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Nucleus (Austin, Tex.) 2011Chromatin remodelers translocate nucleosomes along the DNA chain in an ATP-dependent manner. This catalytic activity is particularly important for DNA replication and...
Chromatin remodelers translocate nucleosomes along the DNA chain in an ATP-dependent manner. This catalytic activity is particularly important for DNA replication and repair since both processes require a significant amount of nucleosome translocations and assembly during DNA synthesis. Recently, we have studied the mobility and interactions of the human ISWI family chromatin remodelers Snf2H and Snf2L as well as Acf1, one of the non-catalytic subunits present in the ACF and CHRAC complexes of Snf2H. We proposed that these protein complexes identify their nucleosomal substrates via a continuous sampling mechanism. It rationalizes the relatively high nuclear mobility and abundance observed for all ISWI proteins in terms of fast target location. According to our model a certain type of ISWI complex visits a given nucleosome in the human genome on the timescale of several seconds to a few minutes. Here, we show that the ISWI proteins Snf2H, Snf2L as well as Acf1 accumulate at UV-induced DNA damage sites within tens of seconds and reach a plateau after a few minutes. These findings corroborate the predictions of the continuous sampling mechanism as an efficient way for targeting chromatin remodelers to sites in the genome that require their activity. In comparison to the mobility of PCNA (proliferating cell nuclear antigen) that also accumulates at DNA repair sites the specifics of substrate location by chromatin remodelers are further characterized.
Topics: Adenosine Triphosphatases; Cell Line, Tumor; Chromatin Assembly and Disassembly; Chromosomal Proteins, Non-Histone; DNA Damage; DNA Repair; DNA-Binding Proteins; Gene Knockout Techniques; Humans; Kinetics; Proliferating Cell Nuclear Antigen; Protein Binding; Transcription Factors; Ultraviolet Rays
PubMed: 21738833
DOI: 10.4161/nucl.2.2.15209 -
The EMBO Journal Jun 1998DNA replication is initiated by binding of initiation factors to the origin of replication. Nucleosomes are known to inhibit the access of the replication machinery to...
DNA replication is initiated by binding of initiation factors to the origin of replication. Nucleosomes are known to inhibit the access of the replication machinery to origin sequences. Recently, nucleosome remodelling factors have been identified that increase the accessibility of nucleosomal DNA to transcription regulators. To test whether the initiation of DNA replication from an origin covered by nucleosomes would also benefit from the action of nucleosome remodelling factors, we reconstituted SV40 DNA into chromatin in Drosophila embryo extracts. In the presence of T-antigen and ATP, a chromatin-associated cofactor allowed efficient replication from a nucleosomal origin in vitro. In search of the energy-dependent cofactor responsible we found that purified 'chromatin accessibility complex' (CHRAC) was able to alter the nucleosomal structure at the origin allowing the binding of T-antigen and efficient initiation of replication. These experiments provide evidence for the involvement of a nucleosome remodelling machine in structural changes at the SV40 origin of DNA replication in vitro.
Topics: Animals; Chromatin; DNA Replication; Drosophila; Histones; Nucleosomes; Replication Origin; Simian virus 40
PubMed: 9628878
DOI: 10.1093/emboj/17.12.3428 -
Cell Stem Cell Jun 2010Regulation of stem cells depends on both tissue-specific transcriptional regulators and changes in chromatin organization, yet the coordination of these events in...
Regulation of stem cells depends on both tissue-specific transcriptional regulators and changes in chromatin organization, yet the coordination of these events in endogenous niches is poorly understood. In the Drosophila testis, local JAK-STAT signaling maintains germline and somatic stem cells (GSCs and cyst progenitor cells, or CPCs) in a single niche. Here we show that epigenetic regulation via the nucleosome-remodeling factor (NURF) complex ensures GSC and CPC maintenance by positively regulating JAK-STAT signaling, thereby preventing premature differentiation. Conversely, NURF is not required in early differentiating daughter cells of either lineage. Because three additional ATP-dependent chromatin remodelers (ACF, CHRAC, and dMi-2/NuRD) are dispensable for stem cell maintenance in the testis, epigenetic regulation of stem cells within this niche may rely primarily on NURF. Thus, local signals cooperate with specific chromatin-remodeling complexes in intact niches to coordinately regulate a common set of target genes to prevent premature stem cell differentiation.
