The INO80 complex is required for damage-induced recombination

The budding yeast INO80 complex has a role in remodeling chromatin structure, and the SWR1 complex replaces a H2A/H2B dimer with a variant dimer, H2A.Z (Htz1)/H2B. It has been reported that these chromatin remodeling complexes contain Arp4 (actin-related protein) and actin in common and are recruited to HO endonuclease-induced DNA double-strand break (DSB) site. Reportedly, Ino80 can evict nucleosomes surrounding a HO-induced DSB; however, it has no apparent role to play in the repair of HO-induced DSB. Here we show that an essential factor for INO80 chromatin remodeling activity, Arp8, is involved in damage-induced sister chromatid recombination and interchromosomal recombination between heteroalleles. In contrast, arp6 mutants are proficient for recombination, indicating that the SWR1 complex does not promote recombination. Our data suggest that the remodeling of chromatin by the INO80 complex facilitates efficient homologous recombination upon DNA damages.     

H2A.Z depletion impairs proliferation and viability but not DNA double-strand breaks repair in human immortalized and tumoral cell lines

In mammalian cells, DNA double-strand breaks (DSB) can be repaired by 2 main pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). To give access to DNA damage to the repair machinery the chromatin structure needs to be relaxed, and chromatin modifications play major roles in the control of these processes. Among the chromatin modifications, changes in nucleosome composition can influence DNA damage response as observed with the H2A.Z histone variant in yeast. In mammals, p400, an ATPase of the SWI/SNF family able to incorporate H2A.Z in chromatin, was found to be important for histone ubiquitination and BRCA1 recruitment around DSB or for HR in cooperation with Rad51. Recent data with 293T cells showed that mammalian H2A.Z is recruited to DSBs and is important to control DNA resection, therefore participating both in HR and NHEJ. Here we show that depletion of H2A.Z in the osteosarcoma U2OS cell line and in immortalized human fibroblasts does not change parameters of DNA DSB repair while affecting clonogenic ability and cell cycle distribution. In addition, no recruitment of H2A.Z around DSB can be detected in U2OS cells either after local laser irradiation or by chromatin immunoprecipitation. These data suggest that the role of H2A.Z in DSB repair is not ubiquitous in mammals. In addition, given that important cellular parameters, such as cell viability and cell cycle distribution, are more sensitive to H2A.Z depletion than DNA repair, our results underline the difficulty to investigate the role of versatile factors such as H2A.Z.     

Mammalian Ino80 mediates double-strand break repair through its role in DNA end strand resection

Chromatin modifications/remodeling are important mechanisms by which cells regulate various functions through providing accessibility to chromatin DNA. Recent studies implicated INO80, a conserved chromatin-remodeling complex, in the process of DNA repair. However, the precise underlying mechanism by which this complex mediates repair in mammalian cells remains enigmatic. Here, we studied the effect of silencing of the Ino80 subunit of the complex on double-strand break repair in mammalian cells. Comet assay and homologous recombination repair reporter system analyses indicated that Ino80 is required for efficient double-strand break repair. Ino80 association with chromatin surrounding double-strand breaks suggested the direct involvement of INO80 in the repair process. Ino80 depletion impaired focal recruitment of 53BP1 but did not impede Rad51 focus formation, suggesting that Ino80 is required for the early steps of repair. Further analysis by using bromodeoxyuridine (BrdU)-labeled single-stranded DNA and replication protein A (RPA) immunofluorescent staining showed that INO80 mediates 5'-3' resection of double-strand break ends.     

Distinct roles for SWR1 and INO80 chromatin remodeling complexes at chromosomal double-strand breaks

INO80 and SWR1 are two closely related ATP-dependent chromatin remodeling complexes that share several subunits. Ino80 was reported to be recruited to the HO endonuclease-induced double-strand break (DSB) at the budding yeast mating-type locus, MAT. We find Swr1 similarly recruited in a manner dependent on the phosphorylation of H2A (gammaH2AX). This is not unique to cleavage at MAT; both Swr1 and Ino80 bind near an induced DSB on chromosome XV. Whereas Swr1 incorporates the histone variant H2A.Z into chromatin at promoters, H2A.Z levels do not increase at DSBs. Instead, H2A.Z, gammaH2AX and core histones are coordinately removed near the break in an INO80-dependent, but SWR1-independent, manner. Mutations in INO80-specific subunits Arp8 or Nhp10 impair the binding of Mre11 nuclease, yKu80 and ATR-related Mec1 kinase at the DSB, resulting in defective end-processing and checkpoint activation. In contrast, Mre11 binding, end-resection and checkpoint activation were normal in the swr1 strain, but yKu80 loading and error-free end-joining were impaired. Thus, these two related chromatin remodelers have distinct roles in DSB repair and checkpoint activation.     

