HP1/ORC complex and heterochromatin assembly

We have used the highly conserved heterochromatin component, heterochromatin protein 1 (HP1), as a molecular tag for purifying other protein components of Drosophila heterochromatin. A complex of HP1 associated with the origin recognition complex (ORC) and an HP1/ORC-associated protein (HOAP) was purified from the maternally loaded cytoplasm of early Drosophila embryo. We propose that the DNA-binding activities of ORC and HOAP function to recruit underphosphorylated isoforms of HP1 to sites of heterochromatin nucleation. The roles of highly phosphorylated HP1, other DNA-binding proteins known to interact with HP1, and histone modifying activities in heterochromatin assembly are also addressed.     

Mutations in the heterochromatin protein 1 (HP1) hinge domain affect HP1 protein interactions and chromosomal distribution

Heterochromatin Protein 1 (HP1) is a conserved component of the highly compact chromatin found at centromeres and telomeres. A conserved feature of the protein is multiple phosphorylation. Hyper-phosphorylation of HP1 accompanies the assembly of cytologically distinct heterochromatin during early embryogenesis. Hypo-phosphorylated HP1 is associated with the DNA-binding activities of the origin recognition complex (ORC) and an HMG-like HP1/ORC-Associated Protein (HOAP). Perturbations in HP1 localization in pericentric and telomeric heterochromatin in mutants for Drosophila ORC2 and HOAP, respectively, indicate roles for these HP1 phosphoisoforms in heterochromatin assembly also. To elucidate the roles of hypo- and hyper-phosphophorylated HP1 in heterochromatin assembly, we have mutated consensus Protein Kinase-A phosphorylation sites in the HP1 hinge domain and examined the mutant proteins for distinct in vitro and in vivo activities. Mutations designed to mimic hyper-phosphorylation render the protein incapable of binding HOAP and the DmORC1 subunit but confer enhanced homo-dimerization and lysine 9-methylated histone H3-binding to the protein. Mutations rendering the protein unphosphorylatable, by contrast, do not affect homo-dimerization or binding to lysine 9-di-methylated histone H3, HOAP, or DmORC1 but do confer novel DmORC2-binding activity to the protein. This mutant protein is ectopically localized throughout the chromosomes when overexpressed in vivo in the presence of a full dose of DmORC2. This ectopic targeting is accompanied by ectopic targeting of lysine 9 tri-methylated histone H3. The distinct activities of these mutant proteins could reflect distinct roles for HP1 phosphoisoforms in heterochromatin structure and function.     

Chromosomal distribution of heterochromatin protein 1 (HP1) in Drosophila: a cytological map of euchromatic HP1 binding sites

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.     

Coordinated methyl and RNA binding is required for heterochromatin localization of mammalian HP1alpha

In mammalian cells, as in Schizosaccharomyces pombe and Drosophila, HP1 proteins bind histone H3 tails methylated on lysine 9 (K9). However, whereas K9-methylated H3 histones are distributed throughout the nucleus, HP1 proteins are enriched in pericentromeric heterochromatin. This observation suggests that the methyl-binding property of HP1 may not be sufficient for its heterochromatin targeting. We show that the association of HP1α with pericentromeric heterochromatin depends not only on its methyl-binding chromo domain but also on an RNA-binding activity present in the hinge region of the protein that connects the conserved chromo and chromoshadow domains. Our data suggest the existence of complex heterochromatin binding sites composed of methylated histone H3 tails and RNA, with each being recognized by a separate domain of HP1α.     

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

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

A rapidly evolving genomic toolkit for Drosophila heterochromatin

Heterochromatin is the enigmatic eukaryotic genome compartment found mostly at telomeres and centromeres. Conventional approaches to sequence assembly and genetic manipulation fail in this highly repetitive, gene-sparse, and recombinationally silent DNA. In contrast, genetic and molecular analyses of euchromatin-encoded proteins that bind, remodel, and propagate heterochromatin have revealed its vital role in numerous cellular and evolutionary processes. Utilizing the 12 sequenced Drosophila genomes, Levine et al¹ took a phylogenomic approach to discover new such protein “surrogates” of heterochromatin function and evolution. This paper reported over 20 new members of what was traditionally believed to be a small and static Heterochromatin Protein 1 (HP1) gene family. The newly identified HP1 proteins are structurally diverse, lineage-restricted, and expressed primarily in the male germline. The birth and death of HP1 genes follows a “revolving door” pattern, where new HP1s appear to replace old HP1s. Here, we address alternative evolutionary models that drive this constant innovation.     

Characterization of a novel chromodomain-containing gene from the silkworm, Bombyx mori

Heterochromatin protein 1 (HP1) is an evolutionarily conserved protein across different eukaryotic species, and is crucial in the establishment and maintenance of heterochromatin. HP1 proteins have two distinct functional domains, an N-terminal chromodomain (CD) and a C-terminal chromoshadow domain (CSD), which are required for the selective binding of HP1 proteins to modified histones. During our screen for HP1-like proteins in the Bombyx mori genome, we found a novel silkworm gene, Bombyx mori chromodomain protein 1 (BmCdp1), encoding a putative chromobox protein with only two CDs. The BmCdp1 family proteins are closely related to the HP1 proteins, and most of them belong to insect lineages. qRT-PCR analysis indicated that BmCdp1 mRNA was most abundantly expressed in early embryos, and relatively higher expression was observed in larval testes, hemocytes, and pupal ovaries. Western blot and immunostaining experiments showed that BmCdp1 was localized mainly in the nucleus of BmN4 cells. We searched BmCdp1-bound loci in the Bombyx genome by ChIP-seq analysis using Flag-tagged BmCdp1-expressing BmN4 cells. Combined with ChIP-qPCR experiments, we identified two reliable BmCdp1-bound loci in the genome. siRNA-mediated knockdown of BmCdp1 in BmN4 cells and early embryos did not affect the expression of the gene located close to the BmCdp1-bound locus.     

