G9a is a SET-domain mammalian histone methyltransferase responsible for mono- and dimethylation of lysine 9 in histone H3 (H3K9) at euchromatic regions. Recently we reported that G9a forms a ...stoichiometric heteromeric complex with another SET-domain-containing molecule, GLP/Eu-HMTase1. Although G9a and GLP can independently methylate H3K9 in vitro, G9a/GLP heteromeric formation seems to be essential for their function as a euchromatic H3K9 methyltransferase in vivo. To further elucidate how G9a/GLP-mediated histone methylation and transcriptional regulation are controlled, we purified and characterized G9a complexes from mouse embryonic stem cells. We identified a novel G9a/GLP-associating zinc finger molecule named Wiz that can interact with G9a and GLP independently but is more stable in the G9a/GLP heteromeric complexes. Interestingly, Wiz small inhibitory RNA knocks down not only Wiz but also G9a. GLP deficiency also decreases G9a levels, suggesting that the Wiz/G9a/GLP tri-complex may protect G9a from degradation and that Wiz plays a major role in G9a/GLP heterodimer formation. Furthermore, amino acid sequence analysis of Wiz predicted two potential CtBP binding sites, and indeed CtBP binding to Wiz and association of CtBP with the Wiz/G9a/GLP complex was observed. These data indicate that Wiz not only contributes to the stability of G9a but also links the G9a/GLP heteromeric complex to the CtBP co-repressor machinery.
How deregulation of chromatin modifiers causes malignancies is of general interest. Here, we show that histone H2A T120 is phosphorylated in human cancer cell lines and demonstrate that this ...phosphorylation is catalyzed by hVRK1. Cyclin D1 was one of ten genes downregulated upon VRK1 knockdown in two different cell lines and showed loss of H2A T120 phosphorylation and increased H2A K119 ubiquitylation of its promoter region, resulting in impaired cell growth. In vitro, H2A T120 phosphorylation and H2A K119 ubiquitylation are mutually inhibitory, suggesting that histone phosphorylation indirectly activates chromatin. Furthermore, expression of a phosphomimetic H2A T120D increased H3 K4 methylation. Finally, both VRK1 and the H2A T120D mutant histone transformed NIH/3T3 cells. These results suggest that histone H2A T120 phosphorylation by hVRK1 causes inappropriate gene expression, including upregulated cyclin D1, which promotes oncogenic transformation.
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•hVRK1 phosphorylates histone H2A T120 in and around promoter regions•H2A pT120 and H2A K119 ubiquitylation are mutually antagonistic•H2A pT120 regulates transcription by affecting histone H3 K4 methylation•The H2A T120D mutation mimics phosphorylation and promotes oncogenic transformation
Aberrant epigenetic changes can promote cancer development. Aihara et al. show that histone H2A T120 is phosphorylated by hVRK1 in and around the CCND1 promoter. H2A T120 phosphorylation antagonizes H2A K119 ubiquitylation and promotes H3 K4 methylation to upregulate cyclin D1 and promote oncogenic transformation.
The dynamic exchange of histones alleviates the nucleosome barrier and simultaneously facilitates various aspects of cellular DNA metabolism, such as DNA repair and transcription. In response to DNA ...damage, the acetylation of Lys5 in the histone variant H2AX, catalyzed by TIP60, plays a key role in promoting histone exchange; however, the detailed molecular mechanism still is unclear. Here, we show that the TIP60 complex includes poly(ADP-ribose) polymerase 1 (PARP-1). PARP-1 is required for the rapid exchange of H2AX on chromatin at DNA damage sites. It is known that PARP-1 binds dynamically to damaged chromatin and is crucial for the subsequent recruitment of other repair factors, and its auto-poly(ADP-ribosyl)ation is required for the dynamics. We also show that the acetylation of histone H2AX at Lys5 by TIP60, but not the phosphorylation of H2AX, is required for the ADP-ribosylation activity of PARP-1 and its dynamic binding to damaged chromatin. Our results indicate the reciprocal regulation of K5 acetylation of H2AX and PARP-1, which could modulate the chromatin structure to facilitate DNA metabolism at damage sites. This could explain the rather undefined roles of PARP-1 in various DNA damage responses.
