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.     

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.     

Distinct Cytoplasmic and Nuclear Fractions of Drosophila Heterochromatin Protein 1: Their Phosphorylation Levels and Associations with Origin Recognition Complex Proteins

The distinct structural properties of heterochromatin accommodate a diverse group of vital chromosome functions, yet we have only rudimentary molecular details of its structure. A powerful tool in the analyses of its structure in Drosophila has been a group of mutations that reverse the repressive effect of heterochromatin on the expression of a gene placed next to it ectopically. Several genes from this group are known to encode proteins enriched in heterochromatin. The best characterized of these is the heterochromatin-associated protein, HP1. HP1 has no known DNA-binding activity, hence its incorporation into heterochromatin is likely to be dependent upon other proteins. To examine HP1 interacting proteins, we isolated three distinct oligomeric species of HP1 from the cytoplasm of early Drosophila embryos and analyzed their compositions. The two larger oligomers share two properties with the fraction of HP1 that is most tightly associated with the chromatin of interphase nuclei: an underphosphorylated HP1 isoform profile and an association with subunits of the origin recognition complex (ORC). We also found that HP1 localization into heterochromatin is disrupted in mutants for the ORC2 subunit. These findings support a role for the ORC-containing oligomers in localizing HP1 into Drosophila heterochromatin that is strikingly similar to the role of ORC in recruiting the Sir1 protein to silencing nucleation sites in Saccharomyces cerevisiae.     

Pericentromeric heterochromatin domains are maintained without accumulation of HP1

The heterochromatin protein 1 (HP1) family is thought to be an important structural component of heterochromatin. HP1 proteins bind via their chromodomain to nucleosomes methylated at lysine 9 of histone H3 (H3K9me). To investigate the role of HP1 in maintaining heterochromatin structure, we used a dominant negative approach by expressing truncated HP1alpha or HP1beta proteins lacking a functional chromodomain. Expression of these truncated HP1 proteins individually or in combination resulted in a strong reduction of the accumulation of HP1alpha, HP1beta, and HP1gamma in pericentromeric heterochromatin domains in mouse 3T3 fibroblasts. The expression levels of HP1 did not change. The apparent displacement of HP1alpha, HP1beta, and HP1gamma from pericentromeric heterochromatin did not result in visible changes in the structure of pericentromeric heterochromatin domains, as visualized by DAPI staining and immunofluorescent labeling of H3K9me. Our results show that the accumulation of HP1alpha, HP1beta, and HP1gamma at pericentromeric heterochromatin domains is not required to maintain DAPI-stained pericentromeric heterochromatin domains and the methylated state of histone H3 at lysine 9 in such heterochromatin domains.     

Heterochromatin formation in mammalian cells: interaction between histones and HP1 proteins

Members of the heterochromatin protein 1 (HP1) family are silencing nonhistone proteins. Here, we show that in P19 embryonal carcinoma (EC) nuclei, HP1 alpha, beta, and gamma form homo- and heteromers associated with nucleosomal core histones. In vitro, all three HP1s bind to tailed and tailless nucleosomes and specifically interact with the histone-fold of histone H3. Furthermore, HP1alpha interacts with the linker histone H1. HP1alpha binds to H3 and H1 through its chromodomain (CD) and hinge region, respectively. Interestingly, the Polycomb (Pc1/M33) CD also interacts with H3, and HP1alpha and Pc1/M33 binding to H3 is severely impaired by CD mutations known to abrogate HP1 and Polycomb silencing in Drosophila. These results define a novel function for the conserved CD and suggest that HP1 self-association and histone binding may play a crucial role in HP1-mediated heterochromatin assembly.     

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.     

Interaction and assembly of murine pre-replicative complex proteins in yeast and mouse cells

Eukaryotic cells coordinate chromosome duplication by the assembly of protein complexes at origins of DNA replication by sequential binding of member proteins of the origin recognition complex (ORC), CDC6, and minichromosome maintenance (MCM) proteins. These pre-replicative complexes (pre-RCs) are activated by cyclin-dependent kinases and DBF4/CDC7 kinase. Here, we carried out a comprehensive yeast two-hybrid screen to establish sequential interactions between two individual proteins of the mouse pre-RC that are probably required for the initiation of DNA replication. The studies revealed multiple interactions among ORC subunits and MCM proteins as well as interactions between individual ORC and MCM proteins. In particular CDC6 was found to bind strongly to ORC1 and ORC2, and to MCM7 proteins. DBF4 interacts with the subunits of ORC as well as with MCM proteins. It was also demonstrated that CDC7 binds to different ORC and MCM proteins. CDC45 interacts with ORC1 and ORC6, and weakly with MCM3, -6, and -7. The three subunits of the single-stranded DNA binding protein RPA show interactions with various ORC subunits as well as with several MCM proteins. The data obtained by yeast two-hybrid analysis were paradigmatically confirmed in synchronized murine FM3A cells by immunoprecipitation of the interacting partners. Some of the interactions were found to be cell-cycle-dependent; however, most of them were cell-cycle-independent. Altogether, 90 protein-protein interactions were detected in this study, 52 of them were found for the first time in any eukaryotic pre-RC. These data may help to understand the complex interplay of the components of the mouse pre-RC and should allow us to refine its structural architecture as well as its assembly in real time.     

