Subunit Of Chromatin Assembly Factor Research Articles

Chromatin assembly factor subunit CHAF1A as a monogenic cause for oculo-auriculo-vertebral spectrum.

Oculo-auriculo-vertebral spectrum (OAVS) is characterized by abnormal development of the 1st and 2nd branchial arches. Despite arguments against a monogenic condition, a few genes have been involved in a minority of cases. We now report heterozygous, presumably loss-of function variants in the CHAF1A gene in 8 individuals, including 3 members of the same family. Four cases fulfill stringent diagnostic criteria for OAVS, including asymmetric ear dysplasia, preauricular tags, mandibular asymmetry +/- vertebral malformations. Two patients also presented with kidney malformations. CHAF1A encodes a subunit of CAF-1 (chromatin assembly factor-1), a heterotrimeric protein complex responsible for the deposition of newly synthesized histones H3-H4 onto the newly synthetized DNA strand during replication. The identification of loss-of-unction variants in CHAF1A is consistent with the hypothesis of early developmental genes dysregulation driving OAVS and other associations recently lumped under the acronym Recurrent Constellations of Embryonic Malformations (RCEM).

The Cac2 subunit is essential for productive histone binding and nucleosome assembly in CAF-1

Nucleosome assembly following DNA replication controls epigenome maintenance and genome integrity. Chromatin assembly factor 1 (CAF-1) is the histone chaperone responsible for histone (H3-H4)2 deposition following DNA synthesis. Structural and functional details for this chaperone complex and its interaction with histones are slowly emerging. Using hydrogen-deuterium exchange coupled to mass spectrometry, combined with in vitro and in vivo mutagenesis studies, we identified the regions involved in the direct interaction between the yeast CAF-1 subunits, and mapped the CAF-1 domains responsible for H3-H4 binding. The large subunit, Cac1 organizes the assembly of CAF-1. Strikingly, H3-H4 binding is mediated by a composite interface, shaped by Cac1-bound Cac2 and the Cac1 acidic region. Cac2 is indispensable for productive histone binding, while deletion of Cac3 has only moderate effects on H3-H4 binding and nucleosome assembly. These results define direct structural roles for yeast CAF-1 subunits and uncover a previously unknown critical function of the middle subunit in CAF-1.

Insights into the molecular architecture and histone H3-H4 deposition mechanism of yeast Chromatin assembly factor 1.

How the very first step in nucleosome assembly, deposition of histone H3-H4 as tetramers or dimers on DNA, is accomplished remains largely unclear. Here, we report that yeast chromatin assembly factor 1 (CAF1), a conserved histone chaperone complex that deposits H3-H4 during DNA replication, binds a single H3-H4 heterodimer in solution. We identify a new DNA-binding domain in the large Cac1 subunit of CAF1, which is required for high-affinity DNA binding by the CAF1 three-subunit complex, and which is distinct from the previously described C-terminal winged-helix domain. CAF1 binds preferentially to DNA molecules longer than 40 bp, and two CAF1-H3-H4 complexes concertedly associate with DNA molecules of this size, resulting in deposition of H3-H4 tetramers. While DNA binding is not essential for H3-H4 tetrasome deposition in vitro, it is required for efficient DNA synthesis-coupled nucleosome assembly. Mutant histones with impaired H3-H4 tetramerization interactions fail to release from CAF1, indicating that DNA deposition of H3-H4 tetramers by CAF1 requires a hierarchical cooperation between DNA binding, H3-H4 deposition and histone tetramerization.

Maintenance of Heterochromatin by the Large Subunit of the CAF-1 Replication-Coupled Histone Chaperone Requires Its Interaction with HP1a Through a Conserved Motif.

In eukaryotic cells, the organization of genomic DNA into chromatin regulates many biological processes, from the control of gene expression to the regulation of chromosome segregation. The proper maintenance of this structure upon cell division is therefore of prime importance during development for the maintenance of cell identity and genome stability. The chromatin assembly factor 1 (CAF-1) is involved in the assembly of H3-H4 histone dimers on newly synthesized DNA and in the maintenance of a higher order structure, the heterochromatin, through an interaction of its large subunit with the heterochromatin protein HP1a. We identify here a conserved domain in the large subunit of the CAF-1 complex required for its interaction with HP1a in the Drosophila fruit fly. Functional analysis reveals that this domain is dispensable for viability but participates in two processes involving heterochromatin: position-effect variegation and long range chromosomal interactions during meiotic prophase. Importantly, the identification in the large subunit of CAF-1 of a domain required for its interaction with HP1 allows the separation of its functions in heterochromatin-related processes from its function in the assembly of H3-H4 dimers onto newly synthesized DNA.

