Interplay between histone deacetylase SIR-2, linker histone H1 and histone methyltransferases in heterochromatin formation

Maintenance of intact heterochromatin structure through epigenetic mechanisms is essential for cell survival. Defects in heterochromatin formation caused by loss of chromatin-modifying enzymes lead to genomic instability and cellular senescence. The NAD+-dependent histone deacetylase SIR-2 and the H1 linker histone are intriguing chromatin elements that are connected to chromatin regulation and cell viability in the single cellular eukaryotic organism yeast. However, it remains an open question how SIR-2 and H1 mediate heterochromatin formation in simple multi-cellular organisms such as C. elegans and in even more complex organisms such as mammals. Recently we have identified SIR-2.1 and the H1 histone subtype, HIS-24 as factors involved in heterochromatin regulation at subtelomeric regions in C. elegans. In addition we show that SIR-2.1, HIS-24, and MES-2, a ortholog to Enhancer of zeste E(Z) are functionally related in heterochromatin formation contributing to fertility and embryogenesis. Here we discuss the interplay between SIR-2, H1 histone and histone methyltransferases in modulation of chromatin structure in further detail.     

Rapid de novo centromere formation occurs independently of heterochromatin protein 1 in C. elegans embryos

DNA injected into the Caenorhabditis elegans germline forms extrachromosomal arrays that segregate during cell division [1, 2]. The mechanisms underlying array formation and segregation are not known. Here, we show that extrachromosomal arrays form de novo centromeres at high frequency, providing unique access to a process that occurs with extremely low frequency in other systems [3-8]. De novo centromerized arrays recruit centromeric chromatin and kinetochore proteins and autonomously segregate on the spindle. Live imaging following DNA injection revealed that arrays form after oocyte fertilization via homologous recombination and nonhomologous end-joining. Individual arrays gradually transition from passive inheritance to active segregation during the early embryonic divisions. The heterochromatin protein 1 (HP1) family proteins HPL-1 and HPL-2 are dispensable for de novo centromerization even though arrays become strongly enriched for the heterochromatin-associated H3K9me3 modification over time. Partial inhibition of HP1 family proteins accelerates the acquisition of segregation competence. In addition to reporting the first direct visualization of new centromere formation in living cells, these findings reveal that naked DNA rapidly builds de novo centromeres in C. elegans embryos in an HP1-independent manner and suggest that, rather than being a prerequisite, HP1-dependent heterochromatin antagonizes de novo centromerization.     

N-terminal phosphorylation of HP1{alpha} promotes its chromatin binding

The phosphorylation of heterochromatin protein 1 (HP1) has been previously described in studies of mammals, but the biological implications of this modification remain largely elusive. Here, we show that the N-terminal phosphorylation of HP1α plays a central role in its targeting to chromatin. Recombinant HP1α prepared from mammalian cultured cells exhibited a stronger binding affinity for K9-methylated histone H3 (H3K9me) than that produced in Escherichia coli. Biochemical analyses revealed that HP1α was multiply phosphorylated at N-terminal serine residues (S11-14) in human and mouse cells and that this phosphorylation enhanced HP1α's affinity for H3K9me. Importantly, the N-terminal phosphorylation appeared to facilitate the initial binding of HP1α to H3K9me by mediating the interaction between HP1α and a part of the H3 tail that was distinct from the methylated K9. Unphosphorylatable mutant HP1α exhibited severe heterochromatin localization defects in vivo, and its prolonged expression led to increased chromosomal instability. Our results suggest that HP1α's N-terminal phosphorylation is essential for its proper targeting to heterochromatin and that its binding to the methylated histone tail is achieved by the cooperative action of the chromodomain and neighboring posttranslational modifications.     

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.     

Dynamic Regulation of Effector Protein Binding to Histone Modifications: The Biology of HP1 Switching

Post-translational modifications of histone proteins, the basic building blocks around which eukaryotic DNA is organized, are crucially involved in the regulation of genome activity as they control chromatin structure and dynamics. The recruitment of specific binding proteins that recognize and interact with particular histone modifications is thought to constitute a fundamental mechanism by which histone marks mediate biological function. For instance, tri-methylation of histone H3 lysine 9 (H3K9me3) is important for recruiting heterochromatin protein 1 (HP1) to discrete regions of the genome, thereby regulating gene expression, chromatin packaging, and heterochromatin formation. Until now, little was known about the regulation of effector-histone mark interactions, and in particular, of the binding of HP1 to H3K9me3. Recently, we and others presented evidence that a "binary methylation-phosphorylation switch" mechanism controls the dynamic release of HP1 from H3K9me3 during the cell cycle: phosphorylation of histone H3 serine 10 (H3S10ph) occurs at the onset of mitosis, interferes with HP1-H3K9me3 interaction, and therefore, ejects HP1 from its binding site. Here, we discuss the biological function of HP1 release from chromatin during mitosis, consider implications why the cell controls HP1 binding by such a methylation-phosphorylation switching mechanism, and reflect on other cellular pathways where binary switching of HP1 might occur.     

HP1a Recruitment to Promoters Is Independent of H3K9 Methylation in Drosophila melanogaster

Heterochromatin protein 1 (HP1) proteins, recognized readers of the heterochromatin mark methylation of histone H3 lysine 9 (H3K9me), are important regulators of heterochromatin-mediated gene silencing and chromosome structure. In Drosophila melanogaster three histone lysine methyl transferases (HKMTs) are associated with the methylation of H3K9: Su(var)3-9, Setdb1, and G9a. To probe the dependence of HP1a binding on H3K9me, its dependence on these three HKMTs, and the division of labor between the HKMTs, we have examined correlations between HP1a binding and H3K9me patterns in wild type and null mutants of these HKMTs. We show here that Su(var)3-9 controls H3K9me-dependent binding of HP1a in pericentromeric regions, while Setdb1 controls it in cytological region 2L:31 and (together with POF) in chromosome 4. HP1a binds to the promoters and within bodies of active genes in these three regions. More importantly, however, HP1a binding at promoters of active genes is independent of H3K9me and POF. Rather, it is associated with heterochromatin protein 2 (HP2) and open chromatin. Our results support a hypothesis in which HP1a nucleates with high affinity independently of H3K9me in promoters of active genes and then spreads via H3K9 methylation and transient looping contacts with those H3K9me target sites.     

The HP1α–CAF1–SetDB1‐containing complex provides H3K9me1 for Suv39‐mediated K9me3 in pericentric heterochromatin

Trimethylation of lysine 9 in histone H3 (H3K9me3) enrichment is a characteristic of pericentric heterochromatin. The hypothesis of a stepwise mechanism to establish and maintain this mark during DNA replication suggests that newly synthesized histone H3 goes through an intermediate methylation state to become a substrate for the histone methyltransferase Suppressor of variegation 39 (Suv39H1/H2). How this intermediate methylation state is achieved and how it is targeted to the correct place at the right time is not yet known. Here, we show that the histone H3K9 methyltransferase SetDB1 associates with the specific heterochromatin protein 1α (HP1α)–chromatin assembly factor 1 (CAF1) chaperone complex. This complex monomethylates K9 on non‐nucleosomal histone H3. Therefore, the heterochromatic HP1α–CAF1–SetDB1 complex probably provides H3K9me1 for subsequent trimethylation by Suv39H1/H2 in pericentric regions. The connection of CAF1 with DNA replication, HP1α with heterochromatin formation and SetDB1 for H3K9me1 suggests a highly coordinated mechanism to ensure the propagation of H3K9me3 in pericentric heterochromatin during DNA replication.