusion also fits well with the discovery that hybrids are highly sensitive to Lhr dosage (Satyaki, 2014).

One area of possible discrepancy is the viability effects and cellular phenotypes associated with Hmr and Lhr mutants versus RNAi knockdown. Thomae (2013) reports a high rate of mitotic defects in Lhr RNAi knockdown tissue culture cells, yet this study found that LhrKO flies are almost fully viable, as are Lhr RNAi knockdown animals. This study also has not observed the lethality or morphological defects in Hmr mutants that are reported for Hmr RNAi cells and animals. For example, Aruna (2009) found reduced longevity but no effect on viability up to eclosion of flies carrying the Df(1)Hmr- allele, a deletion of the 5' end of Hmr. Further work is necessary to determine if these discrepancies reflect phenotypes associated with the use of RNA interference or differences between assaying whole animals versus tissue-culture cells, such as the aneuploid state of cultured cell lines (Satyaki, 2014).

Several HIs involve heterochromatin proteins or heterochromatic sequences, leading to the suggestion that genetic conflicts between selfish DNAs and host fitness are an important force that is driving the evolution of HI (Satyaki, 2014 and references therein).

TE and satellite abundance varies widely among species and is a major contributor to genome-size variation. The evolutionary causes of this variation have been widely debated for many years. When considering genetic conflict theories, it is important to first exclude alternative evolutionary causes of repetitive DNA variation. One explanation is neutrality, with repeat variation governed by mutational processes, in particular the balance between insertions and deletions. Insertion/deletion models are particularly appropriate for inactive and degenerate TEs, and perhaps also for certain classes of satellites that are no longer homogenized by concerted evolution (Satyaki, 2014).

Selectionist models fit better for active repeats, and must be invoked if the adaptive evolution of heterochromatin proteins is proposed to reflect co-evolution with repetitive DNA. One model is that some repeats are co-opted for host functions. Drosophila's telomeric retrotransposons are a relevant example that is discussed below. Three, non-mutually exclusive selective costs associated with repetitive DNA are considered when discussing the evolution of Hmr and Lhr (Satyaki, 2014).

One potential cost arises from the overall load of repetitive DNAs, including increased genome size and instability. A second is direct genetic conflict. Genetic conflict is defined here to refer to fitness costs imposed by selfish DNAs that have evolved specific mechanisms to increase their transmission. Such conflicts could be caused by highly active individual repeats, for example during hybrid dysgenesis caused by introduction of a TE family into naive strains. Finally, genetic conflicts can have indirect costs, such as pleiotropic fertility defects caused by repeat expansions involved in meiotic drive. They infect most genomes, can self-mobilize and increase their copy number, and destabilize genomes via spontaneous mutations, ectopic recombination, and deleterious increases in genome size. Adaptive evolution of TE-defense genes can therefore be readily interpreted as the host species responding to the fitness cost of TEs (Satyaki, 2014).

Like Hmr and Lhr, many piRNA pathway genes are also evolving under positive selection. This raises the possibility that Lhr and Hmr are co-evolving with the piRNA pathway proteins. However, the lack of major perturbations in the piRNA pool in LhrKO suggests that Lhr and Hmr function downstream or independently of piRNA biogenesis. Piwi, guided by piRNA, has been proposed to recruit repressive heterochromatin components including HP1a and histone methyl transferases to transposable elements. One possibility is that Lhr and Hmr function downstream of HP1a to repress TEs via RNA degradation machinery such as the nuclear exosome (Satyaki, 2014).

It is noted that Ago3 is moderately down-regulated in both LhrKO (3.4 fold) and Hmr- (~2 fold), likely because the gene is peri-centromeric. Two results demonstrate that this modest reduction in Ago3 cannot explain the broad effects on TEs in Hmr and Lhr mutants. First, Ago3 expression is unaffected in D. simulans Lhr1, which also shows widespread TE derepression. Second, Ago3 mutants have major disturbances to their piRNA pool, which was not observed in LhrKO (Satyaki, 2014).

