Cellular senescence is an irreversible proliferation arrest, tumor suppression process and likely contributor to tissue aging. Senescence is often characterized by domains of facultative heterochromatin, called senescence-associated heterochromatin foci (SAHF), which repress expression of proliferation-promoting genes. Given its likely contribution to tumor suppression and tissue aging, it is essential to identify all components of the SAHF assembly pathway. Formation of SAHF in human cells is driven by a complex of histone chaperones, namely, HIRA and ASF1a. In yeast, the complex orthologous to HIRA/ASF1a contains two additional proteins, Hpc2p and Hir3p. Using a sophisticated approach to search for remote orthologs conserved in multiple species through evolution, we identified the HIRA-associated proteins, UBN1 and UBN2, as candidate human orthologs of Hpc2p. We show that the Hpc2-related domain of UBN1, UBN2, and Hpc2p is an evolutionarily conserved HIRA/Hir-binding domain, which directly interacts with the N-terminal WD repeats of HIRA/Hir. UBN1 binds to proliferation-promoting genes that are repressed by SAHF and associates with histone methyltransferase activity that methylates lysine 9 of histone H3, a site that is methylated in SAHF. UBN1 is indispensable for formation of SAHF. We conclude that UBN1 is an ortholog of yeast Hpc2p and a novel regulator of senescence.
The mammalian HIRA/UBN1/CABIN1/ASF1a (HUCA) histone chaperone complex deposits the histone H3 variant H3.3 into chromatin, and is linked to gene activation, repression and chromatin assembly in diverse cell contexts. We recently reported that a short N-terminal fragment of UBN1 containing amino acids 1–175 is necessary and sufficient for interaction with the WD repeats of HIRA, and attributed this interaction to a region from residues 120–175 that is highly conserved in a yeast ortholog Hpc2 and so termed the HRD for Hpc2-Related Domain. In this report, through a more comprehensive and refined biochemical and mutational analysis, we identify a smaller and more moderately conserved region within residues 41–77 of UBN1, that we term the NHRD, that is essential for interaction with the HIRA WD repeats; we further demonstrate that the HRD is dispensable for this interaction. We employ analytical ultracentrifugation studies to demonstrate that the NHRD of UBN1 and the WD repeats of HIRA form a tight 1:1 complex with a dissociation constant in the nanomolar range. Mutagenesis experiments identify several key residues in the NHRD that are required for interaction with the HIRA WD repeat domain, stability of the HUCA complex in vitro and in vivo and changes in chromatin organization in primary human cells. Together, these studies implicate the NHRD domain of UBN1 as being an essential region for HIRA interaction and chromatin organization by the HUCA complex.
HIRA; UBN1; Histone Deposition; Chromatin Regulation
Using sensitive protein sequence profile analyses we investigated the evolution of some histone chaperones and showed that Hir3p and Hpc2p have a much wider phyletic pattern than was previously known.
While histone chaperones have been intensely studied, the roles of components of the Hir–Asf1 histone chaperone complex such as Hir3p and Hpc2p are poorly understood. Using sensitive protein sequence profile analyses we investigated the evolution of these proteins and showed that Hir3p and Hpc2p have a much wider phyletic pattern than was previously known. We established the animal histone-deacetylase-complex-interacting proteins, CAIN/CABIN, to be orthologs of Hir3p. They contain a conserved core of around 30 TPR-like bi-helical repeats that are likely to form a super-helical scaffold. We identified a conserved domain, the HUN domain, in all Hpc2p homologs, including animal ubinuclein/yemanuclein and the recently discovered vertebrate cell-cycle regulator FLJ25778. The HUN domain has a characteristic pattern of conserved acidic residues based on which we predict that it is a previously unrecognized histone-tail-binding chaperone. By analyzing various high-throughput data sets, such as RNAi knock-downs, genetic and protein interaction maps and cell-cycle-specific gene expression data, we present evidence that Hpc2p homologs might be deployed in specific processes of chromatin dynamics relating to cell-cycle progression in vertebrates and schizogony in Plasmodium. Beyond the conserved HUN domain these proteins show extensive divergence patterns in different eukaryotic lineages. Hence, we propose that Hpc2p homologs are probably involved in recruitment of the ancient conserved histone-loading Hir–Asf1 complex to different lineage-specific chromatin reorganization processes.
