Understanding chromatin mechanisms involved in DNA damage and chemotherapeutic responses

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dc.contributor.advisorMiller, Kyle M.
dc.contributor.committeeMemberIyer, Vishwanath R
dc.contributor.committeeMemberPaull, Tanya T
dc.contributor.committeeMemberVasquez, Karen M
dc.contributor.committeeMemberXhemalce, Blerta
dc.creatorAgarwal, Poonam
dc.date.accessioned2018-01-30T16:44:05Z
dc.date.available2018-01-30T16:44:05Z
dc.date.created2017-12
dc.date.issued2018-01-24
dc.date.submittedDecember 2017
dc.date.updated2018-01-30T16:44:05Z
dc.description.abstractOne of the hallmarks of cancer is genomic instability driven by DNA damage. Cells respond to these genetic insults through chromatin-based mechanisms that repair the damage. Chromatin plays a pivotal role in protecting cells from genome and epigenome instability that drive cancer progression. Chromatin, a highly dynamic complex of DNA and proteins, undergoes structural and functional changes in response to cellular factors that are essential for replication, transcription, DNA damage responses (DDR) and repair. Chromatin structure and function are highly dependent on histone modifications. Histones are modified on distinct amino acids by post-translational modifications (PTMs). Delineating chromatin-regulated processes are fundamental for understanding the nuclear pathways that regulate access to, and protection of, our genetic and epigenetic information. The first part of my project focused on analyzing the contribution of a chromatin domain, the nucleosome acidic patch in regulating histone H2A/X ubiquitination and the DDR using in vitro and in vivo approaches. I established techniques to biochemically purify human recombinant histones and reconstituted nucleosome core particles (NCPs) containing WT or acidic patch mutant H2A/X for in vitro Ub assays with purified E3 ligases, RNF168 and RING1B/BMI1. Both E3s ubiquitinated H2A/X within WT NCPs but not mutant NCPs. Thus, this assay confirmed our hypothesis that the effect of the acidic patch mutation on H2AX/ H2Aub is direct and that it mediates site-specific ubiquitinations. I showed that the acidic patch interacting peptide LANA could compete with RNF168 and RING1B/BMI1 dependent H2AX/H2A Ub. In the second project, I tested how chromatin alters targeting of an anticancer drug using a cisplatin derivative that acts on the genome. I identified that cotreatment of cisplatin and the clinically approved drug Vorinostat/SAHA generated clusters of lesions that co-localized with translesion synthesis factors. However, I found that activated translesion synthesis no longer acted as a bypass mechanism but instead promoted apoptosis. These results demonstrated that pharmacological alterations of chromatin reprograms genome targeting with platinum drugs and, concomitantly, drug response. The third project for my thesis work involves functional analysis of the bromodomain containing TRIM proteins in DDR. These proteins belong to the bromodomain (BRD) family, which are the readers of PTM acetylation. I identified specific domains in TRIM24 required for its recruitment to damaged DNA and its dependency on other chromatin associated factors, namely, SUV39H1, KAT6B, TRIM28, TRIM33 that regulate TRIM24 dynamics in the context of DNA damage. I validated some interactors of TRIM24, TRIM28 and TRIM33 including the FACT and MCM complex. In summary, knowledge gained from these studies will help to understand how these BRD reader proteins promote the DDR within acetylated chromatin to preserve genome stability.
dc.description.departmentCellular and Molecular Biology
dc.format.mimetypeapplication/pdf
dc.identifierdoi:10.15781/T2K64B91K
dc.identifier.urihttp://hdl.handle.net/2152/63290
dc.language.isoen
dc.subjectDNA damage
dc.subjectChromatin
dc.titleUnderstanding chromatin mechanisms involved in DNA damage and chemotherapeutic responses
dc.typeThesis
dc.type.materialtext
thesis.degree.departmentCellular and Molecular Biology
thesis.degree.disciplineCell and Molecular Biology
thesis.degree.grantorThe University of Texas at Austin
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy

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