While TE repression is typically viewed in terms of genetic conflicts, the relationship between Lhr, Hmr and the telomeric TEs resembles symbiosis. These TEs have been domesticated by Drosophila species for tens of millions of years to serve a vital host function, and thus are not considered selfish DNA. The telomeric TEs were among the most strongly derepressed in Hmr and Lhr mutants, in some cases more than 100 fold. Increases were also observed in HeT-A DNA copy number in Hmr and Lhr stocks. Increased telomeric TE expression does not necessarily increase HeT-A DNA copy number and cause longer telomeres, suggesting that multiple factors control telomere length. If so, then Lhr and Hmr must control multiple processes at the telomere. This is supported by the localization of both proteins to the telomere cap, a protective structure that prevents telomere fusions. The strong reduction in LhrKO of piRNAs from three TAS-repeat containing sub-telomeric piRNA clusters is particularly intriguing. piRNA production from clusters is dependent on them maintaining a heterochromatic state, which could explain why Lhr is required for TAS piRNA expression while it acts as a repressor in most other circumstances (Satyaki, 2014).

This study discovered several striking examples that suggest species-specific co-evolution of Hmr and Lhr with satellite DNAs. D. melanogaster Hmr and Lhr proteins were found to localize to and repress transcripts from GA-rich satellites. GA-rich satellites are ~8 fold less abundant in D. simulans but are cytologically detectable; nevertheless it was found that sim-Lhr does not localize to them. GA-rich satellites also have low abundance in the outgroup species D. erecta, implying that the differential abundance with D. simulans reflects an increase in D. melanogaster. Similarly it was discovered that mel-Lhr-HA localizes to AACAC in D. melanogaster, a repeat that is absent in D. simulans. Furthermore, moderate up-regulation of several other satellite transcripts was detected only in D. melanogaster. These results suggest that Lhr and Hmr may have evolved in D. melanogaster to mitigate the deleterious consequences of satellite expansion, which can include ectopic recombination, increased genome size, and destabilized chromosome segregation (Satyaki, 2014).

Satellite transcripts have been reported from various tissues in wild type D. melanogaster but little is known about their production. They could be products of either non-specific transcription or read-through from adjacent TEs. Increased levels of satellite transcripts are observed in D. melanogaster spn-E mutants, suggesting that RNA interference or piRNA pathways control satellite transcript levels (Satyaki, 2014).

At a broad scale, Lhr and Hmr from both D. melanogaster and D. simulans regulate heterochromatic repetitive DNAs but very few genes. This finding is consistent with previous analyses demonstrating that some functions of these genes are conserved between species. But many of the repeats regulated by Lhr and Hmr are rapidly evolving, raising the question of whether specific repetitive DNAs are directly driving the adaptive evolution of the Lhr and Hmr coding sequences between species. A simple prediction is that D. simulans orthologs should fail to fully repress such repeats when placed into D. melanogaster, a prediction that was tested for Hmr (Satyaki, 2014).

The BS non-LTR retrotransposon is significantly derepressed in D. melanogaster Hmr- and LhrKO, and in D. simulans Lhr1 mutants. Interestingly, BS appears to be transpositionally active in D. melanogaster but inactive in D. simulans. One interpretation is that BS was active in the common ancestor and regulated by Hmr and Lhr. The genes would continue to co-evolve with BS in D. melanogaster, making the sim-Hmr ortholog less effective at repressing BS elements in D. melanogaster. In this scenario Hmr and Lhr are engaged in a recurrent genetic conflict with BS elements that leads to their sequence divergence. Consistent with this prediction, significantly higher expression was found in Hmr-; ø{sim-Hmr-FLAG}/+ compared to Hmr-; ø{mel-Hmr-FLAG}/+ (Satyaki, 2014).

