Human epithelium-specific ETS transcription factor-1 (ESE-1), which is also known as E74-like factor-3 (Elf3) in mice, is strongly expressed in lung during fetal development and in certain lung cancers. The primary goal of the work presented in this thesis was to investigate whether ESE-1 is involved in regeneration of the injured lung epithelium by administering naphthalene to both wild-type (Elf3 +/+) and Elf3-deficient (Elf3 -/-) mice. However, optimal conditions for proper utilization of the naphthalene-induced lung injury model must first be established. Therefore, dose-response studies were initially conducted by administering three different doses of naphthalene to both male and female mice, as described in chapter 2. Although it is shown that the extent of naphthalene-induced Clara cell injury is dose-dependent in both male and female mice, female mice are more sensitive to naphthalene-induced injury than male mice independent of the dose. Furthermore, it is also demonstrated that these gender-dependent differences in naphthalene injury can subsequently influence downstream lung repair kinetics. In light of these findings, lung regeneration was examined in both sexes of both Elf3 +/+ and Elf3 -/- mice. As reported in chapter 3, the kinetics of bronchiolar epithelial cell proliferation and differentiation is delayed considerably in Elf3 -/- mice following naphthalene injury. Moreover, expression of transforming growth factor-beta type II receptor, which is a well-known transcriptional target gene of ESE-1 and is involved in the induction of epithelial cell differentiation, is significantly lower in the bronchiolar airway epithelium of Elf3 -/- mice as compared to Elf3 +/+ mice under steady-state conditions and during repair of naphthalene-induced damage. Collectively, these findings occur to a similar extent in both sexes of both Elf3 +/+ and Elf3 -/- mice, and suggest that ESE-1 plays an important role in regulating the kinetics of airway epithelial regeneration after acute lung injury.

Human epithelium-specific ETS transcription factor-1 (ESE-1), which is also known as E74-like factor-3 (Elf3) in mice, is strongly expressed in lung during fetal development and in certain lung cancers. The primary goal of the work presented in this thesis was to investigate whether ESE-1 is involved in regeneration of the injured lung epithelium by administering naphthalene to both wild-type (Elf3 +/+) and Elf3-deficient (Elf3 -/-) mice. However, optimal conditions for proper utilization of the naphthalene-induced lung injury model must first be established. Therefore, dose-response studies were initially conducted by administering three different doses of naphthalene to both male and female mice, as described in chapter 2. Although it is shown that the extent of naphthalene-induced Clara cell injury is dose-dependent in both male and female mice, female mice are more sensitive to naphthalene-induced injury than male mice independent of the dose. Furthermore, it is also demonstrated that these gender-dependent differences in naphthalene injury can subsequently influence downstream lung repair kinetics. In light of these findings, lung regeneration was examined in both sexes of both Elf3 +/+ and Elf3 -/- mice. As reported in chapter 3, the kinetics of bronchiolar epithelial cell proliferation and differentiation is delayed considerably in Elf3 -/- mice following naphthalene injury. Moreover, expression of transforming growth factor-beta type II receptor, which is a well-known transcriptional target gene of ESE-1 and is involved in the induction of epithelial cell differentiation, is significantly lower in the bronchiolar airway epithelium of Elf3 -/- mice as compared to Elf3 +/+ mice under steady-state conditions and during repair of naphthalene-induced damage. Collectively, these findings occur to a similar extent in both sexes of both Elf3 +/+ and Elf3 -/- mice, and suggest that ESE-1 plays an important role in regulating the kinetics of airway epithelial regeneration after acute lung injury.

Genomic rearrangements, particularly deletions and duplications, are known to cause many genetic disorders. The chromosome 7q11.23 region in humans is prone to recurrent chromosomal rearrangement, due to the presence of low copy repeats that promote non-allelic homologous recombination. The most well characterized rearrangement of 7q11.23 is a hemizygous 1.5 million base pair (Mb) deletion spanning more than 25 genes. This deletion causes Williams-Beuren Syndrome (WBS; OMIM 194050), a multisystem developmental disorder with distinctive physical and behavioural features. Other rearrangements of the region lead to phenotypes distinct from that of WBS. Here we describe the first individual identified with duplication of the same 1.5 Mb region, resulting in severe impairment of expressive language, in striking contrast to people with WBS who have relatively well preserved language skills. We also describe the identification of a new gene for a severe form of childhood epilepsy through the analysis of individuals with deletions on chromosome 7 that extend beyond the boundaries typical for WBS. This gene, MAGI2, is part of the large protein scaffold at the post-synaptic membrane and provides a new avenue of research into both the molecular basis of infantile spasms and the development of effective therapies. Individuals with smaller than typical deletions of 7q11.23 have delineated a minimal critical region for WBS and have implicated two members of the TFII-I transcription factor family. To better understand the contribution of these genes to WBS, I have generated animal models with these genes deleted singly and in combination. Disruption of the first gene, Gtf2ird1, resulted in phenotypes reminiscent of WBS including alterations in social behaviour, natural fear response and anxiety. An alteration in serotonin function was identified in the frontal cortex and may be linked to these behavioural phenotypes. Together with a model for the second gene, Gtf2i, and the double deletion model that was generated using Cre-loxP technology, these resources will permit the study of the individual and additive effects of hemizygosity for Gtf2i and Gtf2ird1 and will greatly expand our understanding of the role the TFII-I gene family in WBS.

