Date of Award

2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Kenneth S. Zaret

Abstract

The genome is thought to be transcriptionally silent during mitosis. Decades of studies have used antibody-based detection of proteins in fixed cells to show that the majority of transcriptional machinery is absent from mitotic chromosomes. In addition, quantification of the incorporation of radio-labeled nucleotides has indicated a large reduction in RNA synthesis during mitosis, suggesting a global decrease in gene expression. Yet technical limitations have prevented the sensitive mapping of transcription during mitosis and mitotic exit. Thus, the means by which the previous interphase pattern of transcription is transduced to daughter cells and the hierarchy of gene reactivation during mitotic exit have been unclear.

Existing methodologies for labeling and sequencing nascent transcripts required lysis of the cell membrane and subsequent isolation of nuclei as the labels were not cell-permeable. In mitosis, the nuclear envelope is dismantled and no longer provides a barrier between the chromatin and the rest of the cell. As such, cell lysis results in loss of chromatin. To circumvent this issue, we used the cell-permeable transcript label, 5-ethynyluridine, to pulse-label nascent transcripts during mitosis and mitotic exit in intact cells. We find that many interphase genes exhibit measurable transcription in mitosis, as confirmed by interphase cell contamination modeling, FITC-UTP labeling, RNA FISH, and RT-qPCR. Further, the first round of transcription immediately following mitosis activates genes that are involved in the growth and rebuilding of daughter cells, rather than genes that promote cell type-specific functions.

Like the majority of transcripts, enhancer usage – as quantified by eRNA transcription rate – was also diminished in mitosis and reactivated in waves during mitotic exit. This observation is in agreement with previous studies that showed that promoter and not enhancer accessibility is retained in mitosis. We therefore propose a promoter-centric model of epigenetic memory during mitosis in which the basic cohort of genes expressed in interphase is largely retained at a low level through mitosis, whereas the relative amplitude of transcription observed in interphase is re-established by enhancers during mitotic exit as higher-order structures reform.

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