Copia shows a different pattern, with ~20-fold up-regulation in LhrKO but only ~2-fold in Lhr1 (and only when mapping to the consensus-sequence database), as well as significant derepression in Hmr-. Copia expression level can be high in D. melanogaster but is variable among populations. In contrast, copia elements in D. simulans typically contain deletions in regulatory elements required for expression, and transcripts are undetectable by Northern blot analysis. These results suggest that Hmr and Lhr could be D. melanogaster host factors that defend against a TE that is currently active within the species. However, copia was fully repressed in Hmr-; ø{sim-Hmr-FLAG}/+, demonstrating that adaptive divergence of Hmr by itself does not affect copia regulation (Satyaki, 2014).

Overall, this study found surprisingly few cases of overexpression associated with Hmr divergence, including no effects on satellite DNAs. It is also noted that most of the TEs identified other than BS and Doc6 are likely transpositionally inactive in D. melanogaster, which makes it more challenging to fit a scenario of direct and recurrent evolution between Hmr and specific TEs (Satyaki, 2014).

Several possible interpretations of these results are suggested. One is that Hmr and Lhr adaptive divergence is in fact driven largely or solely by BS and/or Doc6, a hypothesis that will require understanding the mechanism by which Hmr and Lhr affect expression of these TEs. Second is that Hmr and Lhr may be co-evolving with other genes, and that multiple diverged genes need to be replaced simultaneously in order to detect their effects on other TEs and satellite DNAs. Third is that more sensitive assays are needed, for example monitoring TE transposition rates over multiple generations. A fourth possibility is an alternative to genetic conflict scenarios that arises from population-genetic models. These models suggest that the fitness costs of individual TE families are likely extremely weak under most circumstances. The adaptive evolution of repressor proteins may therefore reflect the cumulative load of repeats within a genome. This alternative view could be applicable to Hmr and Lhr since they repress a large number of TEs and satellites. Finally, Hmr and Lhr may have additional unidentified phenotypes that are also the targets of adaptive evolution (Satyaki, 2014).

D. simulans has a smaller genome with ~4-fold less satellite DNA and significantly fewer TEs compared to D. melanogaster. This large difference in repeat content between D. melanogaster and D. simulans may have wider consequences. Reduced expression from pericentric heterochromatin genes was found in Hmr and Lhr mutants in D. melanogaster. This reduction may reflect the fact that pericentric genes have evolved to use heterochromatin proteins such as Lhr and Hmr to maintain gene expression in a repeat-rich environment. Pericentric genes in species with fewer repeats would presumably not require these proteins. Consistent with this model, this study found that Lhr loss in D. simulans has a negligible impact on pericentric gene expression. This finding suggests that Lhr and Hmr have an adaptive role in blocking effects on gene expression arising from increasing repetitive DNA copy number (Satyaki, 2014).

If each genome is uniquely adapted to its repetitive DNA content, then the shock of hybridization may lead to misregulation of TEs and satellites. TEs are activated in various animal and plant hybrids but the consequences, if any, for hybrid fitness are largely unclear. This study found substantial TE misregulation in hybrid male larvae. Since these hybrids are agametic, this TE expression comes from somatic tissues. The fitness cost of this upregulation is unclear as somatic TE overexpression is not necessarily lethal within D. melanogaster. Comparison of lethal Hmr+ and viable Hmr- hybrid males demonstrates that lethal hybrids have more TE expression than the viable hybrids, which in turn have more TE expression than either of its parents. However, this TE misregulation seems unconnected with Hmr as the TEs differentially expressed between Hmr+ and Hmr- hybrid male larvae are largely distinct from those between Hmr+ and Hmr- D. melanogaster male larvae. Further, while Hmr- causes rampant TE over-expression within D. melanogaster, it is associated with reduced TE levels in hybrids. These observations argue that the TE derepression in hybrids is unrelated to the pure species function of Hmr. This finding is consistent with previous genetic studies that demonstrate that the wild type Hmr+ allele causes hybrid lethality and thus behaves as a gain-of-function allele in hybrids. More generally it underscores the unique nature of the hybrid genetic background. Somatic TE overexpression may result from breakdown in the siRNA or piRNA pathways due to incompatibilities among multiple rapidly evolving TE regulators (Satyaki, 2014).

