cis-Regulation in the Mammalian Rod Photoreceptor

<p>Transcription factors regulate the expression level of target genes by binding to <italic>cis</italic>-regulatory elements (CREs) present in gene promoters. The goal of my thesis research is to define the sequence components of CREs that determine transcriptional output. In order to accomplish this goal, I developed a method to measure the regulatory activity of thousands of CREs in a single experiment. In this method I insert unique barcodes in the 3'UTR of a reporter gene and multiplex expression measurements with RNA sequencing. Using this technique in explanted retinas, I determined the impact of single nucleotide variants in a mammalian promoter by measuring expression controlled by all single nucleotide variants of the Rhodopsin proximal promoter. I found that nearly all (86%) sequence variants drive significantly different activity than the wild-type promoter and that the mechanism of most variants can be interpreted as altered transcription factor binding. In addition, we found that the largest changes in expression resulted from variants located in characterized transcription factor binding site sequences. Next, I explored how combinations of binding sites drive particular levels of gene expression by utilizing a synthetic biology approach. I generated synthetic CREs composed of various combinations of binding sites found in the Rhodopsin promoter and measured the expression driven by these sequences. In this study I found that synthetic CREs containing binding sites for transcriptional activators yielded diverse expression outputs, including both activation and repression of a minimal promoter. Together, these experiments demonstrate that interactions between binding sites and dual regulation of a single binding site can produce diverse gene expression patterns. I conclude that simple <italic>cis</italic>-regulatory elements can produce complex expression outputs due to interactions between transcriptional activators and detailed quantitative models will be necessary to predict expression from these sequences.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biology and Biomedical Sciences: Molecular Genetics and Genomics

Year dc:date.available

2014

Author dc:creator

• Kwasnieski, Jamie C

Contributors dc:contributor

• Barak A Cohen

• cis-regulation
• mammalian photoreceptor
• transcriptional regulation
• transcription factor

Language dc:language

English (en)

DOI

https://doi.org/10.7936/K7R20ZC8

OAI identifier oai:identifier

oai:openscholarship.wustl.edu:etd-2244