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

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

Epigenetic maintenance of telomere identity in Drosophila: Buckle up for the sperm ride

A critical function of telomeres is to prevent the ligation of chromosome ends by DNA repair enzymes. In most eukaryotes, telomeric DNA consists in large arrays of G-rich tandem repeats that are recognized by DNA binding capping proteins. Drosophila telomeres are unusual as they lack short tandem repeats. However, Drosophila capping proteins can bind chromosome extremities in a DNA sequence-independent manner. This epigenetic protection of fly telomeres has been essentially studied in somatic cells where capping proteins such as HOAP or HP1 are essential in preventing chromosome end-to-end fusions. HipHop and K81 are two recently identified paralogous capping proteins with complementary expression patterns. While HipHop is involved in telomere capping in somatic cells, K81 has specialized in the protection of telomeres in post-meiotic male germ cells. Remarkably, K81 is required for the stabilization of HOAP and HP1 at telomeres during the massive paternal chromatin remodeling that occurs during spermiogenesis and at fertilization. We thus propose that the maintenance of capping proteins at Drosophila sperm telomeres is crucial for the transmission of telomere identity to the diploid zygote.     

Histone Variant H2A.Z Regulates Centromere Silencing and Chromosome Segregation in Fission Yeast

The incorporation of histone variant H2A.Z into nucleosomes plays essential roles in regulating chromatin structure and gene expression. A multisubunit complex containing chromatin remodeling protein Swr1 is responsible for the deposition of H2A.Z in budding yeast and mammals. Here, we show that the JmjC domain protein Msc1 is a novel component of the fission yeast Swr1 complex and is required for Swr1-mediated incorporation of H2A.Z into nucleosomes at gene promoters. Loss of Msc1, Swr1, or H2A.Z results in loss of silencing at centromeres and defective chromosome segregation, although centromeric levels of CENP-A, a centromere-specific histone H3 variant that is required for setting up the chromatin structure at centromeres, remain unchanged. Intriguingly, H2A.Z is required for the expression of another centromere protein, CENP-C, and overexpression of CENP-C rescues centromere silencing defects associated with H2A.Z loss. These results demonstrate the importance of H2A.Z and CENP-C in maintaining a silenced chromatin state at centromeres.     

The Drosophila HOAP protein is required for telomere capping

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

The Drosophila Mre11/Rad50 complex is required to prevent both telomeric fusion and chromosome breakage

The MRN complex consists of the two evolutionarily conserved components Mre11 and Rad50 and the third less-conserved component Nbs1/Xrs2. This complex mediates telomere maintenance in addition to a variety of functions in response to DNA double-strand breaks, including homologous recombination, nonhomologous end joining (NHEJ), and activation of DNA damage checkpoints. Mutations in the Mre11 gene cause the human ataxia-telangiectasia-like disorder (ATDL). Here, we show that null mutations in the Drosophila mre11 and rad50 genes cause both telomeric fusion and chromosome breakage. Moreover, we demonstrate that these mutations are in the same epistasis group required for telomere capping and mitotic chromosome integrity. Using an antibody against Rad50, we show that this protein is uniformly distributed along mitotic chromosomes, and that Rad50 is unstable in the absence of its binding partner Mre11. To define the roles of rad50 and mre11 in telomere protection, mutant chromosome preparations were immunostained for both HP1 and HOAP, two proteins that protect Drosophila telomeres from fusion. Cytological analysis revealed that mutations in rad50 and mre11 drastically reduce accumulation of HOAP and HP1 at telomeres. This suggests that the MRN complex protects Drosophila telomeres by facilitating recruitment of HOAP and HP1 at chromosome ends.     

RPA and POT1

Telomere maintenance in cycling cells relies on both DNA replication and capping by the protein complex shelterin. Two single-stranded DNA (ssDNA)-binding proteins, replication protein A (RPA) and protection of telomere 1 (POT1) play critical roles in DNA replication and telomere capping, respectively. While RPA binds to ssDNA in a non-sequence-specific manner, POT1 specifically recognizes singlestranded TTAGGG telomeric repeats. Loss of POT1 leads to aberrant accumulation of RPA at telomeres and activation of the ataxia telangiectasia and Rad3-related kinase (ATR)-mediated checkpoint response, suggesting that POT1 antagonizes RPA binding to telomeric ssDNA. The requirement for both POT1 and RPA in telomere maintenance and the antagonism between the two proteins raises the important question of how they function in concert on telomeric ssDNA. Two interesting models were proposed by recent studies to explain the regulation of POT1 and RPA at telomeres. Here, we discuss how these models help unravel the coordination, and also the antagonism, between POT1 and RPA during the cell cycle.     

The Ku protein complex is involved in length regulation of Drosophila telomeres

Chromosome ends in Drosophila melanogaster can be elongated either by terminal attachment of the telomere-specific retrotransposons HeT-A and TART or by terminal gene conversion. Here we show that a decrease in Ku70 or Ku80 gene dosage causes a sharp increase in the frequency of HeT-A and TART attachments to a broken chromosome end and in terminal DNA elongation by gene conversion. Loss of Ku80 has more pronounced effects than loss of Ku70. However, lower Ku70 concentration reduces the stability of terminally deficient chromosomes. Our results suggest a role of the end-binding Ku complex in the accessibility and length regulation of Drosophila telomeres.     