The human HIRA gene has been named after Hir1p and Hir2p, two corepressors which together appear to act on chromatin structure to control gene transcription in Saccharomyces cerevisiae. HIRA homologs are expressed in a regulated fashion during mouse and chicken embryogenesis, and the human gene is a major candidate for the DiGeorge syndrome and related developmental disorders caused by a reduction to single dose of a fragment of chromosome 22q. Western blot analysis and double-immunofluorescence experiments using a specific antiserum revealed a primary nuclear localization of HIRA. Similar to Hir1p, HIRA contains seven amino-terminal WD repeats and probably functions as part of a multiprotein complex. HIRA and core histone H2B were found to physically interact in a yeast double-hybrid protein interaction trap, in GST pull-down assays, and in coimmunoprecipitation experiments performed from cellular extracts. In vitro, HIRA also interacted with core histone H4. H2B- and H4-binding domains were overlapping but distinguishable in the carboxy-terminal region of HIRA, and the region for HIRA interaction was mapped to the amino-terminal tail of H2B and the second α helix of H4. HIRIP3 (HIRA-interacting protein 3) is a novel gene product that was identified from its HIRA-binding properties in the yeast protein interaction trap. In vitro, HIRIP3 directly interacted with HIRA but also with core histones H2B and H3, suggesting that a HIRA-HIRIP3-containing complex could function in some aspects of chromatin and histone metabolism. Insufficient production of HIRA, which we report elsewhere interacts with homeodomain-containing DNA-binding factors during mammalian embryogenesis, could perturb the stoichiometric assembly of multimolecular complexes required for normal embryonic development.
The HIR/HPC (histone regulation/histone periodic control) negative regulators play important roles in the transcription of six of the eight core histone genes during the Saccharomyces cerevisiae cell cycle. The phenotypes of hir1 and hir2 mutants suggested that the wild-type HIR1 and HIR2 genes encode transcriptional repressors that function in the absence of direct DNA binding. When Hir1p and Hir2p were artificially tethered to yeast promoters, each protein repressed transcription, suggesting that they represent a new class of transcriptional corepressors. The two proteins might function as a complex in vivo: Hir2p required both Hir1p and another Hir protein, Hir3p, to repress transcription when it was tethered to an HTA1-lacZ reporter gene, and Hir1p and Hir2p could be coimmunoprecipitated from yeast cell extracts. Tethered Hir1p also directed the periodic transcription of the HTA1 gene and repressed HTA1 transcription in response to two cell cycle regulatory signals. Thus, it represents the first example of a transcriptional corepressor with a direct role in cell cycle-regulated transcription.
In Saccharomyces cerevisiae, three out of the four histone gene pairs (HTA1-HTB1, HHT1-HHF1, and HHT2-HHF2) are regulated by the HIR co-repressor complex. The histone chaperone Rtt106 has recently been shown to be present at these histone gene loci throughout the cell cycle in a HIR- and Asf1-dependent manner and involved in their transcriptional repression. The SWI/SNF and RSC chromatin remodeling complexes are both recruited to the HIR-dependent histone genes; SWI/SNF is required for their activation in S phase, whereas RSC is implicated in their repression outside of S phase. Even though their presence at the histone genes is dependent on the HIR complex, their specific recruitment has not been well characterized. In this study we focused on characterizing the role played by the histone chaperone Rtt106 in the cell cycle-dependent recruitment of SWI/SNF and RSC complexes to the histone genes.
Using GST pull-down and co-immunoprecipitation assays, we showed that Rtt106 physically interacts with both the SWI/SNF and RSC complexes in vitro and in vivo. We then investigated the function of this interaction with respect to the recruitment of these complexes to HIR-dependent histone genes. Using chromatin immunoprecipitation assays (ChIP), we found that Rtt106 is important for the recruitment of both SWI/SNF and RSC complexes to the HIR-dependent histone genes. Furthermore, using synchronized cell cultures, we showed by ChIP assays that the Rtt106-dependent SWI/SNF recruitment to these histone gene loci is cell cycle regulated and restricted to late G1 phase just before the peak of histone gene expression in S phase.
Overall, these data strongly suggest that the interaction between the histone chaperone Rtt106 and both the SWI/SNF and RSC chromatin remodeling complexes is important for the cell cycle regulated recruitment of these two complexes to the HIR-dependent histone genes.