Topics: Adenosine Triphosphatases; Animals; Chromatin Assembly and Disassembly; Chromosomal Proteins, Non-Histone; Drosophila; Drosophila Proteins; Embryonic Stem Cells; Epigenesis, Genetic; Gene Expression Regulation, Developmental; Janus Kinases; Male; Mutation; RNA, Small Interfering; STAT Transcription Factors; Signal Transduction; Spermatogenesis; Stem Cell Niche; Suppressor of Cytokine Signaling Proteins; Testis; Transcription Factors
PubMed: 20569693
DOI: 10.1016/j.stem.2010.04.018 -
Nucleic Acids Research Jan 2010Nucleosome positioning plays a major role in controlling the accessibility of DNA to transcription factors and other nuclear processes. Nucleosome positions after... (Comparative Study)
Comparative Study
Nucleosome positioning plays a major role in controlling the accessibility of DNA to transcription factors and other nuclear processes. Nucleosome positions after assembly are at least partially determined by the relative affinity of DNA sequences for the histone octamer. Nucleosomes can be moved, however, by a class of ATP dependent chromatin remodeling complexes. We recently showed that the human SWI/SNF remodeling complex moves nucleosomes in a sequence specific manner, away from nucleosome positioning sequences (NPSes). Here, we compare the repositioning specificity of five remodelers of diverse biological functions (hSWI/SNF, the SNF2h ATPase and the hACF, CHRAC and WICH complexes than each contain SNF2h) on 5S rDNA, MMTV and 601 NPS polynucleosomal templates. We find that all five remodelers act similarly to reduce nucleosome occupancy over the strongest NPSes, an effect that could directly contribute to the function of WICH in activating 5S rDNA transcription. While some differences were observed between complexes, all five remodelers were found to result in surprisingly similar nucleosome distributions. This suggests that remodeling complexes may share a conserved repositioning specificity, and that their divergent biological functions may largely arise from other properties conferred by complex-specific subunits.
Topics: Adenosine Triphosphatases; Chromatin Assembly and Disassembly; Chromosomal Proteins, Non-Histone; DNA-Binding Proteins; Humans; Mammary Tumor Virus, Mouse; Nucleoproteins; Nucleosomes; Promoter Regions, Genetic; Protein Subunits; RNA, Ribosomal, 5S; Transcription Factors
PubMed: 19906705
DOI: 10.1093/nar/gkp1030 -
Molecular Cell Dec 2010DNA double-strand breaks (DSBs) are repaired via nonhomologous end-joining (NHEJ) or homologous recombination (HR), but cellular repair processes remain elusive. We...
DNA double-strand breaks (DSBs) are repaired via nonhomologous end-joining (NHEJ) or homologous recombination (HR), but cellular repair processes remain elusive. We show here that the ATP-dependent chromatin-remodeling factors, ACF1 and SNF2H, accumulate rapidly at DSBs and are required for DSB repair in human cells. If the expression of ACF1 or SNF2H is suppressed, cells become extremely sensitive to X-rays and chemical treatments producing DSBs, and DSBs remain unrepaired. ACF1 interacts directly with KU70 and is required for the accumulation of KU proteins at DSBs. The KU70/80 complex becomes physically more associated with the chromatin-remodeling factors of the CHRAC complex, which includes ACF1, SNF2H, CHRAC15, and CHRAC17, after treatments producing DSBs. Furthermore, the frequency of NHEJ as well as HR induced by DSBs in chromosomal DNA is significantly decreased in cells depleted of either of these factors. Thus, ACF1 and its complexes play important roles in DSBs repair.
