Capturing Transcriptional Dynamics using Nascent RNA Sequencing.

Abstract

Transcription plays an essential role in establishing cellular identity and functional control, and each step of the transcriptional process represents a potential point of regulation. Methods that survey RNA abundance within a given cell type can be used to study steady state levels based on the balance between RNA synthesis and turnover. However, a thorough examination of how the transcriptional process contributes to gene expression regulation requires observation of the earliest stages of RNA production. The work presented in this dissertation explored early transcriptional events using Bru-seq nascent RNA sequencing. This technique allows for the detection of changes in RNA synthesis across gene bodies prior to the completion of full length transcripts. Using Bru-seq, I was able to monitor genome-wide expression changes during the first two hours following serum stimulation of human fibroblast cells. This led to the identification of over 2000 genes that demonstrated a transcriptional response to serum activation, including a novel group of genes which were immediately repressed. Response genes were categorized according to distinct transcriptional induction and repression patterns, providing new candidate gene groups for studying common regulatory mechanisms during global transcriptional responses. Additionally, I took advantage of the Bru-seq technique to follow transcription elongation in long genes over time. The dataset revealed how gene length influences transcriptional timing, and demonstrated that a set of genes with different sizes can be simultaneously induced but expressed at various times. Because relative gene size is conserved in mammals, this suggests that gene length plays an important role in maintaining proper temporal expression patterns. Lastly, I used a modification of this technique, BruUV-seq, to identify active enhancer elements based on enhancer RNA production, and to observe the effects of inhibiting BRD4, an important transcriptional coactivator and enhancer chromatin regulator. This dataset indicated that BRD4 inhibition results in immediate disruption of enhancer transcription, and that BRD4 function is required for the maintenance of enhancer activity. Collectively, this work extends our knowledge of how early transcriptional events impact the regulation of gene expression, and provides a foundation for future studies exploring the precise mechanisms which determine cellular identity and functional control.