HP1/Su(var)205


DEVELOPMENTAL BIOLOGY

Maternal depletion of Piwi, a component of the RNAi system, impacts heterochromatin formation in Drosophila

A persistent question in epigenetics is how heterochromatin is targeted for assembly at specific domains, and how that chromatin state is faithfully transmitted. Stable heterochromatin is necessary to silence transposable elements (TEs) and maintain genome integrity. Using reporters subject to Position Effect Variegation (PEV), this study found that depletion of key proteins in the early embryo can lead to loss of silencing assayed at adult stages. The piRNA component Piwi is required in the early embryo for reporter silencing in non-gonadal somatic cells, but knock-down during larval stages has no impact. This implies that Piwi is involved in targeting HP1a when heterochromatin is established at the late blastoderm stage and possibly also during embryogenesis, but that the silent chromatin state created is transmitted through cell division independent of the piRNA system. In contrast, heterochromatin structural protein HP1a is required for both initial heterochromatin assembly and the following mitotic inheritance. Piwi depletion leads to decreased HP1a levels in pericentric heterochromatin, particularly in TEs. The results suggest that the major role of the piRNA system in assembly of heterochromatin in non-gonadal somatic cells occurs in the early embryo during heterochromatin formation, and further demonstrate that failure of heterochromatin formation in the early embryo impacts the phenotype of the adult (Gu, 2013).

These results, coupled with earlier findings, support a model for heterochromatin targeting that utilizes Piwi in the early zygote: it is suggested that Piwi and the associated piRNA system are required (directly or indirectly) to guide HP1a to a subset of TEs, and that the deposition of HP1a further recruits other components to establish H3K9me2-enriched heterochromatin status in those TE regions. Specificity could be achieved via a base-pairing mechanism utilizing piRNAs. Subsequent mitotic transmission of this HP1a/H3K9me2 enriched heterochromatic state during development does not appear to depend on the piRNA system. This targeting mechanism may be of primary importance for TEs in border regions between heterochromatin masses and adjacent euchromatin, the situation for PEV reporters utilized in this study. When Piwi is depleted, the HP1a level is significantly decreased at these sites. Some loss of HP1a is seen in general in heterochromatic regions, presumably because heterochromatin is enriched in TEs and other repetitious elements. Thus the silencing of PEV reporters, which are dependent on the spreading of the local heterochromatin, can be released. The silent chromatin state is apparently transmitted by the heterochromatin system during development, when the piRNA system is largely absent in non-gonadal somatic cells (Gu, 2013).

Using mutant alleles, this study assayed the effect of maternal depletion (which results in depletion in early embryos) and zygotic depletion of HP1a or Piwi on the expression of PEV reporters. Functional HP1a depletion in either the early zygote or developing animals leads to suppression of variegation of the PEV reporters, coupled with decreased levels of HP1a itself as well as the silencing mark H3K9me2 in the reporter regions. This suggests a critical role for HP1a in both early establishment and subsequent maintenance of heterochromatin, and demonstrates that the impact of early depletion can be seen using an adult phenotype, even when wild type alleles of HP1a are present in the developing zygote. In the case of Piwi, only maternal or early zygotic depletion has a significant effect on the reporters in non-gonadal somatic cells. Surprisingly, zygotic Piwi depletion in embryos from wild type mothers does cause a small decrease in PEV silencing of the BL1 reporter in carcasses, and of 118E10 and wm4 reporters in eyes. However, this effect is not as significant as that caused by maternal depletion. A small zygotic effect of Piwi depletion is consistent with prior observations. At the same time, the results of Piwi knock down in the eye lineage argue that Piwi is dispensable for the maintenance of heterochromatin silencing after embryogenesis. Note that the ey-GAL4 driver becomes active in late embryogenesis, much later than the onset of zygotic expression in the 2-hour embryo (Gu, 2013).

Overall, the data demonstrate that Piwi's role in recruiting HP1a and other components to some TE regions happens early in development, while HP1a is essential for heterochromatin formation during every cell cycle. This is in congruence with their expression patterns. Piwi mRNA is present in gonadal cells and early embryos, with little detectable expression in non-gonadal somatic cells, with some exceptions (e.g., larval fat body, possibly nerve cells), while HP1a is expressed in all cells/tissues during development. Thus any HP1a-Piwi interaction likely occurs in gonadal cells and in early embryos, where they are both highly enriched and observed to be nuclear proteins. These stages are also enriched in small RNAs and piRNA pathway components, supporting a model of piRNA-mediated heterochromatin assembly. As it is a structural protein of heterochromatin, one would anticipate that HP1a would be essential for heterochromatin formation in any dividing cell (such as those in the eye imaginal disc) when heterochromatin is re-established after DNA replication as is observed. A second protein found to be important for silencing state maintenance for some reporters is the histone methyltransferase Eggless. Egg has been suggested to be essential for heterochromatin formation in specific regions, including chromosome 4 (in somatic cells) and piRNA clusters (gonads;) (Gu, 2013).

It is of interest that cells in the mature organism 'remember' the loss of HP1a in the early zygote, exhibiting HP1a and H3K9me2 reduction in the reporter promoter region in the adult. The depletion of HP1a at the critical stage of heterochromatin establishment during early development, even when the overall HP1a level is presumably recovered soon after the onset of zygotic transcription, results in diminished heterochromatic regions that apparently cannot be fully re-established, and only partially recover. This implies that both genetic and environmental insults sustained at the critical embryonic stage can have a long-lasting impact on the individual (Gu, 2013).

The reporters exhibiting PEV used in this study either lie near the break point between heterochromatin and euchromatin caused by inversion or translocation (e.g. BL1, BL2 and wm4), or have been inserted into heterochromatic domains by P element transposition (e.g., 118E10). Their silencing is dependent on the spreading of the adjacent heterochromatin structure, making them sensitive to even small changes in the heterochromatin environment and chromatin assembly systems. For example, when Piwi is knocked down in the early embryo, suppression of variegation of the BL2 reporter was observed coupled with significant HP1a loss at the promoter of the reporter. However, no dramatic change of HP1a enrichment was observed in most other heterochromatic regions. The sensitivity of the BL2 reporter to Piwi depletion might be explained by its position at the edge of a heterochromatic mass, and the requirement for spreading of the heterochromatic assembly. The HP1a ChIP-array data in piwi mutant larvae further confirms that depletion of Piwi will lead to a small decrease in the HP1a level at some TE classes, coupled with an overall small decrease of HP1a levels in heterochromatic sequences. However, the data obtained from the ChIP-array includes only the unique probes in the assembled genome sequence, so only a small portion of the TEs have been analyzed. It is possible that the actual overall decrease of HP1a enrichment is greater, as most of the TE sequences are not included in this analysis. Nonetheless, the PEV reporters may be particularly sensitive to Piwi manipulation, either because of their dependency on spreading of heterochromatin, or because the Piwi-dependent response itself is triggered by transcription, more likely to occur in these flanking regions (Gu, 2013).

While these studies have focused on the role of Piwi, the resulting model is consistent with earlier work examining several components of the piRNA pathway (Piwi, Aubergine, Armitage, Spn-E). Mutations in these components are reported to have an impact on the repression of transcription and maintenance of a closed chromatin structure for several TE classes when assayed in the female germ line. This study has demonstrate two additional features: first, that maternal depletion of Piwi has an impact on silencing PEV reporters that can be seen in somatic cells of larvae and adults, and second, that depleting Piwi in early zygotic cells (but not maternally) also impacts PEV assayed in later stages (Gu, 2013).

The results further suggest that the piRNA system observed in this study most likely acts in the context of multiple mechanisms for heterochromatin formation. In the yeast S. pombe the RNAi system is redundant with other heterochromatin protein interaction systems in heterochromatin establishment; such DNA-protein interaction systems have also been inferred in Drosophila. The interplay among these systems remains to be investigated. The system of selective depletion developed in this study should allow further investigation of the role of various components in targeting and maintaining heterochromatin at different heterochromatin domains (Gu, 2013).

Embryonic

There is an enrichment of HP1 in the intensely staining regions near the apical surface of nuclear cycle 10 embryos. At this stage GAGA factor is localized to punctate structures in this same region. This enrichment for HP1 is markedly increased during nuclear cycle 14. Surprisingly, whereas GAGA factor retains its association with the heterochromatin throughout the cell cycle, a significant fraction of HP1 is dispersed throughout the spindle around the segregating chromosomes during mitosis. This dispersed pool of HP1 is observed during mitosis in both early and late Drosophila embryos. Drosophila tissue culture cells prepared by a method which removes soluble protein and avoids fixation of the mitotic chromosomes also show an enrichment for HP1 in the heterochromatin of the chromosomes (Kellum, 1995a).

HP1 is found within the centric beta-heterochromatin, in cytological regions 31, 41 and 80, and throughout polytene chromosome 4. Staining of telomeres is frequently observed, those of chromosome arms 2R and 3R and the X chromosome being the most conspicuous. Staining of intact salivary glands indicates that this rearranged segment of beta-heterochromatin is not associated with the polytene chromocenter, but provides an independent structural reference point. HP1 is not observed in the nuclei of the early syncytial embryo, but becomes concentrated in the nuclei at the syncytial blastoderm stage (ca. nuclear division cycle 10). This suggests that heterochromatin formation occurs at approximately the same stage at which nuclei first become transcriptionally competent (James, 1989).

Cytological evidence is provided for the presence of heterochromatin within a euchromatic chromosome arm by immunolocalization of HP1 to the site of a silenced transgene repeat array. The amount of HP1 associated with arrays in polytene chromosomes is correlated with the array size. Inverted transposons within an array or increased proximity of an array to blocks of naturally occurring heterochromatin may increase transgene silencing without increasing HP1 labeling. Less dense anti-HP1 labeling is found at transposon arrays in which there is no transgene silencing. The results indicate that HP1 targets the chromatin of transposon insertions and binds more densely at a site with repeated sequences susceptible to heterochromatin formation (Fanti, 1998a).

Sex-specific role of Drosophila melanogaster HP1 in regulating chromatin structure and gene transcription

Drosophila melanogaster heterochromatin protein 1 (HP1a or HP1) is believed to be involved in active transcription, transcriptional gene silencing and the formation of heterochromatin. But little is known about the function of HP1 during development. Using a Gal4-induced RNA interference system, it has been shown that conditional depletion of HP1 in transgenic flies results in preferential lethality in male flies. Cytological analysis of mitotic chromosomes shows that HP1 depletion causes sex-biased chromosomal defects, including telomere fusions. The global levels of specific histone modifications, particularly the hallmarks of active chromatin, are preferentially increased in males as well. Expression analysis shows that approximately twice as many genes were specifically regulated by HP1 in males than in females. Furthermore, HP1-regulated genes show a greater enrichment for HP1 binding in males. Taken together, these results indicate that HP1 modulates chromosomal integrity, histone modifications and transcription in a sex-specific manner (Liu, 2005).

Mutations in D. melanogaster Su(var)205 (also called HP1) cause lethality at larval stages, precluding a systematic functional analysis of Su(var)205 during development. To circumvent this limitation, the role of HP1 was studied using a Gal4-inducible RNA interference (RNAi) system, which allows for depletion of HP1 in a tissue- and developmental stage-specific manner. D. melanogaster y w67c23 embryos were transformed with a construct expressing double-stranded RNA from a Su(var)205 cDNA. To deplete HP1, four independent transgenic lines (HP1-2, HP1-11, HP1-21 and HP1-31) were crossed with an act-Gal4 line (y w; +/+; act-Gal4/TM6B) expressing Gal4 ubiquitously during development. Resulting larval progeny from lines HP1-11 and HP1-21 showed a reduction in HP1 levels of ~90%, line HP1-31 showed a 60% reduction and line HP1-2 showed no reduction. Those progeny with a 60%-90% reduction in HP1 generally survived to the third-instar larval stage, but progeny with a 90% reduction rarely survived to the adult stage. Lethality mainly occurred at the pupal stage and seemed to be due to a failure to eclose. Adult progeny of the HP1-31/act-Gal4 line were also viable, but the female:male ratio was highly skewed (2.4:1 versus 0.9:1 for this genotype at the larval stage). An alteration in the sex ratio was also evident in adult flies from the HP1-11/act-Gal4 line: all 21 survivors were female. There were no adult HP1-21/act-Gal4 survivors when progeny were grown at 25°C, but 30 'escapers' were obtained at 18°C, all of which were female. Collectively, these results suggested that there was an association between sex-biased lethality and HP1 dosage (Liu, 2005).

To assess the cause of lethality on depletion of HP1, lines HP1-21 and HP1-11 were crossed with lines inducing RNAi exclusively in eye imaginal discs (ey-Gal4) and in the posterior compartment of developing wings (en-Gal4) and the effect of HP1 depletion in these tissues was examined in third-instar larvae. In HP1-depleted imaginal discs, an increased number of dying cells was consistently found using acridine orange staining, which is often used to detect apoptotic cells. Tissue growth defects were also observed in the eyes and wings of adult flies; defects in both of these tissues were more severe in males than in females. Apoptosis seemed to be mediated through a caspase-dependent pathway; tissue growth defects could be partially rescued by the addition of p35, a cysteine protease apoptosis inhibitor. These results suggest that the observed lethality and growth defects in both sexes are linked to apoptosis (Liu, 2005).

It was next asked whether sex-specific lethality involves specific mitotic chromosome defects, as observed previously in Su(var)205 mutants. A variable number of 'ring-like' chromosomes and other aberrant segregated chromosomes (e.g., chromatin bridges) was found in the metaphase spreads from third-instar larval neuroblast cells of HP1-depleted larvae. The relative frequency of defective mitotic chromosomes in HP1-depleted males was approximately twice that in females, indicating that differential chromosomal segregation defects may underlie sex-biased lethality (Liu, 2005).

