Date of Award

2016

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Gerd A. Blobel

Second Advisor

Arjun Raj

Abstract

Mitosis entails dramatic global alterations to genome structure and regulation, including

chromosome condensation, dissociation of the transcriptional machinery from chromosomes, and transcriptional silencing. Here I report studies that address the macromolecular accessibility of the mitotic genome and the control of transcriptional reactivation upon mitotic exit in a mammalian cell line. The results obtained from measuring the sensitivity of chromatin to DNase I cleavage by sequencing (DNase-seq) in pure mitotic cell populations demonstrate that macromolecular accessibility of the mitotic genome is widely preserved. Thus, steric hindrance from chromatin condensation is insufficient for explaining the eviction of transcription factors from mitotic chromatin. Several lines of evidence point to local modulation of chromatin accessibility; for example, promoters overall exceed predicted distal enhancers in their maintenance of accessibility during mitosis, suggesting the molecular control of these two classes of cis-regulatory elements are differentially susceptible to mitotic perturbation. Other characteristics associated with maintenance of chromatin accessibility during mitosis include the ubiquity of tissue distribution of interphase chromatin accessibility and large domains of DNA hypomethylation, whereas mitotic occupancy of hematopoietic transcription

factor GATA1 is not predictive of the degree of mitotic accessibility. To map the

transcriptional reactivation of the genome upon mitotic exit, anti-RNA polymerase II (Pol

II) chromatin immunoprecipitation-sequencing (ChIP-seq) was performed in a time course

spanning anaphase through late G1. This data enabled unambiguous observation of the

pioneering round of transcription upon mitotic exit, and classification of quantitative postmitotic transcriptional patterns across genes and distal enhancers aided by unsupervised pattern discovery. Surprisingly, the earliest rounds of transcription upon mitotic exit are hyperactive, compared to late G1. This post-mitotic spike in Pol II binding encompasses approximately 50% of active genes and intergenic enhancers, but the post-mitotic spike in gene transcription can be uncoupled from regulation by enhancers. Single-cell imaging by single-molecule RNA fluorescence in situ hybridization (FISH) of nascent and mature mRNAs demonstrates that this post-mitotic transcriptional spike can propagate to cell-to-cell heterogeneity in the mature mRNA levels of key developmental regulators. Together, these findings raise important considerations for the commonly proposed “mitotic bookmark” hypothesis of transcriptional memory, and have broad implications for the stability of gene expression states through cell divisions.

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