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Date of Award

Winter 12-15-2017

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Genetics & Genomics)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Cooperative binding of transcription factors is known to be important in the regulation of gene expression programs conferring cellular identities. However, current methods to measure cooperativity parameters have been laborious and therefore limited to studying only a few sequence variants at a time. This thesis describes a novel method, Coop-seq (cooperativity by sequencing), that is capable of efficiently and accurately determining the cooperativity parameters for hundreds of different DNA sequences in a single experiment. We apply Coop-seq to 12 dimer pairs from the Sox and POU families of transcription factors using 324 unique sequences with changed half-site orientation, altered spacing and discrete randomization within the binding elements. The study reveals specific dimerization profiles of different Sox factors with Oct4. Additionally, we used Spec-seq, which allows for the efficient and accurate determination of relative affinity to a large collection of sequences in parallel to find more preferential differences between cooperative DNA binding of IRF4, IRF8 and BATF family members. We found the IRF proteins binding adjacent to BATF sites increases affinity substantially compared to sequences with spacings between the sites, indicating cooperative binding through protein-protein interactions. These in vitro preferences aid in the understanding of in vivo binding activities.

Language

English (en)

Chair and Committee

Gary Stormo

Committee Members

James J. Havranek, Rob Mitra, Joseph C. Corbo, Henry V. Huang,

Comments

Permanent URL: https://doi.org/10.7936/K73R0S99

Available for download on Wednesday, December 15, 2117

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