One clear example is known where a species-specific difference in a satellite DNA causes incompatibility between Drosophila species. But the toll caused by heterochromatic differences may more commonly be indirect, as heterochromatin proteins diverge in response to changes in heterochromatic DNA repeats. Recent work suggests that hybrid female sterility may be caused by incompatibilities among rapidly evolving piRNA proteins rather than by species-specific differences in TEs. It is suggested that the role of Hmr and Lhr in regulating the activity of three highly dynamic classes of heterochromatin has led to their recurrent adaptive evolution, and secondarily, to their involvement in interspecific hybrid lethality (Satyaki, 2014).

The RNA processing exosome is linked to elongating RNA polymerase II in Drosophila

RNA surveillance factors are involved in heterochromatin regulation in yeast and plants, but less is known about the possible roles of ribonucleases in the heterochromatin of animal cells. This study shows that RRP6, one of the catalytic subunits of the exosome, is necessary for silencing heterochromatic repeats in the genome of Drosophila melanogaster. It was shown that a fraction of RRP6 is associated with heterochromatin, and the analysis of the RRP6 interaction network reveals physical links between RRP6 and the heterochromatin factors HP1a, SU(VAR)3-9 and RPD3. Moreover, genome-wide studies of RRP6 occupancy in cells depleted of SU(VAR)3-9 demonstrates that SU(VAR)3-9 contributes to the tethering of RRP6 to a subset of heterochromatic loci. Depletion of the exosome ribonucleases RRP6 and DIS3 stabilizes heterochromatic transcripts derived from transposons and repetitive sequences, and renders the heterochromatin less compact, as shown by micrococcal nuclease and proximity-ligation assays. Such depletion also increases the amount of HP1a bound to heterochromatic transcripts. Taken together, these results suggest that SU(VAR)3-9 targets RRP6 to a subset of heterochromatic loci where RRP6 degrades chromatin-associated non-coding RNAs in a process that is necessary to maintain the packaging of the heterochromatin (Eberle, 2015).

Approximately 30% of the genome of Drosophila melanogaster is heterochromatic and is made up of transposons, transposon fragments and repetitive sequences with different degrees of complexity. The heterochromatin contains high levels of heterochromatin-specific proteins, such as Heterochromatin Protein 1a (HP1a), and is enriched in certain patterns of post-translational modifications of the histone tails. Heterochromatin formation involves a cascade of histone modifications that are targeted to specific regions of the genome by complex protein-protein and protein-nucleic acid interactions. In the switch from euchromatin to heterochromatin, acetylated H3K9 (H3K9ac) is deacetylated by histone deacetylases such as RPD3/HDAC1. H3K9 is subsequently methylated by histone methyltransferases, and the methylated H3K9 (H3K9me) acts as a binding site for HP1a. The properties of the heterochromatin can spread along the chromatin fiber, and HP1a plays a central role in this process. The ability of HP1a to dimerize, to interact with the methyltransferase SU(VAR)3-9, and to bind H3K9me provides the basis for the spreading of heterochromatin. An additional level of complexity in the establishment of heterochromatic states is provided by the fact that HP1a can also bind RNA in both D. melanogaster and Schizosaccharomyces pombe. Recent studies on Swi6, the HP1a ortholog of S. pombe, have shown that the interaction of Swi6 with RNA interferes with the binding of Swi6 with H3K9me (Eberle, 2015).

Small non-coding RNAs are essential components of the regulation of chromatin packaging in different organisms. Fission yeast uses siRNAs to silence heterochromatic sequences through the recruitment of the H3K9 methyltransferase Clr4. RNAi-dependent mechanisms of heterochromatin assembly exist also in plants, where siRNAs direct de novo DNA methyltransferases to specific genomic sequences. Animal cells use instead the piRNA pathway to trigger heterochromatin assembly and transposon silencing in the germ line. In D. melanogaster, non-coding RNAs transcribed from transposon-rich regions are processed into piRNAs, and a 'Piwi-piRNA guidance hypothesis' has been recently proposed for the recruitment of SU(VAR)3-9 and HP1a to heterochromatin. The Piwi-piRNA system is active during early development and it directs the initial establishment of heterochromatin states not only in the germ line but also in somatic cells. Recent studies suggest that after embryogenesis, the patterns of heterochromatin packaging are maintained through cell divisions via piRNA-independent mechanisms (Eberle, 2015).