The p150N domain of chromatin assembly factor-1 regulates Ki-67 accumulation on the mitotic perichromosomal layer.

Chromatin assembly factor 1 (CAF-1) deposits histones during DNA synthesis. The p150 subunit of human CAF-1 contains an N-terminal domain (p150N) that is dispensable for histone deposition but promotes the localization of specific loci (nucleolar-associated domains [NADs]) and proteins to the nucleolus during interphase. One of the p150N-regulated proteins is proliferation antigen Ki-67, whose depletion also decreases the nucleolar association of NADs. Ki-67 is also a fundamental component of the perichromosomal layer (PCL), a sheath of proteins surrounding condensed chromosomes during mitosis. We show here that a subset of p150 localizes to the PCL during mitosis and that p150N is required for normal levels of Ki-67 accumulation on the PCL. This activity requires the sumoylation-interacting motif within p150N, which is also required for the nucleolar localization of NADs and Ki-67 during interphase. In this manner, p150N coordinates both interphase and mitotic nuclear structures via Ki67.

A DNA binding winged helix domain in CAF-1 functions with PCNA to stabilize CAF-1 at replication forks.

Chromatin assembly factor 1 (CAF-1) is a histone H3–H4 chaperone that deposits newly synthesized histone (H3–H4)2 tetramers during replication-coupled nucleosome assembly. However, how CAF-1 functions in this process is not yet well understood. Here, we report the crystal structure of C terminus of Cac1 (Cac1C), a subunit of yeast CAF-1, and the function of this domain in stabilizing CAF-1 at replication forks. We show that Cac1C forms a winged helix domain (WHD) and binds DNA in a sequence-independent manner. Mutations in Cac1C that abolish DNA binding result in defects in transcriptional silencing and increased sensitivity to DNA damaging agents, and these defects are exacerbated when combined with Cac1 mutations deficient in PCNA binding. Similar phenotypes are observed for corresponding mutations in mouse CAF-1. These results reveal a mechanism conserved in eukaryotic cells whereby the ability of CAF-1 to bind DNA is important for its association with the DNA replication forks and subsequent nucleosome assembly.

Analysis of the Relationships between DNA Double-Strand Breaks, Synaptonemal Complex and Crossovers Using the Atfas1-4 Mutant.

Chromatin Assembly Factor 1 (CAF-1) is a histone chaperone that assembles acetylated histones H3/H4 onto newly synthesized DNA, allowing the de novo assembly of nucleosomes during replication. CAF-1 is an evolutionary conserved heterotrimeric protein complex. In Arabidopsis, the three CAF-1 subunits are encoded by FAS1, FAS2 and MSI1. Atfas1-4 mutants have reduced fertility due to a decrease in the number of cells that enter meiosis. Interestingly, the number of DNA double-strand breaks (DSBs), measured by scoring the presence of γH2AX, AtRAD51 and AtDMC1 foci, is higher than in wild-type (WT) plants, and meiotic recombination genes such AtCOM1/SAE2, AtBRCA1, AtRAD51 and AtDMC1 are overexpressed. An increase in DSBs in this mutant does not have a significant effect in the mean chiasma frequency at metaphase I, nor a different number of AtMLH1 nor AtMUS81 foci per cell compared to WT at pachytene. Nevertheless, this mutant does show a higher gene conversion (GC) frequency. To examine how an increase in DSBs influences meiotic recombination and synaptonemal complex (SC) formation, we analyzed double mutants defective for AtFAS1 and different homologous recombination (HR) proteins. Most showed significant increases in both the mean number of synapsis initiation points (SIPs) and the total length of AtZYP1 stretches in comparison with the corresponding single mutants. These experiments also provide new insight into the relationships between the recombinases in Arabidopsis, suggesting a prominent role for AtDMC1 versus AtRAD51 in establishing interhomolog interactions. In Arabidopsis an increase in the number of DSBs does not translate to an increase in the number of crossovers (COs) but instead in a higher GC frequency. We discuss different mechanisms to explain these results including the possible existence of CO homeostasis in plants.

A separable domain of the p150 subunit of human chromatin assembly factor-1 promotes protein and chromosome associations with nucleoli.