Because the mutated lethal allele Su(var)20502 does not result in telomeric fusions, however, lethality cannot be solely due to this cause. To explore whether additional mechanisms are involved in the sex-biased lethality, the impact of HP1 depletion on core histone modifications was measured, since increases in histone acetylation can cause apoptosis. Using cell extracts from larval imaginal discs of HP1 RNAi mutants and control larvae, the global levels were compared of several core histone modifications in males and females. The levels of acetylation at Lys8 of histone H4 (H4K8ac), methylation at Lys4 of histone H3 (H3K4me) and methylation at Lys79 of histone H3 (H3K79me; all hallmarks of active chromatin) were all increased in males after HP1 depletion. But levels of methylation at Lys20 of histone H4 (H4K20me) and methylation at Lys9 of histone H3 (H3K9me; both hallmarks of heterochromatin) showed a global decrease when cells were lysed in 300 mM salt buffer. No change was observed in H4K20me or H3K9me when cells were lysed in SDS buffer, suggesting that the changes in histone modifications associated with the active state may have a role in the observed lethality. These effects were not caused by misregulation of genes encoding known histone-modifying enzymes, including histone methylases, acetylases or deacetylases, as these were unaffected by HP1 depletion (Liu, 2005).

It was asked whether any change in histone H3K9me occurs on chromatin, since this histone modification is interdependent with the dynamics of HP1. In polytene chromosomes from HP1-depleted mutants, H3K9me remains at the pericentric heterochromatin region in both sexes. But the intensity of the pericentric H3K9me signal in males is lower than that in females, a modification linked to X-chromosome dosage compensation in males. This staining showed no obvious changes that were dependent on HP1. To test the possibility that the HP1-induced preferential lethality in males is linked to the disruption of specific functional genes in males, total RNA isolated from two independent populations of male and female third-instar larvae of line HP1-21/act-Gal4 was compared using microarray analysis. More than 200 predicted transcripts or genes were specifically affected in males, but only 119 were specifically affected in females; 127 genes seemed to be affected in both males and females. Among the affected genes with known function, those essential for DNA replication, such as mus209 and Mcm6, were downregulated in both sexes; wrinkled (W) and Rep4, both regulators of apoptosis, were upregulated. Notably, a number of genes encoding cell cycle regulators, such as fizzy (fzy), pimples (pim), cyclin-dependent kinase subunit (Cks30A) and the DNA replication initiation inhibitor geminin, were all specifically affected only in males, suggesting that these genes have a role in the observed differential lethality. Transcription of genes known to regulate the sex ratio, such as msl-1 (also called MSL), was not affected (Liu, 2005).

Of the 127 genes affected in both males and females, transcription of nearly two-thirds were upregulated in the absence of HP1. In addition, 22 of 24 genes that had lower expression in wild-type females were upregulated in the female RNAi mutants. These observations are consistent with the known role of HP1 in transcriptional gene silencing. In addition, however, nearly one-half of the affected genes were downregulated in the absence of HP1, supporting the possibility that HP1 may have a role in both negative and positive regulation of transcription (Liu, 2005).

To determine whether the sex-biased effects of HP1 on histone modification and transcription were due to a differential distribution of HP1 on chromatin in males and females, chromatin immunoprecipitation (ChIP) analysis was carried out. Sonicated chromatin extracts of nuclei isolated from male and female third-instar y w67c23 larvae were immmunoprecipitated with polyclonal antibodies against D. melanogaster HP1. Among the eight genes tested that were affected in both males and females, four showed similar levels of HP1 binding enrichment in the two sexes, implying a direct role for HP1 in their transcription. Of 12 genes specifically affected in females, six were enriched in HP1 binding to similar levels in both sexes, and the other six were HP1-negative. Notably, 11 of 18 genes specifically affected in males showed a severalfold enrichment of HP1 binding in males compared with females; 5 were similarly enriched in both sexes, and 2 were not associated with HP1. Although the ChIP results indicated that genes specifically affected in males seemed to be enriched in HP1 binding in males compared with females, genes specifically affected in females did not have a 'female-specific' HP1 binding pattern, indicating that HP1 might invoke sex-specific mechanisms in the regulation of chromatin or transcription (Liu, 2005).

These results show that HP1 has rather different roles in males versus females. RNAi knock-down of HP1 resulted in sex-biased defective chromosome segregation, alterations in histone modifications, specific changes in transcription and a skewed sex ratio in surviving progeny. Two recent studies suggest that chromosomal segregation defects, particularly telomeric fusion, may have a key role in the apoptosis and sex-biased lethality observed in this study. Overexpression of the heterochromatin protein Su(var)3-7 also induces lethality in males, with a shortened or condensed X chromosome. But the morphology of the X chromosome and the global level and distribution of H4K16ac seem unaffected in male HP1 knock-down progeny, suggestive of an alternative mechanism (Liu, 2005).

The differential change in H3K9me on chromatin may be due to an alteration in Su(var)3-9 localization, since HP1 is essential for maintaining its dynamics. Changes in global histone acetylation and phosphorylation could result from an HP1-induced global change in chromatin structure or from secondary effects; the absence of a Su(var)3-9 homolog in mammals also caused changes in different histone modifications, in addition to H3K9me. Notably, all these changes occur in a sex-biased manner. This is attributed to the sex-specific distribution of HP1 on chromatin, shown by ChIP analysis. This hypothesis suggests that the male genome, relatively enriched in HP1, is subject to more changes in histone modifications, more chromosome segregation defects and more changes in transcription in the absence of HP1, which seems to be the case. The heterochromatic Y chromosome in males may be also involved in the sex-biased distribution of HP1 in the genome, for example, by altering the distribution of remaining HP1 and other heterochromatin proteins (Liu, 2005).

A previous cytological study of mealybugs identified a conspicuous HP1-associated 'mass/aggregate' structure in male chromosomes, contrasting with a scattered localization along female chromosomes. This result and the results presented in this study support the hypothesis that HP1 has a distinct regulatory role in male versus female chromatin. Whether the sex-specific distribution of HP1 on chromatin directly regulates the sex-biased differences in global transcription, showing relatively lower transcription in males than in females, is not known. The facts that HP1 is involved in transcriptional gene silencing and that depletion of HP1 results in upregulation of some male genes, normally transcribed at a lower level in males than in females, seem to support the idea that HP1 has a role in the phenomenon. But these sex-biased regulation mechanisms also seem to require other sex-specific regulators (e.g., proteins or RNA). Future studies are required to define those regulators and to understand their role in the organization of sex-biased chromatin and transcriptional regulation. Understanding the mechanisms of this regulation may also yield important clues about the basis of sexual dimorphism in animals (Liu, 2005).

The endogenous siRNA pathway is involved in heterochromatin formation in Drosophila

A new class of small RNAs (endo-siRNAs) produced from endogenous double-stranded RNA (dsRNA) precursors was recently shown to mediate transposable element (TE) silencing in the Drosophila soma. These endo-siRNAs might play a role in heterochromatin formation. This has been shown in S. pombe for siRNAs derived from repetitive sequences in chromosome pericentromeres. To address this possibility, the viral suppressors of RNA silencing B2 and P19 were used. These proteins normally counteract the RNAi host defense by blocking the biogenesis or activity of virus-derived siRNAs. It was hypothesized that both proteins would similarly block endo-siRNA processing or function, thereby revealing the contribution of endo-siRNA to heterochromatin formation. Accordingly, P19 as well as a nuclear form of P19 expressed in Drosophila somatic cells were found to sequester TE-derived siRNAs whereas B2 predominantly bound their longer precursors. Strikingly, B2 or the nuclear form of P19, but not P19, suppressed silencing of heterochromatin gene markers in adult flies, and altered histone H3-K9 methylation as well as chromosomal distribution of histone methyl transferase Su(var)3-9 and Heterochromatin Protein 1 in larvae. Similar effects were observed in dcr2, r2d2, and ago2 mutants. These findings provide evidence that a nuclear pool of TE-derived endo-siRNAs is involved in heterochromatin formation in somatic tissues in Drosophila (Fagegaltier, 2009).

This study implicates components of the RNAi pathway in heterochromatin silencing during late Drosophila development. The study also provides correlative evidence supporting a functional link between endo-siRNAs and the formation or maintenance of somatic heterochromatin in flies. The viral proteins NLS-P19 and B2 suppress the silencing of PEV markers and induce aberrant distribution of H3m2K9 and H3m3K9 heterochromatic marks as well as histone H3 methylase Su(var)3-9 in larval tissues. Dcr2 and Ago2 mutations have similar effects. In striking contrast, cytoplasmic P19 has no noticeable effect on chromatin. It is proposed that B2 inhibits Dcr2-mediated processing of double-stranded TE read-through transcripts in the cytoplasm; it is further proposed that NLS-P19 sequesters TE-derived siRNA duplexes. This model implies that part of the cytoplasmic pool of TE-derived endo-siRNA (which might be involved in PTGS events) is translocated back into the nucleus to exert chromatin-based functions. In C. elegans, silencing of nuclear-localized transcripts involves nuclear transport of siRNAs by an NRDE-3 Argonaute protein. A similar siRNA nuclear translocation system, possibly mediated by Ago2, may also exist in flies. Alternatively, an as yet unidentified siRNA duplex transporter may be involved. Deep sequencing analyses show that the fraction of siRNAs sequestered by NLS-P19 is smaller as compared with the one bound by P19 in the cytoplasm. Thus, the poor effects of P19 on nuclear gene silencing may be explained if the cytoplasmic pool of siRNA competes with the pool of siRNA to be translocated in the nucleus (Fagegaltier, 2009).

The Dcr-1 partner Loquacious (Loqs), but not the Dcr-2 partner R2D2, was unexpectedly found to be required for biogenesis of siRNA derived from fold-back genes that form dsRNA hairpins. By contrast, it is noteworthy that loqs mutations had little or no impact on the accumulation of siRNA derived from TE. The finding that r2d2 but not loqs mutation suppresses the silencing of PEV reporters and delocalizes H3m2K9 and H3m3K9 heterochromatic marks agrees with these results and further suggests that siRNA involved in heterochromatin formation and siRNA derived from endogenous hairpins arise from distinct r2d2- and loqs-dependent pathways, respectively. One possible mechanism by which TE- or repeat-derived endo-siRNAs could promote heterochromatin formation is by tethering complementary nascent TE transcripts and guiding Su(var)3-9 recruitment and H3K9 methylation. Identifying which enzymes tether siRNAs to chromatin in animals is a future challenge. In addition, some endo-siRNAs could also impact on heterochromatin formation by posttranscriptionaly regulating the expression of chromatin modifiers, such as Su(var)3-9. In any case, the current results demonstrate the value of viral silencing suppressor proteins in linking siRNAs to heterochromatin silencing in the fly soma, as established in S. pombe and higher plants. Because silencing suppressors are at the core of the viral counterdefensive arsenal against antiviral RNA silencing in fly, whether they also induce epigenetic changes in chromatin states during natural infections by viruses deserves further investigation (Fagegaltier, 2009).

Chromatin remodeling in the aging genome of Drosophila

Chromatin structure affects the accessibility of DNA to transcription, repair, and replication. Changes in chromatin structure occur during development, but less is known about changes during aging. This study examined the state of chromatin structure and its effect on gene expression during aging in Drosophila at the whole genome and cellular level using whole-genome tiling microarrays of activation and repressive chromatin marks, whole-genome transcriptional microarrays and single-cell immunohistochemistry. Dramatic reorganization of chromosomal regions was found with age. Mapping of H3K9me3 and HP1 signals to fly chromosomes reveals in young flies the expected high enrichment in the pericentric regions, the 4th chromosome, and islands of facultative heterochromatin dispersed throughout the genome. With age, there is a striking reduction in this enrichment resulting in a nearly equivalent level of H3K9me3 and HP1 in the pericentric regions, the 4th chromosome, facultative heterochromatin, and euchromatin. These extensive changes in repressive chromatin marks are associated with alterations in age-related gene expression. Large-scale changes in repressive marks with age are further substantiated by single-cell immunohistochemistry that shows changes in nuclear distribution of H3K9me3 and HP1 marks with age. Such epigenetic changes are expected to directly or indirectly impinge upon important cellular functions such as gene expression, DNA repair, and DNA replication. The combination of genome-wide approaches such as whole-genome chromatin immunoprecipitation and transcriptional studies in conjunction with single-cell immunohistochemistry as shown in this study provide a first step toward defining how changes in chromatin may contribute to the process of aging in metazoans (Wood, 2010).

Effects of Mutation or Deletion

Drosophila heterochromatin-associated protein 1 (HP1) is an abundant component of heterochromatin, a highly condensed compartment of the nucleus that comprises a major fraction of complex genomes. Some organisms have been shown to harbor multiple HP1-like proteins, each exhibiting spatially distinct localization patterns within interphase nuclei. The subnuclear localization patterns of two newly discovered Drosophila HP1-like proteins (HP1b and HP1c) have been characterized, comparing them with that of the originally described fly HP1 protein (here designated HP1a). While HP1a targets heterochromatin, HP1b localizes to both heterochromatin and euchromatin and HP1c is restricted exclusively to euchromatin. All HP1-like proteins contain an amino-terminal chromo domain, a connecting hinge, and a carboxyl-terminal chromo shadow domain. Truncated and chimeric HP1 proteins were expressed in vivo to determine which of these segments might be responsible for heterochromatin-specific and euchromatin-specific localization. Both the HP1a hinge and chromo shadow domain independently target heterochromatin, while the HP1c chromo shadow domain is implicated solely in euchromatin localization. Comparative sequence analyses of HP1 homologs reveal a conserved sequence block within the hinge that contains an invariant sequence (KRK) and a nuclear localization motif. This block is not conserved in the HP1c hinge, possibly accounting for its failure to function as an independent targeting segment. It is concluded that sequence variations within the hinge and shadow account for HP1 targeting distinctions. It is proposed that these targeting features allow different HP1 complexes to be distinctly sequestered in organisms that harbor multiple HP1-like proteins (Smothers, 2001).

