Jongbum Kwon

Mammalian SWI/SNF complexes facilitate DNA double‐strand break repair by promoting γ‐H2AX induction

Although mammalian SWI/SNF chromatin remodeling complexes have been well established to play important role in transcription, their role in DNA repair has remained largely unexplored. Here we show that inactivation of the SWI/SNF complexes and downregulation of the catalytic core subunits of the complexes both result in inefficient DNA double‐strand break (DSB) repair and increased DNA damage sensitivity as well as a large defect in H2AX phosphorylation (γ‐H2AX) and nuclear focus formation after DNA damage. The expression of most DSB repair genes remains unaffected and DNA damage checkpoints are grossly intact in the cells inactivated for the SWI/SNF complexes. Although the SWI/SNF complexes do not affect the expression of ATM, DNA‐PK and ATR, or their activation and/or recruitment to DSBs, they rapidly bind to DSB‐surrounding chromatin via interaction with γ‐H2AX in the manner that is dependent on the amount of DNA damage. Given the crucial role for γ‐H2AX in efficient DSB repair, these results suggest that the SWI/SNF complexes facilitate DSB repair, at least in part, by promoting H2AX phosphorylation by directly acting on chromatin.

A cooperative activation loop among SWI/SNF, γ-H2AX and H3 acetylation for DNA double-strand break repair

Although recent studies highlight the importance of histone modifications and ATP‐dependent chromatin remodelling in DNA double‐strand break (DSB) repair, how these mechanisms cooperate has remained largely unexplored. Here, we show that the SWI/SNF chromatin remodelling complex, earlier known to facilitate the phosphorylation of histone H2AX at Ser‐139 (S139ph) after DNA damage, binds to γ‐H2AX (the phosphorylated form of H2AX)‐containing nucleosomes in S139ph‐dependent manner. Unexpectedly, BRG1, the catalytic subunit of SWI/SNF, binds to γ‐H2AX nucleosomes by interacting with acetylated H3, not with S139ph itself, through its bromodomain. Blocking the BRG1 interaction with γ‐H2AX nucleosomes either by deletion or overexpression of the BRG1 bromodomain leads to defect of S139ph and DSB repair. H3 acetylation is required for the binding of BRG1 to γ‐H2AX nucleosomes. S139ph stimulates the H3 acetylation on γ‐H2AX nucleosomes, and the histone acetyltransferase Gcn5 is responsible for this novel crosstalk. The H3 acetylation on γ‐H2AX nucleosomes is induced by DNA damage. These results collectively suggest that SWI/SNF, γ‐H2AX and H3 acetylation cooperatively act in a feedback activation loop to facilitate DSB repair.

A proteomics approach for the identification of nucleophosmin and heterogeneous nuclear ribonucleoprotein C1/C2 as chromatin-binding proteins in response to DNA double-strand breaks.

Double-strand breaks (DSBs) of chromosomal DNA trigger the cellular response that activates the pathways for DNA repair and cell-cycle checkpoints, and sometimes the pathways leading to cell death if the damage is too severe to be tolerated. Evidence indicates that, upon generation of DNA DSBs, many nuclear proteins that are involved in DNA repair and checkpoints are recruited to chromatin around the DNA lesions. In the present study we used a proteomics approach to identify DNA-damage-induced chromatin-binding proteins in a systematic way. Two-dimensional gel analysis for protein extracts of chromatin from DNA-damage-induced and control HeLa cells identified four proteins as the candidates for DNA-damage-induced chromatin-binding proteins. MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS analysis identified these proteins to be NPM (nucleophosmin), hnRNP (heterogeneous nuclear ribonucleoprotein) C1, hnRNP C2 and 37-kDa laminin-receptor precursor, and the identity of these proteins was further confirmed by immunoblot analysis with specific antibodies. We then demonstrated with chromatin-binding assays that NPM and hnRNP C1/C2, the abundant nuclear proteins with pleiotropic functions, indeed bind to chromatin in a DNA-damage-dependent manner, implicating these proteins in DNA repair and/or damage response. Immunofluorescence experiments showed that NPM, normally present in the nucleoli, is mobilized into the nucleoplasm after DNA damage, and that neither NPM nor hnRNP C1/C2 is actively recruited to the sites of DNA breaks. These results suggest that NPM and hnRNP C1/C2 may function at the levels of the global context of chromatin, rather than by specifically targeting the broken DNA.

Mammalian SWI/SNF chromatin remodeling complexes are required to prevent apoptosis after DNA damage.