Chromatin assembly factor-1 (CAF-1) is a three-subunit protein complex conserved throughout eukaryotes that deposits histones during DNA synthesis. Here we present a novel role for the human p150 subunit in regulating nucleolar macromolecular interactions. Acute depletion of p150 causes redistribution of multiple nucleolar proteins and reduces nucleolar association with several repetitive element-containing loci. Of note, a point mutation in a SUMO-interacting motif (SIM) within p150 abolishes nucleolar associations, whereas PCNA or HP1 interaction sites within p150 are not required for these interactions. In addition, acute depletion of SUMO-2 or the SUMO E2 ligase Ubc9 reduces α-satellite DNA association with nucleoli. The nucleolar functions of p150 are separable from its interactions with the other subunits of the CAF-1 complex because an N-terminal fragment of p150 (p150N) that cannot interact with other CAF-1 subunits is sufficient for maintaining nucleolar chromosome and protein associations. Therefore these data define novel functions for a separable domain of the p150 protein, regulating protein and DNA interactions at the nucleolus.

Chromatin Fractionation Assay in Fission Yeast

The protein recruitment onto chromatin is a critical process for DNA metabolism, including DNA replication, DNA repair and DNA recombination. Especially DNA modification enzymes and checkpoint proteins are loaded onto DNA damage sites in a context-dependent manner. In our recent study (Kunoh and Habu, 2014), the chromatin association of Pcf1, a large subunit of Chromatin Assembly Factor-1 (CAF-1), was monitored after exposure of cells to hydroxyurea which slowed down the DNA replication. Results of the chromatin fractionation assay provided evidence that Pcf1 was recruited to chromatin upon DNA replication stress. A similar procedure enabled to reveal the chromatin association of Orp1, Mcm proteins, and Swi6 (Sadaie et al., 2008; Ogawa et al., 1999). This assay allows us to fractionate chromatin-bound and -unbound proteins from living cells. The following immunoblot of the respective fractions provides the information concerning the chromatin binding status of our target proteins.

The ATM-Dependent DNA Damage Response Acts as an Upstream Trigger for Compensation in thefas1Mutation during Arabidopsis Leaf Development

During leaf development, a decrease in cell number often triggers an increase in cell size. This phenomenon, called compensation, suggests that some system coordinates cell proliferation and cell expansion, but how this is mediated at the molecular level is still unclear. The fugu2 mutants in Arabidopsis (Arabidopsis thaliana) exhibit typical compensation phenotypes. Here, we report that the FUGU2 gene encodes FASCIATA1 (FAS1), the p150 subunit of Chromatin Assembly Factor1. To uncover how the fas1 mutation induces compensation, we performed microarray analyses and found that many genes involved in the DNA damage response are up-regulated in fas1. Our genetic analysis further showed that activation of the DNA damage response and the accompanying decrease of cell number in fas1 depend on ATAXIA TELANGIECTASIA MUTATED (ATM) but not on ATM AND RAD3 RELATED. Kinematic analysis suggested that the delay in the cell cycle leads to a decrease in cell number in fas1 and that loss of ATM partially restores this phenotype. Consistently, both cell size phenotypes and high ploidy phenotypes of fas1 are also suppressed by atm, supporting that the ATM-dependent DNA damage response leads to these phenotypes. Altogether, these data suggest that the ATM-dependent DNA damage response acts as an upstream trigger in fas1 to delay the cell cycle and promote entry into the endocycle, resulting in compensated cell expansion.

Interplay between mismatch repair and chromatin assembly

Single strand nicks and gaps in DNA have been reported to increase the efficiency of nucleosome loading mediated by chromatin assembly factor 1 (CAF-1). However, on mismatch-containing substrates, these strand discontinuities are utilized by the mismatch repair (MMR) system as loading sites for exonuclease 1, at which degradation of the error-containing strand commences. Because packaging of DNA into chromatin might inhibit MMR, we were interested to learn whether chromatin assembly is differentially regulated on heteroduplex and homoduplex substrates. We now show that the presence of a mismatch in a nicked plasmid substrate delays nucleosome loading in human cell extracts. Our data also suggest that, once the mismatch is removed, repair of the single-stranded gap is accompanied by efficient nucleosome loading. We postulated that the balance between MMR and chromatin assembly might be governed by proliferating cell nuclear antigen (PCNA), the processivity factor of replicative DNA polymerases, which is loaded at DNA termini and which interacts with the MSH6 subunit of the mismatch recognition factor MutSα, as well as with CAF-1. We now show that this regulation might be more complex; MutSα and CAF-1 interact not only with PCNA, but also with each other. In vivo this interaction increases during S-phase and may be controlled by the phosphorylation status of the p150 subunit of CAF-1.

HP1α 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.