Two allelic dominant suppressors of position-effect variegation (PEV) are found to contain mutations within the gene encoding HP-1. The site of mutation for each allele is given: one converts Lys169 into a nonsense (ochre) codon, while the other is a frameshift after Ser10. In flies heterozygous for nonsense codon, a truncated HP-1 protein is detected by western blot analysis. An HP-1 minigene under control of an Hsp70 heat-inducible promoter, was transduced into flies by germ line transformation. Heat-shock driven expression of this minigene results in elevated HP-1 protein level and enhancement of position-effect variegation. Levels of variegating gene expression appear to depend upon the level of expression of this heterochromatin-specific protein. It is thought that PEV arises from alterations in mass action-drive heterochromatin assembly and a requirement for a precise stoichiometry of heterochromatin protein subunits (Eissenberg, 1992 and references).

Point mutations in the HP1 chromo domain abolish the ability of HP1 to promote gene silencing (Platero, 1995).

Heat shock-driven HP1 cDNA is capable of fully rescuing the recessive lethality associated with HP1 mutations. If heat shock-induced HP1 expression is delayed for as long as 5 days, more than half of the mutant flies still survive until adulthood, consistent with a substantial maternal contribution to embryonic and larval viability. Elevating HP1 levels as late as 7-8 days of development is sufficient to enhance variegation three-fold, suggesting that the extent of heterochromatic position effect can be modified subsequent to the initial appearance of HP1 in the nuclei of syncytial blastoderm embryos (Eissenberg, 1993).

The insertion of a heterochromatin segment into a euchromatic gene (brown, an eye color locus), results in position-effect variegation of brown. The insertion of heterochromatin also causes the aberrant cytological association of the gene and its homologous copy to heterochromatin. The cytological association of the heterochromatic region is affected by chromosomal distance from heterochromatin and by genic modifiers of PEV. Thus HP1 mutations, which can result in position-effect variegation, suppress trans-inactivation of the heterochromatinized euchromatic gene. When HP-1 is present in three doses, PEV of brown is enhanced (Csink, 1996).

Transgenes inserted into the telomeric regions of Drosophila melanogaster chromosomes exhibit position effect variegation (PEV), a mosaic silencing characteristic of euchromatic genes brought into juxtaposition with heterochromatin. Telomeric transgenes on the second and third chromosomes are flanked by telomeric associated sequences (TAS), while fourth chromosome telomeric transgenes are most often associated with repetitious transposable elements. Telomeric PEV on the second and third chromosomes is suppressed by mutations in Su(z)2, but not by mutations in Su(var)2-5 (encoding HP1), while the converse is true for telomeric PEV on the fourth chromosome. This genetic distinction allows for a spatial and molecular analysis of telomeric PEV. Reciprocal translocations between the fourth chromosome telomeric region containing a transgene and a second chromosome telomeric region result in a change in nuclear location of the transgene. While the variegating phenotype of the white transgene is suppressed, sensitivity to a mutation in HP1 is retained. Corresponding changes in the chromatin structure and inducible activity of an associated hsp26 transgene are observed. The data indicate that both nuclear organization and local chromatin structure play a role in this telomeric PEV (Cryderman, 1999).

The Su(var)2-5 locus, an essential gene in Drosophila, encodes the heterochromatin-associated protein HP1. The Su(var)2-5 lethal period is late third instar. Maternal HP1 is still detectable in first instar larvae, but disappears by third instar, suggesting that developmentally late lethality is probably the result of depletion of maternal protein. Heterochromatic silencing of a normally euchromatic reporter gene is completely lost by third instar in zygotically HP1 mutant larvae, implying a defect in heterochromatin-mediated transcriptional regulation in these larvae. However, expression of the essential heterochromatic genes rolled and light is reduced in Su(var)2-5 mutant larvae, suggesting that reduced expression of essential heterochromatic genes could underlie the recessive lethality of Su(var)2-5 mutations. These results also show that HP1, initially recognized as a transcriptional silencer, is required for the normal transcriptional activation of heterochromatic genes (Lu, 2000).

Both the dominant and recessive phenotypes of mutations in HP1 were examined to look for an essential requirement for HP1 in development. It is proposed that reduced expression of one or more essential heterochromatic genes results in the recessive late larval lethality of Su(var)2-5. In support of this hypothesis, the essential heterochromatic genes rolled and light are misregulated in Su(var)2-5 mutants. rolled transcription at its normal chromosomal location is reduced in Su(var)2-5 mutant flies. Since no maternal Rolled protein is detectable in third instar larvae homozygous for rolled deficiencies, the RNA levels that are detected in mutant larvae and adults reflect zygotic gene expression. In the case of the heteroallelic mutant larvae, it should be emphasized that at the time the larvae were collected for Northern analysis, the Su(var)2-5 mutant larvae appeared healthy and would have lived on for several more days as third instar larvae before dying; indeed, a further decline in rolled RNA preceding larval death cannot be ruled out. Thus, reduced expression of rolled could contribute to the defects associated with loss of HP1. Of course, reduced expression of other heterochromatic genes probably also contributes to lethality due to HP1 deficiency (Lu, 2000 and references therein).

light also experiences variegated inactivation in Su(var)2-5 larval Malpighian tubules, and light transcripts are dramatically reduced overall in Su(var)2-5 mutant larvae. It is important to stress that the repressed light locus in these experiments is also in its normal chromosomal location. It is concluded that silencing of light in these experiments is a direct consequence of HP1 depletion, depriving the light locus of the heterochromatin context required for its normal expression. Several other genes reside in heterochromatin, and it will be interesting to see whether dependence on HP1 is a general attribute of gene expression in heterochromatin (Lu, 2000).

Mutations in rolled, like Su(var)2-5 mutations, lead to late larval or early pupal lethality with defective or missing imaginal discs. At the cytological level, rolled mutations cause defects in mitosis, including overcondensed and/or lagging anaphase chromosomes. Intriguingly, neuroblasts of larvae doubly mutant for hypomorphic alleles of rl and abnormal spindles (encodes a microtubule-associated protein) show telomeric stickiness and increased frequency of aneuploid mitotic figures. These phenotypes are also seen in neuroblasts of larvae heteroallelic for Su(var)2-5 mutations; indeed, the highest frequency of defects occurs in larvae heteroallelic for the Su(var)2-5205 allele, which is carried on a chromosome marked with a hypomorphic rl allele. Therefore, reduced expression of rolled caused by loss of HP1 could contribute to mitotic defects in HP1 mutant larval brains (Lu, 2000).

How can HP1 be required both for activation of heterochromatic genes and silencing of euchromatic genes? It has been proposed that certain heterochromatin-associated proteins function to support normal transcription of heterochromatic genes when those genes are at their normal chromosomal sites and that position effects result when heterochromatic genes are deprived of such essential heterochromatic proteins by displacement away from heterochromatin 'compartments' where such proteins are in high concentration. Such context-dependent regulatory activity has also been described for yeast RAP1 (repressor/activator protein 1); RAP1 is required for high-level expression of many ribosomal protein and glycolytic enzyme genes, but it promotes position-effect silencing at the HM silent mating type cassettes and telomeres. Genetic evidence suggests that RAP1 has distinct activator and silencing domains that could recruit or stabilize distinct chromosomal complexes at distinct chromosomal sites. Similarly, HP1 could interact with different proteins or protein complexes to promote silencing or activation in different chromosomal contexts. Another possibility is that HP1 may contribute to the formation of a particular chromatin structure that interferes with activation of euchromatic genes but to which heterochromatic genes have become adapted and dependent. Loss of HP1 would deplete the nucleus of this particular chromatin conformation, releasing silenced genes from repression while simultaneously depriving the resident heterochromatin genes of their functional context (Lu, 2000).

HP1 is required for correct chromosome segregation in Drosophila embryos (Kellem, 1995b). HP1 has been reported to cause recessive embryonic lethality associated with defects in chromosomal morphology and mitotic segregation (Kellem, 1995b). The conclusion reached by Kellum (1995b) that HP1/Su(var)2-5 is a recessive embryonic lethal has been reported to be incorrect. An intimation of this may be found in the Fanti (1998) where they mention in passing that they recovered about 20% heteroallelic Su(var)2-5 mutant third instars (indeed, this whole paper would have been impossible if Su(var)2-5 were a recessive embryonic lethal, since the cytology involves third instar neuroblasts). For five different Su(var)2-5 alleles in six different allelic combinations, heteroallelic flies survive in Mendelian proportions to the end of third instar, and die at or prior to pupariation. This was done using genetically marked larvae. Kellum and Alberts (1995b) finding cannot at this time be explained, but their conclusion was based on the observation of a reduced hatch rate and the assumption that the unhatched embryos must be HP1 homozygous mutant. In unpublished work, Eissenberg reports also finding mitotic defects in flies from such crosses, but using blue balancers, could show that the mitotic defects were not correlated with genotype (Lu, 2000).

Telomeres of Drosophila melanogaster contain arrays of the retrotransposon-like elements HeT-A and TART. Their transposition to broken chromosome ends has been implicated in chromosome healing and telomere elongation. A genetic system has been developed which enables the determination of the frequency of telomere elongation events and their mechanism. The frequency differs among lines with different genotypes, suggesting that several genes are in control. The Su(var)2-5 gene encoding heterochromatin protein 1 (HP1) is involved in regulation of telomere length. Different Su(var)2-5 mutations in the heterozygous state increase the frequency of HeT-A and TART attachment to the broken chromosome end by more than a hundred times. The attachment occurs through either HeT-A/TART transposition or recombination with other telomeres. Terminal DNA elongation by gene conversion is greatly enhanced by Su(var)2-5 mutations only if the template for DNA synthesis is on the same chromosome but not on the homologous chromosome. The Drosophila lines bearing the Su(var)2-5 mutations maintain extremely long telomeres consisting of HeT-A and TART for many generations. Thus, HP1 plays an important role in the control of telomere elongation in Drosophila (Savitsky, 2002).

Two highly conserved histone deacetylases, Sir2 and Rpd3, have been linked to caloric restriction and the extension of longevity. Because the Drosophila forms of each protein can silence genes in either euchromatin or heterochromatin, it was determined whether longevity extension is mediated by silencing in the latter domain. When silencing was increased and decreased using mutations that affect heterochromatin protein 1 (HP1), but have no direct effect upon Sir2 or Rpd3, lifespan was unaffected. Heterochromatin-mediated gene silencing was then modulated without directly influencing HP1 as well as the deacetylases, again yielding no effect on lifespan. Mortality rates were unchanged by all manipulations, indicating that euchromatic targets are likely to be the effectors of deacetylase-mediated longevity extension in Drosophila (Frankel, 2005).

Loss of the modifiers of variegation Su(var)3-7 or HP1 impacts male X polytene chromosome morphology and dosage compensation

Loss of Su(var)3-7 or HP1 suppresses the genomic silencing of position-effect variegation, whereas over-expression enhances it. In addition, loss of Su(var)3-7 results in preferential male lethality. In polytene chromosomes deprived of Su(var)3-7, a specific bloating of the male X chromosome is observed, leading to shortening of the chromosome and to blurring of its banding pattern. In addition, the chromocenter, where heterochromatin from all polytene chromosomes fuses, appears decondensed. The same chromosomal phenotypes are observed as a result of loss of HP1. Mutations of Su(var)3-7 or of Su(var)2-5, the gene encoding HP1, also cause developmental defects, including a spectacular increase in size of the prothoracic gland and its polytene chromosomes. Thus, although structurally very different, the two proteins cooperate closely in chromosome organization and development. Finally, bloating of the male X chromosome in the Su(var)3-7 mutant depends on the presence of a functional dosage compensation complex on this chromosome. This observation reveals a new and intriguing genetic interaction between epigenetic silencing and compensation of dose (Spierer, 2005).

Su(var)3-7 function is still poorly understood. It encodes a large protein associated with pericentric heterochromatin, telomeres and a few euchromatic sites on interphase polytene chromosomes. Seven widely spaced zinc fingers stand out in the sequence of the N-terminal half. In vitro, the zinc finger region of Su(var)3-7 has affinity for DNA, and preferentially for some satellite sequences. There is also evidence for direct binding of Su(var)3-7 with DNA in vivo. The N-terminal half of Su(var)3-7 interacts nonspecifically in vivo with heterochromatin and euchromatin, whereas the C-terminal half promotes interaction with itself, and with pericentric heterochromatin. Su(var)3-7 also interacts genetically and physically with HP1 and with Su(var)3-9, as determined in yeast by the two-hybrid assay and in vivo. To decipher the function of Su(var)3-7, mutants were generated by homologous recombination, and a detailed examination was undertaken of their phenotype. Su(var)3-7 was shown to be essential, the maternal contribution being sufficient for viability. Interestingly, males are more sensitive than females to the lack of Su(var)3-7. The cause of this lethality is unknown (Spierer, 2005).

This study reports the building of a new mutant of Su(var)3-7 by homologous recombination: described are the phenotypes of mutations on polytene chromosome morphology and on the organism -- these are similar to phenotypes resulting from mutational loss of HP1. The male X chromosome is more sensitive to these effects, leading to an understanding of an interaction between the modifier of PEV Su(var)3-7 and the dosage compensation machinery. It is concluded that the importance of the roles and partnership of Su(var)3-7 and HP1 extend beyond genomic silencing in the maintenance of chromosome integrity and function, including the male X-specific chromosome-wide mechanism of dosage compensation (Spierer, 2005).

