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1.  Damaged DNA-binding protein down-regulates epigenetic mark H3K56Ac through histone deacetylase 1 and 2 
Mutation research  2015;776:16-23.
Acetylated histone H3 lysine 56 (H3K56Ac) is one of the reversible histone post-translational modifications (PTMs) responsive to DNA damage. We previously described a biphasic decrease and increase of epigenetic mark H3K56Ac in response to ultraviolet radiation (UVR)-induced DNA damage. Here, we report a new function of UV damaged DNA-binding protein (DDB) in deacetylation of H3K56Ac through specific histone deacetylases (HDACs). We show that simultaneous depletion of HDAC1/2 compromises the deacetylation of H3K56Ac, while depletion of HDAC1 or HDAC2 alone has no effect on H3K56Ac. The H3K56Ac deacetylation does not require functional nucleotide excision repair (NER) factors XPA and XPC, but depends on the function of upstream factors DDB1 and DDB2. UVR enhances the association of DDB2 with HDAC1 and, enforced DDB2 expression leads to translocation of HDAC1 to UVR-damaged chromatin. HDAC1 and HDAC2 are recruited to UVR-induced DNA damage spots, which are visualized by anti-XPC immunofluorescence. Dual HDAC1/2 depletion decreases XPC ubiquitination, but does not affect the recruitment of DDB2 to DNA damage. By contrast, the local accumulation of γH2AX at UVR-induced DNA damage spots was compromised upon HDAC1 as well as dual HDAC1/2 depletions. Additionally, UVR-induced ATM activation decreased in H12899 cells expressing H3K56Ac-mimicing H3K56Q. These results revealed a novel role of DDB in H3K56Ac deacetylation during early step of NER and the existence of active functional cross-talk between DDB-mediated damage recognition and H3K56Ac deacetylation.
PMCID: PMC5053336  PMID: 26255936
Histone post-translational modifications; Nucleotide excision repair; Damaged DNA-binding protein 2; Histone deacetylases; Acetylated histone H3 lysine 56; Xeroderma pigmentosum group E
2.  NER Initiation Factors, DDB2 and XPC, Regulate UV Radiation Response by Recruiting ATR and ATM Kinases to DNA Damage Sites 
DNA repair  2013;12(4):273-283.
ATR and ATM kinases are central to the checkpoint activation in response to DNA damage and replication stress. However, the nature of the signal, which initially activates these kinases in response to UV damage, is unclear. Here, we have shown that DDB2 and XPC, two early UV damage recognition factors, are required for the damage-specific ATR and ATM recruitment and phosphorylation. ATR and ATM physically interacted with XPC and promptly localized to the UV damage sites. ATR and ATM recruitment and their phosphorylation were negatively affected in cells defective in DDB2 or XPC functions. Consequently, the phosphorylation of ATR and ATM substrates, Chk1, Chk2, H2AX, and BRCA1 was significantly reduced or abrogated in mutant cells. Furthermore, UV exposure of cells defective in DDB2 or XPC resulted in a marked decrease in BRCA1 and Rad51 recruitment to the damage site. Conversely, ATR- and ATM-deficiency failed to affect the recruitment of DDB2 and XPC to the damage site, and therefore did not influence the NER efficiency. These findings demonstrate a novel function of DDB2 and XPC in maintaining a vital cross-talk with checkpoint proteins, and thereby coordinating subsequent repair and checkpoint activation.
PMCID: PMC4174315  PMID: 23422745
ATM; ATR; XPC; DDB2; UV irradiation; checkpoint activation; nucleotide excision repair
3.  p38 MAPK- and Akt-mediated p300 phosphorylation regulates its degradation to facilitate nucleotide excision repair 
Nucleic Acids Research  2012;41(3):1722-1733.
Besides the primary histone acetyltransferase (HAT)-mediated chromatin remodeling function, co-transcriptional factor, p300, is also known to play a distinct role in DNA repair. However, the exact mechanism of p300 function in DNA repair has remained unclear and difficult to discern due to the phosphorylation and degradation of p300 in response to DNA damage. Here, we have demonstrated that p300 is only degraded in the presence of specific DNA lesions, which are the substrates of nucleotide excision repair (NER) pathway. In contrast, DNA double-strand breaks fail to degrade p300. Degradation is initiated by phosphorylation of p300 at serine 1834, which is catalyzed by the cooperative action of p38 mitogen-activated protein kinases and Akt kinases. In depth, functional analysis revealed that (i) p300 and CBP act redundantly in repairing ultraviolet (UV) lesions, (ii) the phosphorylation of p300 at S1834 is critical for efficient removal of UV-induced cyclobutane pyrimidine dimers and (iii) p300 is recruited to DNA damage sites located within heterochromatin. Taken together, we conclude that phosphorylated p300 initially acetylates histones to relax heterochromatin to allow damage recognition factors access to damage DNA. Thereupon, p300 is promptly degraded to allow the sequential recruitment of downstream repair proteins for successful execution of NER.
PMCID: PMC3561975  PMID: 23275565
4.  Targeting zymogen activation to control the matriptase-prostasin proteolytic cascade 
Journal of medicinal chemistry  2011;54(21):7567-7578.