The p150 subunit of CAF-1 causes association of SUMO2/3 with the DNA replication foci

The small ubiquitin-related modifier 2/3 (SUMO2/3) can be post-translationally conjugated to a wide variety of proteins constituting chromatin, the platform for genetic and epigenetic regulation. Nevertheless, it is unclear how SUMO2/3 and SUMO2/3-modified proteins are delivered to the chromatin fibers. Here we report that the largest subunit of chromatin assembly factor 1 (CAF-1), human p150, interacts directly and preferentially with SUMO2/3. Amino acid residue of 98–105 in p150 is essential and sufficient for SUMO2/3 interaction. p150-SUMO2/3 interaction coincided with regions that replicate chromatin fibers, because accumulation of the proliferating cell nuclear antigen (PCNA), and incorporation of bromodeoxyuridine (BrdU) were detected at foci co-localized with both p150 and SUMO2/3 during the S-phase in a cell line expressing epitope-tagged p150. Although inhibition of SUMO2/3 expression had only a small effect on p150 deposition on the replication sites, depletion of p150 led to delocalization of SUMO2/3 from the replication foci. Furthermore, p150 mutants deficient in SUMO2/3 interaction, caused a major reduction of SUMO2/3 at the replication foci. Thus, our findings suggest an expanded role of p150 as a SUMO2/3-interacting factor, and raise the intriguing possibility that p150 plays a role in promoting delivery of SUMO2/3 or SUMO2/3-modified proteins (or both) on chromatin fibers during replication.

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 1alpha (HP1alpha)-chromatin assembly factor 1 (CAF1) chaperone complex. This complex monomethylates K9 on non-nucleosomal histone H3. Therefore, the heterochromatic HP1alpha-CAF1-SetDB1 complex probably provides H3K9me1 for subsequent trimethylation by Suv39H1/H2 in pericentric regions. The connection of CAF1 with DNA replication, HP1alpha with heterochromatin formation and SetDB1 for H3K9me1 suggests a highly coordinated mechanism to ensure the propagation of H3K9me3 in pericentric heterochromatin during DNA replication.

The HP1–p150/CAF-1 interaction is required for pericentric heterochromatin replication and S-phase progression in mouse cells

The heterochromatin protein 1 (HP1)-rich heterochromatin domains next to centromeres are crucial for chromosome segregation during mitosis. This mitotic function requires their faithful reproduction during the preceding S phase, a process whose mechanism and regulation are current puzzles. Here we show that p150, a subunit of chromatin assembly factor 1, has a key role in the replication of pericentric heterochromatin and S-phase progression in mouse cells, independently of its known function in histone deposition. By a combination of depletion and complementation assays in vivo, we link this unique function of p150 to its ability to interact with HP1. Absence of this functional interaction triggers S-phase arrest at the time of replication of pericentromeric heterochromatin, without eliciting known DNA-based checkpoint pathways. Notably, in cells lacking the histone methylases Suv39h, in which pericentric domains do not show HP1 accumulation, p150 is dispensable for S-phase progression.

Structural Basis for the Recognition of Histone H4 by the Histone-Chaperone RbAp46

SummaryRbAp46 and RbAp48 (pRB-associated proteins p46 and p48, also known as RBBP7 and RBBP4, respectively) are highly homologous histone chaperones that play key roles in establishing and maintaining chromatin structure. We report here the crystal structure of human RbAp46 bound to histone H4. RbAp46 folds into a seven-bladed β propeller structure and binds histone H4 in a groove formed between an N-terminal α helix and an extended loop inserted into blade six. Surprisingly, histone H4 adopts a different conformation when interacting with RbAp46 than it does in either the nucleosome or in the complex with ASF1, another histone chaperone. Our structural and biochemical results suggest that when a histone H3/H4 dimer (or tetramer) binds to RbAp46 or RbAp48, helix 1 of histone H4 unfolds to interact with the histone chaperone. We discuss the implications of our findings for the assembly and function of RbAp46 and RbAp48 complexes.