Polytene chromosomes are affected similarly by severe loss of Su(var)3-7 or HP1. In both cases, the main mutant phenotype is a bloated X in males, and an expanded chromocenter in males and females. Why is chromosome morphology modified when HP1 or Su(var)3-7 amounts are strongly reduced? There are several possible explanations. Su(var)3-7 and HP1 are both required for stability of chromatid association, and reduction of dose could lead to dissociation. This mechanism has been suggested for similar phenotypes in other conditions. This hypothesis could be tested by determining whether a phenomenon based on chromatid association, such as transvection, is affected in Su(var)3-7 or HP1 mutants. Su(var)3-7 and HP1 are required for compaction of intercalary heterochromatin on euchromatic arms. The loss of this compaction, similar to what is seen at the chromocenter, could lead to bloating and disruption of the banding pattern. If indeed Su(var)3-7 and HP1 are instrumental in chromosome compaction, then one could expect that excess amounts of the proteins lead in turn to an excess of compaction. This is actually the case for Su(var)3-7; increasing amounts of Su(var)3-7 first affect the male X chromosome, which becomes strongly compacted. Furthermore, targeting HP1 to an ectopic site promotes chromosomal loops linking this ectopic site with sites of intercalary heterochromatin. The question remains of the particular sensitivity of the male X chromosome to loss and excess of Su(var)3-7 and to loss of HP1 (Spierer, 2005).

That the male X chromosome is affected first and most severely could result from association of this chromosome with the dosage compensation complex (DCC). Chromatin relaxation triggered by the DCC in the male X would render it more sensitive to variations of the amount of chromatin-associated proteins. Indeed, male X bloating and shortening has been observed in several conditions, and has been named the 'pompon' phenotype and described as resulting from specific environmental aggressions or mutations. Male X bloating was described as resulting from the loss of several chromatin-modifying factors such as Jil-1 or the Nurf complex. The various environmental and genetic conditions in which bloating of the male X occurs underline the peculiar sensitivity of the phenotype, and could explain the differences of phenotype intensity seen using different X chromosomes (Spierer, 2005).

Finally, the X-chromosome-specific phenotype might result from a direct interaction between the DCC and silencing factors. This paper indeed demonstrates a genetic interaction between an essential gene of the dosage compensation machinery, mle, and Su(var)3-7. However, in the wild type, preferential association of Su(var)3-7 with the polytene male X chromosome has not been detected using either a polyclonal antibody raised against Su(var)3-7 sequences, or a monoclonal antibody raised against the tag of HA-Su(var)3-7. However, preferential association with the male X is seen when Su(var)3-7 is over-expressed from a transgene. At this point it is not possible to distinguish between two possibilities: either Su(var)3-7 modulates the transcription level of the X chromosome by counteracting the DCC relaxing effect, or it protects the X-linked genes that do not need to be dosage compensated. The role of HP1 also remains to be explored. No preferential association of HP1 with the male X polytene chromosome has been seen. Nevertheless, when Su(var)3-7 is over-expressed, HP1 is found associated preferentially with the male X (Spierer, 2005).

In conclusion, Su(var)3-7 and HP1 participate in chromocenter and male X polytene chromosome integrity. The similarity of the phenotypes seen in mutations of either one, the partial compensation of the loss of dose in one by an increase of dose in the other in PEV, and the physical interaction between Su(var)3-7 and HP1 seen in vitro and in vivo (Delattre, 2000) all point to the same conclusion. These two structurally very different proteins cooperate closely in chromosome organization. An interaction also existes between Su(var)3-7 and compensation of dose. This interaction between the genomic silencing of PEV dependent on Su(var)3-7 association, and hyperactivation dependent on association of the DCC, needs to be unravelled (Spierer, 2005).

HP1 is distributed within distinct chromatin domains at Drosophila telomeres

Telomeric regions in Drosophila are composed of three subdomains. A chromosome cap distinguishes the chromosome end from a DNA double-strand break; an array of retrotransposons, HeT-A, TART, and TAHRE (HTT), maintains telomere length by targeted transposition to chromosome ends; and telomere-associated sequence (TAS), which consists of a mosaic of complex repeated sequences, has been identified as a source of gene silencing. Heterochromatin protein 1 (HP1) and HP1-ORC-associated protein (HOAP) are major protein components of the telomere cap in Drosophila and are required for telomere stability. Besides the chromosome cap, HP1 is also localized along the HTT array and in TAS. Mutants for Su(var)205, the gene encoding HP1, have decreased the HP1 level in the HTT array and increased transcription of individual HeT-A elements. This suggests that HP1 levels directly affect HeT-A activity along the HTT array, although they have little or no effect on transcription of a white reporter gene in the HTT. Chromatin immunoprecipitation to identify other heterochromatic proteins indicates that TAS and the HTT array may be distinct from either heterochromatin or euchromatin (Frydrychova, 2008).

On the basis of expression of telomeric white and yellow transgenes Drosophila telomeres have been proposed to have two distinct domains: TAS, which resembles heterochromatin and the HTT array, which behaves like euchromatin. According to the pattern of chromatin proteins revealed by immunostaining of extended polytene chromosomes in a Tel mutant, telomeres consist of three distinct and nonoverlapping domains: the chromosome cap, the HTT array, and TAS. The immunostaining results indicate that HP1 in telomeres is restricted to the cap region (Frydrychova, 2008).

Using ChIP, this study has shown that HP1 is also present along the HTT array outside of the cap as well as in TAS. The difference between these observations and previous reports might be due to a higher abundance of HP1 in the telomere cap than in the internal HTT region or better accessibility of antibodies to the telomere cap, and thus the difference in the reports may be explained by higher sensitivity of ChIP compared to immmnostaining of polytene chromosomes. The difference may be caused also by different properties of long telomeres of a Tel mutant or different biological properties of polytene salivary chromosomes compared to diploid or other polyploid cells. In any case, ChIP data on whole animals are more likely to be generalizable than immunostaining data on a specific cell type (Frydrychova, 2008).

Su(var)205 belongs to a group of suppressor of variegation [Su(var)] genes, many of which encode chromosomal proteins or modifiers of chromosomal proteins. Mutations in Su(var) genes lead to suppression of position-effect variegation (PEV), which is repressed and variegated expression of genes placed in or near pericentric heterochromatin. Despite phenotypic similarities between PEV and telomere position effect (TPE), TPE does not respond to Su(var) mutations. Although TAS was identified as a source of telomeric silencing, and the retrotransposon array genetically resembles euchromatin, comparable levels of HP1 were found at transgenes inserted in these two telomeric domains. The levels of other marks for silent chromatin, such as histone H2A.v and MeK9H3, however, did vary between these two regions in a manner consistent with proposals in previous reports that HTT is associated with open chromatin and TAS is associated with closed chromatin. TPE may thus be caused by a silencing system different from HP1-mediated heterochromatin. One candidate is Polycomb silencing; Polycomb group proteins were found associated with TAS. Since levels of the chromatin markers in all tested regions, including euchromatin and pericentric heterochromatin, showed significant differences, interpretation of HTT and TAS as either heterochromatin or euchromatin is rather difficult. It may suggest that HTT and TAS are in a category of some transitional type of chromatin between euchromatin and heterochromatin, such as closed/inactive euchromatin, or it suggests the existence of additional chromatin types (Frydrychova, 2008).

The relatively high level of HP1 on a transgene inserted into pericentric heterochromatin compared with transgenes in either HTT or TAS may suggest that failure of telomeric HP1 to silence telomeric transgenes is caused by its relative paucity. HP1, however, is a negative regulator of telomere length; its mutations lead to an increase in the transcriptional activity of HeT-A and TART, as well as an accumulation of these elements at the chromosome end. The promoter activity of a telomeric w transgene inserted between the HTT array and TAS significantly exceeds the activity of a single HeT-A promoter. This study shows that that Su(var)205 mutations lead to a severalfold increase in the transcriptional activity of HeT-A, however no increase is seen in transcription of a w gene inserted into the HTT array. In particular, using HeT-A/P-element readthrough transcripts in three P-element insertion lines, it was found that Su(var)205 mutations lead to stimulation of HeT-A elements along the HTT array in all regions assayed. With regard to the low level of HP1 in telomeric regions compared to pericentric heterochromatin, as observed by ChIP experiments, it is conceivable that the relatively weak HeT-A promoter is more sensitive to HP1 concentration than the more robust w promoter. However, HP1 per se cannot be considered as a signal for silencing. An analysis of genomewide correlations between the HP1 binding pattern and the pattern of gene expression revealed that recruitment of the protein is not sufficient to repress transcription completely. Moreover, some euchromatic genes in Drosophila are activated by the presence of HP1. With respect to these observations, it is difficult to predict the effect of HP1 recruitment on the transcription pattern in any specific region (Frydrychova, 2008).

HP1, by interaction with HOAP, forms capping complexes at the ends of Drosophila chromosomes. Formation or maintenance of the HP1-HOAP capping complex requires ATM. Loss of ATM reduces localization of HP1 and HOAP at telomeres and leads to frequent telomeric fusions. tefu and cav mutations, however, did not lead to a profound increase in HeT-A transcription, as was observed in Su(var)205 mutants. This suggests that HP1 presence in the cap does not significantly participate in overall HeT-A transcriptional activity, and that HeT-A transcription is regulated mainly by HP1 in the HTT array outside the cap. The data are consistent with previous studies that suggested two distinct mechanisms for HP1 control of telomere capping and telomere elongation by retroelement transcription. It was proposed that the capping function of HP1 is due to its direct binding to telomeric DNA, while the silencing of telomeric sequences and control of transcription of telomeric retroelements is due to interaction of HP1 with MeK9H3 and spreading of HP1 and repressive chromatin along the telomere (Frydrychova, 2008).

Collectively, these data show that HP1 is present along the HTT array as well as in TAS and plays a role as a negative regulator of transcription of telomeric retroelements. The present data also support the observation that the HeT-A promoter is relatively weak compared with a mini-w promoter and more sensitive to local HP1 concentration and suggest that telomeric chromatin in Drosophila may be distinct from either euchromatin or heterochromatin (Frydrychova, 2008).

The epigenetic trans-silencing effect in Drosophila involves maternally-transmitted small RNAs whose production depends on the piRNA pathway and HP1

The study of P transposable element repression in Drosophila led to the discovery of the Trans-Silencing Effect (TSE), a homology-dependent repression mechanism by which a P-transgene inserted in subtelomeric heterochromatin (Telomeric Associated Sequences, 'TAS') has the capacity to repress in trans, in the female germline, a homologous P-lacZ transgene located in euchromatin. Phenotypic and genetic analysis have shown that TSE exhibits variegation in ovaries, displays a maternal effect as well as epigenetic transmission through meiosis and involves heterochromatin (including HP1) and RNA silencing. This study shows that mutations in squash and zucchini, which are involved in the piwi-interacting RNA (piRNA) silencing pathway, strongly affect TSE. In addition, a molecular analysis of TSE was carried out and it was shown that silencing is correlated to the accumulation of lacZ small RNAs in ovaries. Finally, the production of these small RNAs was shown to be sensitive to mutations affecting squash and zucchini, as well as to the dose of HP1. Thus, these results indicate that the TSE represents a bona fide piRNA-based repression. In addition, the sensitivity of TSE to HP1 dose suggests that in Drosophila, as previously shown in Schizosaccharomyces pombe, an RNA silencing pathway can depend on heterochromatin components (Todeschini, 2010)

Trans-silencing has been shown to be strongly impaired by mutations affecting several components of the piRNA silencing pathway (aubergine, armitage, homeless, piwi). By contrast, TSE was not impaired by mutations affecting R2D2, a component of the siRNA pathway, or Loquacios, a component of both the miRNA and endo-siRNA pathways. This indicates that TSE likely involves the piRNA silencing pathway, a hypothesis which is consistent with the fact that TSE is restricted to the germline, the tissue in which the “canonical” piRNA pathway functions. Further, Squash and Zucchini were found to interact with Aubergine and to localize to the nuage, a cytoplasmic organelle surrounding the nurse cell nuclei, which also contains Aubergine and Armitage and appears to be involved in RNA silencing. squ and zuc mutations were also shown to affect piRNA production in ovaries at the cytological 42AB repetitive sequence cluster, a typical piRNA-producing genomic region. Regarding TE repression in the germline, squ and zuc mutants were found to derepress transcription of the telomeric retrotransposons Het-A and TART and of the I factor, a retrotransposon involved in a Drosophila system of hybrid dysgenesis. It is noteworthy that the I factor and the Het-A retrotransposons have also been found to be sensitive to aub, armi and hls (spn-E). The genetic analysis reported in this study shows that TSE is also highly sensitive to zuc and squ mutations. TSE is therefore sensitive to mutations affecting all the genes of the germline piRNA pathway tested and thus appears to represents a bona fide piRNA-based repression (Todeschini, 2010)

The presence of lacZ small RNAs in ovaries of females carrying a TSE silencer was therefore investigated using RNase protection analysis. In addition, paternal vs maternal transmission of the telomeric silencer was compared. Indeed, TSE was previously shown to have a maternal effect, i.e. strong repression occurs only when the telomeric silencer is maternally inherited, whereas a paternally-inherited telomeric silencer has weak or null repression capacities. More precisely, it was shown genetically that TSE requires inheritance of two components, a maternal cytoplasmic component plus a chromosomal copy of the transgene, but these two components can be transmitted separately. Indeed, a paternally-inherited telomeric transgene can be 'potentiated' by a maternally-inherited cytoplasm from a female bearing a silencer. This interaction also functions between telomeric silencers located on different chromosomal arms. The RNase protection analysis reported in this study shows that: (1) P-1152, a telomeric P-lacZ silencer, produces small lacZ RNAs in ovaries; (2) P-1152 lacZ small RNA accumulation is negatively affected in squ and zuc mutants; (3) maternal transmission of P-1152 leads to accumulation of higher levels of these small RNAs than that observed upon paternal P-1152 transmission. These results were reproduced with independent RNAse protection assays. The size of the small RNAs detected in this study appears smaller (around 22-23 nt) than that corresponding to piRNAs as characterized by deep sequencing (23-28 nt), but they are consistent with piRNAs as detected by RNAse protection assays in other studies: this can result from the RNAse protection protocol which tends to reduce the size of the RNAs detected. In conclusion, the results strongly suggest that the lacZ small RNAs in P-1152 oocytes may correspond to cytoplasmically-transmitted piRNAs mediating the maternal effect of TSE, as well potentiating a paternally-inherited telomeric silencer (Todeschini, 2010)

TSE was previously shown to be sensitive to mutations affecting HP1 since a negative, dose-dependent, effect on TSE was found with two loss of function alleles of Su(var)205 (including Su(var)2-505). RNase protection analysis shows here that lacZ small RNA accumulation is also negatively affected by the dose of HP1. Although it cannot be excluded that this effect may be indirect, this opens the possibility that some piRNA-producing loci depend on the presence of HP1 itself at the locus to produce piRNAs. A similar model was recently proposed for rhino, a HP1 homolog, mutations of which strongly reduce the production of piRNAs by dual strand piRNA-producing loci (Klattenhoff, 2009). That study proposed that rhino is required for the production of the long precursor RNAs which are further processed to produce primary piRNAs. Note that in that study, rhino mutants were shown to have a drastic effect on the production of piRNAs by the X-chromosome TAS locus. A similar situation may therefore exist for HP1 at this locus and, if so, it would be interesting to characterize more precisely the function of HP1 in the production of piRNAs at the TAS locus (Todeschini, 2010).