Topics: Adenosine Triphosphatases; Antigens, Nuclear; Cells, Cultured; Chromosomal Proteins, Non-Histone; DNA Breaks, Double-Stranded; DNA Polymerase III; DNA Repair; DNA-Binding Proteins; Green Fluorescent Proteins; Humans; Kinetics; Ku Autoantigen; Nucleoproteins; Transcription Factors; Ultraviolet Rays
PubMed: 21172662
DOI: 10.1016/j.molcel.2010.12.003 -
The EMBO Journal Oct 2004The nucleosome remodelling ATPase ISWI resides in several distinct protein complexes whose subunit composition reflects their functional specialization. Association of...
The nucleosome remodelling ATPase ISWI resides in several distinct protein complexes whose subunit composition reflects their functional specialization. Association of ISWI with ACF1, the largest subunit of CHRAC and ACF complexes, improves the efficiency of ISWI-induced nucleosome mobilization by an order of magnitude and also modulates the reaction qualitatively. In order to understand the principle by which ACF1 improves the efficiency of ISWI, we mapped their mutual interaction requirements and generated a series of ACF complexes lacking conserved ACF1 domains. Deletion of the C-terminal PHD finger modules of ACF1 or their disruption by zinc chelation profoundly affected the nucleosome mobilization capability of associated ISWI in trans. Interactions of the PHD fingers with the central domains of core histones contribute significantly to the binding of ACF to the nucleosome substrate, suggesting a novel role for PHD modules as nucleosome interaction determinants. Connecting ACF to histones may be prerequisite for efficient conversion of ATP-dependent conformational changes of ISWI into translocation of DNA relative to the histones during nucleosome mobilization.
Topics: Adenosine Triphosphatases; Animals; Baculoviridae; Chromatin; Drosophila; Drosophila Proteins; Escherichia coli; Glutathione Transferase; Histones; Insect Proteins; Insecta; Models, Biological; Nucleosomes; Protein Structure, Tertiary; Recombinant Fusion Proteins; Transcription Factors
PubMed: 15457208
DOI: 10.1038/sj.emboj.7600382 -
Cell Reports Apr 2014Chromatin reorganization and the incorporation of specific histone modifications during DNA damage response are essential steps for the successful repair of any DNA...
Chromatin reorganization and the incorporation of specific histone modifications during DNA damage response are essential steps for the successful repair of any DNA lesion. Here, we show that the histone-fold protein CHRAC14 plays an essential role in response to DNA damage in Drosophila. Chrac14 mutants are hypersensitive to genotoxic stress and do not activate the G2/M cell-cycle checkpoint after damage induction. Even though the DNA damage repair process is activated in the absence of CHRAC14, lesions are not repaired efficiently. In the absence of CHRAC14, the centromere-specific histone H3 variant CENP-A localizes to sites of DNA damage, causing ectopic kinetochore formation and genome instability. CENP-A and CHRAC14 are able to interact upon damage. Our data suggest that CHRAC14 modulates chromatin composition in response to DNA damage, which is required for efficient DNA damage repair in Drosophila.
Topics: Animals; Autoantigens; Centromere Protein A; Chromatin; Chromosomal Proteins, Non-Histone; DNA Damage; DNA Repair; Drosophila; Drosophila Proteins; G2 Phase Cell Cycle Checkpoints; Genomic Instability; Kinetochores; Nucleoproteins
PubMed: 24703848
DOI: 10.1016/j.celrep.2014.03.008 -
Genes & Development Jun 1999The assembly of core histones and DNA into periodic nucleosome arrays is mediated by ACF, an ISWI-containing factor, and NAP-1, a core histone chaperone, in an...
