Essential role of chromatin assembly factor-1-mediated rapid nucleosome assembly for DNA replication and cell division in vertebrate cells

Chromatin assembly factor-1 (CAF-1), a complex consisting of p150, p60, and p48 subunits, is highly conserved from yeast to humans and facilitates nucleosome assembly of newly replicated DNA in vitro. To investigate roles of CAF-1 in vertebrates, we generated two conditional DT40 mutants, respectively, devoid of CAF-1p150 and p60. Depletion of each of these CAF-1 subunits led to delayed S-phase progression concomitant with slow DNA synthesis, followed by accumulation in late S/G2 phase and aberrant mitosis associated with extra centrosomes, and then the final consequence was cell death. We demonstrated that CAF-1 is necessary for rapid nucleosome formation during DNA replication in vivo as well as in vitro. Loss of CAF-1 was not associated with the apparent induction of phosphorylations of S-checkpoint kinases Chk1 and Chk2. To elucidate the precise role of domain(s) in CAF-1p150, functional dissection analyses including rescue assays were preformed. Results showed that the binding abilities of CAF-1p150 with CAF-1p60 and DNA polymerase sliding clamp proliferating cell nuclear antigen (PCNA) but not with heterochromatin protein HP1-gamma are required for cell viability. These observations highlighted the essential role of CAF-1-dependent nucleosome assembly in DNA replication and cell proliferation through its interaction with PCNA.     

HP1alpha recruitment to DNA damage by p150CAF-1 promotes homologous recombination repair

Heterochromatin protein 1 (HP1), a major component of constitutive heterochromatin, is recruited to DNA damage sites. However, the mechanism involved in this recruitment and its functional importance during DNA repair remain major unresolved issues. Here, by characterizing HP1α dynamics at laser-induced damage sites in mammalian cells, we show that the de novo accumulation of HP1α occurs within both euchromatin and heterochromatin as a rapid and transient event after DNA damage. This recruitment is strictly dependent on p150CAF-1, the largest subunit of chromatin assembly factor 1 (CAF-1), and its ability to interact with HP1α. We find that HP1α depletion severely compromises the recruitment of the DNA damage response (DDR) proteins 53BP1 and RAD51. Moreover, HP1α depletion leads to defects in homologous recombination-mediated repair and reduces cell survival after DNA damage. Collectively, our data reveal that HP1α recruitment at early stages of the DDR involves p150CAF-1 and is critical for proper DNA damage signaling and repair.     

An analysis of CAF-1-interacting proteins reveals dynamic and direct interactions with the KU complex and 14-3-3 proteins

CAF-1 is essential in human cells for the de novo deposition of histones H3 and H4 at the DNA replication fork. Depletion of CAF-1 from various cell lines causes replication fork arrest, activation of the intra-S phase checkpoint, and global defects in chromatin structure. CAF-1 is also involved in coordinating inheritance of states of gene expression and in chromatin assembly following DNA repair. In this study, we generated cell lines expressing RNAi-resistant versions of CAF-1 and showed that the N-terminal 296 amino acids are dispensable for essential CAF-1 function in vivo. N-terminally truncated CAF-1 p150 was deficient in proliferating cell nuclear antigen (PCNA) binding, reinforcing the existence of two PCNA binding sites in human CAF-1, but the defect in PCNA binding had no effect on the recruitment of CAF-1 to chromatin after DNA damage or to resistance to DNA-damaging agents. Tandem affinity purification of CAF-1-interacting proteins under mild conditions revealed that CAF-1 was directly associated with the KU70/80 complex, part of the DNA-dependent protein kinase, and the phosphoserine/threonine-binding protein 14-3-3 ζ. CAF-1 was a substrate for DNA-dependent protein kinase, and the 14-3-3 interaction in vitro is dependent on DNA-dependent protein kinase phosphorylation. These results highlight that CAF-1 has prominent interactions with the DNA repair machinery but that the N terminus is dispensable for the role of CAF-1 in DNA replication- and repair-coupled chromatin assembly.     