The HIR complex, which is comprised of the four proteins Hir1, Hir2, Hir3 and Hpc2, was first characterized as a repressor of three of the four histone gene loci in Saccharomyces cerevisiae. Using a bioinformatical approach, previous studies have identified a region of Hpc2 that is conserved in Schizosaccharomyces pombe and humans. Using a similar approach, we identified two additional domains, CDI and CDII, of the Hpc2 protein that are conserved amongst yeast species related to S. cerevisiae. We showed that the N terminal CDI domain (spanning amino acids 63–79) is dispensable for HIR complex assembly, but plays an essential role in the repression of the histone genes by recruiting the HIR complex to the HIR-dependent histone gene loci. The second conserved domain, CDII (spanning amino acids 452–480), is required for the stability of the Hpc2 protein itself as well as for the assembly of the HIR complex. In addition, we report a novel separation-of-function mutation within CDI of Hpc2, which causes derepression of the histone genes but does not confer other reported hir/hpc-phenotypes (such as Spt phenotypes, heterochromatin silencing defects and repression of cryptic promoters). This is the first direct demonstration that a separation-of-function mutation exists within the HIR complex.
histone gene; HIR complex; HPC2; transcription; chromatin; yeast
The orderly deposition of histones onto DNA is mediated by conserved assembly complexes, including Chromatin Assembly Factor-1 (CAF-1) and the Hir proteins [1–4]. CAF-1 and the Hir proteins operate in distinct but functionally overlapping histone deposition pathways in vivo [5, 6]. The Hir proteins and CAF-1 share a common partner, the highly conserved histone H3/H4-binding protein Asf1, which binds the middle subunit of CAF-1 as well as to Hir proteins [7–11]. Asf1 binds to newly synthesized histones H3/H4  and this complex stimulates histone deposition by CAF-1 [7, 12, 13]. In yeast, Asf1 is required for the contribution of the Hir proteins to gene silencing [7, 14]. Here, we demonstrate that Hir1, Hir2, Hir3 and Hpc2 comprise the HIR complex, which co-purifies with histone deposition protein Asf1. Together, the HIR complex and Asf1 deposit histones onto DNA in a replication-independent manner. Histone deposition by the HIR complex and Asf1 is impaired by a mutation in Asf1 that inhibits HIR binding. These data indicate that the HIR complex and Asf1 proteins function together as a conserved eukaryotic pathway for histone replacement throughout the cell cycle.
chromatin assembly; HIR; Asf1; histones
HIRA-like (Hir) proteins are evolutionarily conserved and are implicated in the assembly of repressive chromatin. In Saccharomyces cerevisiae, Hir proteins contribute to the function of centromeres. However, S. cerevisiae has point centromeres that are structurally different from the complex centromeres of metazoans. In contrast, Schizosaccharomyces pombe has complex centromeres whose domain structure is conserved with that of human centromeres. Therefore, we examined the functions of the fission yeast Hir proteins Slm9 and the previously uncharacterised protein Hip1. Deletion of hip1+ resulted in phenotypes that were similar to those described previously for slm9Δ cells: a cell cycle delay, synthetic lethality with cdc25-22, and poor recovery from nitrogen starvation. However, while it has previously been shown that Slm9 is not required for the periodic expression of histone H2A, we found that loss of Hip1 led to derepression of core histone genes expression outside of S phase. Importantly, we found that deletion of either hip1+ or slm9+ resulted in increased rates of chromosome loss, increased sensitivity to spindle damage, and reduced transcriptional silencing in the outer centromeric repeats. Thus, S. pombe Hir proteins contribute to pericentromeric heterochromatin, and our data thus suggest that Hir proteins may be required for the function of metazoan centromeres.
Human HIRA, ASF1a, ASF1b and CAF-1 are evolutionally conserved histone chaperones that form multiple functionally distinct chromatin assembly complexes, with roles linked to diverse nuclear process, such as DNA replication and formation of heterochromatin in senescent cells. We report the crystal structure of an ASF1a/HIRA heterodimer and a biochemical dissection of ASF1a's mutually exclusive interactions with HIRA and the p60 subunit of CAF-1. The HIRA B-domain forms an antiparallel β-hairpin that binds perpendicular to the strands of the β-sandwich of ASF1a, via β-sheet, salt-bridge and van der Waals contacts. The N- and C-terminal regions of ASF1a and ASF1b determine the different affinities of these two proteins for HIRA, by contacting regions outside the HIRA B-domain. CAF-1 p60 also employs B-domain-like motifs for binding to ASF1a, thereby competing with HIRA. Together, these studies begin to define the molecular determinants of assembly of functionally diverse macromolecular histone chaperone complexes.