An important player in the regulation of non-coding RNAs is the exosome, a multiprotein complex with ribonucleolytic activity. In D. melanogaster, the core of the exosome associates with two catalytic active subunits, RRP6 and DIS3. In the cell nucleus, the exosome is involved in the processing of many non-coding RNAs, including pre-rRNAs, and in the quality control of mRNA biogenesis. The exosome ribonucleases also degrade a large variety of unstable, non-coding RNAs in various organisms including S. cerevisiae, plants, and animals. Moreover, recent studies have revealed that RRP6 participates in the regulation of enhancer RNAs and in the degradation of unstable transcripts synthesized at DNA double-strand breaks (Eberle, 2015).

The exosome has been functionally linked to the methylation of H3K9 in heterochromatin. In S. pombe, RRP6 participates in the assembly of centromeric heterochromatin through an RNAi-independent mechanism, and collaborates with the RNAi machinery to silence developmentally regulated loci and retrotransposons. Much less is known about the possible links between RRP6 and heterochromatin in animals. This study found that a fraction of RRP6 is associated with heterochromatin in the genome of D. melanogaster, and physical interactions has been identifiedf between RRP6 and several heterochromatin factors, including HP1a, SU(VAR)3-9, and RPD3. These results show that SU(VAR)3-9 promotes the targeting of RRP6 to transposon-rich heterochromatic loci. In these loci, RRP6 contributes to maintaining the structure of the heterochromatin by degrading non-coding RNAs that would otherwise compromise the packaging of the chromatin (Eberle, 2015).

This study shows that RRP6 interacts physically with HP1a and SU(VAR)3-9, and that RRP6 is associated with a subset of heterochromatic regions of the genome. Less RRP6 is bound to the heterochromatin in cells with reduced levels of SU(VAR)3-9, which indicates that SU(VAR)3-9 contributes to the targeting of RRP6 to heterochromatin. Although the RNAi experiments do not reveal whether the effect of SU(VAR)3-9 knockdown on RRP6 occupancy is direct or indirect, the fact that RRP6 and SU(VAR)3-9 colocalize and can be co-immunoprecipitated suggests that SU(VAR)3-9 facilitates the recruitment of RRP6 to the heterochromatin, or stabilizes the interaction of RRP6 with other chromatin components, through a physical interaction (Eberle, 2015).

This study has focused on RRP6, and the existence of multiple exosome subcomplexes in cells of D. melanogaster makes it difficult to establish whether the entire exosome has a role in the heterochromatin. However, two observations suggest that this is the case. Firstly, the simultaneous depletion of both catalytic subunits of the exosome, RRP6 and DIS3, gave additive effects on the levels of chromatin-associated RNAs and on the association of HP1a to heterochromatic RNAs. Secondly, it was previously shown that a fraction of RRP4, a core exosome subunit, is also associated with chromatin. Altogether, these observations suggest that the entire exosome, not RRP6 alone, is targeted to heterochromatic loci through an interaction with SU(VAR)3-9 (Eberle, 2015).

Depletion of RRP6 or simultaneous depletion of RRP6 and DIS3 led to a local increase in heterochromatic transcripts associated with subtelomeric and pericentromeric regions, without a significant increase in the density of RNA Pol-II at those regions. This suggests that under normal conditions the RRP6 and DIS3 degrade pervasive RNAs that are transcribed from the heterochromatin. Direct MNase assays and PLA-based assays designed to measure the compaction of the chromatin revealed that the depletion of the exosome ribonucleases loosens the structure of the heterochromatin in the regions that accumulate heterochromatic non-coding RNAs, without affecting the levels of H3K9 methylation or the association of SU(VAR)3-9 with the chromatin. In S. pombe, deletion of the rrp6 gene leads to a derepression of heterochromatin, and this effect is partly due to the fact that in the absence of RRP6 activity, aberrant RNA species accumulate in S. pombe and recruit the siRNA machinery in competition with the RNAi-dependent pathways of H3K9 methylation. The situation is different in D. melanogaster, as no change in H3K9me2 or SU(VAR)3-9 recruitment occurred when RRP6 and DIS3 were depleted (Eberle, 2015).

