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Centromeres are epigenetically determined chromatin structures that specify the assembly site of the kinetochore, the multiprotein machinery that binds microtubules and mediates chromosome segregation during mitosis and meiosis. The centromeric protein A (CENP-A) and its Drosophila orthologue centromere identifier (Cid) are H3 histone variants that replace the canonical H3 histone in centromeric nucleosomes of eukaryotes. CENP-A/Cid is required for recruitment of other centromere and kinetochore proteins and its deficiency disrupts chromosome segregation. Despite the many components that are known to cooperate in centromere function, the complete network of factors involved in CENP-A recruitment remains to be defined. In Drosophila, the Trx-G proteins localize along the heterochromatin with specific patterns and some of them localize to the centromeres of all chromosomes. Here, we show that the Trx, Ash1, and CBP proteins are required for the correct chromosome segregation and that Ash1 and CBP mediate for Cid/CENP-A recruitment at centromeres through post-translational histone modifications. We found that centromeric H3 histone is consistently acetylated in K27 by CBP and that nej and ash1 silencing respectively causes a decrease in H3K27 acetylation and H3K4 methylation along with an impairment of Cid loading.

A very important open question in stem cells regulation is how the fine balance between GSCs self-renewal and differentiation is orchestrated at the molecular level. In the past several years much progress has been made in understanding the molecular mechanisms underlying intrinsic and extrinsic controls of GSC regulation but the complex gene regulatory networks that regulate stem cell behavior are only partially understood. HP1 is a dynamic epigenetic determinant mainly involved in heterochromatin formation, epigenetic gene silencing and telomere maintenance. Furthermore, recent studies have revealed the importance of HP1 in DNA repair, sister chromatid cohesion and, surprisingly, in positive regulation of gene expression. Here, we show that HP1 plays a crucial role in the control of GSC homeostasis in Drosophila. Our findings demonstrate that HP1 is required intrinsically to promote GSC self-renewal and progeny differentiation by directly stabilizing the transcripts of key genes involved in GSCs maintenance.

HP1 is a conserved prototype protein that plays an essential role in heterochromatin formation and epigenetic gene silencing through its interaction with histone methyltransferase enzymes (HMTases) and the histone H3 at lysine 9 (H3-MeK9). HP1 is also involved in telomere capping and, more surprisingly, in positive regulation of gene expression. Recently, a wide expression analysis, using a RIP-chip assays (RNA-immunoprecipitation on microarrays), has shown that HP1 associates with the transcripts of more than one hundred euchromatic genes and interacts with the heterogeneous nuclear ribonucleoproteins (hnRNPs) that are known to be involved in RNA processing. By these results, HP1 seems to be part of a complex that stabilizes RNA transcripts. Though previously unsuspected, it was also found that HP1-interacting hnRNPs have a functional role in heterochromatin formation. These proteins bind heterochromatin and are dominant suppressors of position effect variegation. Taken together, the results in the paper by Piacentini et al. open a window on a possible new conceptual landscape in which similar epigenetic mechanisms could have a significant role, both in the metabolism of RNA transcripts and in heterochromatin formation, producing opposite functional effects. These data seem to establish a functional link between euchromatin and heterochromatin.

Heterochromatin Protein 1 (HP1a) is a well-known conserved protein involved in heterochromatin formation and gene silencing in different species including humans. A general model has been proposed for heterochromatin formation and epigenetic gene silencing in different species that implies an essential role for HP1a. According to the model, histone methyltransferase enzymes (HMTases) methylate the histone H3 at lysine 9 (H3K9me), creating selective binding sites for itself and the chromodomain of HP1a. This complex is thought to form a higher order chromatin state that represses gene activity. It has also been found that HP1a plays a role in telomere capping. Surprisingly, recent studies have shown that HP1a is present at many euchromatic sites along polytene chromosomes of Drosophila melanogaster, including the developmental and heat-shock-induced puffs, and that this protein can be removed from these sites by in vivo RNase treatment, thus suggesting an association of HP1a with the transcripts of many active genes. To test this suggestion, we performed an extensive screening by RIP-chip assay (RNA-immunoprecipitation on microarrays), and we found that HP1a is associated with transcripts of more than one hundred euchromatic genes. An expression analysis in HP1a mutants shows that HP1a is required for positive regulation of these genes. Cytogenetic and molecular assays show that HP1a also interacts with the well known proteins DDP1, HRB87F, and PEP, which belong to different classes of heterogeneous nuclear ribonucleoproteins (hnRNPs) involved in RNA processing. Surprisingly, we found that all these hnRNP proteins also bind heterochromatin and are dominant suppressors of position effect variegation. Together, our data show novel and unexpected functions for HP1a and hnRNPs proteins. All these proteins are in fact involved both in RNA transcript processing and in heterochromatin formation. This suggests that, in general, similar epigenetic mechanisms have a significant role on both RNA and heterochromatin metabolisms., Competing Interests: The authors have declared that no competing interests exist.

In Drosophila, the Polycomb group and trithorax group proteins play a critical role in controlling the expression states of homeotic gene complexes during development. The common view is that these two classes of proteins bind to the homeotic complexes and regulate transcription at the level of chromatin. In the present work, we tested the involvement of both groups in mitotic heterochromatin formation in Drosophila. Using specific antibodies, we show that some of the tested Pc-G proteins are present in heterochromatin, while all the tested trx-G proteins localize to specific regions of heterochromatin in both mitotic chromosomes and interphase nuclei. We also observed that mutations in trx-G genes are recessive enhancers of position-effect variegation and are able to repress the transcription of heterochromatic genes. These results strongly suggest that trx-G proteins, along with some Pc-G proteins, play an active role in heterochromatin formation in Drosophila.

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

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

The Heterochromatin Protein 1 (HP1) is a conserved protein which is best known for its strong association with the heterochromatin of Drosophila melanogaster. We previously demonstrated that another important property of HP1 is its localization to the telomeres of Drosophila, a feature that reflects its critical function as a telomere capping protein. Here we report our analysis of the euchromatic sites to which HP1 localizes. Using an anti-HP1 antibody, we compared immunostaining patterns on polytene chromosomes of the Ore-R wild type laboratory strain and four different natural populations. HP1 was found to accumulate at specific euchromatic sites, with a subset of the sites conserved among strains. These sites do not appear to be defined by an enrichment of known repetitive DNAs. Comparisons of HP1 patterns among several Drosophila species revealed that association with specific euchromatic regions, heterochromatin and telomeres is a conserved characteristic of HP1. Based on these results, we argue that HP1 serves a broader function than typically postulated. In addition to its role in heterochromatin assembly and telomere stability, we propose that HP1 plays an important role in regulating the expression of many different euchromatic regions.