Histone Deposition; Chromatin Regulation; Histone Chaperones; ASF1; HIRA; CAF-1
Chromatin assembly factor I (CAF-I) is a three-subunit histone-binding complex conserved from the yeast Saccharomyces cerevisiae to humans. Yeast cells lacking CAF-I (cacΔ mutants) have defects in heterochromatic gene silencing. In this study, we showed that deletion of HIR genes, which regulate histone gene expression, synergistically reduced gene silencing at telomeres and at the HM loci in cacΔ mutants, although hirΔ mutants had no silencing defects when CAF-I was intact. Therefore, Hir proteins are required for an alternative silencing pathway that becomes important in the absence of CAF-I. Because Hir proteins regulate expression of histone genes, we tested the effects of histone gene deletion and overexpression on telomeric silencing and found that alterations in histone H3 and H4 levels or in core histone stoichiometry reduced silencing in cacΔ mutants but not in wild-type cells. We therefore propose that Hir proteins contribute to silencing indirectly via regulation of histone synthesis. However, deletion of combinations of CAC and HIR genes also affected the growth rate and in some cases caused partial temperature sensitivity, suggesting that global aspects of chromosome function may be affected by the loss of members of both gene families.
Substrates of cyclin-cdk2 kinases contain two distinct primary sequence motifs: a cyclin-binding RXL motif and one or more phosphoacceptor sites (consensus S/TPXK/R or S/TP). To identify novel cyclin-cdk2 substrates, we searched the database for proteins containing both of these motifs. One such protein is human HIRA, the homologue of two cell cycle-regulated repressors of histone gene expression in Saccharomyces cerevisiae, Hir1p and Hir2p. Here we demonstrate that human HIRA is an in vivo substrate of a cyclin-cdk2 kinase. First, HIRA bound to and was phosphorylated by cyclin A- and E-cdk2 in vitro in an RXL-dependent manner. Second, HIRA was phosphorylated in vivo on two consensus cyclin-cdk2 phosphoacceptor sites and at least one of these, threonine 555, was phosphorylated by cyclin A-cdk2 in vitro. Third, phosphorylation of HIRA in vivo was blocked by cyclin-cdk2 inhibitor p21cip1. Fourth, HIRA became phosphorylated on threonine 555 in S phase when cyclin-cdk2 kinases are active. Fifth, HIRA was localized preferentially to the nucleus, where active cyclin A- and E-cdk2 are located. Finally, ectopic expression of HIRA in cells caused arrest in S phase and this is consistent with the notion that it is a cyclin-cdk2 substrate that has a role in control of the cell cycle.
In Saccharomyces cerevisiae, the histone chaperone Rtt106 binds newly synthesized histone proteins and mediates their delivery into chromatin during transcription, replication, and silencing. Rtt106 is also recruited to histone gene regulatory regions by the HIR histone chaperone complex to ensure S-phase-specific expression. Here we showed that this Rtt106:HIR complex included Asf1 and histone proteins. Mutations in Rtt106 that reduced histone binding reduced Rtt106 enrichment at histone genes, leading to their increased transcription. Deletion of the chromatin boundary element Yta7 led to increased Rtt106:H3 binding, increased Rtt106 enrichment at histone gene regulatory regions, and decreased histone gene transcription at the HTA1-HTB1 locus. These results suggested a unique regulatory mechanism in which Rtt106 sensed the level of histone proteins to maintain the proper level of histone gene transcription. The role of these histone chaperones and Yta7 differed markedly among the histone gene loci, including the two H3-H4 histone gene pairs. Defects in silencing in rtt106 mutants could be partially accounted for by Rtt106-mediated changes in histone gene repression. These studies suggested that feedback mediated by histone chaperone complexes plays a pivotal role in regulating histone gene transcription.