Membrane-associated serine protease matriptase has been implicated in human diseases, and might be a drug target. In the present study, a novel class of matriptase inhibitors targeting zymogen activation is developed by a combination of the screening of compound library using a cell-based matriptase activation assay and a computer-aided search of commercially available analogs of a selected compound. Four structurally related compounds are identified that can inhibit matriptase activation with IC50 at low μM in both intact-cell and cell-free systems, suggesting that these inhibitors target the matriptase autoactivation machinery rather than the intracellular signaling pathways. These activation inhibitors can also inhibit prostasin activation, a downstream event that occurs in lockstep with matriptase activation. In contrast, the matriptase catalytic inhibitor CVS-3983 at a concentration 300-fold higher than its Ki fails to inhibit activation of either protease. Our results suggest that inhibiting matriptase activation is an efficient way to control matriptase function.
PMCID: PMC3214968  PMID: 21966950
5.  ASF1A and ATM regulate H3K56-mediated cell-cycle checkpoint recovery in response to UV irradiation 
Nucleic Acids Research  2011;39(18):7931-7945.
Successful DNA repair within chromatin requires coordinated interplay of histone modifications, chaperones and remodelers for allowing access of repair and checkpoint machineries to damaged sites. Upon completion of repair, ordered restoration of chromatin structure and key epigenetic marks herald the cell’s normal function. Here, we demonstrate such a restoration role of H3K56 acetylation (H3K56Ac) mark in response to ultraviolet (UV) irradiation of human cells. A fast initial deacetylation of H3K56 is followed by full renewal of an acetylated state at ~24–48 h post-irradiation. Histone chaperone, anti-silencing function-1 A (ASF1A), is crucial for post-repair H3K56Ac restoration, which in turn, is needed for the dephosphorylation of γ-H2AX and cellular recovery from checkpoint arrest. On the other hand, completion of DNA damage repair is not dependent on ASF1A or H3K56Ac. H3K56Ac restoration is regulated by ataxia telangiectasia mutated (ATM) checkpoint kinase. These cross-talking molecular cellular events reveal the important pathway components influencing the regulatory function of H3K56Ac in the recovery from UV-induced checkpoint arrest.
PMCID: PMC3185425  PMID: 21727091
6.  Human SNF5/INI1, a Component of the Human SWI/SNF Chromatin Remodeling Complex, Promotes Nucleotide Excision Repair by Influencing ATM Recruitment and Downstream H2AX Phosphorylation▿  
Molecular and Cellular Biology  2009;29(23):6206-6219.
Recent studies have implicated the role of the SWI/SNF ATP-dependent chromatin remodeling complex in nuclear excision repair (NER), but the mechanism of its function has remained elusive. Here, we show that the human SWI/SNF component human SNF5 (hSNF5) interacts with UV damage recognition factor XPC and colocalizes with XPC at the damage site. Inactivation of hSNF5 did not affect the recruitment of XPC but affected the recruitment of ATM checkpoint kinase to the damage site and ATM activation by phosphorylation. Consequently, hSNF5 deficiency resulted in a defect in H2AX and BRCA1 phosphorylation at the damage site. However, recruitment of ATR checkpoint kinase to the damage site was not affected by hSNF5 deficiency, supporting that hSNF5 functions downstream of ATR. Additionally, ATM/ATR-mediated Chk2/Chk1 phosphorylation was not affected in hSNF5-depleted cells in response to UV irradiation, suggesting that the cell cycle checkpoint is intact in these cells. Taken together, the results indicate that the SWI/SNF complex associates with XPC at the damage site and thereby facilitates the access of ATM, which in turn promotes H2AX and BRCA1 phosphorylation. We propose that the SWI/SNF chromatin remodeling function is utilized to increase the DNA accessibility of NER machinery and checkpoint factors at the damage site, which influences NER and ensures genomic integrity.
PMCID: PMC2786693  PMID: 19805520
7.  Transcription of Human Resistin Gene Involves an Interaction of Sp1 with Peroxisome Proliferator-Activating Receptor Gamma (PPARγ) 
PLoS ONE  2010;5(3):e9912.
Resistin is a cysteine rich protein, mainly expressed and secreted by circulating human mononuclear cells. While several factors responsible for transcription of mouse resistin gene have been identified, not much is known about the factors responsible for the differential expression of human resistin.
Methodology/Principal Finding
We show that the minimal promoter of human resistin lies within ∼80 bp sequence upstream of the transcriptional start site (−240) whereas binding sites for cRel, CCAAT enhancer binding protein α (C/EBP-α), activating transcription factor 2 (ATF-2) and activator protein 1 (AP-1) transcription factors, important for induced expression, are present within sequences up to −619. Specificity Protein 1(Sp1) binding site (−276 to −295) is also present and an interaction of Sp1 with peroxisome proliferator activating receptor gamma (PPARγ) is necessary for constitutive expression in U937 cells. Indeed co-immunoprecipitation assay demonstrated a direct physical interaction of Sp1 with PPARγ in whole cell extracts of U937 cells. Phorbol myristate acetate (PMA) upregulated the expression of resistin mRNA in U937 cells by increasing the recruitment of Sp1, ATF-2 and PPARγ on the resistin gene promoter. Furthermore, PMA stimulation of U937 cells resulted in the disruption of Sp1 and PPARγ interaction. Chromatin immunoprecipitation (ChIP) assay confirmed the recruitment of transcription factors phospho ATF-2, Sp1, Sp3, PPARγ, chromatin modifier histone deacetylase 1 (HDAC1) and the acetylated form of histone H3 but not cRel, C/EBP-α and phospho c-Jun during resistin gene transcription.
Our findings suggest a complex interplay of Sp1 and PPARγ along with other transcription factors that drives the expression of resistin in human monocytic U937 cells.
PMCID: PMC2848011  PMID: 20360975

Results 1-7 (7)