Phosphorylation of the linker histone H1 by CDK regulates its binding to HP1alpha

Two key components of mammalian heterochromatin that play a structural role in higher order chromatin organization are the heterochromatin protein 1alpha (HP1alpha) and the linker histone H1. Here, we show that these proteins interact in vivo and in vitro through their hinge and C-terminal domains, respectively. The phosphorylation of H1 by CDK2, which is required for efficient cell cycle progression, disrupts this interaction. We propose that phosphorylation of H1 provides a signal for the disassembly of higher order chromatin structures during interphase, independent of histone H3-lysine 9 (H3-K9) methylation, by reducing the affinity of HP1alpha for heterochromatin.     

Structural basis of HP1/PXVXL motif peptide interactions and HP1 localisation to heterochromatin

HP1 family proteins are adaptor molecules, containing two related chromo domains that are required for chromatin packaging and gene silencing. Here we present the structure of the chromo shadow domain from mouse HP1beta bound to a peptide containing a consensus PXVXL motif found in many HP1 binding partners. The shadow domain exhibits a novel mode of peptide recognition, where the peptide binds across the dimer interface, sandwiched in a beta-sheet between strands from each monomer. The structure allows us to predict which other shadow domains bind similar PXVXL motif-containing peptides and provides a framework for predicting the sequence specificity of the others. We show that targeting of HP1beta to heterochromatin requires shadow domain interactions with PXVXL-containing proteins in addition to chromo domain recognition of Lys-9-methylated histone H3. Interestingly, it also appears to require the simultaneous recognition of two Lys-9-methylated histone H3 molecules. This finding implies a further complexity to the histone code for regulation of chromatin structure and suggests how binding of HP1 family proteins may lead to its condensation.     

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.     

Epitope analysis of chromo antigen and clinical features in a subset of patients with anti-centromere antibodies

Heterochromatin protein 1 (HP1) is one of the nonhistone chromosomal components tightly associated with the pericentromeric heterochromatic region in Drosophila. The human homologue of HP1 is recognized by a subpopulation of anti-centromere antibodies (ACA). Such autoantibodies recognize a group of several nuclear proteins with Mr of 23–25 kDa and have been termed anti-chromo antibodies (AChA) because an evolutionarily conserved N-terminal half called the chromo domain of HP1 is the epitope. In this study, 84 ACA sera were examined by immunoblotting with recombinant 25-kDa chromo protein (p25). The p25 antigen was expressed as a glutathione S-transferase-fusion protein in E. coli and purified with glutathione-sepharose. Except for one serum specimen, AChA-positive sera reacted with the N-terminus (a.a 16–106) and/or the C-terminus (a.a. 83–191) of p25. Autoimmune response against the N-terminus of p25 in 33 patients was significantly associated with systemic lupus erythematosus and significantly related to leukopenia, thrombocytopenia and elevated erythrocyte sedimentation rate; C-terminal reactivity in 30 patients was significantly associated with primary Sjogren's syndrome and related to leukopenia. The internal 64-amino acid stretch (a.a. 43–106) with DNA-binding activity was not autoantigenic. p25 has two separate homologous regions to Drosophila HP1 at the N- and C-termini; the chromo domain and the chromo shadow domain. Patients with autoimmune responses against these conserved domains might form a clinical subset of patients positive for ACA.Abbreviations ACA anti-centromere antibodies - AChA anti-chromo antibodies - CENP centromere protein - DM dermatomyositis - ESR erythrocyte sedimentation rate - GST glutathione S-transferase - HP1 heterochromatin protein 1 - p25 25-kDa chromo protein - SLE systemic lupus erythematosus - SS Sjogren's syndrome - SSc systemic sclerosis - WBC white blood cell     

Hsk1-Dfp1 is required for heterochromatin-mediated cohesion at centromeres

Heterochromatin performs a central role in chromosome segregation and stability by promoting cohesion at centromeres. Establishment of both heterochromatin-mediated silencing and cohesion requires passage through S phase, although the mechanism is unknown. Here we demonstrate that Schizosaccharomyces pombe Hsk1 (CDC7), a conserved Dbf4-dependent protein kinase (DDK) that regulates replication initiation, interacts with and phosphorylates the heterochromatin protein 1 (HP1) equivalent Swi6 (ref. 6). Hsk1 and its regulatory subunit Dfp1 function downstream of Swi6 localization to promote heterochromatin function and cohesion specifically at centromeres. This role for Hsk1-Dfp1 is separable from its replication initiation activity, providing a temporal link between S phase and centromere cohesion that is mediated by heterochromatin.