DNA and histone synthesis are both triggered at the beginning of S phase by cyclin/cdk2 activity. Previous studies showed that inhibition of DNA synthesis with hydroxyurea or cytosine arabinoside (AraC) triggers a concerted repression of histone synthesis, indicating that sustained histone synthesis depends on continued DNA synthesis. Here we show that ectopic expression of HIRA, the likely human ortholog of two cell cycle-regulated repressors of histone gene transcription in yeast (Hir1p and Hir2p), represses transcription of histones and that this, in turn, triggers a concerted block of DNA synthesis. Thus, in mammalian cells sustained DNA synthesis and histone synthesis are mutually dependent on each other during S phase. Although cyclin/cdk2 activity drives activation of both DNA and histone synthesis at the G1/S transition of cycling cells, concerted repression of DNA or histone synthesis in response to inhibition of either one of these is not accompanied by prolonged inhibition of cyclin A/cdk2 or E/cdk2 activity. Therefore, during S phase coupling of DNA and histone synthesis occurs, at least in part, through a mechanism that is independent of cyclin/cdk2 activity. Coupling of DNA and histone synthesis in S phase presumably contributes to the prompt and orderly assembly of newly replicated DNA into chromatin.
Budding yeast centromeres are comprised of ~125-bp DNA sequences that direct formation of the kinetochore, a specialized chromatin structure that mediates spindle attachment to chromosomes. We report here a novel role for the histone deposition complex chromatin assembly factor I (CAF-I) in building centromeric chromatin. The contribution of CAF-I to kinetochore function overlaps that of the Hir proteins, which have also been implicated in nucleosome formation and heterochromatic gene silencing. cacΔ hirΔ double mutant cells lacking both CAF-I and Hir proteins are delayed in anaphase entry in a spindle assembly checkpoint-dependent manner. Further, cacΔ and hirΔ deletions together cause increased rates of chromosome missegregation, genetic synergies with mutations in kinetochore protein genes, and alterations in centromeric chromatin structure. Finally, CAF-I subunits and Hir1 are enriched at centromeres, indicating that these proteins make a direct contribution to centromeric chromatin structures.
Centromere; kinetochore; histone; yeast; checkpoint; chromatin
The eukaryotic genome is packaged together with histone proteins into chromatin following DNA replication. Recent studies have shown that histones can also be assembled into chromatin independently of DNA replication and that this dynamic exchange of histones may be biased toward sites undergoing transcription. Here we show that epitope-tagged histone H4 can be incorporated into nucleosomes throughout the budding yeast (Saccharomyces cerevisiae) genome regardless of the phase of the cell cycle, the transcriptional status, or silencing of the region. Direct comparisons reveal that the amount of histone incorporation that occurs in G1-arrested cells is similar to that occurring in cells undergoing DNA replication. Additionally, we show that this histone incorporation is not dependent on the histone H3/H4 chaperones CAF-1, Asf1, and Hir1 individually. This study demonstrates that DNA replication and transcription are not necessary prerequisites for histone exchange in budding yeast, indicating that chromatin is more dynamic than previously thought.
Histone chaperones have been implicated in nucleosome assembly and disassembly as well as histone modification. ASF1 is a highly conserved histone H3/H4 chaperone that synergizes in vitro with two other histone chaperones, chromatin assembly factor 1 (CAF-1) and histone repression A factor (HIRA), in DNA synthesis-coupled and DNA synthesis-independent nucleosome assembly. Here, we identify mutants of histones H3.1 and H3.3 that are unable to interact with human ASF1A and ASF1B isoforms but that are still competent to bind CAF-1 and HIRA, respectively. We show that these mutant histones are inefficiently deposited into chromatin in vivo. Furthermore, we found that both ASF1A and ASF1B participate in the DNA synthesis-independent deposition of H3.3 in HeLa cells, thus highlighting an unexpected role for ASF1B in this pathway. This pathway does not require interaction of ASF1 with HIRA. We provide the first direct determination that ASF1A and ASF1B play a role in the efficiency of nucleosome assembly in vivo in human cells.
Chromatin assembly factor I (CAF-I) is a conserved histone H3/H4 deposition complex. Saccharomyces cerevisiae mutants lacking CAF-I subunit genes (CAC1 to CAC3) display reduced heterochromatic gene silencing. In a screen for silencing-impaired cac1 alleles, we isolated a mutation that reduced binding to the Cac3p subunit and another that impaired binding to the DNA replication protein PCNA. Surprisingly, mutations in Cac1p that abolished PCNA binding resulted in very minor telomeric silencing defects but caused silencing to be largely dependent on Hir proteins and Asf1p, which together comprise an alternative silencing pathway. Consistent with these phenotypes, mutant CAF-I complexes defective for PCNA binding displayed reduced nucleosome assembly activity in vitro but were stimulated by Asf1p-histone complexes. Furthermore, these mutant CAF-I complexes displayed a reduced preference for depositing histones onto newly replicated DNA. We also observed a weak interaction between Asf1p and Cac2p in vitro, and we hypothesize that this interaction underlies the functional synergy between these histone deposition proteins.
