Until recently, complete understanding of the endogenous activity of pathologically relevant genes was out of reach and research was confined to in situ work, plasmid-based constructs and artificial model systems. The development and expansion of the CRISPR/Cas9 genome editing technique has enabled us to explore the molecular underpinnings of gene activation using the cell's own endogenous regulatory environment. In this work, we report on the development of a novel tool to monitor the endogenous activity of a causative gene in Parkinson's disease, a-synuclein. We use CRISPR/Cas9 to insert a highly sensitive engineered luciferase at the C-terminal of a-synuclein and assessed its responses to stimuli. Our system responds to epigenetic stimuli, which was unable to be recapitulated by previously available gene activity assays. After development of a sensitive detection tool for epigenetic stimuli, we focused on developed a modular suite of epigenetic writers and erasers by modification of the SunTag protein tagging system and used catalytically dead Cas9 (dCas9) to direct our modular epigenetic toolkit to individual genes. We show that our toolkit of epigenetic effectors successfully writes epigenetic information in a site-specific manner. Using the sensitive a-synuclein reporter we previously developed, we screen the promoter region of this pathologically relevant gene at high resolution and identify the most effective areas for epigenetic intervention in this cell line. These tools allow us to dissect and understand the endogenous regulatory mechanisms of almost any gene targetable by Cas9 in ways that were not previously available may prove to be an effective strategy for persistently altering pathologic transcriptional activity. This system offers a strong tool for to dissect and understand underlying epigenetic architecture and opens potential new avenues for therapeutic strategies for various disease conditions.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / DNA sequencing techniques have evolved to the point where one can sequence millions of bases per minute, while our capacity to use this information has been left behind. One particularly notorious example is in the area of gene regulatory networks. A molecular study of gene regulation proceeds one protein at a time, requiring bench scientists months of work purifying transcription factors and performing DNA footprinting studies. Massive scale options like ChIP-Seq and microarrays are a step up, but still require considerable resources in terms of manpower and materials. While computational biologists have developed methods to predict protein function from sequence, gene locations from sequence, and even metabolic networks from sequence, the space of regulatory network reconstruction from sequence remains virtually untouched. Part of the reason comes from the fact that the components of a regulatory interaction, such as transcription factors and binding sites, are difficult to detect. The other, more prominent reason, is that there exists no "recognition code" to determine which transcription factors will bind which sites. I've created a pipeline to reconstruct regulatory networks starting from an unannotated complete genomic sequence for a prokaryotic organism. The pipeline predicts necessary information, such as gene locations and transcription factor sequences, using custom tools and third party software. The core step is to determine the likelihood of interaction between a TF and a binding site using a black box style recognition code developed by applying machine learning methods to databases of prokaryotic regulatory interactions. I show how one can use this pipeline to reconstruct the virtually unknown regulatory network of Bacillus anthracis. / 2999-01-01

Social insects are ecologically dominant because of their specialized, cooperative castes. Reproductive queens lay eggs, while workers take part in brood rearing, nest defense, and foraging. These cooperative castes are a prime example of phenotypic plasticity, whereby a single genetic code gives rise to variation in form and function based on environmental differences. Thus, social insects are well suited for studying mechanisms which give rise to and maintain phenotypic plasticity. At the molecular level, phenotypic plasticity coincides with the differential expression of genes. This dissertation examines the molecular evolution of genes with differential expression between discrete phenotypic or environmental contexts, represented chiefly by female queen and worker castes in social insects. The studies included herein examine evolution at three important levels of biological information: (i) gene expression, (ii) modifications to DNA in the form of methylation, and (iii) protein-coding sequence. From these analyses, a common theme has emerged: genes with differential expression among castes frequently exhibit signatures of relaxed selective constraint relative to ubiquitously expressed genes. Thus, genes associated with phenotypic plasticity paradoxically exhibit modest importance to overall fitness but exceptional evolutionary potential, as illustrated by the success of the social insects.

Autism is a neurodevelopmental disorder with an estimated prevalence of 1 in 150 children which makes it more common than childhood cancer and juvenile diabetes. It is estimated that there are more than 100,000 individuals affected by autism in Canada and tens of millions worldwide. It is well established that genetic factors play important role in the pathophysiology of autism; still, our current understanding of these genetic factors is limited and cause of autism remains an important question. During the past decade, after completion of human genome, several new high throughput genome scan technologies have been developed such as microarrays. In the present study, we undertook the challenge of identifying X-chromosomal genes involved in autism by performing genome-wide copy number variation analysis of more than 400 probands with autism using Affymetrix 500K single nucleotide polymorphism (SNP) microarrays. We identified copy number variants implicating several genes on the chromosome X such as PTCHD1, IL1RAPL1, IL1RAPL2 and TSPAN7 as autism candidate genes. We also demonstrated that autism and intellectual disability may share some of these genes as etiologic factors. We performed a comprehensive analysis of PTCHD1 locus and showed that mutations at this locus are associated with autism in ~1 % of the cases. This study also demonstrated that PTCHD1 mutations can cause intellectually disability with or without autism, and that the PTCHD1 protein may act as a receptor in Hedgehog signaling pathway. We have also carried out a detailed analysis of TSPAN7 and IL1RAPL1 to explore the contributions of these genes in autism. We identified one family with intronic deletion of IL1RAPL1 and another case with a missense mutation in this gene, thus implicating this known intellectual disability gene in autism. Our findings highlight the importance of the X chromosome in the etiology of autism, and demonstrate the power of copy number variation analysis coupled with other technologies in identification of disease genes, in particular for complex genetic disorders such as autism.