HP1 was shown to be present at TAS. A first possibility would be that HP1 stimulates transcription of the TAS locus as a classical transcription factor, independent of any heterochromatic role at this locus. Consistent with this, it was shown that PIWI, a partner of HP1 (Brower-Toland, 2007), promotes euchromatin histone modification and piRNA transcription at the third chromosome TAS. The precise status of TAS, however, remains complex since some studies have shown that TAS exhibit some of the properties attributed to heterochromatin and carry primarily heterochromatic histone tags. Therefore, a second possibility would be that HP1 enhances the heterochromatic status of TAS in the germline, such that production of aberrant transcripts being processed into piRNAs is enhanced. This would result in a 'heterochromatin-dependent RNA silencing pathway'. Examples of heterochromatin formation that depends on RNA silencing ('RNA-dependent heterochromatin formation') have been described in numerous species including yeast and plants. In Drosophila, this type of interaction has been described for variegation of pigment production in the eye linked to the insertion of the white gene in different types of heterochromatin structures, as well as for heterochromatin formation at telomeres in the germline. Therefore, telomeric regions in fly may be submitted to both RNA-dependent heterochromatin formation and heterochromatin-dependent RNA silencing. RNA silencing may favor heterochromatin formation that in turn potentiates RNA silencing, resulting in a functional positive loop between transcriptional gene silencing and post-transcriptional gene silencing. In such cases, RNA silencing and heterochromatin may not only reinforce each other but may also be functionally interdependent. Such bidirectional reinforcement between RNA silencing and heterochromatin formation was demonstrated in S. pombe since: (1) deletion of genes involved in RNA silencing were shown to derepress transcriptional silencing from centromeric heterochromatic repeats, accompanied by loss of Histone 3 Lysine 9 methylation and Swi6 (a HP1 homolog) delocalization; (2) Swi6 was found to be required for the propagation and the maintenance of the RNA Induced Transcriptional Silencing (RITS) complex at the mat locus, a complex involved in amplification of RNA silencing. A positive loop between RNA silencing and heterochromatin formation may therefore also be at play in the Drosophila germline. According to this model, the epigenetic transmission of TSE through meiosis, ( i.e., six generations of maternal transmission of the silencer are required to elicit a strong TSE following maternal inheritance of a cytoplasm devoid of lacZ piRNAs would underlie progressive establishment of this loop. Note that RNAi-dependent DNA methylation in Arabidopsis thaliana has been shown to occur progressively over several consecutive generations (Todeschini, 2010)

Since TSE can be considered as a sub-phenomenon within P regulation, it may underlie epigenetic transmission of the P element repression. P element mobilization is responsible for a syndrome of germline abnormalities, known as the 'P-M' system of hybrid dysgenesis which includes a high mutation rate, chromosomal rearrangements, male recombination and an agametic temperature-sensitive sterility called GD sterility (Gonadal Dysgenesis). P-induced hybrid dysgenesis is repressed by a maternally inherited cellular state called the 'P cytotype'. The absence of P-repression is called M cytotype. G1 females produced from the cross (P cytotype females × M cytotype males) present a strong capacity for repression, whereas females produced from the reciprocal cross present a weak capacity for repression. In the subsequent generations, cytotype is progressively determined by the chromosomal P elements but the influence of the initial maternal inheritance can be detected for up to five generations. Therefore, P cytotype exhibits partial epigenetic transmission through meiosis. Furthermore, the identification and use of telomeric P elements as P cytotype determinants, has made it possible to show that P cytotype (like TSE) involves a strictly-maternally inherited component (called the pre-P cytotype), is sensitive to mutations affecting HP1 and aubergine and is correlated to maternal deposition of piRNAs. Some of these properties are also found for the I factor which is responsible for the occurrence of another system of hybrid dysgenesis ('I-R' system). TSE therefore parallels germline regulation of TEs (P, I), and does not resemble regulation of TEs in the somatic follicle cells (gypsy, ZAM, Idefix) for which no epigenetic transmission of repression capacities through meiosis has been described so far. It will be interesting to test if previously described cases of RNA-dependent heterochromatin formation show the reciprocal dependence, thus being able to form a positive loop (Todeschini, 2010 and references therein)

piRNA-mediated regulation of transposon alternative splicing in the soma and germ line

Transposable elements can drive genome evolution, but their enhanced activity is detrimental to the host and therefore must be tightly regulated. The Piwi-interacting small RNA (piRNA) pathway is vital for the regulation of transposable elements, by inducing transcriptional silencing or post-transcriptional decay of mRNAs. This study shows that piRNAs and piRNA biogenesis components regulate precursor mRNA splicing of P-transposable element transcripts in vivo, leading to the production of the non-transposase-encoding mature mRNA isoform in Drosophila germ cells. Unexpectedly, it was shown that the piRNA pathway components do not act to reduce transcript levels of the P-element transposon during P-M hybrid dysgenesis, a syndrome that affects germline development in Drosophila. Instead, splicing regulation is mechanistically achieved together with piRNA-mediated changes to repressive chromatin states, and relies on the function of the Piwi-piRNA complex proteins Asterix (also known as Gtsf1) and Panoramix (Silencio), as well as Heterochromatin protein 1a [HP1a; encoded by Su(var)205]. Furthermore, this machinery, together with the piRNA Flamenco cluster, not only controls the accumulation of Gypsy retrotransposon transcripts but also regulates the splicing of Gypsy mRNAs in cultured ovarian somatic cells, a process required for the production of infectious particles that can lead to heritable transposition events. These findings identify splicing regulation as a new role and essential function for the Piwi pathway in protecting the genome against transposon mobility, and provide a model system for studying the role of chromatin structure in modulating alternative splicing during development (Teixeira, 2017).

Hybrid dysgenesis is a syndrome that affects progeny in a non-reciprocal fashion, being normally restricted to the offspring of crosses in which males carry transposable elements but which females lack. In Drosophila, the dysgenic traits triggered by the P-element DNA transposon are restricted to the germ line and include chromosomal rearrangements, high rates of mutation, and sterility. The impairment is most prominent when hybrids are grown at higher temperatures, with adult dysgenic females being completely sterile at 29°C. Despite the severe phenotypes, little is known about the development of germ cells during P-M dysgenesis. To address this, germline development was characterized in the progeny obtained from reciprocal crosses between w1118 (P-element-devoid strain) and Harwich (P-element-containing strain) flies at 29°C. In non-dysgenic progeny, germline development progressed normally throughout embryonic and larval stages, leading to fertile adults. Although the development of dysgenic germline cells was not disturbed during embryogenesis, germ cells decreased in number during early larval stages, leading to animals with no germ cells by late larval stages. These results indicate that the detrimental effects elicited by P-element activity are triggered early on during primordial germ cell (PGC) development in dysgenic progeny, leading to premature germ cell death (Teixeira, 2017).

Maternally deposited small RNAs cognate to the P-element are thought to provide the 'P-cytotype' by conferring the transgenerationally inherited ability to protect developing germ cells against P-elements. Small RNA-based transposon regulation is typically mediated by either transcriptional silencing or post-transcriptional clearance of mRNAs, both of which result in a decrease in the accumulation of transposon mRNA. To understand how maternally provided small RNAs control P-elements in germ cells, this study focused on embryonic PGCs sorted from 4- to 20-h-old embryos generated from reciprocal crosses between w1118 and Harwich strains. Surprisingly, the accumulation of P-element RNA as measured by quantitative reverse transcription PCR (RT-qPCR) showed no change in dysgenic PGCs when compared to non-dysgenic PGCs. This indicates that P-cytotype small RNAs exert their function by means other than regulating P-element mRNA levels (Teixeira, 2017).

P-element activity relies on production of a functional P-element transposase protein, the expression of which requires precursor mRNA (pre-mRNA) splicing of three introns. To analyse P-element RNA splicing in germ cells during hybrid dysgenesis, primers were designed that specifically anneal to spliced mRNA transcripts. The accumulation of spliced forms for the first two introns (IVS1 and IVS2) did not show changes in dysgenic PGCs when compared to non-dysgenic PGCs. By contrast, the accumulation of spliced transcripts for the third intron (IVS3) was substantially increased in dysgenic germ cells. Given that the overall accumulation of P-element mRNA showed no changes, the results indicate that the maternally provided P-cytotype can negatively regulate P-element IVS3 splicing and therefore inhibits the production of functional P-transposase in germ cells (Teixeira, 2017).

Analysis of publically available small RNA sequencing data from 0-2-h-old embryos laid by Harwich females indicated that two classes of small RNAs cognate to the P-element are maternally transmitted: small interfering RNAs (siRNAs, 20-22-nucleotides long) and piRNAs (23-29 nucleotides long). To test the role of distinct small RNA populations on P-element expression, mutants were analyzed uniquely affecting each small RNA biogenesis pathway in the Harwich background. Mutations that disrupt siRNA biogenesis components Dicer-2 (Dcr-2) and Argonaute 2 (AGO2), or mutations ablating components of the piRNA biogenesis pathway, such as the Argonautes piwi, aubergine (aub), and Argonaute 3 (AGO3), as well as the RNA helicase vasa (vas) and spindle E (spn-E), did not affect P-element mRNA accumulation in adult ovaries as measured by RT-qPCR. However, mutations that disrupted piRNA biogenesis, and not the siRNA pathway, led to a strong and specific increase in the accumulation of IVS3-spliced mRNAs. RNA sequencing (RNA-seq) analysis on poly(A)-selected RNAs from aub and piwi mutant adult ovaries confirmed the specific effect on IVS3 splicing. To examine transposon expression in tissue, RNA fluorescent in situ hybridization (FISH) was performed using probes specific for the P-element and for the Burdock retrotransposon, a classic target of the germline piRNA pathway. In mutants affecting piRNA biogenesis, increased abundance of Burdock RNA was readily observed in germline tissues, with most of the signal accumulating close to the oocyte. By contrast, no difference was detected in the P-element RNA FISH signal in piRNA biogenesis mutants compared to control. Nuclear RNA foci observed in nurse cells were of similar intensity and number regardless of the genotype, and cytoplasmic signal showed no detectable difference. Therefore, the results indicate that in germ cells, piRNAs specifically modulate IVS3 splicing. This regulation is reminiscent of the well-documented mechanism that restricts P-element activity to germline tissues, which involves the expression of a host-encoded RNA binding repressor protein that negatively regulates IVS3 splicing in somatic tissues (Teixeira, 2017).

In somatic tissues, P-element alternative splicing regulation is mediated by the assembly of a splicing repressor complex on an exonic splicing silencer element directly upstream of IVS3. To test whether the P-element IVS3 and flanking exon sequences were sufficient to trigger the piRNA-mediated splicing regulation in germ cells, a transgenic reporter system for IVS3 splicing was used in which a heterologous promoter (Hsp83) drives the expression of an IVS3-lacZ-neo fusion mRNA specifically in the germ line. Using RT-qPCR, the F1 progeny from reciprocal crosses between w1118 and Harwich flies were analyzed in the presence of the hsp83-IVS3-lacZ-neo reporter. The fraction of spliced mRNAs produced from the transgenic reporter was substantially increased in dysgenic compared to non-dysgenic adult ovaries, in agreement with previously reported results. Most importantly, genetic experiments confirmed that the repression of IVS3 splicing in germ cells relies on piRNA biogenesis, as the splicing repression observed with this reporter in non-dysgenic progeny was specifically abolished in adult ovaries of aub and vas mutants (Teixeira, 2017).

Mechanistically, piRNA-mediated splicing regulation may be achieved through direct action of piRNA complexes on target pre-mRNAs carrying the IVS3 sequence or indirectly by piRNA-mediated changes in chromatin states. Piwi-interacting proteins such as Asterix (Arx) and Panoramix (Panx) are dispensable for piRNA biogenesis but are essential for establishing Piwi-mediated chromatin changes, possibly by acting as a scaffold to recruit histone-modifying enzymes and chromatin-binding proteins to target loci. To test the role of these chromatin regulators on P-element splicing, germline-specific RNA interference (RNAi) knockdown experiments were performed in the Harwich background. Similar to what was observed for the piRNA biogenesis components, germline knockdown of Arx and Panx showed no change in the accumulation of P-element RNA, but a strong and specific effect on IVS3 splicing in adult ovaries. The same pattern on IVS3 splicing was observed in the germline knockdown of HP1a and Maelstrom (Mael), both of which act downstream of Piwi-mediated targeting to modulate chromatin structure. The same genetic requirement for Panx for IVS3 splicing control was also confirmed when using the transgenic IVS3 splicing reporter, further indicating that Piwi-mediated chromatin changes at the target locus are involved in IVS3 splicing regulation. At target loci, Piwi complexes are known to mediate the deposition of the classic heterochromatin mark histone H3 lysine 9 trimethylation (H3K9me3). To assess the effect of piRNA-targeting on P-element chromatin marks directly, H3K9me3 chromatin immunoprecipitation was performed followed by sequencing (ChIP-seq) or quantitative PCR on adult ovaries of progeny from reciprocal crosses between w1118 and Harwich strains (to avoid developmental defects, ChIP was performed on F1 progeny raised at 18°C. This analysis revealed a specific loss of global H3K9me3 levels over P-element insertions in dysgenic progeny when compared to non-dysgenic progeny (Teixeira, 2017).