A CAF-1 dependent pool of HP1 during heterochromatin duplication

To investigate how the complex organization of heterochromatin is reproduced at each replication cycle, we examined the fate of HP1-rich pericentric domains in mouse cells. We find that replication occurs mainly at the surface of these domains where both PCNA and chromatin assembly factor 1 (CAF-1) are located. Pulse-chase experiments combined with high-resolution analysis and 3D modeling show that within 90 min newly replicated DNA become internalized inside the domain. Remarkably, during this time period, a specific subset of HP1 molecules (alpha and gamma) coinciding with CAF-1 and replicative sites is resistant to RNase treatment. Furthermore, these replication-associated HP1 molecules are detected in Suv39 knockout cells, which otherwise lack stable HP1 staining at pericentric heterochromatin. This replicative pool of HP1 molecules disappears completely following p150CAF-1 siRNA treatment. We conclude that during replication, the interaction of HP1 with p150CAF-1 is essential to promote delivery of HP1 molecules to heterochromatic sites, where they are subsequently retained by further interactions with methylated H3-K9 and RNA.     

Heterochromatin dynamics in mouse cells: interaction between chromatin assembly factor 1 and HP1 proteins.

Mechanisms contributing to the maintenance of heterochromatin in proliferating cells are poorly understood. We demonstrate that chromatin assembly factor 1 (CAF-1) binds to mouse HP1 proteins via an N-terminal domain of its p150 subunit, a domain dispensable for nucleosome assembly during DNA replication. Mutations in p150 prevent association with HP1 in heterochromatin in cells that are not in S phase and the formation of CAF-1-HP1 complexes in nascent chromatin during DNA replication in vitro. We suggest that CAF-1 p150 has a heterochromatin-specific function distinct from its nucleosome assembly function during S phase. Just before mitosis, CAF-1 p150 and some HP1 progressively dissociate from heterochromatin concomitant with histone H3 phosphorylation. The HP1 proteins reassociate with chromatin at the end of mitosis, as histone H3 is dephosphorylated.     

The chromatin assembly factor subunit FASCIATA1 is involved in homologous recombination in plants

DNA replication in cycling eukaryotic cells necessitates the reestablishment of chromatin after nucleosome redistribution from the parental to the two daughter DNA strands. Chromatin assembly factor 1 (CAF-1), a heterotrimeric complex consisting of three subunits (p150/p60/p48), is one of the replication-coupled assembly factors involved in the reconstitution of S-phase chromatin. CAF-1 is required in vitro for nucleosome assembly onto newly replicated chromatin in human cells and Arabidopsis thaliana, and defects in yeast (Saccharomyces cerevisiae) affect DNA damage repair processes, predominantly those involved in genome stability. However, in vivo chromatin defects of caf-1 mutants in higher eukaryotes are poorly characterized. Here, we show that fasciata1-4 (fas1-4), a new allele of the Arabidopsis fas1 mutant defective in the p150 subunit of CAF-1, has a severe developmental phenotype, reduced heterochromatin content, and a more open conformation of euchromatin. Most importantly, homologous recombination (HR), a process involved in maintaining genome stability, is increased dramatically in fas1-4, as indicated by a 96-fold stimulation of intrachromosomal HR. Together with the open conformation of chromatin and the nearly normal expression levels of HR genes in the mutant, this result suggests that chromatin is a major factor restricting HR in plants.     

Alanine scanning of Arp1 delineates a putative binding site for Jnm1/dynamitin and Nip100/p150Glued

Arp1p is the only actin-related protein (ARP) known to form actin-like filaments. Unlike actin, Arp1p functions with microtubules, as part of the dynein regulator, dynactin. Arp1p's dissimilar functions imply interactions with a distinct set of proteins. To distinguish surface features relating to Arp1p's core functions and to identify the footprint of protein interactions essential for dynactin function, we performed the first complete charge-cluster-to-alanine scanning mutagenesis of an ARP and compared the results with a similar study of actin. The Arp1p mutations revealed three nonoverlapping surfaces with distinct genetic properties. One of these surfaces encompassed a region unique to Arp1p that is crucial for Jnm1p (dynamitin/p50) and Nip100p (p150^Glued) association as well as pointed-end associations. Unlike the actin mutations, none of the ARP1 alleles disrupt filament formation; however, one pointed-end allele delayed the elution of Arp1p on gel filtration, consistent with loss of additional subunits.     