INO80 promotes H2A.Z occupancy to regulate cell fate transition in pluripotent stem cells

The INO80 chromatin remodeler is involved in many chromatin-dependent cellular functions. However, its role in pluripotency and cell fate transition is not fully defined. We examined the impact of Ino80 deletion in the naïve and primed pluripotent stem cells. We found that Ino80 deletion had minimal effect on self-renewal and gene expression in the naïve state, but led to cellular differentiation and de-repression of developmental genes in the transition toward and maintenance of the primed state. In the naïve state, INO80 pre-marked gene promoters that would adopt bivalent histone modifications by H3K4me3 and H3K27me3 upon transition into the primed state. In the primed state, in contrast to its known role in H2A.Z exchange, INO80 promoted H2A.Z occupancy at these bivalent promoters and facilitated H3K27me3 installation and maintenance as well as downstream gene repression. Together, our results identified an unexpected function of INO80 in H2A.Z deposition and gene regulation. We showed that INO80-dependent H2A.Z occupancy is a critical licensing step for the bivalent domains, and thereby uncovered an epigenetic mechanism by which chromatin remodeling, histone variant deposition and histone modification coordinately control cell fate.     

H2AX post-translational modifications in the ionizing radiation response and homologous recombination

Histone H2AX phosphorylation on a C-terminal serine residue to form "γ-H2AX" is a critical early event in the chromatin response to chromosomal DNA double strand breaks in eukaryotes. In mammalian cells, γ-H2AX is formed when H2AX is phosphorylated on serine 139 by ATM or by other DNA damage response kinases. H2AX prevents genomic instability and tumorigenesis, and supports class-switch recombination at immunoglobulin heavy chain loci in mammals. We showed previously that H2AX controls double strand break repair by homologous recombination (HR) between sister chromatids. The HR functions of H2AX are mediated by interaction of γ-H2AX with the chromatin-associated adaptor protein MDC1. H2AX is potentially subject to additional post-translational modifications associated with the DNA damage response and with other chromatin functions. To test this idea, we used mass spectroscopy to identify H2AX residues additional to serine 139 that are post-translationally modified following exposure of cells to ionizing radiation (IR) and identified several new IR-responsive residues of H2AX. We determined the impact of IR-responsive H2AX residues on cellular resistance to IR and on H2AX-dependent homologous recombination, and also analyzed the contribution to HR of other known or potential post-translationally modified residues of H2AX. The results suggest that the HR and IR-resistance functions of H2AX are controlled in large part by specific MDC1-interacting residues of H2AX, but that additional H2AX residues modulate these core functions of H2AX.     

Anp32e,a higher eukaryotic histone chaperone directs preferential recognition for H2A.Z

H2A.Z is a highly conserved histone variant in all species. The chromatin deposition of H2A.Z is specifically catalyzed by the yeast chromatin remodeling complex SWR1 and its mammalian counterpart SRCAP. However, the mechanism by which H2A.Z is preferentially recognized by non-histone proteins remains elusive. Here we identified Anp32e, a novel higher eukaryote-specific histone chaperone for H2A.Z. Anp32e preferentially associates with H2A.Z-H2B dimers rather than H2A-H2B dimers in vitro and in vivo and dissociates non-nucleosomal aggregates formed by DNA and H2A-H2B. We determined the crystal structure of the Anp32e chaperone domain (186-232) in complex with the H2A.Z-H2B dimer. In this structure, the region containing Anp32e residues 214-224, which is absent in other Anp32 family proteins, specifically interacts with the extended H2A.Z αC helix, which exhibits an unexpected conformational change. Genome-wide profiling of Anp32e revealed a remarkable co-occupancy between Anp32e and H2A.Z. Cells overexpressing Anp32e displayed a strong global H2A.Z loss at the +1 nucleosomes, whereas cells depleted of Anp32e displayed a moderate global H2A.Z increase at the +1 nucleosomes. This suggests that Anp32e may help to resolve the non-nucleosomal H2A.Z aggregates and also facilitate the removal of H2A.Z at the +1 nucleosomes, and the latter may help RNA polymerase II to pass the first nucleosomal barrier.     

H2AX post-translational modifications in the ionizing radiation response and homologous recombination

Histone H2AX phosphorylation on a C-terminal serine residue to form “γ-H2AX” is a critical early event in the chromatin response to chromosomal DNA double strand breaks in eukaryotes. In mammalian cells, γ-H2AX is formed when H2AX is phosphorylated on serine 139 by ATM or by other DNA damage response kinases. H2AX prevents genomic instability and tumorigenesis, and supports class-switch recombination at immunoglobulin heavy chain loci in mammals. We showed previously that H2AX controls double strand break repair by homologous recombination (HR) between sister chromatids. the HR functions of H2AX are mediated by interaction of γ-H2AX with the chromatin-associated adaptor protein MDC1. H2AX is potentially subject to additional post-translational modifications associated with the DNA damage response and with other chromatin functions. To test this idea, we used mass spectroscopy to identify H2AX residues additional to serine 139 that are post-translationally modified following exposure of cells to ionizing radiation (IR) and identified several new IR-responsive residues of H2AX. We determined the impact of IR-responsive H2AX residues on cellular resistance to IR and on H2AX-dependent HR, and also analyzed the contribution to HR of other known or potential post-translationally modified residues of H2AX. The results suggest that the HR and IR-resistance functions of H2AX are controlled in large part by specific MDC1-interacting residues of H2AX, but that additional H2AX residues modulate these core functions of H2AX.Key words: H2AX, homologous recombination, ionizing radiation, double strand break repair, histone, histone code, post-translational modification, chromatin, DNA repair     