To analyse the chromatin structure at individual P-element insertions, DNA sequencing (DNA-seq) data was used to identify all euchromatic insertions in the Harwich strain, and RNA-seq analysis was used to define transcriptionally active insertions. At transcriptionally active P-element euchromatic insertions, the spreading of H3K9me3 into the flanking genomic regions was readily observed in non-dysgenic progeny, but was completely absent in dysgenic offspring. Similarly, a reduction in H3K9me3 modification levels was also observed over the IVS3 transgenic reporter in dysgenic progeny when compared to non-dysgenic progeny. Interestingly, euchromatic insertions with no evidence of transcriptional activity were devoid of an H3K9me3 signal in both non-dysgenic and dysgenic crosses, providing further evidence for a model initially suggested in yeast and more recently proposed for Drosophila and mammals, in which H3K9me3 deposition by piRNA complexes would require transcription of the target loci. Mechanistically different from the well-described somatic repression, the results uncovered the existence of an unexpected piRNA-mediated, chromatin-based mechanism regulating IVS3 alternative splicing in germ cells (Teixeira, 2017).

To expand the analysis, the literature was searched for other cases of transposon splicing regulation. Drosophila Gypsy elements are retrotransposons that have retrovirus-like, infective capacity owing to their envelope (Env) protein. These elements are expressed in somatic ovarian cells, in which they are regulated by the flamenco locus, a well-known piRNA cluster that is a soma-specific source of antisense piRNAs cognate to Gypsy. Interestingly, it has been shown that mutations in flamenco not only elicited the accumulation of Gypsy RNA, but also modulated pre-mRNA splicing, favouring the production of the env mRNA and therefore germline infection. To test whether the piRNA pathway, in addition to its role in regulating the accumulation of Gypsy RNA, is also responsible for modulating the splicing of Gypsy elements in somatic tissues, publically available RNA-seq data from poly(A)-selected RNAs extracted from in vivo cultures of ovarian somatic cells (OSCs) was analyzed. The analysis indicates that piwi knockdown was sufficient to modulate Gypsy splicing, favouring the accumulation of env-encoding mRNA. In agreement with a chromatin-mediated regulation of alternative splicing, RNAi depletion of Arx, Panx, HP1a and Mael, as well as knockdown of the histone linker H1, was sufficient to favour Gypsy splicing, recapitulating the effect caused by Piwi depletion. Notably, this was also the case for the H3K9 methyltransferase Setdb1, but not for the H3K9 methyltransferases Su(var)3-9 and G9a, indicating specific genetic requirements. Taken together, the results indicate that the piRNA pathway, through its role in mediating changes in chromatin states, regulates the splicing of transposon pre-mRNAs in both somatic and germline tissues (Teixeira, 2017).

Using P-M hybrid dysgenesis as a model, this study hasa uncovered splicing regulation elicited by chromatin changes as a previously unknown mechanism by which the piRNA pathway protects the genome from the detrimental effects of transposon activity. Splicing control at piRNA-target loci is likely to be mechanistically different from what has been observed for germline piRNA clusters given the low enrichment of the HP1 homologue Rhino (also known as HP1D) protein, which is required for piRNA cluster RNA processing, over the endogenous P-element insertions in the Harwich genome or over the transgenic IVS3 splicing reporter in non-dysgenic and dysgenic progeny (as measured by ChIP-qPCR). Because small RNA-based systems leading to chromatin mark changes at target loci are pervasive in eukaryotes, it is expected that this new type of targeted regulation is of importance in settings far beyond the scope of the piRNA pathway and Drosophila. Indeed, small RNA-guided DNA methylation over the LINE retrotransposon Karma was recently shown to modulate alternative splicing in oil palm, disrupting nearby gene expression and ultimately affecting crop yield. In this context, small RNA-based control of chromatin structure may be crucially important in genomes with a high content of intronic transposon insertions, such as the human genome, by providing a mechanism to suppress exonization of repeat elements. Although the means by which piRNA-mediated changes in chromatin states could regulate alternative splicing remain to be determined, it is tempting to speculate that piRNA pathway components do so by co-transcriptionally modulating interactions between RNA polymerase II and the spliceosome (Teixeira, 2017).


REFERENCES

Aagaard, L., et al. (1999). Functional mammalian homologues of the Drosophila PEV-modifier Su(var)3-9 encode centromere-associated proteins which complex with the heterochromatin component M31. EMBO J. 18: 1923-1938. 10202156

Aasland, R. and Stewart, A. F. (1995). The chromo shadow domain, a second chromo domain in heterochromatin-binding protein 1, HP1. Nucleic Acids Res 23: 3168-3173. PubMed Citation: 7667093

Agirre, E., Bellora, N., Allo, M., Pages, A., Bertucci, P., Kornblihtt, A. R. and Eyras, E. (2015). A chromatin code for alternative splicing involving a putative association between CTCF and HP1alpha proteins. BMC Biol 13: 31. PubMed ID: 25934638

Alekseyenko, A. A., Gorchakov, A. A., Zee, B. M., Fuchs, S. M., Kharchenko, P. V. and Kuroda, M. I. (2014). Heterochromatin-associated interactions of Drosophila HP1a with dADD1, HIPP1, and repetitive RNAs. Genes Dev 28: 1445-1460. PubMed ID: 24990964

Andrulis, E. D., Werner, J., Nazarian, A., Erdjument-Bromage, H., Tempst, P. and Lis, J. T. (2002). The RNA processing exosome is linked to elongating RNA polymerase II in Drosophila. Nature 420: 837-841. PubMed ID: 12490954

Ayyanathan, K., et al. (2003). Regulated recruitment of HP1 to a euchromatic gene induces mitotically heritable, epigenetic gene silencing: a mammalian cell culture model of gene variegation. Genes Dev. 17: 1855-1869. 12869583

Azzaz, A. M., Vitalini, M. W., Thomas, A. S., Price, J., Blacketer, M. J., Cryderman, D. E., Zirbel, L. N., Woodcock, C. L., Elcock, A. H., Wallrath, L. L. and Shogren-Knaak, M. A. (2014). HP1Hsalpha promotes nucleosome associations that drive chromatin condensation. J Biol Chem. [Epub ahead of print] PubMed ID: 24415761

Badugu, R. Shareef, M. M. and Kellum, R. (2003). Novel Drosophila Heterochromatin protein 1 (HP1)/Origin recognition complex-associated protein (HOAP) repeat motif in HP1/HOAP interactions and chromocenter associations. J. Biol. Chem. 278: 34491-34498. 12826664

Ball, L. J., et al. (1997). Structure of the chromatin binding (chromo) domain from mouse modifier protein 1. EMBO J 16 (9): 2473-2481. PubMed Citation: 9171360

Bannister, A. J., et al. (2001). Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature 410: 120-124. 11242054

Belyaeva, E. S., et al. (1993). Cytogenetic and molecular aspects of position-effect variegation in Drosophila melanogaster. V. Heterochromatin-associated protein HP1 appears in euchromatic chromosomal regions that are inactivated as a result of position-effect variegation. Chromosoma 102: 583-90. PubMed Citation: 8243169

Brasher, S. V., et al. (2000). The structure of mouse HP1 suggests a unique mode of single peptide recognition by the shadow chromo domain dimer. EMBO J. 19: 1587-1597. 10747027

Brower-Toland, B., et al. (2007). Drosophila PIWI associates with chromatin and interacts directly with HP1a. Genes Dev. 21(18): 2300-11. PubMed citation; Online text

Cammas, F., et al. (2004). Association of the transcriptional corepressor TIF1ß with heterochromatin protein 1 (HP1): an essential role for progression through differentiation. Genes Dev. 18: 2147-2160. 15342492

Cardoso, C., et al. (2005). XNP-1/ATR-X acts with RB, HP1 and the NuRD complex during larval development in C. elegans. Dev. Biol. 278(1): 49-59. 15649460

Cenci, G., et al. (1997). UbcD1, a Drosophila ubiquitin-conjugating enzyme required for proper telomere behavior. Genes Dev. 11: 863-875. PubMed Citation: 9106658

Cenci, G., Siriaco, G., Raffa, G.D., Kellum, R. and Gatti, M. (2003). The Drosophila HOAP protein is required for telomere capping. Nat. Cell Biol. 5: 82-84. 12510197

Chavez, J., Murillo-Maldonado, J. M., Bahena, V., Cruz, A. K., Castaneda-Sortibran, A., Rodriguez-Arnaiz, R., Zurita, M. and Valadez-Graham, V. (2017). dadd1 and dxnp prevent genome instability by maintaining HP1a localization at Drosophila telomeres. Chromosoma [Epub ahead of print]. PubMed ID: 28688038

Cheutin, T., et al. (2003). Maintenance of stable heterochromatin domains by dynamic HP1 binding. Science 299(5607): 721-5. 12560555

Clark, R. F. and Elgin, S. C. (1992). Heterochromatin protein 1, a known suppressor of position-effect variegation, is highly conserved in Drosophila. Nucleic Acids Res 20: 6067-74. PubMed Citation: 1461737

Cléard, F., Delattre, M., and Spierer, P. (1997). SU(VAR)3-7, a Drosophila heterochromatin-associated protein and companion of HP1 in the genomic silencing of position-effect variegation. EMBO J. 16: 5280-5288. PubMed Citation: 9311988

Clynes, D., Jelinska, C., Xella, B., Ayyub, H., Scott, C., Mitson, M., Taylor, S., Higgs, D. R. and Gibbons, R. J. (2015). Suppression of the alternative lengthening of telomere pathway by the chromatin remodelling factor ATRX. Nat Commun 6: 7538. PubMed ID: 26143912

Cowieson, N. P., et al. (2000). Dimerisation of a chromo shadow domain and distinctions from the chromodomain as revealed by structural analysis. Curr. Biol. 10: 517-525. 10801440

Cryderman, D. E., et al. (1999). Silencing at Drosophila telomeres: nuclear organization and chromatin structure play critical roles. EMBO J. 18(13): 3724-35. PubMed Citation: 10393187

Csink, A. K. and Henikoff, S. (1996). Genetic modification of heterochromatic association and nuclear organization in Drosophila. Nature 381: 529-531. PubMed Citation: 8632827

Danzer, J. R. and Wallrath, L. L. (2004). Mechanisms of HP1-mediated gene silencing in Drosophila. Development 131: 3571-3580. 15215206

Dawson, M. A., et al. (2009). JAK2 phosphorylates histone H3Y41 and excludes HP1alpha from chromatin. Nature 461(7265): 819-22. PubMed Citation: 19783980

Delattre, M., et al. (2000). The genomic silencing of position-effect variegation in Drosophila melanogaster: interaction between the heterochromatin-associated proteins Su(var)3-7 and HP1. J. Cell Sci. 113: 4253-61. 11069770

Demakova, O. V., et al. (2007). The SU(VAR)3-9/HP1 complex differentially regulates the compaction state and degree of underreplication of X chromosome pericentric heterochromatin in Drosophila melanogaster. Genetics 175(2): 609-20. PubMed Citation: 17151257

de Wit, E., Greil, F. and van Steensel, B. (2005). Genome-wide HP1 binding in Drosophila: Developmental plasticity and genomic targeting signals. Genome Res. 15(9): 1265-73. 16109969

Dialynas, G. K., Makatsori, D., Kourmouli, N., Theodoropoulos, P. A., McLean, K., Terjung, S., Singh, P. B. and Georgatos, S. D. (2006). Methylation-independent binding to histone H3 and cell cycle-dependent incorporation of HP1beta into heterochromatin. J Biol Chem 281: 14350-14360. PubMed ID: 16547356

Dubruille, R., et al. (2010). Specialization of a Drosophila capping protein essential for the protection of sperm telomeres. Curr. Biol. 20(23): 2090-9. PubMed Citation: 21093267

Eberle, A.B., Jordán-Pla, A., Gañez-Zapater, A., Hessle, V., Silberberg, G., von Euler, A., Silverstein, R.A. and Visa, N. (2015). An interaction between RRP6 and SU(VAR)3-9 targets RRP6 to heterochromatin and contributes to heterochromatin maintenance in Drosophila melanogaster. PLoS Genet 11: e1005523. PubMed ID: 26389589

Eissenberg, J. C., et al. (1992). The heterochromatin-associated protein HP-1 is an essential protein in Drosophila with dosage-dependent effects on position-effect variegation. Genetics: 131: 345-352. PubMed Citation: 1644277

Eissenberg, J. C. and Hartnett, T. (1993). A heat shock-activated cDNA rescues the recessive lethality of mutations in the heterochromatin-associated protein HP1 of Drosophila melanogaster. Mol. Gen. Genet. 240: 333-8. PubMed Citation: 8413181

Eissenberg, J. C., Ge, Y. W. and Hartnett, T. (1994). Increased phosphorylation of HP1, a heterochromatin-associated protein of Drosophila, is correlated with heterochromatin assembly. J. Biol. Chem. 269: 21315-21321. PubMed Citation: 8063756