Chromosomal translocations are hallmarks of various types of cancers and leukemias. However, the molecular mechanisms of chromosome translocations remain largely unknown. The ataxia-telangiectasia ...mutated (ATM) protein, a DNA damage signaling regulator, facilitates DNA repair to prevent chromosome abnormalities. Previously, we showed that ATM deficiency led to the 11q23 chromosome translocation, the most frequent chromosome abnormalities in secondary leukemia. Here, we show that ARP8, a subunit of the INO80 chromatin remodeling complex, is phosphorylated after etoposide treatment. The etoposide-induced phosphorylation of ARP8 is regulated by ATM and ATR, and attenuates its interaction with INO80. The ATM-regulated phosphorylation of ARP8 reduces the excessive loading of INO80 and RAD51 onto the breakpoint cluster region. These findings suggest that the phosphorylation of ARP8, regulated by ATM, plays an important role in maintaining the fidelity of DNA repair to prevent the etoposide-induced 11q23 abnormalities.
The reorganization of damaged chromatin plays an important role in the regulation of the DNA damage response. A recent study revealed the presence of 2 vertebrate H2A.Z isoforms, H2A.Z-1 and H2A.Z-2. ...However, the roles of the vertebrate H2A.Z isoforms are still unclear. Thus, in this study we examined the roles of the vertebrate H2A.Z isoforms in chromatin reorganization after the induction of DNA double-strand breaks (DSBs).
To examine the dynamics of H2A.Z isoforms at damaged sites, we constructed GM0637 cells stably expressing each of the green fluorescent protein (GFP)-labeled H2A.Z isoforms, and performed fluorescence recovery after photobleaching (FRAP) analysis and inverted FRAP analysis in combination with microirradiation. Immunofluorescence staining using an anti-RAD51 antibody was performed to study the kinetics of RAD51 foci formation after 2-Gy irradiation of wild-type (WT), H2A.Z-1- and H2A.Z-2-deficient DT40 cells. Colony-forming assays were also performed to compare the survival rates of WT, H2A.Z-1-, and H2A.Z-2-deficient DT40 cells with control, and H2A.Z-1- and H2A.Z-2-depleted U2OS cells after irradiation.
FRAP analysis revealed that H2A.Z-2 was incorporated into damaged chromatin just after the induction of DSBs, whereas H2A.Z-1 remained essentially unchanged. Inverted FRAP analysis showed that H2A.Z-2 was released from damaged chromatin. These findings indicated that H2A.Z-2 was exchanged at DSB sites immediately after the induction of DSBs. RAD51 focus formation after ionizing irradiation was disturbed in H2A.Z-2-deficient DT40 cells but not in H2A.Z-1-deficient cells. The survival rate of H2A.Z-2-deficient cells after irradiation was lower than those of WT and H2A.Z-1- DT40 cells. Similar to DT40 cells, H2A.Z-2-depleted U2OS cells were also radiation-sensitive compared to control and H2A.Z-1-depleted cells.
We found that vertebrate H2A.Z-2 is involved in the regulation of the DNA damage response at a very early stage, via the damaged chromatin reorganization required for RAD51 focus formation.
The Fanconi anemia (FA) pathway is critically involved in the maintenance of hematopoietic stem cells and the suppression of carcinogenesis. A key FA protein, FANCD2, is monoubiquitinated and ...accumulates in chromatin in response to DNA interstrand crosslinks (ICLs), where it coordinates DNA repair through mechanisms that are still poorly understood. Here, we report that CtIP protein directly interacts with FANCD2. A region spanning amino acids 166 to 273 of CtIP and monoubiquitination of FANCD2 are both essential for the FANCD2-CtIP interaction and mitomycin C (MMC)-induced CtIP foci. Remarkably, both FANCD2 and CtIP are critical for MMC-induced RPA2 hyperphosphorylation, an event that accompanies end resection of double-strand breaks. Collectively, our results reveal a role of monoubiquitinated FANCD2 in end resection that depends on its binding to CtIP during ICL repair.
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•CtIP protein directly interacts with FANCD2•CtIP forms MMC-induced foci that are dependent on association with FANCD2•FANCD2-CtIP interaction requires FANCD2 monoubiquitination in vivo•Both CtIP and FANCD2 are necessary for efficient DNA-end resection during ICL repair
Upon DNA crosslink damage, the DNA repair/replication protein FANCD2 is activated/monoubiquitinated and removes replication fork blockades. Its deficiency causes Fanconi anemia, which is associated with cancer and hematopoietic stem cell failure. Here, Unno et al. uncover an additional role of FANCD2 in a step of homologous recombination, expanding its function as a master coordinator of crosslink repair. Specifically, monoubiquitinated FANCD2 directly binds CtIP protein and promotes its chromatin recruitment, facilitating nucleolytic end resection during DNA repair.
