HP1/Su(var)205
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).
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)
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).
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 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).
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 (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).
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).
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).
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).
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).
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).
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 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).
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).
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 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).
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).
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).
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).
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).
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).
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).
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