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Identification of Novel Genes in BRCA1-Regulated Pathways

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Identification of Novel Genes in BRCA1-Regulated Pathways DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Shweta Kotian, M.S. Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2013 Dissertation Committee: Professor Jeffrey D. Parvin, Advisor Professor Joanna L. Groden Professor Denis C. Guttridge Professor Mark R. Parthun Copyright by Shweta Kotian 2013 Abstract BRCA1 is an important breast- and ovarian-specific tumor suppressor gene. It is important in various cellular functions in the body including transcriptional regulation, cell cycle checkpoint activation, DNA damage response, and maintenance of genomic stability. Approximately 40% of hereditary breast cancers have mutations in BRCA1 or BRCA2, and it is unclear how the remaining cases are caused, presumably by mutations or in the alteration of expression of unknown genes. We hypothesize that these unknown genes or ‘missing BRCAs’ can be identified by bio-informatics methods. Performing gene co-expression analysis, we have compared the expression profiles of hundreds of genes across publicly available breast cancer microarray datasets, with those of BRCA1 and BRCA2. We propose that the genes, whose expression is highly correlated with BRCA1 and BRCA2, might function together in the same pathways. They could also potentially interact with each other. The main aim of this study is to identify these genes, and then biologically validate them in functional BRCA1-regulated pathways including homologous recombination and centrosome duplication. We also hope to find any mechanistic ii associations between these genes and BRCA1. Eventually, we hope to evaluate if these genes contribute to the process of carcinogenesis. As a result of the informatics data mining, we narrowed down the list of genes co- expressed with BRCA1 to a few dozen. Of these genes, we decided to choose the histone deacetylases (HDACs) and NUSAP1 for biological validation. We had several criteria for choosing these, such as 1) the presence of enzymatic domains in the case of the HDAC proteins, 2) an unknown association with BRCA1, 3) a paucity of the literature about this gene/protein; and 4) cancer- associated changes in gene expression. Biological validation was performed using two main assays: homology-directed repair and centrosome duplication. BRCA1 is important for proper DNA repair, as well as regulation of centrosome numbers. When BRCA1 is depleted from cells in these assays using RNA interference, homologous recombination is impaired, and there is aberrant centrosome duplication causing formation of supernumerary centrosomes. Likewise, we were interested in determining if the HDACs or NUSAP1 would be important for these processes as well. The HDAC family catalyzes the deacetylation of histones on chromatin causing chromatin compaction. This is turn, suppresses transcription. In this way, HDACs help regulate the expression of key developmental, cell cycle, and transcription factors. We observed that multiple members of the HDAC family of genes were co-expressed with BRCA1 and BRCA2 in multiple gene expression datasets. Dysregulation of HDACs has been observed in several cancers, and there is iii much interest in the development of HDAC inhibitor drugs. These drugs inhibit the growth of cancer cells, but it is not known how. Thus, we decided to test HDAC function in homologous recombination using these inhibitors. A tissue- culture based homology directed repair (HDR) assay was used in which repair of a double-stranded break, by homologous recombination, results in gene conversion of an inactive GFP allele to an active GFP gene. Homologous recombination events were then readily scored by measuring GFP positive cells by FACS. We found that treatment with HDAC inhibitors significantly reduced homologous recombination levels. Upon carrying out an individual depletion of HDACs 1 to 11 by RNA interference, we found that depletions of HDAC9 and HDAC10 specifically reduce homologous recombination levels. This was corroborated as HDAC9 and HDAC10 depletion sensitized cells to the interstrand crosslinker mitomycin C, indicating faulty homologous recombination. We think that HDAC9 and HDAC10 catalyze a crucial deacetylation step at the site of the double strand break that is important for homologous recombination. NUSAP1 is important for proper mitotic spindle assembly and cytokinesis. It is also aberrantly expressed in several cancers. We performed NUSAP1 depletion in the HDR assay, and saw that homologous recombination was impaired. In addition, depletion of NUSAP1 in the centrosome assay, caused centrosome amplification. Over-expression of BRCA1 reversed the