The rice FLATTENED SHOOT MERISTEM, encoding CAF-1 p150 subunit, is required for meristem maintenance by regulating the cell-cycle period

We isolated flattened shoot meristem (fsm) mutants in rice that showed defective seedling growth and died in the vegetative phase. Since most fsm plants had flat and small shoot apical meristems (SAMs), we suggest that FSM is required for proper SAM maintenance. FSM encodes a putative ortholog of Arabidopsis FASCIATA1 (FAS1) that corresponds to the p150 subunit of chromatin assembly factor-1 (CAF-1). FSM is expressed patchily in tissues with actively dividing cells, suggesting a tight association of FSM with specific cell-cycle phases. Double-target in situ hybridization counterstained with cell-cycle marker genes revealed that FSM is expressed mainly in the G1 phase. In fsm, expressions of the two marker genes representing S- and G2- to M-phases were enhanced in SAM, despite a reduced number of cells in SAM, suggesting that S- and G2-phases are prolonged in fsm. In addition, developmental events in fsm leaves took place at the proper time, indicating that the temporal regulation of development occurs independently of the cell-cycle period. In contrast to the fasciated phenotype of Arabidopsis fas1, fsm showed size reduction of SAM. The opposite phenotypes between fsm and fas1 indicate that the SAM maintenance is regulated differently between rice and Arabidopsis.

Interaction between Lim15/Dmc1 and the homologue of the large subunit of CAF‐1 – a molecular link between recombination and chromatin assembly during meiosis

In eukaryotes, meiosis leads to genetically variable gametes through recombination between homologous chromosomes of maternal and paternal origin. Chromatin organization following meiotic recombination is critical to ensure the correct segregation of homologous chromosomes into gametes. However, the mechanism of chromatin organization after meiotic recombination is unknown. In this study we report that the meiosis-specific recombinase Lim15/Dmc1 interacts with the homologue of the largest subunit of chromatin assembly factor 1 (CAF-1) in the basidiomycete Coprinopsis cinerea (Coprinus cinereus). Using C. cinerea LIM15/DMC1 (CcLIM15) as the bait in a yeast two-hybrid screen, we have isolated the C. cinerea homologue of Cac1, the largest subunit of CAF-1 in Saccharomyces cerevisiae, and named it C. cinerea Cac1-like (CcCac1L). Two-hybrid assays confirmed that CcCac1L binds CcLim15 in vivo. beta-Galactosidase assays revealed that the N-terminus of CcCac1L preferentially interacts with CcLim15. Co-immunoprecipitation experiments showed that these proteins also interact in the crude extract of meiotic cells. Furthermore, we demonstrate that, during meiosis, CcCac1L interacts with proliferating cell nuclear antigen (PCNA), a component of the DNA synthesis machinery recently reported as an interacting partner of Lim15/Dmc1. Taken together, these results suggest a novel role of the CAF-1-PCNA complex in meiotic events. We propose that the CAF-1-PCNA complex modulates chromatin assembly following meiotic recombination.

The Werner syndrome protein is required for recruitment of chromatin assembly factor 1 following DNA damage

The Werner syndrome protein (WRN) and chromatin assembly factor 1 (CAF-1) are both involved in the maintenance of genome stability. In response to DNA-damaging signals, both of these proteins relocate to sites where DNA synthesis occurs. However, the interaction between WRN and CAF-1 has not yet been investigated. In this report, we show that WRN interacts physically with the largest subunit of CAF-1, hp150, in vitro and in vivo. Although hp150 does not alter WRN catalytic activities in vitro, and the chromatin assembly activity of CAF-1 is not affected in the absence of WRN in vivo, this interaction may have an important role during the cellular response to DNA replication fork blockage and/or DNA damage signals. In hp150 RNA-mediated interference (RNAi) knockdown cells, WRN partially formed foci following hydroxyurea (HU) treatment. However, in the absence of WRN, hp150 did not relocate to form foci following exposure to HU and ultraviolet light. Thus, our results demonstrate that WRN responds to DNA damage before CAF-1 and suggest that WRN may recruit CAF-1, via interaction with hp150, to DNA damage sites during DNA synthesis.

Increased frequency of homologous recombination and T-DNA integration in Arabidopsis CAF-1 mutants

Chromatin assembly factor 1 (CAF-1) is involved in nucleo some assembly following DNA replication and nucleotide excision repair. In Arabidopsis thaliana, the three CAF-1 subunits are encoded by FAS1, FAS2 and, most likely, MSI1, respectively. In this study, we asked whether genomic stability is altered in fas1 and fas2 mutants that are lacking CAF-1 activity. Depletion of either subunit increased the frequency of somatic homologous recombination (HR) in planta approximately 40-fold. The frequency of transferred DNA (T-DNA) integration was also elevated. A delay in loading histones onto newly replicated or repaired DNA might make these DNA stretches more accessible, both to repair enzymes and to foreign DNA. Furthermore, fas mutants exhibited increased levels of DNA double-strand breaks, a G2-phase retardation that accelerates endoreduplication, and elevated levels of mRNAs coding for proteins involved in HR-all factors that could also contribute to upregulation of HR frequency in fas mutants.