The ubiquitin (Ub)-conjugating enzyme Ubc13 is implicated in Rad6/Rad18-dependent postreplication repair (PRR) in budding yeast, but its function in vertebrates is not known. We show here that ...disruption or siRNA depletion of
UBC13 in chicken DT40 or human cells confers severe growth defects due to chromosome instability, and hypersensitivity to both UV and ionizing radiation, consistent with a conserved role for Ubc13 in PRR. Remarkably, Ubc13-deficient cells are also compromised for DNA double-strand break (DSB) repair by homologous recombination (HR). Recruitment and activation of the E3 Ub ligase function of BRCA1 and the subsequent formation of the Rad51 nucleoprotein filament at DSBs are abolished in Ubc13-deficient cells. Furthermore, generation of ssDNA/RPA complexes at DSBs is severely attenuated in the absence of Ubc13. These data reveal a critical and unexpected role for vertebrate Ubc13 in the initiation of HR at the level of DSB processing.
It is well known that histone acetylases are important chromatin modifiers and that they play a central role in chromatin transcription. Here, we present evidence for novel roles of histone ...acetylases. The TIP60 histone acetylase purifies as a multimeric protein complex. Besides histone acetylase activity on chromatin, the TIP60 complex possesses ATPase, DNA helicase, and structural DNA binding activities. Ectopic expression of mutated TIP60 lacking histone acetylase activity results in cells with defective double-strand DNA break repair. Importantly, the resulting cells lose their apoptotic competence, suggesting a defect in the cells' ability to signal the existence of DNA damage to the apoptotic machinery. These results indicate that the histone acetylase TIP60-containing complex plays a role in DNA repair and apoptosis.
A deficiency in the SMN gene product causes the motor neuron degenerative disease spinal muscular atrophy. GEMIN2 was identified as an SMN-interacting protein, and the SMN–GEMIN2 complex constitutes ...part of the large SMN complex, which promotes the assembly of the spliceosomal small nuclear ribonucleoprotein (snRNP). In addition to its splicing function, we previously found that GEMIN2 alone stimulates RAD51-mediated recombination in vitro, and functions in DNA double-strand-break (DSB) repair through homologous recombination in vivo. However, the function of SMN in homologous recombination has not been reported. In the present study, we successfully purified the SMN–GEMIN2 complex as a fusion protein. The SMN–GEMIN2 fusion protein complemented the growth-defective phenotype of GEMIN2-knockout cells. The purified SMN–GEMIN2 fusion protein enhanced the RAD51-mediated homologous pairing much more efficiently than GEMIN2 alone. SMN–GEMIN2 possessed DNA-binding activity, which was not observed with the GEMIN2 protein, and significantly stimulated the secondary duplex DNA capture by the RAD51-single-stranded DNA complex during homologous pairing. These results provide the first evidence that the SMN–GEMIN2 complex plays a role in homologous recombination, in addition to spliceosomal snRNP assembly.
Bach2 is a B cell-specific transcription repressor whose deficiency in mice causes a reduced class switch recombination and a reduced somatic hypermutation of immunoglobulin genes. Little is known ...about the direct target genes of Bach2 in B cells. By analyzing various B cell and plasma cell lines, we showed that the expression patterns of Bach2 and Blimp-1 (B lymphocyte-induced maturation protein 1), a master regulator of plasma cell differentiation, are mutually exclusive. The reporter gene of the Blimp-1 gene (Prdm1) was repressed by the overexpression of Bach2 in B cell lines. The heterodimer of Bach2/MafK bound to the Maf recognition element located upstream of the Prdm1 promoter in an electrophoretic mobility shift assay. The binding of MafK in B cells to the Prdm1 Maf recognition element was confirmed by chromatin immunoprecipitation assays. When MafK was purified from the BAL17 B cell line, a significant portion of it was present as a heterodimer with Bach2, with no apparent formation of MafK homodimer. These results strongly suggest that Bach2 represses the expression of Blimp-1 together with MafK in B cells prior to plasma cell differentiation. Accordingly, the knockdown of Bach2 mRNA using short hairpin RNA in BAL17 cells resulted in higher levels of Prdm1 expression after the stimulation of B cell receptor by surface IgM cross-linking. Induction of Prdm1 was more robust and faster in primary Bach2-deficient B cells than in wild-type control B cells upon lipopolysaccharide stimulation. Therefore, the Prdm1 regulation in B cells involves the repression by Bach2, which may be cancelled upon terminal plasma cell differentiation.
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