Actin-Related Protein Arp6 Influences H2A.Z-Dependent and -Independent Gene Expression and Links Ribosomal Protein Genes to Nuclear Pores

Actin-related proteins are ubiquitous components of chromatin remodelers and are conserved from yeast to man. We have examined the role of the budding yeast actin-related protein Arp6 in gene expression, both as a component of the SWR1 complex (SWR-C) and in its absence. We mapped Arp6 binding sites along four yeast chromosomes using chromatin immunoprecipitation from wild-type and swr1 deleted (swr1Δ) cells. We find that a majority of Arp6 binding sites coincide with binding sites of Swr1, the catalytic subunit of SWR-C, and with the histone H2A variant Htz1 (H2A.Z) deposited by SWR-C. However, Arp6 binding detected at centromeres, the promoters of ribosomal protein (RP) genes, and some telomeres is independent of Swr1 and Htz1 deposition. Given that RP genes and telomeres both show association with the nuclear periphery, we monitored the ability of Arp6 to mediate the localization of chromatin to nuclear pores. Arp6 binding is sufficient to shift a randomly positioned locus to nuclear periphery, even in a swr1Δ strain. Arp6 is also necessary for the pore association of its targeted RP promoters possibly through cell cycle-dependent factors. Loss of Arp6, but not Htz1, leads to an up-regulation of these RP genes. In contrast, the pore-association of GAL1 correlates with Htz1 deposition, and loss of Arp6 reduces both GAL1 activation and peripheral localization. We conclude that Arp6 functions both together with the nucleosome remodeler Swr1 and also without it, to mediate Htz1-dependent and Htz1-independent binding of chromatin domains to nuclear pores. This association is shown to have modulating effects on gene expression.     

Telomere capping in Drosophila: dealing with chromosome ends that most resemble DNA breaks

Telomere caps prevent chromosome ends from being recognized as DNA double-strand breaks (DSBs). Unlike most organisms studied, the telomere-capping function of Drosophila does not require a specific sequence. Without this sequence component, Drosophila telomeres most resemble DNA breaks and, thus, represent a simpler system for the study of telomere capping. I review recent progress in Drosophila telomere studies, and challenge the notion that Drosophila may not be a relevant model for the study of telomere maintenance. This review article is part of a series dedicated to the eighth International conference on Drosophila heterochromatin, held in Gubbio (Italy), June 2007.     

DNA-PKcs phosphorylates hnRNP-A1 to facilitate the RPA-to-POT1 switch and telomere capping after replication

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) has been implicated in telomere protection and telomerase activation. Recent evidence has further demonstrated that hnRNP-A1 plays a crucial role in maintaining newly replicated telomeric 3′ overhangs and facilitating the switch from replication protein A (RPA) to protection of telomeres 1 (POT1). The role of hnRNP-A1 in telomere protection also involves DNA-dependent protein kinase catalytic subunit (DNA-PKcs), although the detailed regulation mechanism has not been clear. Here we report that hnRNP-A1 is phosphorylated by DNA-PKcs during the G2 and M phases and that DNA-PK-dependent hnRNP-A1 phosphorylation promotes the RPA-to-POT1 switch on telomeric single-stranded 3′ overhangs. Consequently, in cells lacking hnRNP-A1 or DNA-PKcs-dependent hnRNP-A1 phosphorylation, impairment of the RPA-to-POT1 switch results in DNA damage response at telomeres during mitosis as well as induction of fragile telomeres. Taken together, our results indicate that DNA-PKcs-dependent hnRNP-A1 phosphorylation is critical for capping of the newly replicated telomeres and prevention of telomeric aberrations.     

Monoubiquitylation of H2A.Z distinguishes its association with euchromatin or facultative heterochromatin

H2A.Z is a histone H2A variant that is essential for viability in organisms such as Tetrahymena thermophila, Drosophila melanogaster, and mice. In Saccharomyces cerevisiae, loss of H2A.Z is tolerated, but proper regulation of gene expression is affected. Genetics and genome-wide localization studies show that yeast H2A.Z physically localizes to the promoters of genes and functions in part to protect active genes in euchromatin from being silenced by heterochromatin spreading. To date, the function of H2A.Z in mammalian cells is less clear, and evidence so far suggests that it has a role in chromatin compaction and heterochromatin silencing. In this study, we found that the bulk of H2A.Z is excluded from constitutive heterochromatin in differentiated human and mouse cells. Consistent with this observation, analyses of H2A.Z- or H2A-containing mononucleosomes show that the H3 associated with H2A.Z has lower levels of K9 methylation but higher levels of K4 methylation than those associated with H2A. We also found that a fraction of mammalian H2A.Z is monoubiquitylated and that, on the inactive X chromosomes of female cells, the majority of this histone variant is modified by ubiquitin. Finally, ubiquitylation of H2A.Z is mediated by the RING1b E3 ligase of the human polycomb complex, further supporting a silencing role of ubiquitylated H2A.Z. These new findings suggest that mammalian H2A.Z is associated with both euchromatin and facultative heterochromatin and that monoubiquitylation is a specific mark that distinguishes the H2A.Z associated with these different chromatin states.