Autism is a neurodevelopmental disorder with an estimated prevalence of 1 in 150 children which makes it more common than childhood cancer and juvenile diabetes. It is estimated that there are more than 100,000 individuals affected by autism in Canada and tens of millions worldwide. It is well established that genetic factors play important role in the pathophysiology of autism; still, our current understanding of these genetic factors is limited and cause of autism remains an important question. During the past decade, after completion of human genome, several new high throughput genome scan technologies have been developed such as microarrays. In the present study, we undertook the challenge of identifying X-chromosomal genes involved in autism by performing genome-wide copy number variation analysis of more than 400 probands with autism using Affymetrix 500K single nucleotide polymorphism (SNP) microarrays. We identified copy number variants implicating several genes on the chromosome X such as PTCHD1, IL1RAPL1, IL1RAPL2 and TSPAN7 as autism candidate genes. We also demonstrated that autism and intellectual disability may share some of these genes as etiologic factors. We performed a comprehensive analysis of PTCHD1 locus and showed that mutations at this locus are associated with autism in ~1 % of the cases. This study also demonstrated that PTCHD1 mutations can cause intellectually disability with or without autism, and that the PTCHD1 protein may act as a receptor in Hedgehog signaling pathway. We have also carried out a detailed analysis of TSPAN7 and IL1RAPL1 to explore the contributions of these genes in autism. We identified one family with intronic deletion of IL1RAPL1 and another case with a missense mutation in this gene, thus implicating this known intellectual disability gene in autism. Our findings highlight the importance of the X chromosome in the etiology of autism, and demonstrate the power of copy number variation analysis coupled with other technologies in identification of disease genes, in particular for complex genetic disorders such as autism.

The past few decades have witnessed an array of advances in DNA science including the introduction of genomics and bioinformatics. The quest for complete genome sequences has driven the development of microarray and massively parallel sequencing technologies at a rapid pace, yielding numerous scientific discoveries. My thesis applies several of these genome-scale technologies to understand genomic response to perturbation as well as chromatin structure, and it is divided into three major studies. The first study describes a method I developed to identify drug targets by overexpressing human genes in yeast. This chemical genomic assay makes use of the human ORFeome collection and oligonucleotide microarrays to identify potential novel human drug targets. My second study applies genome resequencing of yeast that have evolved resistance to antifungal drug combinations. Using massively parallel genomic sequencing, I identified novel genomic variations that were responsible for this resistance and it was confirmed in vivo. Lastly, I report the characterization of chromatin structure in a non-eukaryotic species, an archaeon. The conservation of the nucleosomal landscape in archaea suggests that chromatin is not solely a hallmark of eukaryotes, and that its role in transcriptional regulation is ancient. Together, these 3 studies illustrate how maturation of genomic technology for research applications has great utility for the identification of potential human and antifungal drug targets and offers an encompassing glance at the structure of genomes.

The past few decades have witnessed an array of advances in DNA science including the introduction of genomics and bioinformatics. The quest for complete genome sequences has driven the development of microarray and massively parallel sequencing technologies at a rapid pace, yielding numerous scientific discoveries. My thesis applies several of these genome-scale technologies to understand genomic response to perturbation as well as chromatin structure, and it is divided into three major studies. The first study describes a method I developed to identify drug targets by overexpressing human genes in yeast. This chemical genomic assay makes use of the human ORFeome collection and oligonucleotide microarrays to identify potential novel human drug targets. My second study applies genome resequencing of yeast that have evolved resistance to antifungal drug combinations. Using massively parallel genomic sequencing, I identified novel genomic variations that were responsible for this resistance and it was confirmed in vivo. Lastly, I report the characterization of chromatin structure in a non-eukaryotic species, an archaeon. The conservation of the nucleosomal landscape in archaea suggests that chromatin is not solely a hallmark of eukaryotes, and that its role in transcriptional regulation is ancient. Together, these 3 studies illustrate how maturation of genomic technology for research applications has great utility for the identification of potential human and antifungal drug targets and offers an encompassing glance at the structure of genomes.

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (p. 118-126).

In eukaryotic cells, transcription factors and polymerases must access DNA in the context of nucleosomes and chromatin. The accessibility of DNA sequences to such trans-acting factors is an important feature of gene regulatory elements, including promoters. In vertebrates, the majority of gene promoters coincide with CpG islands (CGIs), which remain free from DNA methylation and exhibit elevated CpG densities. This hypomethylated and CpG-rich state at CGI promoters is associated not only with transcriptional activity, but also with high levels of chromatin accessibility. However, the causes and consequences of such chromatin accessibility remain unclear. To address this, I have profiled chromatin accessibility in mouse embryonic stem cells (ESCs). In addition to confirming that CGI accessibility is independent of transcriptional activity, I was able to demonstrate that the loss of DNA methylation in ESCs resulted in increased chromatin accessibility at a subset of CpG-rich repetitive elements, suggesting that non-methylated CpG-rich sequences may, at least partially, facilitate open chromatin states. This was supported by preliminary work targeting bacterial CpG-rich sequences into the mouse genome, where they were sufficient to establish novel regions of chromatin accessibility. To examine potential mechanisms by which hypomethylated DNA could serve to promote chromatin accessibility, I profiled chromatin accessibility in mouse ESCs lacking various chromatin-modifying proteins which are normally enriched at CGIs, with the histone demethylases KDM2A/B linked to maintaining open chromatin at CGIs. As an alternative approach to understanding the causes of chromatin accessibility in mouse ESCs, I examined the mechanism by which the pioneer transcription factor OCT4 is able to access previously inaccessible chromatin, and reveal that it requires the chromatin remodeller BRG1 to remodel chromatin and facilitate transcription factor binding at distal regulatory elements. Ultimately, this work provides an insight into some of the molecular determinants of chromatin accessibility in mouse ESCs, although many of the consequences of such chromatin states remain unclear.