DEVELOPMENT AND EXPLORATION OF TECHNIQUES TO STUDY THE SPATIAL ORGANIZATION OF THE HUMAN GENOME
Kushner, Max Garrett
Gene expression is regulated by a number of different mechanisms, but of particular interest recently is the regulation of gene expression through spatial organization of the genome. Various techniques have been developed and continue to be developed to further characterize and study the dependence of transcriptional regulation on genome organization. In this dissertation, I will discuss a series of projects aimed at designing a novel method to examine the relationship between transcriptional activity and genomic contacts.I will discuss efforts to characterize photoactivatable compounds including members of the psoralen family for use as a tool to examine genomic contacts. I will explain the determination of many different photochemical properties of psoralen compounds. I will also mention optimization and the use of psoralen compounds with two-photon excitation. Here I present a technique we call Femto-Seq. This technique utilizes a photoactivatable DNA crosslinking compound with an affinity tag to take a snapshot of the DNA sequences spatially around a genomic locus of interest. After allowing the photoactivatable compound to intercalate, two-photon excitation is used to covalently bind sequences in a nuclear volume of interest (around a fluorescently labeled gene for example). The affinity tag on the crosslinker is used to enrich for sequences from the irradiated volume which are then analyzed through sequencing. I will present pilot experiments designed to examine the efficacy of such a method by looking at the enrichment of a targeted transgene. We report a 15-fold enrichment of the transgene matching expected enrichment based on the nuclear volume of interest. I will describe potential applications and extensions of such a technique. Many parts of the Femto-Seq method can be further optimized. Since the technique involves fluorescently labeling a genomic locus of interest, fluorescently labeled engineerable DNA binding proteins are particularly valuable. While many exist, we were particularly interested in Transcription Activator Like Effectors (TALEs). In order to understand the mechanisms of TALE binding we utilized a variety of techniques including DNA curtains, single molecule co-localization and Fluorescent Correlation Spectroscopy. I will also discuss the potential use of microfluidic platforms to increase throughput of the pulldown and cleanup processes.
Zipfel, Warren R.
Lis, John T.; Wang, Michelle D.
Ph. D., Biophysics
Doctor of Philosophy
dissertation or thesis