defective phenotypes seen in both these assays upon NUSAP1 depletion. While NUSAP1 depletion reduced BRCA1 protein levels in the soluble extracts, and specifically in the iv chromatin fractions, there was no change in total BRCA1 protein levels. This indicated that NUSAP1 controls BRCA1 localization. We further confirmed this by determining that NUSAP1 depletion inhibited recruitment of BRCA1 to DNA damage-induced foci upon ionizing irradiation. In addition to BRCA1, RAD51 recruitment to the DNA damage foci was affected. Thus, NUSAP1 impacts the DNA damage response through control of BRCA1 localization. Thus, we have successfully identified and validated three novel genes- HDAC9, HDAC10, NUSAP1 as important in the BRCA1-regulated pathway of homologous recombination. NUSAP1 is also important for centrosome amplification. Since, these genes are implicated in cancer; we hope that our study will help shed light on exactly how they are important for tumorigenesis. Further work will determine whether these proteins may be useful as biomarkers for breast cancer. v Dedication I dedicate this document to my grandparents, the late Mr. Rajeeva Suvarna and the late Mrs. Vimala Suvarna. Words cannot describe how truly grateful I am for their loving care and support in my crucial, formative years. vi Acknowledgments I would like to offer my humble gratitude to my advisor Dr. Jeffrey D. Parvin. Not only is he an immense source of knowledge, but he also has immense patience. I am thankful to him for his invaluable guidance, but most of all for not giving up on me during a very difficult phase in my life. Thanks are due to the members of my lab who have always offered help willingly when needed. I would also like to make a special mention of my colleague, Ms. Mansi Arora, for offering both professional and personal support. I would like to sincerely thank my committee members, Dr. Joanna L. Groden, Dr. Mark R. Parthun, and Dr. Denis C. Guttridge, for offering their valuable time and expertise to help guide me with my dissertation. I would also like to thank my program chair, Dr. David Bisaro, for always being willing to lend a listening ear to even a graduate student’s petty problems, and offer practical advice. This dissertation would not be complete without acknowledging some key people in my life. I will always be grateful to my best friend and partner, Mr. Daniel Stanojevic, who has provided tremendous emotional support to me during this journey. Last, but not least, I am eternally indebted to my mother, Ms. Shashikala vii Kotian, whose fortitude and personal sacrifices, have made it possible for me to reach this personal milestone in my life. viii Vita 2004 ....................................................... B.S. Biochemistry, University of Mumbai 2006 ....................................................... M.S. Biochemistry, University of Mumbai 2006 to present ...................................... Graduate Research Associate, Department of Biomedical Informatics, The Ohio State University Publications Kotian S, Parvin JD. NUSAP1 Influences the DNA damage response by controlling BRCA1 localization. Manuscript in preparation Zhang J, Lu K, Xiang Y, Islam M, Kotian S, Kais Z, Lee C, Arora A, Liu H, Parvin JD, Huang K (2012). Weighted frequent gene co-expression network mining to identify genes involved in genome stability. PLoS Comput Biol. 8(8):e1002656. Epub 2012 Aug 30. Kotian S, Liyanarachchi S, Zelent A, Parvin JD (2011). Histone deacetylases 9 and 10 are required for homologous recombination. J Biol Chem, 286(10):7722-6 ix Fields of Study Major Field: Molecular, Cellular and Developmental Biology x Table of Contents Abstract .................................................................................................................ii Dedication ............................................................................................................vi Acknowledgments ............................................................................................... vii Vita .......................................................................................................................ix Publications ..........................................................................................................ix Fields of Study ...................................................................................................... x Table of Contents .................................................................................................xi List of Tables ...................................................................................................... xvi List of Figures .................................................................................................... xvii Chapter 1:
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