Identification of an ubinuclein 1 region required for stability and function of the human HIRA/UBN1/CABIN1/ASF1a histone H3.3 chaperone complex

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 with the 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, which 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.     

Human UBN1 Is an Ortholog of Yeast Hpc2p and Has an Essential Role in the HIRA/ASF1a Chromatin-Remodeling Pathway in Senescent Cells

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 histone chaperone complex HIR maintains nucleosome occupancy and counterbalances impaired histone deposition in CAF‐1 complex mutants

Chromatin organization is essential for coordinated gene expression, genome stability, and inheritance of epigenetic information. The main components involved in chromatin assembly are specific complexes such as Chromatin Assembly Factor 1 (CAF‐1) and Histone Regulator (HIR), which deposit histones in a DNA synthesis‐dependent or ‐independent manner, respectively. Here, we characterize the role of the plant orthologs Histone Regulator A (HIRA), Ubinuclein (UBN) and Calcineurin Binding protein 1 (CABIN1), which constitute the HIR complex. Arabidopsis loss‐of‐function mutants for the various subunits of the complex are viable, but hira mutants show reduced fertility. We show that loss of HIRA reduces extractable histone H3 protein levels and decreases nucleosome occupancy at both actively transcribed genes and heterochromatic regions. Concomitantly, HIRA contributes to maintenance of silencing of pericentromeric repeats and certain transposons. A genetic analysis based on crosses between mutants deficient in subunits of the CAF‐1 and HIR complexes showed that simultaneous loss of both the CAF‐1 and HIR histone H3 chaperone complexes severely affects plant survival, growth and reproductive development. Our results suggest that HIRA partially rescues impaired histone deposition in fas mutants to preserve nucleosome occupancy, implying plasticity in histone variant interaction and deposition.     

In vivo study of the nucleosome assembly functions of ASF1 histone chaperones in human cells

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.     

Yeast histone deposition protein Asf1p requires Hir proteins and PCNA for heterochromatic silencing

BACKGROUND: Position-dependent gene silencing in yeast involves many factors, including the four HIR genes and nucleosome assembly proteins Asf1p and chromatin assembly factor I (CAF-I, encoded by the CAC1-3 genes). Both cac Delta asfl Delta and cac Delta hir Delta double mutants display synergistic reductions in heterochromatic gene silencing. However, the relationship between the contributions of HIR genes and ASF1 to silencing has not previously been explored. RESULTS: Our biochemical and genetic studies of yeast Asf1p revealed links to Hir protein function. In vitro, an active histone deposition complex was formed from recombinant yeast Asf1p and histones H3 and H4 that lack a newly synthesized acetylation pattern. This Asf1p/H3/H4 complex generated micrococcal nuclease--resistant DNA in the absence of DNA replication and stimulated nucleosome assembly activity by recombinant yeast CAF-I during DNA synthesis. Also, Asf1p bound to the Hir1p and Hir2p proteins in vitro and in cell extracts. In vivo, the HIR1 and ASF1 genes contributed to silencing the heterochromatic HML locus via the same genetic pathway. Deletion of either HIR1 or ASF1 eliminated telomeric gene silencing in combination with pol30--8, encoding an altered form of the DNA polymerase processivity factor PCNA that prevents CAF-I from contributing to silencing. Conversely, other pol30 alleles prevented Asf1/Hir proteins from contributing to silencing. CONCLUSIONS: Yeast CAF-I and Asf1p cooperate to form nucleosomes in vitro. In vivo, Asf1p and Hir proteins physically interact and together promote heterochromatic gene silencing in a manner requiring PCNA. This Asf1/Hir silencing pathway functionally overlaps with CAF-I activity.     

The essential role of Drosophila HIRA for de novo assembly of paternal chromatin at fertilization

In many animal species, the sperm DNA is packaged with male germ line–specific chromosomal proteins, including protamines. At fertilization, these non-histone proteins are removed from the decondensing sperm nucleus and replaced with maternally provided histones to form the DNA replication competent male pronucleus. By studying a point mutant allele of the Drosophila Hira gene, we previously showed that HIRA, a conserved replication-independent chromatin assembly factor, was essential for the assembly of paternal chromatin at fertilization. HIRA permits the specific assembly of nucleosomes containing the histone H3.3 variant on the decondensing male pronucleus. We report here the analysis of a new mutant allele of Drosophila Hira that was generated by homologous recombination. Surprisingly, phenotypic analysis of this loss of function allele revealed that the only essential function of HIRA is the assembly of paternal chromatin during male pronucleus formation. This HIRA-dependent assembly of H3.3 nucleosomes on paternal DNA does not require the histone chaperone ASF1. Moreover, analysis of this mutant established that protamines are correctly removed at fertilization in the absence of HIRA, thus demonstrating that protamine removal and histone deposition are two functionally distinct processes. Finally, we showed that H3.3 deposition is apparently not affected in Hira mutant embryos and adults, suggesting that different chromatin assembly machineries could deposit this histone variant.     

