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theses

Genetic information is stored in DNA sequences, referred to as the genome. Decoding the meaning of these sequences is a critical challenge in biology. The genome contains functional components that modulate information in the genome into cell type specific functions. Of these components, Cis-regulatory modules (CRMs) integrate transcription factor inputs to causally affect gene expression. The genomic locations and sequences of CRMs are useful for resolving interactions within gene regulatory networks, and are therefore useful to address fundamental questions pertaining to development, evolution, cancer, and genetic disease. My work addressed major methodological limitations in CRM analysis. Previously, there was no functional method to identify CRMs at genome-scale. Thus, I developed a Genome-scale Reporter Assay Method for CRMs, or GRAMc, that utilizes random 25bp DNA barcodes (N25s) as reporters to quantitatively and reproducibly identify CRMs across an entire genome. The method was applied in cultured human liver cells and in sea urchin embryos. Due to the limited delivery rate of reporter constructs into embryos, it is often advantageous to test GRAMc libraries containing long (>1kb) genomic inserts. Previously, there was no efficient way to characterize genome-scale reporter libraries containing long inserts. To overcome this limitation, I developed a bi-directional mate-pair library approach to characterize long insert containing libraries of genome-scale magnitude. Although genome-scale identification of CRMs is critical to increase cis-regulatory analysis, in embryos it is also necessary to characterize the spatial activity of CRMs. Available methods to identify the spatial activity of CRMs rely on image analysis. Due to the limited number of optically distinct reporter genes, image-based methods are limited to testing only a few CRMs at a time. To address this challenge, I developed a method for Multiplex and Mosaic Observation of Spatial information encoded In CRMs, or MMOSAIC, that increases throughput of spatial cis-regulatory analysis in embryos by several orders of magnitude over traditional imaging based approaches. Together, these tools for cis-regulatory analysis overcome several major limitations for the study of functional genomics

ETD_8939

Ph.D.

Includes bibliographical references

by Catherine L. Guay

doi:10.7282/T38G8Q21

ETD doctoral

The author owns the copyright to this work.