Date of Award

2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biology

First Advisor

Hua-Ying Fan

Abstract

Cockayne syndrome is a devastating premature aging disorder characterized by extreme sun sensitivity and severe neurological and developmental defects. The majority of Cockayne syndrome cases are due to mutations within the ATP-dependent chromatin remodeler Cockayne syndrome protein B (CSB). CSB functions in transcription regulation and is required for transcription-coupled nucleotide excision repair (TC-NER). Importantly, CSB is also critical for the relief of oxidative stress. Studies suggest CSB may participate in the major repair pathway for oxidative DNA damage repair, base excision repair (BER), yet direct evidence supporting this hypothesis remains elusive and exactly how CSB functions in this process is unknown. CSB is also suggested to function in the relief of oxidative stress by regulating transcription. We demonstrated CSB’s interaction with chromatin is stabilized by oxidative stress and the goal of this work is to understand the role of CSB in the relief of oxidative stress by characterizing the mechanisms underlying CSB’s oxidative stress-induced chromatin interaction. Using chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) we characterized for the first time CSB’s genome-wide occupancy following oxidative stress. This revealed CSB is enriched at the binding motif for the CCCTC-binding factor (CTCF), and further analysis demonstrated CSB and CTCF directly interact and regulate each other’s targeting to specific genomic loci in response to oxidative stress. This, in addition to an increase in CSB’s occupancy at promoters, suggests CSB and CTCF may cooperate in regulating transcription in response to oxidative stress, perhaps by mediating long-range chromatin interactions. Analysis of CSB mutant constructs revealed CSB’s oxidative stress-induced chromatin association occurs by a mechanism distinct from that of its interaction upon UV irradiation, where it is required for TC-NER, in that it does not require CSB’s ATP hydrolysis activity and is mediated by it’s ATPase and C-terminal domains. While the BER proteins OGG1 and APE1 did not impact CSB-chromatin association upon oxidative stress, another DNA repair protein, poly(ADP-ribose) polymerase 1 (PARP1) significantly enhances CSB’s interaction with chromatin in both a global and locus-specific manner. Together this work suggests CSB, CTCF, and PARP1 cooperate in the relief of oxidative stress by regulating transcription and facilitating DNA repair.

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