Chromatin disassembly and reassembly, mediated by histone chaperones such as anti-silencing function 1 (Asf1), are likely to accompany all nuclear processes that occur on the DNA template. In order to gain insight into the functional conservation of Asf1 across eukaryotes, we have replaced the budding yeast Asf1 protein with Drosophila Asf1 (dAsf1) or either of the two human Asf1 (hAsf1a and hAsf1b) counterparts. We found that hAsf1b is best able to rescue the growth defect of Saccharomyces cerevisiae lacking Asf1. Moreover, dAsf1 and hAsf1b but not hAsf1a can replace the role of yeast Asf1 in protecting against replicational stress and activating the PHO5 gene, while only hAsf1a can replace the role of Asf1 in protecting against double-stranded-DNA-damaging agents. Furthermore, it appears that the interaction between Asf1 and the DNA damage checkpoint protein Rad53 is not required for Asf1's role in maintaining genomic integrity. In addition to indicating the functional conservation of the Asf1 proteins across species, these studies suggest distinct roles for the two human Asf1 proteins.
HIRA (or Hir) proteins are conserved histone chaperones that function in multi-subunit complexes to mediate replication-independent nucleosome assembly. We have previously demonstrated that the Schizosaccharomyces pombe HIRA proteins, Hip1 and Slm9, form a complex with a TPR repeat protein called Hip3. Here we have identified a new subunit of this complex.
To identify proteins that interact with the HIRA complex, rapid affinity purifications of Slm9 were performed. Multiple components of the chaperonin containing TCP-1 complex (CCT) and the 19S subunit of the proteasome reproducibly co-purified with Slm9, suggesting that HIRA interacts with these complexes. Slm9 was also found to interact with a previously uncharacterised protein (SPBC947.08c), that we called Hip4. Hip4 contains a HRD domain which is a characteristic of the budding yeast and human HIRA/Hir-binding proteins, Hpc2 and UBN1. Co-precipitation experiments revealed that Hip4 is stably associated with all of the other components of the HIRA complex and deletion of hip4+ resulted in the characteristic phenotypes of cells lacking HIRA function, such as temperature sensitivity, an elongated cell morphology and hypersensitivity to the spindle poison, thiabendazole. Moreover, loss of Hip4 function alleviated the heterochromatic silencing of reporter genes located in the mating type locus and centromeres and was associated with increased levels of non-coding transcripts derived from centromeric repeat sequences. Hip4 was also found to be required for the distinct form of silencing that controls the expression of Tf2 LTR retrotransposons.
Overall, these results indicate that Hip4 is an integral component of the HIRA complex that is required for transcriptional silencing at multiple loci.
Cellular senescence is an irreversible proliferation arrest of primary cells and an important tumor suppression process. Senescence is often characterized by domains of facultative heterochromatin, called Senescence-Associated Heterochromatin Foci (SAHF), which repress expression of proliferation-promoting genes. Formation of SAHF is driven by a complex of histone chaperones, HIRA and ASF1a, and depends upon prior localization of HIRA to PML nuclear bodies. However, how the SAHF assembly pathway is activated in senescent cells is not known. Here we show that expression of the canonical Wnt2 ligand and downstream canonical Wnt-signals are repressed in senescent human cells. Repression of Wnt2 occurs early in senescence and independent of the pRB and p53 tumor suppressor proteins, and drives relocalization of HIRA to PML bodies, formation of SAHF and senescence, likely through GSK3β-mediated phosphorylation of HIRA. These results have major implications for our understanding of both Wnt-signaling and senescence in tissue homeostasis and cancer progression.
Heterochromatin impacts various nuclear processes by providing a recruiting platform for diverse chromosomal proteins. In fission yeast, HP1 proteins Chp2 and Swi6, which bind to methylated histone H3 lysine 9, associate with SHREC (Snf2/HDAC repressor complex) and Clr6 histone deacetylases (HDACs) involved in heterochromatic silencing. However, heterochromatic silencing machinery is not fully defined. We describe a histone chaperone complex containing Asf1 and HIRA that spreads across silenced domains via its association with Swi6 to enforce transcriptional silencing. Asf1 function in concert with a Clr6 HDAC complex to silence heterochromatic repeats, and it suppresses antisense transcription by promoting histone deacetylation. Furthermore, we demonstrate that Asf1 and SHREC facilitate nucleosome occupancy at heterochromatic regions but TFIIIC transcription factor binding sites within boundary elements are refractory to these factors. These analyses uncover a role for Asf1 in global histone deacetylation and suggest that HP1-associated histone chaperone promote nucleosome occupancy to assemble repressive heterochromatin.