Although SWI/SNF chromatin remodeling complexes play important roles in transcription, recent studies suggest that they also participate directly in DNA repair. In yeast, SWI/SNF and related RSC complexes have been shown to be recruited to the sites of DNA double strand breaks (DSBs) to facilitate DNA repair. We recently have shown that mammalian SWI/SNF complexes contribute to DBS repair by direct mechanisms of stimulating the phosphorylation of histone H2AX at DSB-surrounding chromatin. Here we investigated the role of mammalian SWI/SNF complexes in cell survival after DNA damage. When SWI/SNF was inactivated by means of dominant negativity or its catalytic subunit BRG1 was knockdowned by small interfering RNA, cells became highly susceptible to DNA damage-induced apoptosis. SWI/SNF inactivation had no effect on the activation and establishment of G2/M DNA damage checkpoint. However, SWI/SNF-defective cells could not sustain the G2/M checkpoint long enough to survive DNA damage, and rather underwent apoptosis before entering mitosis. We also found that, although the basal state and DNA damage-triggered activation of p53 were normal, the kinetics of p53 downregulation was significantly delayed in SWI/SNF-defective cells. Finally, the sustained p53 activation in SWI/SNF-defective cells was accompanied by accumulation of unrepaired DSBs owing to inefficient DNA repair. These results suggest that mammalian SWI/SNF complexes prevent DNA damage-induced apoptosis in part by facilitating efficient repair and thereby ensuring timely elimination of unrepaired DSBs that could otherwise lead to excessive prolongation of p53 activation.

Roles of human INO80 chromatin remodeling enzyme in DNA replication and chromosome segregation suppress genome instability

Although INO80 chromatin remodeling enzyme has been shown in yeast to play roles in non-transcriptional nuclear processes such as DNA replication, its cellular functions in higher eukaryotes have remained largely unexplored. Here, we provide evidence that human INO80 (hINO80) participates in both DNA replication and chromosome segregation during the normal cell division cycle. hINO80 binds to chromatin localizing at replication forks during the S-phase, and is required for efficient DNA synthesis and S-phase progression. Unexpectedly, hINO80 associates with spindle microtubule during mitosis, and its deficiency leads to defective microtubule assembly and abnormal chromosome segregation. Consistent with these results, hINO80 is critical for suppressing aneuploidy and structural chromosome abnormalities. This work therefore not only emphasizes the evolutionary importance of INO80 in DNA replication but also reveals a new role for this remodeler in chromosome segregation, both of which likely come into play in maintaining the genome integrity.

Stabilization and targeting of INO80 to replication forks by BAP1 during normal DNA synthesis

The INO80 chromatin-remodelling complex has been implicated in DNA replication during stress in yeast. However, its role in normal DNA replication and its underlying mechanisms remain unclear. Here, we show that INO80 binds to replication forks and promotes fork progression in human cells under unperturbed, normal conditions. We find that Ino80, which encodes the catalytic ATPase of INO80, is essential for mouse embryonic DNA replication and development. Ino80 is recruited to replication forks through interaction with ubiquitinated H2A--aided by BRCA1-associated protein-1 (BAP1), a tumour suppressor and nuclear de-ubiquitinating enzyme that also functions to stabilize Ino80. Importantly, Ino80 is downregulated in BAP1-defective cancer cells due to the lack of an Ino80 stabilization mechanism via BAP1. Our results establish a role for INO80 in normal DNA replication and uncover a mechanism by which this remodeler is targeted to replication forks, suggesting a molecular basis for the tumour-suppressing function of BAP1.

Multiplex analysis of cytokines in the serum and cerebrospinal fluid of patients with Alzheimer's disease by color-coded bead technology.

Background and purpose The availability and promise of effective treatments for neurodegenerative disorders are increasing the importance of early diagnosis. Having molecular and biochemical markers of Alzheimers disease (AD) would complement clinical approaches, and further the goals of early and accurate diagnosis. Combining multiple biomarkers in evaluations significantly increases the sensitivity and specificity of the biochemical tests. Methods In this study, we used color-coded bead-based Luminex technology to test the potential of using chemokines and cytokines as biochemical markers of AD. We measured the levels of 22 chemokines and cytokines in the serum and cerebrospinal fluid (CSF) of 32 de novo patients (13 controls, 11 AD, and 8 Parkinsons disease [PD]). Results MCP-1 was the only cytokine detectable in CSF, and its levels did not differ between control and disease groups. However, the serum concentration of eotaxin was significantly higher in AD patients than in the control group. Conclusions The analysis of multiple inflammatory mediators revealed marginal differences in their CSF and serum concentrations for the differential diagnosis of AD and PD. These results provide evidence that immunological responses are not major contributors to the pathogenesis of AD and PD.

