Abstract:
Gene expression is a fundamental process in living organisms that allows genetic information to be converted into a functional product. These gene products define cellular function, and so each cell type exhibits a unique expression pattern corresponding with its role. During the flow of information from gene to product, transcription is a critical first step. Importantly, each step of the transcription process represents a potential point of regulation. Researchers often survey all of the RNAs which are present in a given cell type in order to study transcription and gene expression. However, this approach usually defines RNA abundance based on a balance between the synthesis of complete transcripts and their degradation. A more thorough examination of how transcription is contributing to the regulation of gene expression requires observation of the earliest stages of RNA production.
The work presented here explores these early steps of transcription using Bru-seq nascent RNA sequencing. Through the use of this technique, I was able to observe complex patterns of productive initiation during cellular activation, resulting in global gene expression changes. This data exposed a multitude of different induction and repression patterns, which is likely related to efficient protein production for optimal function within response pathways. Additionally, I took advantage of the ability of the Bru-seq technique to monitor transcription across genes to follow transcription elongation over time. This data revealed the major influence of gene length in transcriptional timing, and how it plays a role in constructing elaborate patterns of gene expression. Lastly, I used a modification of this technique, BruUV-seq, to explore the role of transcriptional activity at enhancer elements in the regulation of gene transcription. This data includes the identification and examination of putative active enhancers, and demonstrated the effects of disrupting their activity on RNA production at enhancers and associated genes. 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 mechanisms which determine cellular identity and functional control.