Phosphorylation of human INO80 is involved in DNA damage tolerance

Double strand breaks (DSBs) are the most serious type of DNA damage. DSBs can be generated directly by exposure to ionizing radiation or indirectly by replication fork collapse. The DNA damage tolerance pathway, which is conserved from bacteria to humans, prevents this collapse by overcoming replication blockages. The INO80 chromatin remodeling complex plays an important role in the DNA damage response. The yeast INO80 complex participates in the DNA damage tolerance pathway. The mechanisms regulating yINO80 complex are not fully understood, but yeast INO80 complex are necessary for efficient proliferating cell nuclear antigen (PCNA) ubiquitination and for recruitment of Rad18 to replication forks. In contrast, the function of the mammalian INO80 complex in DNA damage tolerance is less clear. Here, we show that human INO80 was necessary for PCNA ubiquitination and recruitment of Rad18 to DNA damage sites. Moreover, the C-terminal region of human INO80 was phosphorylated, and overexpression of a phosphorylation-deficient mutant of human INO80 resulted in decreased ubiquitination of PCNA during DNA replication. These results suggest that the human INO80 complex, like the yeast complex, was involved in the DNA damage tolerance pathway and that phosphorylation of human INO80 was involved in the DNA damage tolerance pathway. These findings provide new insights into the DNA damage tolerance pathway in mammalian cells.     

Distinct roles of the histone chaperones NAP1 and NRP and the chromatin‐remodeling factor INO80 in somatic homologous recombination in Arabidopsis thaliana

Homologous recombination (HR) of nuclear DNA occurs within the context of a highly complex chromatin structure. Despite extensive studies of HR in diverse organisms, mechanisms regulating HR within the chromatin context remain poorly elucidated. Here we investigate the role and interplay of the histone chaperones NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1) and NAP1‐RELATED PROTEIN (NRP) and the ATP‐dependent chromatin‐remodeling factor INOSITOL AUXOTROPHY80 (INO80) in regulating somatic HR in Arabidopsis thaliana. We show that simultaneous knockout of the four AtNAP1 genes and the two NRP genes in the sextuple mutant m123456‐1 barely affects normal plant growth and development. Interestingly, compared with the respective AtNAP1 (m123‐1 and m1234‐1) or NRP (m56‐1) loss‐of‐function mutants, the sextuple mutant m123456‐1 displays an enhanced plant hypersensitivity to UV or bleomycin treatments. Using HR reporter constructs, we show that AtNAP1 and NRP act in parallel to synergistically promote somatic HR. Distinctively, the AtINO80 loss‐of‐function mutation (atino80‐5) is epistatic to m56‐1 in plant phenotype and telomere length but hypostatic to m56‐1 in HR determinacy. Further analyses show that expression of HR machinery genes and phosphorylation of H2A.X (γ‐H2A.X) are not impaired in the mutants. Collectively, our study indicates that NRP and AtNAP1 synergistically promote HR upstream of AtINO80‐mediated chromatin remodeling after the formation of γ‐H2A.X foci during DNA damage repair.     

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex''s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.     

Interaction of Saccharomyces cerevisiae HMO2 domains with distorted DNA

The Saccharomyces cerevisiae high mobility group protein HMO2 is a component of the chromatin remodeling complex INO80. In this capacity, it has been shown to direct INO80 to DNA double-strand breaks, thereby contributing to double-strand break repair. Consistent with such function, HMO2 binds DNA ends, protecting them from exonucleolytic degradation. We show here that both domains of HMO2, HMO2-BoxA and HMO2-BoxB, bind preferentially to distorted DNA, with HMO2-BoxA binding preferentially to four-way DNA junctions and DNA with tandem mismatches and HMO2-BoxB binding four-way junctions as well as DNA with stem-loop structures, tandem mismatches, and abasic sites. As previously reported for mammalian high mobility group proteins, the acidic C-terminal extension significantly attenuates DNA binding. Notably, the unique ability of HMO2 to protect DNA ends is conferred by the Box A domain. Considering the reported roles for INO80 in other events such as recovery of stalled replication forks and nucleotide excision repair, we assessed the effect of DNA damaging agents on an hmo2Δ strain; while modest growth inhibition is seen upon exposure to UV light, exposure to hydroxyurea, which causes replication fork arrest, induces severe growth deficiency. These data suggest that HMO2 may also participate in directing the INO80 complex to sites such as stalled replication forks; the preferred binding of HMO2 domains to damaged DNA and intermediates in homologous recombination is consistent with such function.