Genome-Wide Screen of Human Bromodomain-Containing Proteins Identifies Cecr2 as a Novel DNA Damage Response Protein

The formation of γ-H2AX foci after DNA double strand breaks (DSBs) is crucial for the cellular response to this lethal DNA damage. We previously have shown that BRG1, a chromatin remodeling enzyme, facilitates DSB repair by stimulating γ-H2AX formation, and this function of BRG1 requires the binding of BRGI to acetylated histone H3 on γ-H2AX-containing nucleosomes using its bromodomain (BRD), a protein module that specifically recognizes acetyl-Lys moieties. We also have shown that the BRD of BRG1, when ectopically expressed in cells, functions as a dominant negative inhibitor of the BRG1 activity to stimulate γ-H2AX and DSB repair. Here, we found that BRDs from a select group of proteins have no such activity, suggesting that the γ-H2AX inhibition activity of BRG1 BRD is specific. This finding led us to search for more BRDs that exhibit γ-H2AX inhibition activity in the hope of finding additional BRD-containing proteins involved in DNA damage responses. We screened a total of 52 individual BRDs present in 38 human BRD-containing proteins, comprising 93% of all human BRDs. We identified the BRD of cat eye syndrome chromosome region candidate 2 (Cecr2), which recently was shown to form a novel chromatin remodeling complex with unknown cellular functions, as having a strong γ-H2AX inhibition activity. This activity of Cecr2 BRD is specific because it depends on the chromatin binding affinity of Cecr2 BRD. Small interfering RNA knockdown experiments showed that Cecr2 is important for γ-H2AX formation and DSB repair. Therefore, our genomewide screen identifies Cecr2 as a novel DNA damage response protein.

Targeting BRG1 Chromatin Remodeler via Its Bromodomain for Enhanced Tumor Cell Radiosensitivity In Vitro and In Vivo

Radiotherapy treats cancer by inducing DNA double-strand breaks (DSB) in tumor cells using ionizing radiation. However, DNA repair in tumor cells often leads to radioresistance and unsuccessful outcome. Inhibition of DNA repair by targeting repair proteins can increase radiosensitivity of tumor cells. The BRG1 chromatin remodeling enzyme assists DSB repair by stimulating γ-H2AX formation and BRG1 binding to acetylated histones at DSBs via bromodomain (BRD) is critical for this activity. Here, we show that ectopic expression of BRG1-BRD inhibited γ-H2AX and DSB repair after irradiation and increased the radiosensitivity in various human cancer cells, including HT29 colon cancer. Dimerization of BRG1-BRD, increasing its chromatin binding affinity, aggravated the defects in γ-H2AX and DSB repair and further enhanced the radiosensitivity. While little affecting the upstream ATM activation, BRG1-BRD in irradiated HT29 cells inhibited the recruitment of 53BP1 to damaged chromatin, the downstream event of γ-H2AX, and compromised the G2–M checkpoint and increased apoptosis. Importantly, in a xenograft mouse model, BRG1-BRD increased the radiosensitivity of HT29 tumors, which was further enhanced by dimerization. These data suggest that BRG1-BRD radiosensitizes tumor cells by a dominant negative activity against BRG1, which disrupts γ-H2AX and its downstream 53BP1 pathways, leading to inefficient DNA repair, G2–M checkpoint defect, and increased apoptosis. This work therefore identifies BRG1-BRD as a novel tumor radiosensitizer and its action mechanism, providing the first example of chromatin remodeler as a target for improving cancer radiotherapy. Mol Cancer Ther; 14(2); 597–607. ©2014 AACR.

Genome-wide reorganization of histone H2AX toward particular fragile sites on cell activation

γH2AX formation by phosphorylation of the histone variant H2AX is the key process in the repair of DNA lesions including those arising at fragile sites under replication stress. Here we demonstrate that H2AX is dynamically reorganized to preoccupy γH2AX hotspots on increased replication stress by activated cell proliferation and that H2AX is enriched in aphidicolin-induced replisome stalling sites in cycling cells. Interestingly, H2AX enrichment was particularly found in genomic regions that replicate in early S phase. High transcription activity, a hallmark of early replicating fragile sites, was a determinant of H2AX localization. Subtelomeric H2AX enrichment was also attributable to early replication and high gene density. In contrast, late replicating and infrequently transcribed regions, including common fragile sites and heterochromatin, lacked H2AX enrichment. In particular, heterochromatin was inaccessible to H2AX incorporation, maybe partly explaining the cause of mutation accumulation in cancer heterochromatin. Meanwhile, H2AX in actively dividing cells was intimately colocalized with INO80. INO80 silencing reduced H2AX levels, particularly at the INO80-enriched sites. Our findings suggest that active DNA replication is accompanied with the specific localization of H2AX and INO80 for efficient damage repair or replication-fork stabilization in actively transcribed regions.