The assembly of core histones and DNA into periodic nucleosome arrays is mediated by ACF, an ISWI-containing factor, and NAP-1, a core histone chaperone, in an ATP-dependent process. We describe the isolation of Drosophila acf1 cDNA, which encodes the p170 and p185 forms of the Acf1 protein in ACF. Acf1 is a novel protein that contains two PHD fingers, one bromodomain, and two new conserved regions. Human WSTF, which is encoded by one of multiple genes that is deleted in Williams syndrome individuals, is the only currently known mammalian protein with each of the conserved motifs in Acf1. Purification of the native form of Acf1 led to the isolation of ACF comprising Acf1 (both p170 and p185 forms) and ISWI. Native Acf1 did not copurify with components of NURF or CHRAC, which are other ISWI-containing complexes in Drosophila. Purified recombinant ACF, consisting of Acf1 (either p185 alone or both p170 and p185) and ISWI, catalyzes the deposition of histones into extended periodic nucleosome arrays. Notably, the Acf1 and ISWI subunits function synergistically in the assembly of chromatin. ISWI alone exhibits a weak activity that is approximately 3% that of ACF. These results indicate that both Acf1 and ISWI participate in the chromatin assembly process and suggest further that the Acf1 subunit confers additional functionality to the general 'motor' activity of ISWI.
Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Amino Acid Sequence; Animals; Base Sequence; Catalysis; Chromatin; Chromosomal Proteins, Non-Histone; DNA, Complementary; Drosophila; Drosophila Proteins; Humans; Molecular Sequence Data; Nucleosomes; Proteins; Rabbits; Transcription Factors
PubMed: 10385622
DOI: 10.1101/gad.13.12.1529 -
Molecular Cell Feb 2000Drosophila ISWI, a highly conserved member of the SWI2/SNF2 family of ATPases, is the catalytic subunit of three chromatin-remodeling complexes: NURF, CHRAC, and ACF. To...
Drosophila ISWI, a highly conserved member of the SWI2/SNF2 family of ATPases, is the catalytic subunit of three chromatin-remodeling complexes: NURF, CHRAC, and ACF. To clarify the biological functions of ISWI, we generated and characterized null and dominant-negative ISWI mutations. We found that ISWI mutations affect both cell viability and gene expression during Drosophila development. ISWI mutations also cause striking alterations in the structure of the male X chromosome. The ISWI protein does not colocalize with RNA Pol II on salivary gland polytene chromosomes, suggesting a possible role for ISWI in transcriptional repression. These findings reveal novel functions for the ISWI ATPase and underscore its importance in chromatin remodeling in vivo.
Topics: Acetylation; Adenosine Triphosphatases; Animals; Cell Survival; Chromatin; Chromosomes; DNA-Binding Proteins; Drosophila; Drosophila Proteins; Euchromatin; Female; Fluorescent Antibody Technique; Gene Expression; Genes, Essential; Heterochromatin; Homeodomain Proteins; Male; Mitosis; Mutation; Phenotype; Transcription Factors; X Chromosome
PubMed: 10882076
DOI: 10.1016/s1097-2765(00)80430-x -
The Journal of Biological Chemistry Jul 2007Histone fold proteins Dpb4 and Dls1 are components of the yeast ISW2 chromatin remodeling complex that resemble the smaller subunits of the CHRAC (Chromatin...
Histone fold proteins Dpb4 and Dls1 are components of the yeast ISW2 chromatin remodeling complex that resemble the smaller subunits of the CHRAC (Chromatin Accessibility Complex) complex found in Drosophila and humans. DNA photoaffinity labeling found that the Dpb4 subunit contacts extranucleosomal DNA 37-53 bp away from the entry/exit site of the nucleosome. Binding of Dpb4 to Isw2 and Itc2, the two largest subunits of ISW2, was found to require Dls1. Even after remodeling and nucleosome movement, Dpb4 tends to remain bound to its original binding site and likely serves as an anchor point for ISW2 on DNA. In vitro, only minor differences can be detected in the nucleosome binding and mobilization properties of ISW2 with or without Dpb4 and Dls1. Changes in the contacts of the largest subunit Itc1 with extranucleosomal DNA have, however, been found upon deletion of the Dpb4 and Dls1 dimer that may affect the nucleosome spacing properties of ISW2.
Topics: Adenosine Triphosphatases; Animals; DNA, Fungal; Dimerization; Drosophila; Humans; Nuclear Proteins; Nucleosomes; Protein Binding; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcription Factors
PubMed: 17491017
DOI: 10.1074/jbc.M700640200