HP1-mediated formation of alternative lengthening of telomeres-associated PML bodies requires HIRA but not ASF1a

Approximately 10% of cancers use recombination-mediated Alternative Lengthening of Telomeres (ALT) instead of telomerase to prevent telomere shortening. A characteristic of cells that utilize ALT is the presence of ALT-associated PML nuclear bodies (APBs) containing (TTAGGG)n DNA, telomere binding proteins, DNA recombination proteins, and heterochromatin protein 1 (HP1). The function of APBs is unknown and it is possible that they are functionally heterogeneous. Most ALT cells lack functional p53, and restoration of the p53/p21 pathway in these cells results in growth arrest/senescence and a substantial increase in the number of large APBs that is dependent on two HP1 isoforms, HP1α and HP1γ. Here we investigated the mechanism of HP1-mediated APB formation, and found that histone chaperones, HIRA and ASF1a, are present in APBs following activation of the p53/p21 pathway in ALT cells. HIRA and ASF1a were also found to colocalize inside PML bodies in normal fibroblasts approaching senescence, providing evidence for the existence of a senescence-associated ASF1a/HIRA complex inside PML bodies, consistent with a role for these proteins in induction of senescence in both normal and ALT cells. Moreover, knockdown of HIRA but not ASF1a significantly reduced p53-mediated induction of large APBs, with a concomitant reduction of large HP1 foci. We conclude that HIRA, in addition to its physical and functional association with ASF1a, plays a unique, ASF1a-independent role, which is required for the localization of HP1 to PML bodies and thus for APB formation.     

Replication-independent histone deposition by the HIR complex and Asf1

The orderly deposition of histones onto DNA is mediated by conserved assembly complexes, including chromatin assembly factor-1 (CAF-1) and the Hir proteins . CAF-1 and the Hir proteins operate in distinct but functionally overlapping histone deposition pathways in vivo . 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 . Asf1 binds to newly synthesized histones H3/H4 , and this complex stimulates histone deposition by CAF-1 . In yeast, Asf1 is required for the contribution of the Hir proteins to gene silencing . Here, we demonstrate that Hir1, Hir2, Hir3, and Hpc2 comprise the HIR complex, which copurifies with the 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.     

Structure of a human ASF1a-HIRA complex and insights into specificity of histone chaperone complex assembly

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 beta-hairpin that binds perpendicular to the strands of the beta-sandwich of ASF1a, via beta-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 uses 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.     

Definition of pRB- and p53-dependent and -independent steps in HIRA/ASF1a-mediated formation of senescence-associated heterochromatin foci

Cellular senescence is an irreversible proliferation arrest triggered by short chromosome telomeres, activated oncogenes, and cell stress and mediated by the pRB and p53 tumor suppressor pathways. One of the earliest steps in the senescence program is translocation of a histone chaperone, HIRA, into promyelocytic leukemia (PML) nuclear bodies. This relocalization precedes other markers of senescence, including the appearance of specialized domains of facultative heterochromatin called senescence-associated heterochromatin foci (SAHF) and cell cycle exit. SAHF represses expression of proliferation-promoting genes, thereby driving exit from the cell cycle. HIRA bound to another histone chaperone, ASF1a, drives formation of SAHF. Here, we show that HIRA's translocation to PML bodies occurs in response to all senescence triggers tested. Dominant negative HIRA mutants that block HIRA's localization to PML bodies prevent formation of SAHF, as does a PML-RARalpha fusion protein which disrupts PML bodies, directly supporting the idea that localization of HIRA to PML bodies is required for formation of SAHF. Significantly, translocation of HIRA to PML bodies occurs in the absence of functional pRB and p53 tumor suppressor pathways. However, our evidence indicates that downstream of HIRA's localization to PML bodies, the HIRA/ASF1a pathway cooperates with pRB and p53 to make SAHF, with the HIRA/ASF1a and pRB pathways acting in parallel. We present evidence that convergence of the HIRA/ASF1a and pRB pathways occurs through a DNAJ-domain protein, DNAJA2.     

Hira基因与发育：从酵母到人类

Hir/Hira基因产物HIR/HIRA为组蛋白的伴侣蛋白,最先作为组蛋白基因表达的一种负调节因子从酵母中被鉴定出来。现已证实,HIRA包含一组保守的蛋白家族,广泛存在于低等真核生物、无脊椎动物和脊椎动物等多种生物体当中,为生命发育所必需。Hir/Hira基因功能突变对酵母以及高等真核生物的发育都有非常严重的影响。结合研究组的工作,综述了组蛋白调节基因Hir/Hira在不同生物体发育过程中的作用,以及该领域的研究方向之一 ¾ HIRA作用机理的最新进展。     

Contribution of CAF-I to anaphase-promoting-complex-mediated mitotic chromatin assembly in Saccharomyces cerevisiae

The anaphase-promoting complex (APC) is required for mitotic progression and genomic stability. Recently, we demonstrated that the APC is also required for mitotic chromatin assembly and longevity. Here, we investigated the role the APC plays in chromatin assembly. We show that apc5(CA) mutations genetically interact with the CAF-I genes as well as ASF1, HIR1, and HIR2. When present in multiple copies, the individual CAF-I genes, CAC1, CAC2, and MSI1, suppress apc5(CA) phenotypes in a CAF-1- and Asf1p-independent manner. CAF-I and the APC functionally overlap, as cac1delta cac2delta msi1delta (caf1delta) cells expressing apc5(CA) exhibit a phenotype more severe than that of apc5(CA) or caf1delta. The Ts- phenotypes observed in apc5(CA) and apc5(CA) caf mutants may be rooted in compromised histone metabolism, as coexpression of histones H3 and H4 suppressed the Ts- defects. Synthetic genetic interactions were also observed in apc5(CA) asf1delta cells. Furthermore, increased expression of genes encoding Asf1p, Hir1p, and Hir2p suppressed the apc5(CA) Ts- defect in a CAF-I-dependent manner. Together, these results suggest the existence of a complex molecular mechanism controlling APC-dependent chromatin assembly. Our data suggest the APC functions with the individual CAF-I subunits, Asf1p, and the Hir1p and Hir2p proteins. However, Asf1p and an intact CAF-I complex are dispensable for CAF-I subunit suppression, whereas CAF-I is necessary for ASF1, HIR1, and HIR2 suppression of apc5(CA) phenotypes. We discuss the implications of our observations.