The essential WD-repeat protein Rsa4p is required for rRNA processing and intra-nuclear transport of 60S ribosomal subunits

We report the characterization of a novel factor, Rsa4p (Ycr072cp), which is essential for the synthesis of 60S ribosomal subunits. Rsa4p is a conserved WD-repeat protein that seems to localize in the nucleolus. In vivo depletion of Rsa4p results in a deficit of 60S ribosomal subunits and the appearance of half-mer polysomes. Northern hybridization and primer extension analyses of pre-rRNA and mature rRNAs show that depletion of Rsa4p leads to the accumulation of the 27S, 25.5S and 7S pre-rRNAs, resulting in a reduction of the mature 25S and 5.8S rRNAs. Pulse–chase analyses of pre-rRNA processing reveal that, at least, this is due to a strong delay in the maturation of 27S pre-rRNA intermediates to mature 25S rRNA. Furthermore, depletion of Rsa4p inhibited the release of the pre-60S ribosomal particles from the nucleolus to the nucleoplasm, as judged by the predominantly nucleolar accumulation of the large subunit Rpl25-eGFP reporter construct. We propose that Rsa4p associates early with pre-60S ribosomal particles and provides a platform of interaction for correct processing of rRNA precursors and nucleolar release of 60S ribosomal subunits.     

Chromatin restoration following nucleotide excision repair involves the incorporation of ubiquitinated H2A at damaged genomic sites

Restoration of functionally intact chromatin structure following DNA damage processing is crucial for maintaining genetic and epigenetic information in human cells. Here, we show the UV-induced uH2A foci formation in cells lacking XPC, DDB2, CSA or CSB, but not in cells lacking XPA, XPG or XPF indicating that uH2A incorporation relied on successful damage repair occurring through either GGR or TCR sub-pathway. In contrast, XPA, XPG or XPF were not required for formation of γH2AX foci in asynchronous cells. Notably, the H2A ubiquitin ligase Ring1B, a component of Polycomb repressor complex 1, did not localize at DNA damage sites. However, histone chaperone CAF-1 showed distinct localization to the damage sites. Knockdown of CAF-1 p60 abolished CAF-1 as well as uH2A foci formation. CAF-1 p150 was found to associate with NER factors TFIIH, RPA p70 and PCNA in chromatin. These data demonstrate that successful NER of genomic lesions and prompt CAF-1-mediated chromatin restoration link uH2A incorporation at the sites of damage repair within chromatin.     

Mutual interactions between subunits of the human RNase MRP ribonucleoprotein complex

The eukaryotic ribonuclease for mitochondrial RNA processing (RNase MRP) is mainly located in the nucleoli and belongs to the small nucleolar ribonucleoprotein (snoRNP) particles. RNase MRP is involved in the processing of pre-rRNA and the generation of RNA primers for mitochondrial DNA replication. A closely related snoRNP, which shares protein subunits with RNase MRP and contains a structurally related RNA subunit, is the pre-tRNA processing factor RNase P. Up to now, 10 protein subunits of these complexes have been described, designated hPop1, hPop4, hPop5, Rpp14, Rpp20, Rpp21, Rpp25, Rpp30, Rpp38 and Rpp40. To get more insight into the assembly of the human RNase MRP complex we studied protein–protein and protein–RNA interactions by means of GST pull-down experiments. A total of 19 direct protein–protein and six direct protein–RNA interactions were observed. The analysis of mutant RNase MRP RNAs showed that distinct regions are involved in the direct interaction with protein subunits. The results provide insight into the way the protein and RNA subunits assemble into a ribonucleoprotein particle. Based upon these data a new model for the architecture of the human RNase MRP complex was generated.     

黑腹果蝇dCAF-1-p55突变引起果蝇发育迟缓和染色体不稳定性

在真核生物中高度保守的染色质装配因子1(chromatin assembly factor 1,CAF-1)是染色质装配过程中的组蛋白分子伴侣之一.dCAF-1-p55是果蝇中CAF-1复合物中的最小亚基,它与另外两个亚基dCAF-1-p180及dCAF-1-p105一起负责将组蛋白H3/H4组装到新合成的DNA上.除了CAF-1复合物,dCAF-1-p55还参与其他多个复合物的形成,如NURF、PRC2及Sin3-HDAC1.dCAF-1-p55的这一广泛参与性提示了其功能的多样性和重要性.为了研究dCAF-1-p55的体内功能,我们利用基因靶向敲除技术制备了果蝇dCAF-1-p55突变体.实验结果表明,dCAF-1-p55的缺失导致果蝇发育迟缓并且最终致死.进一步研究发现,在dCAF-1-p55突变细胞中,中期染色体较为松散,姐妹染色单体连接异常,后期染色体不能正常分离.这些缺陷都是与癌症发生密切相关的染色体不稳定性(chromosome instability,CIN)的典型特征.综上所述,我们的研究表明了dCAF-1-p55在果蝇发育过程及维持染色体稳定性方面的重要作用,同时提示该基因具有保护细胞免遭CIN和癌变的潜在功能.     

