Date of Award

2018

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Gerd A. Blobel

Abstract

Transcription factors (TFs) occupy chromatin to coordinate widespread regulation of gene expression programs that define cellular identity. These proteins bind diverse DNA sequences genome-wide and in a non-uniform manner across the cell cycle, yet the spatial and temporal determinants of TF chromatin occupancy are largely unknown. First, to define DNA sequence determinants of in vivo TF binding, we developed an approach that exploits natural genetic variation between highly similar erythroid cell lines. From ChIP-seq data we were able to directly identify extensive single nucleotide variants that discriminate these cell lines from each other. By measuring the impact of these variants on TF ChIP-seq binding intensities, we defined at single nucleotide resolution the binding determinants of the GATA1, TAL1, and CTCF factors. We also identified contextual sequences that, in addition to a TF’s core DNA motif, dictate TF specificity and modulate TF binding when mutated. Together, these studies present new approaches and biological insights regarding the DNA sequence requirements for TF binding to chromatin. Next, we tested whether TF chromatin occupancy is dynamic across the cell cycle. In particular, we asked whether the BRD4 TF binds chromatin in erythroid cells during mitosis, and whether it might function as a bookmark of transcriptional programs across mitosis. While we find that BRD4 is preferentially enriched during mitosis at erythroid-specific genes in the G1E-ER4 erythroid cell line, transient removal of BRD4 from chromatin during mitosis does not impair the reactivation of erythroid-specific programs following mitosis. Thus, BRD4 does not function as a bookmark of transcription, and instead we considered that it might passively bind to acetylated histones during mitosis. Given that the histone PTM landscape during mitosis has not been previously characterized, we used histone mass spectrometry and genome-wide location analysis to find extensive preservation of H3K14ac, H3K122ac, and H4K16ac on mitotic chromatin. Furthermore, these marks are predictive of BRD4 and Pol II binding to mitotic chromatin and are preferentially enriched at erythroid-specific genes during mitosis, suggestive of a role in mitotic bookmarking. Together, these studies reveal new insights about the mechanisms by which transcription factors occupy chromatin.

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