Ekwall, K., et al. (1996). Mutations in the fission yeast silencing factors clr4+ and rik1+ disrupt the localisation of the chromo domain protein Swi6p and impair centromere function. J. Cell Sci. 109: 2637-48. 8937982

Elgin, S. C. R. (1996). Heterochromatin and gene regulation in Drosophila. Curr. Opin. Genet. Dev. 6: 193-202. PubMed Citation: 8722176

Emelyanov, A. V., Konev, A. Y., Vershilova, E. and Fyodorov, D. V. (2010). Protein complex of Drosophila ATRX/XNP and HP1a is required for the formation of pericentric beta-heterochromatin in vivo. J. Biol. Chem. 285(20): 15027-37. PubMed ID: 20154359

Fagegaltier, D., et al. (2009). The endogenous siRNA pathway is involved in heterochromatin formation in Drosophila. Proc. Natl. Acad. Sci. 106(50): 21258-63. PubMed Citation: 19948966

Fanti, L., et al. (1998a). Heterochromatin protein 1 binds transgene arrays. Chromosoma 107(5): 286-92. PubMed Citation: 9880761

Fanti, L., Giovinazzo, G., Berloco, M. and Pimpinelli, S. (1998b). The heterochromatin protein 1 prevents telomere fusions in Drosophila. Mol. Cell 2(5): 527-38. PubMed Citation: 9844626

Festenstein, R., et al. (1999). Heterochromatin protein 1 modifies mammalian PEV in a dose- and chromosomal-context- dependent manner. Nat. Genet. 23: 457-461. PubMed Citation: 10581035

Figueiredo, M. L., Philip, P., Stenberg, P. and Larsson, J. (2012). HP1a recruitment to promoters is independent of H3K9 methylation in Drosophila melanogaster. PLoS Genet 8: e1003061. PubMed ID: 23166515

Filion, G. J., van Bemmel, J. G., Braunschweig, U., Talhout, W., Kind, J., Ward, L. D., Brugman, W., de Castro, I. J., Kerkhoven, R. M., Bussemaker, H. J. and van Steensel, B. (2010). Systematic protein location mapping reveals five principal chromatin types in Drosophila cells. Cell 143: 212-224. PubMed ID: 20888037

Fischle, W., et al. (2005). Regulation of HP1-chromatin binding by histone H3 methylation and phosphorylation. Nature 438(7071): 1116-22. 16222246

Frankel, S., et al. (1997). An actin-related protein in Drosophila colocalizes with heterochromatin protein 1 in pericentric heterochromatin. J. Cell Sci. 110: 1999-2012. PubMed Citation: 9378752

Frankel, S. and Rogina, B. (2005). Drosophila longevity is not affected by heterochromatin-mediated gene silencing. Aging Cell. 4(1):53-6. 15659213

Frydrychova, R. C., Mason, J. M. and Archer, T. K. (2008). HP1 is distributed within distinct chromatin domains at Drosophila telomeres. Genetics 180(1): 121-31. PubMed Citation: 18723888

Gao, G., Walser, J. C., Beaucher, M. L., Morciano, P., Wesolowska, N., Chen, J. and Rong, Y. S. (2010). HipHop interacts with HOAP and HP1 to protect Drosophila telomeres in a sequence-independent manner. EMBO J. 29(4): 819-29. PubMed Citation: 20057353

Gilbert, N., et al. (2003). Formation of facultative heterochromatin in the absence of HP1. EMBO J. 22: 5540-5550. 14532126

Greil, F., et al. (2003). Distinct HP1 and Su(var)3-9 complexes bind to sets of developmentally coexpressed genes depending on chromosomal location. Genes Dev. 17: 2825-2838. 14630943

Greil, F., de Wit, E., Bussemaker, H. J., van Steensel, B. (2007). HP1 controls genomic targeting of four novel heterochromatin proteins in Drosophila. Embo J. 26: 741-751. PubMed Citation: 17255947

Gu, T. and Elgin, S. C. (2013). Maternal depletion of Piwi, a component of the RNAi system, impacts heterochromatin formation in Drosophila. PLoS Genet 9: e1003780. PubMed ID: 24068954

Hirota, T., Lipp, J. J., Toh, B. H. and Peters, J. M. (2005). Histone H3 serine 10 phosphorylation by Aurora B causes HP1 dissociation from heterochromatin. Nature. 438(7071): 1176-80. 16222244

Huang, D. W., et al. (1998). Distinct cytoplasmic and nuclear fractions of Drosophila heterochromatin protein 1: their phosphorylation levels and associations with origin recognition complex proteins. J. Cell Biol. 142(2): 307-18. PubMed Citation: 9679132

Huang, H., et al. (1999). A nonessential HP1-like protein affects starvation-induced assembly of condensed chromatin and gene expression in macronuclei of Tetrahymena thermophila. Mol. Cell. Biol. 19: 3624-3634. PubMed Citation: 10207086

Huang, X. A., Yin, H., Sweeney, S., Raha, D., Snyder, M. and Lin, H. (2013). A major epigenetic programming mechanism guided by piRNAs. Dev Cell 24: 502-516. PubMed ID: 23434410

Hwang, K.-K., Eissenberg, J. C. and Worman, H. J. (2001). Transcriptional repression of euchromatic genes by Drosophila heterochromatin protein 1 and histone modifiers. Proc. Natl. Acad. Sci. 98: 11423-11427. 11562500

Ivanova, A. V., et al. (1998). The chromo and SET domains of the Clr4 protein are essential for silencing in fission yeast. Nature Genet. 19: 192-195. PubMed Citation: 9620780

Jacobs, S. A., et al. (2001). Specificity of the HP1 chromo domain for the methylated N-terminus of histone H3. EMBO J. 20. 5232-5241. 11566886

Jacobs, S. A. and Khorasanizadeh, S. (2002) Structure of a HP1 chromodomain bound to a lysine 9-methylted histone H3 tail. Science, 295: 2080-2083. 11859155

James, T. C., et al. (1989). Distribution patterns of HP1, a heterochromatin-associated nonhistone chromosomal protein of Drosophila. Eur. J. Cell Biol. 50: 170-80

Johansson, A. M., Stenberg, P., Pettersson, F. and Larsson, J. (2007). POF and HP1 bind expressed exons, suggesting a balancing mechanism for gene regulation. PLoS Genet 3: e209. PubMed ID: 18020713

Joppich, C., Scholz, S., Korge, G. and Schwendemann, A. (2009). Umbrea, a chromo shadow domain protein in Drosophila melanogaster heterochromatin, interacts with Hip, HP1 and HOAP. Chromosome Res. 17(1): 19-36. PubMed Citation: 19190990

Josse, T., et al. (2007). Telomeric trans-silencing: an epigenetic repression combining RNA silencing and heterochromatin formation. PLoS Genet. 3(9): 1633-43. PubMed citation; Online text

Juang, B. T., Gu, C., Starnes, L., Palladino, F., Goga, A., Kennedy, S. and L'Etoile N, D. (2013). Endogenous Nuclear RNAi Mediates Behavioral Adaptation to Odor. Cell 154: 1010-1022. PubMed ID: 23993094

Kappes, F., et al. (2011). The DEK oncoprotein is a Su(var) that is essential to heterochromatin integrity. Genes Dev. 25(7): 673-8. PubMed Citation: 21460035

Kellum, R., Raff, J. W. and Alberts, B. M. (1995a). Heterochromatin protein 1 distribution during development and during the cell cycle in Drosophila embryos. J Cell Sci 108: 1407-1418. PubMed Citation: 7615662

Kellum, R. and Alberts, B.M. (1995b). Heterochromatin protein 1 is required for correct chromosome segregation in Drosophila embryos, J Cell Sci 108: 1419-31. PubMed Citation: 7615663

Kharchenko, P. V., (2011). Comprehensive analysis of the chromatin landscape in Drosophila melanogaster. Nature 471: 480-485. PubMed ID: 21179089

Kourmouli, N., et al. (2000). Dynamic associations of heterochromatin protein 1 with the nuclear envelope. EMBO J. 19: 6558-6568. PubMed Citation: 11101528

Klattenhoff, C., et al. (2009). The Drosophila HP1 homolog Rhino is required for transposon silencing and piRNA production by dual-strand clusters. Cell 138: 1137-1149. PubMed Citation: 19732946

Lachner, M., et al. (2001). Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 410(6824): 116-20. 11242053

Lagarou, A., et al. (2008). dKDM2 couples histone H2A ubiquitylation to histone H3 demethylation during Polycomb group silencing. Genes Dev. 22: 2799-2810. PubMed Citation: 18923078

Lehming, N., et al. (1998). Chromatin components as part of a putative transcriptional repressing complex. Proc. Natl. Acad. Sci. 95(13): 7322-7326. PubMed Citation: 9636147

Levine, M. T., McCoy, C., Vermaak, D., Lee, Y. C., Hiatt, M. A., Matsen, F. A. and Malik, H. S. (2012). Phylogenomic analysis reveals dynamic evolutionary history of the Drosophila heterochromatin protein 1 (HP1) gene family. PLoS Genet 8: e1002729. Pubmed: 22737079

Li, H., et al. (2011). Cooperative and antagonistic contributions of two heterochromatin proteins to transcriptional regulation of the Drosophila sex determination decision. PLoS Genet. 7(6): e1002122. PubMed Citation: 21695246

Li, Y., et al. (2003). Effects of tethering HP1 to euchromatic regions of the Drosophila genome. Development 130: 1817-1824. 12642487

Li, Y., et al. (2006). A novel histone deacetylase pathway regulates mitosis by modulating Aurora B kinase activity. Genes Dev. 20(18): 2566-79. Medline abstract: 16980585

Lin, C. H., Paulson, A., Abmayr, S. M. and Workman, J. L. (2012). HP1a targets the Drosophila KDM4A demethylase to a subset of heterochromatic genes to regulate H3K36me3 levels. PLoS One 7: e39758. PubMed Citation: 22761891

Linder, B., Gerlach, N. and Jäckle, H. (2001). The Drosophila homolog of the human AF10 is an HP1-interacting suppressor of position effect variegation. EMBO Rep. 2: 211-216. 11266362

Liu, L.-P., et al. (2005). Sex-specific role of Drosophila melanogaster HP1 in regulating chromatin structure and gene transcription. Nature Genetics 37: 1361-1366. 16258543

Lorentz, A., et al. (1994). Switching gene swi6, involved in repression of silent mating-type loci in fission yeast, encodes a homologue of chromatin-associated proteins from Drosophila and mammals. Gene 143: 139-43. PubMed Citation: 8200530

Loyola, A., et al. (2001). Reconstitution of recombinant chromatin establishes a requirement for histone-tail modifications during chromatin assembly and transcription. Genes Dev. 15: 2837-2851. 11691835

Lu, B. Y., et al. (2000). Heterochromatin protein 1 is required for the normal expression of two heterochromatin genes in Drosophila. Genetics 155: 699-708. PubMed Citation: 10835392

Lundberg, L. E., Stenberg, P. and Larsson, J. (2013). HP1a, Su(var)3-9, SETDB1 and POF stimulate or repress gene expression depending on genomic position, gene length and expression pattern in Drosophila melanogaster. Nucleic Acids Res 41: 4481-4494. PubMed ID: 23476027

Marshall, O. J. and Brand, A. H. (2017). Chromatin state changes during neural development revealed by in vivo cell-type specific profiling. Nat Commun 8(1): 2271. PubMed ID: 29273756

Meehan, R. R., Kao, C.-F. and Pennings, S. (2003). HP1 binding to native chromatin in vitro is determined by the hinge region and not by the chromodomain EMBO J. 22: 3164-3174. 12805230

Mellone, B. G., et al. (2003). Centromere silencing and function in fission yeast is governed by the amino terminus of histone H3. Curr. Biol. 13: 1748-1757. 14561399

Mendez, D. L., Kim, D., Chruszcz, M., Stephens, G. E., Minor, W., Khorasanizadeh, S. and Elgin, S. C. (2011). The HP1a disordered C terminus and chromo shadow domain cooperate to select target peptide partners. Chembiochem 12: 1084-1096. PubMed ID: 21472955

Miklos, G. L. G. and Cotsell, J. N. (1991). Chromosome structure at interfaces between major chromatin types: alpha- and beta-heterochromatin. Bioessays 12: 1-6

Minc, E., Allory, V., Worman, H. J., Courvalin, J. C. and Buendia, B. (1999). Localization and phosphorylation of HP1 proteins during the cell cycle in mammalian cells. Chromosoma 108: 220-234. PubMed Citation: 10460410

Muchardt, C., Guilleme, M., Seeler, J. S., Trouche, D., Dejean, A. and Yaniv, M. (2002). Coordinated methyl and RNA binding is required for heterochromatin localization of mammalian HP1alpha. EMBO Rep. 3: 975-981. 12231507

Murzina, N., et al. (1999). Heterochromatin dynamics in mouse cells: interaction between chromatin assembly factor 1 and HP1 proteins. Mol. Cell 4(4): 529-40. PubMed Citation: 10549285

Nakayama, J.-i., Klar, A. J. S. and Grewa, S. I. S. (2000). A chromodomain protein, Swi6, performs imprinting functions in fission yeast during mitosis and meiosis. Cell 101: 307-317. 10847685

Nakayama, J., et al. (2001). Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly. Science 292(5514): 110-3. 11283354

Napier, C. E., Huschtscha, L. I., Harvey, A., Bower, K., Noble, J. R., Hendrickson, E. A. and Reddel, R. R. (2015). ATRX represses alternative lengthening of telomeres. Oncotarget 6(18): 16543-16558. PubMed ID: 26001292

Narita, M., et al. (2003). Rb-mediated heterochromatin formation and silencing of E2F target genes during cellular senescence. Cell 113: 703-716. 12809602

Nielsen, A. L., et al. (1999). Interaction with members of the heterochromatin protein 1 (HP1) family and histone deacetylation are differentially involved in transcriptional silencing by members of the TIF1 family. EMBO J. 18(22): 6385-6395. 10562550