CAF-1-induced oligomerization of histones H3/H4 and mutually exclusive interactions with Asf1 guide H3/H4 transitions among histone chaperones and DNA

Anti-silencing function 1 (Asf1) and Chromatin Assembly Factor 1 (CAF-1) chaperone histones H3/H4 during the assembly of nucleosomes on newly replicated DNA. To understand the mechanism of histone H3/H4 transfer among Asf1, CAF-1 and DNA from a thermodynamic perspective, we developed and employed biophysical approaches using full-length proteins in the budding yeast system. We find that the C-terminal tail of Asf1 enhances the interaction of Asf1 with CAF-1. Surprisingly, although H3/H4 also enhances the interaction of Asf1 with the CAF-1 subunit Cac2, H3/H4 forms a tight complex with CAF-1 exclusive of Asf1, with an affinity weaker than Asf1–H3/H4 or H3/H4–DNA interactions. Unlike Asf1, monomeric CAF-1 binds to multiple H3/H4 dimers, which ultimately promotes the formation of (H3/H4)₂ tetramers on DNA. Thus, transition of H3/H4 from the Asf1-associated dimer to the DNA-associated tetramer is promoted by CAF-1-induced H3/H4 oligomerization.     

CAF-1 is essential for heterochromatin organization in pluripotent embryonic cells

During mammalian development, chromatin dynamics and epigenetic marking are important for genome reprogramming. Recent data suggest an important role for the chromatin assembly machinery in this process. To analyze the role of chromatin assembly factor 1 (CAF-1) during pre-implantation development, we generated a mouse line carrying a targeted mutation in the gene encoding its large subunit, p150CAF-1. Loss of p150CAF-1 in homozygous mutants leads to developmental arrest at the 16-cell stage. Absence of p150CAF-1 in these embryos results in severe alterations in the nuclear organization of constitutive heterochromatin. We provide evidence that in wild-type embryos, heterochromatin domains are extensively reorganized between the two-cell and blastocyst stages. In p150CAF-1 mutant 16-cell stage embryos, the altered organization of heterochromatin displays similarities to the structure of heterochromatin in two- to four-cell stage wild-type embryos, suggesting that CAF-1 is required for the maturation of heterochromatin during preimplantation development. In embryonic stem cells, depletion of p150CAF-1 using RNA interference results in the mislocalization, loss of clustering, and decondensation of pericentric heterochromatin domains. Furthermore, loss of CAF-1 in these cells results in the alteration of epigenetic histone methylation marks at the level of pericentric heterochromatin. These alterations of heterochromatin are not found in p150CAF-1-depleted mouse embryonic fibroblasts, which are cells that are already lineage committed, suggesting that CAF-1 is specifically required for heterochromatin organization in pluripotent embryonic cells. Our findings underline the role of the chromatin assembly machinery in controlling the spatial organization and epigenetic marking of the genome in early embryos and embryonic stem cells.     

Molecular and Functional Basis for the Scaffolding Role of the p50/Dynamitin Subunit of the Microtubule-associated Dynactin Complex

p50/dynamitin (DM) is a major subunit of the microtubule-associated dynactin complex that is required for stabilization and attachment of its two distinct structural domains, namely the Arp1 rod and the shoulder/sidearm. Here, we define the determinants of p50/DM required for self-oligomerization of the protein and for interactions with other subunits of the dynactin complex. Whereas the N-terminal 1–91-amino acid region of the protein is required and sufficient for binding to the Arp1 rod, additional determinants contained within the first half of the protein are required for optimal recruitment of the p150^Glued subunit of the shoulder/sidearm. Overexpression experiments confirmed that the N-terminal 1–91-amino acid region of p50/DM is critical for dynactin functionality, because this fragment acts as a dominant negative to inhibit both dynein-dependent and -independent functions of the complex. Furthermore, the first two predicted coiled-coil motifs of p50/DM contain determinants that mediate self-association of the protein. Interestingly, p50/DM self-association does not contribute to p50/DM-induced disruption of the dynactin complex, but most likely participates in the stabilization of the complex.