DF/HCC Program Affiliation

Cancer Cell Biology
Cancer Genetics

Research Abstract

Down regulating DNA repair- phosphatases and micro RNAs

Activation of DNA repair pathways have been well studied for decades with very limited research on down regulation. Most importantly, the physiological relevance of down modulating DNA repair is still unclear. We have focused on two aspects of down regulation, one is dephosphorylation of repair proteins via phosphatases, and second is decreased expression of repair factors via micro RNAs (miRNAs).

One of the earliest events in the double stranded DNA break (DSB) response is the phosphorylation of the histone H2A variant, H2AX. The phosphorylated form of H2AX (gamma-H2AX) has a role in repair, replication, recombination of DNA and regulating cell cycle. Although the formation of g-H2AX has been well studied very little is known about its down regulation. In two parallel studies – one in mammals, the other in S. cerevisiae – we identified roles for PP2A family phosphatases in g-H2AX dephosphorylation. We found that PP2AC specifically removes g-H2AX foci formed in mammalian cells in response to exogenous DNA damage. At the same time in a collaborative study we identified a Pph3 phosphatase-containing trimeric complex in yeast that regulates basal g-H2AX levels. Although both PP2AC and Pph3 efficiently dephosphorylate g-H2AX in vitro, their roles in cells appeared to be distinct leading to the speculation that more than one phosphatase might be involved in g-H2AX removal in mammalian cells. Investigating this question we found a trimeric PP4C-containing phosphatase complex in mammalian cells that controls basal levels ofg-H2AX in the absence of exogenous DNA damage. Although several putative PP4C-containing complexes have been identified, their biological function is not well defined. We are continuing to investigate the cellular roles of PP4.

MiRNAs are small non-coding RNAs that typically dampen gene expression and are mis-expressed in many cancer cells. DNA repair proteins represent a group of genes whose inappropriate downregulation leads to genomic instability, the underlying cause of cancer. We hypothesize that miRNAs downregulate the expression of DNA repair proteins. When do normal cells attenuate DNA repair? There are two scenarios, one in terminally differentiated cells where overall DSB repair is downregulated with decreased levels of repair factors and second, in different phases of the cell cycle where specific DSB pathways are selectively suppressed. Using microarrays we have identified miRNAs whose levels change radically during differentiation and/or in the course of the cell cycle, potentially influencing the choice of repair pathway. We use a combination of computational tools and a novel biochemical strategy to identify targets of these miRNAs. As proof of principle and method, we find that in blood cells miR-24 gets overexpressed with differentiation, downregulates H2AX, and significantly impacts DNA repair. In a complementary approach a miRNA expression library is being screened to identify miRNAs that affect the repair of a single DSB using a reporter system. A role for miRNAs in DNA repair is pertinent for understanding DNA replication and recombination.

Publications

• Wang B, Zhao A, Sun L, Zhong X, Zhong J, Wang H, Cai M, Li J, Xu Y, Liao J, Sang J, Chowdhury D, Pfeifer GP, Yen Y, Xu X.Protein phosphatase PP4 is overexpressed in human breast and lung tumors.Cell Res 2008 Aug 12.
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• Chowdhury D, Xu X, Zhong X, Ahmed F, Zhong J, Liao J, Dykxhoorn DM, Weinstock DM, Pfeifer GP, Lieberman J.A PP4-phosphatase complex dephosphorylates gamma-H2AX generated during DNA replication.Mol Cell 2008 Jul 11;31(1):33-46.
  18614045
• Dykxhoorn DM, Chowdhury D, Lieberman J.RNA interference and cancer: endogenous pathways and therapeutic approaches.Adv Exp Med Biol 2008;615:299-329.
  18437900
• Chowdhury D, Beresford PJ, Zhu P, Zhang D, Sung JS, Demple B, Perrino FW, Lieberman J.The exonuclease TREX1 is in the SET complex and acts in concert with NM23-H1 to degrade DNA during granzyme A-mediated cell death.Mol Cell 2006 Jul 7;23(1):133-42.
  16818237
• Chowdhury D, Keogh MC, Ishii H, Peterson CL, Buratowski S, Lieberman J.gamma-H2AX dephosphorylation by protein phosphatase 2A facilitates DNA double-strand break repair.Mol Cell 2005 Dec 9;20(5):801-9.
  16310392
• Keogh MC, Kim JA, Downey M, Fillingham J, Chowdhury D, Harrison JC, Onishi M, Datta N, Galicia S, Emili A, Lieberman J, Shen X, Buratowski S, Haber JE, Durocher D, Greenblatt JF, Krogan NJ.A phosphatase complex that dephosphorylates gammaH2AX regulates DN
  16299494
• Chowdhury D, Novina CD.RNAi and RNA-Based Regulation of Immune System Function.Adv Immunol 2005;88:267-92.
  16227093