The mechanism of telomere protection: a comparison between Drosophila and humans

Drosophila telomeres are maintained by transposition of specialized retrotransposons rather than by telomerase activity, and their stability is independent of the sequence of DNA termini. Recent studies have identified several proteins that protect Drosophila telomeres from fusion events. These proteins include the telomere capping factors HP1/ORC-associated protein (HOAP) and heterochromatin protein 1 (HP1), the Rad50 and Mre11 DNA repair proteins that are required for HOAP and HP1 localization at telomeres, and the ATM kinase. Another telomere-protecting factor identified in Drosophila is UbcD1, a polypeptide highly homologous to class I ubiquitin-conjugating E2 enzymes. In addition, it has been shown that HP1 and both components of the Drosophila Ku70/80 heterodimer act as negative regulators of telomere length. Except for HOAP, all these proteins are conserved in humans and are associated with human telomeres. Collectively, these results indicate that Drosophila is an excellent model system for the analysis of the mechanisms of telomere maintenance. In past and current studies, 15 Drosophila genes have been identified that prevent telomeric fusion, and it has been estimated that the Drosophila genome contains at least 40 genes required for telomere protection. We believe that the molecular characterization of these genes will lead to identification of many novel human genes with roles in telomere maintenance.     

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

Association of the highly conserved heterochromatin protein, HP1, with the specialized chromatin of centromeres and telomeres requires binding to a specific histone H3 modification of methylation on lysine 9. This modification is catalyzed by the Drosophila Su(var)3-9 gene product and its homologues. Specific DNA binding activities are also likely to be required for targeting this activity along with HP1 to specific chromosomal regions. The Drosophila HOAP protein is a DNA-binding protein that was identified as a component of a multiprotein complex of HP1 containing Drosophila origin recognition complex (ORC) subunits in the early Drosophila embryo. Here we show direct physical interactions between the HOAP protein and HP1 and specific ORC subunits. Two additional HP1-like proteins (HP1b and HP1c) were recently identified in Drosophila, and the unique chromosomal distribution of each isoform is determined by two independently acting HP1 domains (hinge and chromoshadow domain) (47). We find heterochromatin protein 1/origin recognition complex-associated protein (HOAP) to interact specifically with the originally described predominantly heterochromatic HP1a protein. Both the hinge and chromoshadow domains of HP1a are required for its interaction with HOAP, and a novel peptide repeat located in the carboxyl terminus of the HOAP protein is required for the interaction with the HP1 hinge domain. Peptides that interfere with HP1a/HOAP interactions in co-precipitation experiments also displace HP1 from the heterochromatic chromocenter of polytene chromosomes in larval salivary glands. A mutant for the HOAP protein also suppresses centric heterochromatin-induced silencing, supporting a role for HOAP in centric heterochromatin.     

Histones H3/H4 form a tight complex with the inner nuclear membrane protein LBR and heterochromatin protein 1

We have recently shown that heterochromatin protein 1 (HP1) interacts with the nuclear envelope in an acetylation-dependent manner. Using purified components and in vitro assays, we now demonstrate that HP1 forms a quaternary complex with the inner nuclear membrane protein LBR and a sub-set of core histones. This complex involves histone H3/H4 oligomers, which mediate binding of LBR to HP1 and cross-link these two proteins that do not interact directly with each other. Consistent with previous observations, HP1 and LBR binding to core histones is strongly inhibited when H3/H4 are modified by recombinant CREB-binding protein, revealing a new mechanism for anchoring domains of under-acetylated chromatin to the inner nuclear membrane.     

Drosophila Heterochromatin Protein 1 (HP1)/Origin Recognition Complex (ORC) Protein Is Associated with HP1 and ORC and Functions in Heterochromatin-induced Silencing

Heterochromatin protein 1 (HP1) is a conserved component of the highly compact chromatin of higher eukaryotic centromeres and telomeres. Cytogenetic experiments in Drosophila have shown that HP1 localization into this chromatin is perturbed in mutants for the origin recognition complex (ORC) 2 subunit. ORC has a multisubunit DNA-binding activity that binds origins of DNA replication where it is required for origin firing. The DNA-binding activity of ORC is also used in the recruitment of the Sir1 protein to silence nucleation sites flanking silent copies of the mating-type genes in Saccharomyces cerevisiae. A fraction of HP1 in the maternally loaded cytoplasm of the early Drosophila embryo is associated with a multiprotein complex containing Drosophila melanogaster ORC subunits. This complex appears to be poised to function in heterochromatin assembly later in embryonic development. Here we report the identification of a novel component of this complex, the HP1/ORC-associated protein. This protein contains similarity to DNA sequence-specific HMG proteins and is shown to bind specific satellite sequences and the telomere-associated sequence in vitro. The protein is shown to have heterochromatic localization in both diploid interphase and mitotic chromosomes and polytene chromosomes. Moreover, the gene encoding HP1/ORC-associated protein was found to display reciprocal dose-dependent variegation modifier phenotypes, similar to those for mutants in HP1 and the ORC 2 subunit.