Nielsen, A. L., et al. (2001). Heterochromatin formation in mammalian cells: Interaction between histones and HP1 proteins. Molec. Cell 7: 729-739

Nielsen, A. L., et al. (2002). Selective interaction between the chromatin-remodeling factor BRG1 and the heterochromatin-associated protein HP1alpha. EMBO J: 21: 5797-5806. 12411497

Nielsen, P. R., et al. (2002). Structure of the HP1 chromodomain bound to histone H3 methylated at lysine 9. Nature 416: 103-107. 11882902

Nielsen, S. J., et al. (2001). Rb targets histone H3 methylation and HP1 to promoters. Nature 412: 561-565. 11484059

Pak, D.T., et al. (1997). Association of the origin recognition complex with heterochromatin and HP1 in higher eukaryotes. Cell 91(3): 311-323

Paro, R. and Hogness, D. S. (1991). The Polycomb protein shares a homologous domain with a heterochromatin-associated protein of Drosophila. Proc Natl Acad Sci 88: 263-7

Pak, D. T. S., Pflumm, M., Chesnokov, I., Huang, D. W., Kellum, R., Marr, J., Romanowski, P. and Botchan, M. R. (1997). Association of the origin recognition complex with heterochromatin and HP1 in higher eukaryotes. Cell 91: 311-323

Perrini, B., et al. (2004). HP1 controls telomere capping, telomere elongation, and telomere silencing by two different mechanisms in Drosophila. Molec. Cell 15: 467-476. 15304225

Piacentini, L., et al. (2003). Heterochromatin protein 1 (HP1) is associated with induced gene expression in Drosophila euchromatin. J. Cell Biol. 161: 707-714. 12756231

Platero, J.S., Hartnett, T. and Eissenberg, J.C. (1995). Functional analysis of the chromo domain of HP1. EMBO J. 14: 3977-3986

Platero, J. S., et al. (1996). In vivo assay for protein-protein interactions using Drosophila chromosomes. Chromosoma 104: 393-404

Powers, J. A. and Eissenberg, J. C. (1993). Overlapping domains of the heterochromatin-associated protein HP1 mediate nuclear localization and heterochromatin binding. J. Cell Biol. 120: 291-299

Ramamoorthy, M. and Smith, S. (2015). Loss of ATRX Suppresses Resolution of Telomere Cohesion to Control Recombination in ALT Cancer Cells. Cancer Cell 28(3): 357-369. PubMed ID: 26373281

Reiss, D., Josse, T., Anxolabehere, D. and Ronsseray, S. (2004). Aubergine mutations in Drosophila melanogaster impair P cytotype determination by telomeric P elements inserted in heterochromatin. Mol. Genet. Genomics 272: 336-343. PubMed citation: 15372228

Roche, S. E. and Rio, D. C. (1998). Trans-silencing by P elements inserted in subtelomeric heterochromatin involves the Drosophila Polycomb group gene, Enhancer of zeste. Genetics 149: 1839-1855. PubMed citation: 9691041

Ronsseray, S., Lehmann, M., Nouaud, D. and Anxolabehere, D. (1996). The regulatory properties of autonomous subtelomeric P elements are sensitive to a Suppressor of variegation in Drosophila melanogaster. Genetics 143: 1663-1674. PubMed citation: 8844154

Ronsseray, S., Boivin, A. and Anxolabehere, D. (2001). P-Element repression in Drosophila melanogaster by variegating clusters of P-lacZ-white transgenes. Genetics 159: 1631-1642. PubMed citation: 11779802

Ryan, R. F., et al. (1999). KAP-1 corepressor protein interacts and colocalizes with heterochromatic and euchromatic HP1 Proteins: a potential role for Krüppel-associated box-zinc finger proteins in heterochromatin-mediated gene silencing. Mol. Cell. Biol. 19: 4366-4378

Ryu, H. W., Lee, D. H., Florens, L., Swanson, S. K., Washburn, M. P. and Kwon, S. H. (2014). Analysis of the heterochromatin protein 1 (HP1) interactome in Drosophila. J Proteomics 102C:137-147. PubMed ID: 24681131

Satyaki, P. R., Cuykendall, T. N., Wei, K. H., Brideau, N. J., Kwak, H., Aruna, S., Ferree, P. M., Ji, S. and Barbash, D. A. (2014). The Hmr and Lhr hybrid incompatibility genes suppress a broad range of heterochromatic repeats. PLoS Genet 10(3): e1004240. PubMed ID: 24651406

Saveliev, A., et al. (2003). DNA triplet repeats mediate heterochromatin-protein-1-sensitive variegated gene silencing. Nature 422(6934): 909-13. 12712207

Savitsky, M., Kravchuk, O., Melnikova, L. and Georgiev, P., (2002). Heterochromatin protein 1 is involved in control of telomere elongation in Drosophila melanogaster. Mol. Cell. Biol. 22: 3204-3218. 11940677

Schneiderman, J. I., Sakai, A., Goldstein, S. and Ahmad, K. (2009). The XNP remodeler targets dynamic chromatin in Drosophila. Proc Natl Acad Sci U S A 106: 14472-14477. PubMed ID: 19706533

Schotta, G., et al. (2002). Central role of Drosophila SU(VAR)3-9 in histone H3-K9 methylation and heterochromatic gene silencing. EMBO J. 21: 1121-1131. 11867540

Schotta, G., et al. (2004). A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin. Genes Dev. 18: 1251-1262. 15145825

Schultz, D. C., et al. (2002). SETDB1: a novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins. Genes Dev. 16: 919-932. 11959841

Schwendemann, A., et al. (2008). Hip, an HP1-interacting protein, is a haplo- and triplo-suppressor of position effect variegation. Proc. Natl. Acad. Sci. 105: 204-209. PubMed Citation: 18162556

Seeler, J. S., et al. (1998). Interaction of SP100 with HP1 proteins: a link between the promyelocytic leukemia-associated nuclear bodies and the chromatin compartment. Proc. Natl. Acad. Sci. 95(13): 7316-7321

Seum, C., et al. (2001). Ectopic HP1 promotes chromosome loops and variegated silencing in Drosophila. EMBO J. 20: 812-818. 11179225

Shaffer, C. D., et al. (2002). Heterochromatin protein 2 (HP2), a partner of HP1 in Drosophila heterochromatin. Proc. Natl. Acad. Sci. 99(22): 14332-7. 12376620

Sharma, G. G., et al. (2003). Human heterochromatin protein 1 isoforms HP1(Hsalpha) and HP1(Hsbeta) interfere with hTERT-telomere interactions and correlate with changes in cell growth and response to ionizing radiation. Mol. Cell. Biol. 23: 8363-8376. Medline abstract: 14585993

Shermoen, A. W., McCleland, M. L. and O'Farrell, P. H. (2010). Developmental control of late replication and S phase length. Curr. Biol. 20(23): 2067-77. PubMed Citation: 21074439

Shevelyov, Y. Y., Lavrov, S. A., Mikhaylova, L. M., Nurminsky, I. D., Kulathinal, R. J., Egorova, K. S., Rozovsky, Y. M. and Nurminsky, D. I. (2009). The B-type lamin is required for somatic repression of testis-specific gene clusters. Proc Natl Acad Sci U S A 106: 3282-3287. PubMed ID: 19218438

Shi, S., et al. (2006). JAK signaling globally counteracts heterochromatic gene silencing. Nat. Genet. 38(9): 1071-6. Medline abstract: 16892059

Shimada, A., et al. (2009). Phosphorylation of Swi6/HP1 regulates transcriptional gene silencing at heterochromatin. Genes Dev. 23(1): 18-23. PubMed Citation: 19136623

Smith, M. B. and Weiler, K. S. (2010). Drosophila D1 overexpression induces ectopic pairing of polytene chromosomes and is deleterious to development. Chromosoma 119: 287-309. PubMed ID: 20127347

Smothers, J. F. and Henikoff, S. (2000). The HP1 chromo shadow domain binds a consensus peptide pentamer. Curr. Biol. 10: 27-30. PubMed Citation: 10660299

Smothers, J. F. and Henikoff, S. (2001). The hinge and chromo shadow domain impart distinct targeting of HP1-like proteins. Mol. Cell. Bio. 21: 2555-2569. 11259603

Spierer, A., Seum, C., Delattre, M. and Spierer, P. (2005). Loss of the modifiers of variegation Su(var)3-7 or HP1 impacts male X polytene chromosome morphology and dosage compensation. J. Cell Sci. 118(Pt 21): 5047-57. 16234327

Stokes, D. G., Tartof, K. D. and Perry, R. P. (1996). CHD1 is concentrated in interbands and puffed regions of Drosophila polytene chromosomes. Proc. Natl. Acad. Sci. 93(14): 7137-7142

Sugimoto, K., et al. (1996). Human homolog of Drosophila heterochromatin-associated protein 1 (HP1) is a DNA-binding protein which possesses a DNA-binding motif with weak similarity to that of human centromere protein C (CENP-C). J. Biochem. (Tokyo) 120(1): 153-9

Sun, F. L., Haynes, K., Simpson, C. L., Lee, S. D., Collins, L., Wuller, J., Eissenberg, J. C. and Elgin, S. C. (2004). cis-Acting determinants of heterochromatin formation on Drosophila melanogaster chromosome four. Mol. Cell. Biol. 24(18): 8210-20. 15340080

Swaminathan J., Baxter, E. M. and Corces, V. G. (2005). The role of histone H2Av variant replacement and histone H4 acetylation in the establishment of Drosophila heterochromatin. Genes Dev. 19(1): 65-76. 15630020

Tatsuke, T., Zhu, L., Li, Z., Mitsunobu, H., Yoshimura, K., Mon, H., Lee, J. M. and Kusakabe, T. (2014). Roles of Piwi proteins in transcriptional regulation mediated by HP1s in cultured silkworm cells. PLoS One 9: e92313. PubMed ID: 24637637

Terada, Y., Tatsuka, M., Suzuki, F., Yasuda, Y., Fujita, S. and Otsu, M. (1998). AIM-1: a mammalian midbody-associated protein required for cytokinesis. EMBO J. 17: 667-676. 9450992

Teixeira, F. K., Okuniewska, M., Malone, C. D., Coux, R. X., Rio, D. C. and Lehmann, R. (2017). piRNA-mediated regulation of transposon alternative splicing in the soma and germ line. Nature 552(7684): 268-272. PubMed ID: 29211718

Todeschini, A. L., Teysset, L., Delmarre, V. and Ronsseray, S. (2010). The epigenetic trans-silencing effect in Drosophila involves maternally-transmitted small RNAs whose production depends on the piRNA pathway and HP1. PLoS One. 5(6): e11032. PubMed Citation: 20559422

Tschiersch, B., et al. (1994). The protein encoded by the Drosophila position-effect variegation suppressor gene Su(var)3-9 combines domains of antagonistic regulators of homeotic gene complexes. EMBO J 13: 3822-3831

van der Vlag, J., et al. (2000). Transcriptional repression mediated by Polycomb group proteins and other chromatin-associated repressors is selectively blocked by insulators. J Biol Chem. 275: 697-704

Verní, F. and Cenci, G. (2015). The Drosophila histone variant H2A.V works in concert with HP1 to promote kinetochore-driven microtubule formation. Cell Cycle 14(4):577-88. PubMed ID: 25591068

Wang, G., et al. (2001). Conservation of heterochromatin protein 1 function. Mol. Cell. Biol. 20(18): 6970-83. 10958692

Wood, J. G., Hillenmeyer, S., Lawrence, C., Chang, C., Hosier, S., Lightfoot, W., Mukherjee, E., Jiang, N., Schorl, C., Brodsky, A. S., Neretti, N. and Helfand, S. L. (2010). Chromatin remodeling in the aging genome of Drosophila. Aging Cell 9: 971-978. PubMed ID: 20961390

Woodage, T., et al. (1997). Characterization of the CHD family of proteins. Proc. Natl. Acad..Sci. 94(21): 11472-11477

Wreggett, K. A., et al (1994). A mammalian homologue of Drosophila heterochromatin protein 1 (HP1) is a component of constitutive heterochromatin. Cytogenet Cell Genet 66: 99-103

Yamaguchi, K., Hidema, S. and Mizuno, S. (1998). Chicken chromobox proteins: cDNA cloning of CHCB1, -2, -3 and their relation to W-heterochromatin. Exp. Cell Res. 242(1): 303-314

Ye, Q. and Worman, H. J. (1996). Interaction between an integral protein of the nuclear envelope inner membrane and human chromodomain proteins homologous to Drosophila HP1. J. Biol. Chem. 271: 14653-14656

Yin, H. and Lin, H. (2007). An epigenetic activation role of Piwi and a Piwi-associated piRNA in Drosophila melanogaster. Nature 450(7167): 304-8. PubMed citation; Online text

Zhao, T. and Eissenberg, J. C. (1999). Phosphorylation of heterochromatin protein 1 by casein Kinase II is required for efficient heterochromatin binding in Drosophila. J. Biol. Chem. 274(21): 15095-15100. 10329715

Zhao, T., Heyduk, T., Allis, C. D. and Eissenberg, J. C. (2000). Heterochromatin protein 1 binds to nucleosomes and DNA in vitro. J. Biol. Chem.275: 36

Zhimulev, I. F., Belyaeva, E. S., Makunin, I. V., Pirrotta, V., Volkova, E. I., Alekseyenko, A. A., Andreyeva, E. N., Makarevich, G. F., Boldyreva, L. V., Nanayev, R. A. and Demakova, O. V. (2003). Influence of the SuUR gene on intercalary heterochromatin in Drosophila melanogaster polytene chromosomes. Chromosoma 111: 377-398. PubMed ID: 12644953


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

date revised: 25 April 2018

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