What is then the mechanism by which the exosome ribonucleases influence the compaction of the heterochromatin in D. melanogaster? The HP1a ortholog in S. pombe, Swi6, is an RNA-binding protein, and non-coding RNAs can cause the eviction of Swi6 from the S. pombe heterochromatin by competing with H3K9me for Swi6. The HP1a protein of D. melanogaster interacts with several RNA-binding proteins and can bind directly to RNA. This study has shown that depletion of RRP6 and DIS3 results in increased levels of non-coding transcripts associated with heterochromatin in D. melanogaster cells. HP1a-RIP signals at selected heterochromatic loci are also increased in cells depleted of RRP6 and DIS3. Altogether, these observations are consistent with a model in which RRP6, and perhaps also DIS3, participate in the degradation of heterochromatic non-coding RNAs that, if stabilized, would outcompete the binding of HP1a to the methylated H3K9 and would thereby disrupt the packaging of the heterochromatin (Eberle, 2015).

Heterochromatin domains are characterized by high levels of H3K9me2 and by the presence of HP1a and SU(VAR)3-9. These results show that RRP6 interacts with SU(VAR)3-9 and that this interaction is important to tether RRP6 to the heterochromatin. Transcripts derived from sporadic transcription of heterochromatic repeat sequences are kept at low levels by RRP6 degradation. Failure to degrade such transcripts results in increased levels of chromatin-associated transcripts, increased binding of HP1 to the chromatin-associated transcripts, and chromatin decondensation (Eberle, 2015).

Specialized protein-protein interactions target RRP6 to different chromatin environments RRP6 and the exosome act on many different types of transcripts and participate in many essential biological processes. The existence of multiple mechanisms to target RRP6 to different types of transcripts, or even to different nuclear compartments, is thus not unexpected. The association of the exosome-or exosome subunits- with genes transcribed by RNA polymerase II (Pol-II) is mediated by interactions with different types of proteins. Co-immunoprecipitation experiments in D. melanogaster identified SPT5 and SPT6, two transcription elongation factors, as interaction partners for the exosome, which led to the proposal that the exosome is tethered to the transcription machinery during transcription elongation. In D. melanogaster, the exosome is also tethered to protein-coding loci through interactions with the hnRNP protein HRP59/RUMP. In human cells, a NEXT complex containing MTR4, the Zn-knuckle protein ZCCHC8, and the putative RNA binding protein RBM7 mediates an interaction between the exosome and Pol-II transcripts through the nuclear cap-binding complex. In many cases, these intermolecular interactions target the exosome to genomic loci that produce relatively stable transcripts, for instance protein-coding transcripts or stable non-coding RNAs. In these loci, the role of the exosome is primarily linked to RNA surveillance, not turnover (Eberle, 2015).

Much less is known about the mechanisms that target the exosome or its individual subunits to non-protein coding RNAs in the heterochromatin. This study of the RRP6 interactome in cells of D. melanogaster has revealed interactions between RRP6 and heterochromatin factors, and has established an important role for SU(VAR)3-9 in determining RRP6 occupancy. Depletion of SU(VAR)3-9 has a profound effect on the association of RRP6 with a subset of chromatin regions, including many transposon loci. The present findings suggest that these regions, that can be referred to as 'SUV-dependent', produce transcripts that are actively degraded by RRP6. SU(VAR)3-9 has less impact on the targeting of RRP6 to euchromatic protein-coding genes, where interactions with the Pol-II machinery and with mRNA-binding proteins play instead a decisive role. Altogether, the picture that emerges from many studies is that specialized protein-protein interactions target RRP6 to specific genomic environments where RRP6 participates in the processing, surveillance or degradation of specific RNA substrates (Eberle, 2015).

HP1/Su(var)205: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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