DNA encodes the genetic material to instruct all cellular process and to establish cellular identity. Cellular identity is established by both genetic content and regulation of gene expression. Gene expression is regulated by many factors including chromatin structure in eukaryotes. Chromatin structure consists of nucleosomes, comprised of ~150 bp DNA wrapped around a histone octamer. This structure regulates several DNA dependent processes including transcription and DNA damage repair. Understanding the mechanisms that regulate chromatin structure is key to understanding how biological systems are controlled in the cell. DNA dependent cellular process are known to be regulated by chromatin remodelers. Chromatin remodelers couple the energy of ATP hydrolysis to slide nucleosomes, transfer histones, and/or distort the octamer? activities that are essential to regulate the chromatin state. A unique chromatin remodeler, INO80, requires both the DEAD family ATPase and accessory subunits to slide mononucleosomes in vitro. In vivo, INO80 plays a role in both transcription and DNA damage repairs. However, it is unclear how INO80 sliding activity contributes to its diverse biological roles. Recent work from our lab revealed the following about the INO80 mechanism: (1) INO80 is regulated by two nucleosome cues: flanking DNA length and an acidic patch on histones; (2) INO80 remodeling reaction has at least one intermediate state and transition between this intermediate to a sliding product is regulated by DNA length; (3) the Nhp10 accessory module regulates the transition between intermediate state and sliding activity. With these findings, I hypothesize that INO80 senses various nucleosome cues through the accessory subunits and that these interactions contribute to the kinetic steps of a INO80 remodeling reaction. I also hypothesize that distributing the regulation of INO80 across different nucleosome cues and accessory subunits allows INO80 to respond accordingly to nucleosome cues specific to various biological contexts. This research will first investigate the intermediate nucleosome states generated by INO80 remodeling and investigate the coordination between nucleosome substrate cues and accessory modules in regulating the kinetic steps in INO80 reaction. I specifically aim to (1) determine the intermediate nucleosome structures generated by INO80; (2) determine how the intermediate step of flanking DNA unpeeling regulates the INO80 reaction; (3) determine how INO80 accessory modules regulate the ability of INO80 to sense and respond to flanking DNA length; and (4) determine how the INO80 accessory modules regulate the ability of INO80 to sense and respond to histone cues.

Public Health Relevance

DNA encodes the genetic material essential for cellular identity, hence regulating the delivery and maintenance of the genetic code is essential. Chromatin structure and dynamics, mediated by chromatin remodelers such as INO80, regulate DNA dependent processes like transcription and DNA damage repair. Understanding the biochemical mechanisms that regulate INO80 chromatin remodeling activity will bridge our understanding how of changes in chromatin dynamics contribute to biological function.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1)
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Brown, Patrick
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University of California San Francisco
Schools of Medicine
San Francisco
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