CAF-1 and HIR are highly conserved histone chaperone protein complexes that function in the assembly of nucleosomes onto chromatin. CAF-1 is characterized as having replication-coupled nucleosome activity, whereas the HIR complex can assemble nucleosomes independent of replication. Histone H3K56 acetylation, controlled by the acetyltransferase Rtt109 and deacetylase Hst3, also plays a significant role in nucleosome assembly. In this study, we generated a set of deletion mutants to genetically characterize pathway-specific and overlapping functions of CAF-1 and HIR in C. albicans. Their roles in epigenetic maintenance of cell type were examined by using the white-opaque switching system in C. albicans. We show that CAF-1 and HIR play conserved roles in UV radiation recovery, repression of histone gene expression, correct chromosome segregation, and stress responses. Unique to C. albicans, the cac2Δ/Δ mutant shows increased sensitivity to the Hst3 inhibitor nicotinamide, while the rtt109Δ/Δ cac2Δ/Δ and hir1Δ/Δ cac2Δ/Δ mutants are resistant to nicotinamide. CAF-1 plays a major role in maintaining cell types, as the cac2Δ/Δ mutant exhibited increased switching frequencies in both directions and switched at a high frequency to opaque in response to nicotinamide. Like the rtt109Δ/Δ mutant, the hir1Δ/Δ cac2Δ/Δ double mutant is defective in maintaining the opaque cell fate and blocks nicotinamide-induced opaque formation, and the defects are suppressed by ectopic expression of the master white-opaque regulator Wor1. Our data suggest an overlapping function of CAF-1 and HIR in epigenetic regulation of cell fate determination in an H3K56 acetylation-associated manner.
The mammalian genome encodes multiple variants of histone H3 including H3.1/H3.2 and H3.3. In contrast to H3.1/H3.2, H3.3 is enriched in the actively transcribed euchromatin and the telomeric heterochromatins. However, the mechanism for H3.3 to incorporate into the different domains of chromatin is not known. Here, taking the advantage of well-defined transcription analysis system of yeast, we attempted to understand the molecular mechanism of selective deposition of human H3.3 into actively transcribed genes. We show that there are systemic H3 substrate-selection mechanisms operating even in yeasts, which encode a single type of H3. Yeast HIR complex mediated H3-specific recognition specificity for deposition of H3.3 in the transcribed genes. A critical component of this process was the H3 A-IG code composed of amino acids 87, 89 and 90. The preference toward H3.3 was completely lost when HIR subunits were absent and partially suppressed by human HIRA. Asf1 allows the influx of H3, regardless of H3 type. We propose that H3.3 is introduced into the active euchromatin by targeting the recycling pathway that is mediated by HIRA (or HIR), and this H3-selection mechanism is highly conserved through the evolution. These results also uncover an unexpected role of RI chaperones in evolution of variant H3s.
The histone genes are an important group of cell cycle regulated genes whose transcription is activated during the G1/S transition and repressed in early G1, late S, and G2/M. The HIR complex, comprised of Hir1, Hir2, Hir3 and Hpc2, regulates three of the four histone gene loci. While relief of repression at the G1/S boundary involves the HIR complex, as well as other cofactors, the mechanism by which this derepression occurs remains unknown. To better understand how transcriptional repression contributes to periodic expression in the cell cycle, we sought to identify the cell cycle signals required to alleviate HIR-mediated repression of the histone genes. By measuring histone gene transcription in strains with various combinations of clb mutations, we found that the mitotic Clb1/Clb2 cyclins are required to alleviate Hir-mediated repression during the G1/S transition and that Clb2 physically interacts with the HIR complex. While the HIR complex regulates histone genes transcription in combination with two other histone H3/H4 chaperones, Asf1 and Rtt106, our data demonstrate that the mitotic Clb cyclins are necessary to specifically alleviate the repressive action of the HIR complex itself in order to allow proper expression of the histone genes in late G1/early S phase.
Clb cyclins; HIR complex; Asf1; Rtt106; histone genes; cell cycle