Gene regulation mediated by transcription factors (TFs) is essential for all organisms. The functionality of TFs can largely be described by the fraction of time they occupy their regulatory binding sites on the chromosome. DNA-binding proteins have been shown to find their targets through facilitated diffusion in vitro. In its simplest form this means that the protein combines a random 3D search in the cytoplasm with 1D sliding along DNA. This has been proposed to speed up target location. It is difficult to mimic the in vivo conditions for gene regulation in biochemistry experiments; i.e. the ionic strength, chromosomal structure, and the presence of other DNA-binding macromolecules.    In this thesis single molecule imaging assays for live cell measurements were developed to study the kinetics of the Escherichia coli transcription factor LacI. The low copy number LacI, in fusion with a fluorescent protein (Venus) is detected as a localized near-diffraction limited spot when being DNA-bound for longer than the exposure time. An allosteric inducer is used to control binding and release. Using this method we can measure the time it takes for LacI to bind to different operator sequences. We then extend the assay and show that LacI slides in to and out from the operator site, and that it is obstructed by another DNA-binding protein positioned next to its target. We present a new model where LacI redundantly passes over the operator many times before binding.    By combining experiments with molecular dynamics simulations we can characterize the details of non-specific DNA-binding. In particular, we validate long-standing assumptions that the non-specific association is diffusion-controlled. In addition it is seen that the non-specifically bound protein diffuses along DNA in a helical path.    Using microfluidics we design a chase assay to measure in vivo dissociation rates for the LacI-Venus dimer. Based on the comparison of these rates with association rates and equilibrium binding data we suggest that there might be a short time following TF dissociation when transcription initiation is silenced. This implies that the fraction of time the operator is occupied is not enough to describe the regulatory range of the promoter.

Regulation of transcription is a central mechanism in all living cells that now can be investigated with high-throughput technologies. Data produced from such experiments give new insights to how transcription factors (TFs) coordinate the gene transcription and thereby regulate the amounts of proteins produced. These studies are also important from a medical perspective since TF proteins are often involved in disease. To learn more about transcriptional regulation, we have developed strategies for analysis of data from microarray and massively parallel sequencing (MPS) experiments. Our computational results consist of methods to handle the steadily increasing amount of data from high-throughput technologies. Microarray data analysis tools have been assembled in the LCB-Data Warehouse (LCB-DWH) (paper I), and other analysis strategies have been developed for MPS data (paper V). We have also developed a de novo motif search algorithm called BCRANK (paper IV). The analysis has lead to interesting biological findings in human liver cells (papers II-V). The investigated TFs appeared to bind at several thousand sites in the genome, that we have identified at base pair resolution. The investigated histone modifications are mainly found downstream of transcription start sites, and correlated to transcriptional activity. These histone marks are frequently found for pairs of genes in a bidirectional conformation. Our results suggest that a TF can bind in the shared promoter of two genes and regulate both of them. From a medical perspective, the genes bound by the investigated TFs are candidates to be involved in metabolic disorders. Moreover, we have developed a new strategy to detect single nucleotide polymorphisms (SNPs) that disrupt the binding of a TF (paper IV). We further demonstrated that SNPs can affect transcription in the immediate vicinity. Ultimately, our method may prove helpful to find disease-causing regulatory SNPs.

<p>Uncovering the DNA regulatory logic in complex organisms has been one of the important goals of modern biology in the post-genomic era. The sequencing of multiple genomes in combination with the advent of DNA microarrays and, more recently, of massively parallel high-throughput sequencing technologies has made possible the adoption of a global perspective to the inference of the regulatory rules governing the context-specific interpretation of the genetic code that complements the more focused classical experimental approaches. Extracting useful information and managing the complexity resulting from the sheer volume and the high-dimensionality of the data produced by these genomic assays has emerged as a major challenge which we attempt to address in this work by developing computational methods and tools, specifically designed for the study of the gene regulatory processes in this new global genomic context. </p><p>First, we focus on the genome-wide discovery of physical interactions between regulatory sequence regions and their cognate proteins at both the DNA and RNA level. We present a motif analysis framework that leverages the genome-wide</p><p>evidence for sequence-specific interactions between trans-acting factors and their preferred cis-acting regulatory regions. The utility of the proposed framework is demonstarted on DNA and RNA cross-linking high-throughput data.</p><p>A second goal of this thesis is the development of scalable approaches to dimension reduction based on spectral decomposition and their application to the study of population structure in massive high-dimensional genetic data sets. We have developed computational tools and have performed theoretical and empirical analyses of their statistical properties with particular emphasis on the analysis of the individual genetic variation measured by Single Nucleotide Polymorphism (SNP) microrarrays.</p> / Dissertation

No description available.

Thesis (Ph. D.)--Cornell University, January, 2007. / Vita. Includes bibliographical references (leaves 92-106).

Thesis (Ph. D.)--Cornell University, August, 2006. / Vita. Includes bibliographical references (leaves 116-143).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 69-77).