published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy

published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy

No description available.

I present a method that regroups cis-regulatory modules by shared sequences motifs. The goal of this approach is to search for clusters of modules that may share some function, using only sequence similarity. The proposed similarity measure is based on a variable-order Markov model likelihood scoring of sequences. I also introduce an extension of the variable-order Markov model which could better perform the required task. Results. I show that my method may recover subsets of sequences sharing a pattern in a set of generated sequences. I found that the proposed approach is successful in finding groups of modules that shared a type of transcription factor binding site.

Transcription and translation are essential cellular processes that are coupled in bacteria. Though it was well-known that the rate of translation matches the rate of transcription, only in 2010 did evidence suggest direct physical coupling between the transcribing RNA polymerase (RNAP) and the translating ribosome. Nuclear magnetic resonance spectroscopy data showed that the RNAP-binding, transcription factor NusG could bind to the small ribosomal subunit protein, S10, through its C-terminal domain, thus, suggesting a model in which NusG simultaneously binds the transcription and translation machineries. In Chapter Two, I describe my investigations of the mechanism through which NusG-mediated transcription-translation coupling is established in bacteria, and how this coupling is regulated during gene expression. Specifically, I employed cell extract-based luciferase assays and purified C-terminal NusG mutants to show that the NusG N-terminal domain (NTD) and NusG F165A both inhibit transcription. This inhibitory effect was suppressed in an extract derived from a backtracking-resistant RNAP mutant strain, indicating that preventing backtracking by linking RNAP to the lead ribosome is a key function of NusG. While working with the cell extract-based luciferase assay system used to study NusG, I observed that deleting the cryptic rac prophage resulted in cell extracts with extremely low luciferase activity despite the strain having no phenotype in vivo. This initial observation grew into the project described in Chapter Three in which I explore the possibility of viral control of host genes by the poorly characterized rac prophage protein, RacR, through a combination of biochemical methods, structural modeling, bioinformatic analysis, and next-generation, transcriptome-wide, deep RNA sequencing. Taken together, the results reveal overlap between computationally predicted host gene targets and messenger RNA expression levels and suggest that RacR can function as a DNA-binding transcriptional regulator of host genes.

A defining contribution of systems biology has been to reveal how cellular circuitry works to govern the state of a cell. Often, cell-state is determined by the activity of a small number of hyperconnected transcriptional regulators (TRs; e.g., transcription factors, (de)acetylases, (de)methylases, and other genes that act at the level of DNA to affect transcription). The activity of these TRs can be detected from the transcription of their targets, but doing so requires accurate gene regulatory networks (GRNs). The best way to construct GRNs is by combining computationally inferred networks with experimental perturbation data, but until recently this has not been feasible in human cells. As a first step in that direction, I undertook to perform a large-scale Transcriptional REgulator Knock-down (TREK), at two timepoints, in two cancer cell lines, at single-cell level, and to use the resulting data to improve our ability to infer the regulatory state of the cell. In all, I constructed regulons for over 900 TRs and described the dynamics both over time and across contexts. I have significantly improved our GRNs and, consequently, our ability to measure protein activity and identify cell-state regulators.

Mutations in genes that cause transcriptional dysregulation, such as genes that encode DNA and RNA-binding proteins (RNABPs), are a well-described cause of neurodevelopmental syndromes such as autism and epilepsy. Heterozygous de novo mutations involving the gene HNRNPU, which encodes the heterogeneous nuclear ribonuclear protein U, have been implicated in a neurodevelopmental syndrome most commonly characterized by epileptic encephalopathy. Although hnRNP U is a highly-abundant and ubiquitously-expressed DNA- and RNA-binding protein involved in a variety of important nuclear processes—most notably gene expression regulation—the role it plays in neurological disease is unclear and has yet to be studied. The work presented here examines a precision medicine approach for epilepsies thought to have a transcriptomic basis, starting with a thorough neurophysiological characterization of a heterozygous loss-of-function Hnrnpu mouse model (Hnrnpu+/113DEL), followed by a comprehensive and region-specific single-cell transcriptomic study, and finally the validation of implicated brain regions. Characterization of the Hnrnpu+/113DEL mouse line revealed an increased susceptibility to seizures in Hnrnpu+/113DEL mice, along with an increased perinatal mortality, global developmental delay and gait abnormalities. Gene expression profiling, including bulk RNA-sequencing of neocortex and single cell RNA-sequencing of both neocortex and hippocampus, revealed widespread, yet modest, dysregulation of gene expression that was largely inversely correlated to gene-length, and involved important, neurodevelopmental disease genes. In particular, pyramidal neurons of the subiculum displayed greater transcriptional burden upon heterozygous loss of Hnrnpu, with the known epilepsy gene Mef2c as a clear outlier showing greater than 50% reduction in expression. Follow-up investigation into whether this region- and cell-type specific gene dysregulation correlated to differences in neuronal function using c-Fos immunostaining, revealed an overall decrease in neuronal activity within the ventral subiculum in Hnrnpu+/113DEL mice. In summary, our data validates the presence of neurodevelopmental defects upon heterozygous loss of Hnrnpu and supports the notion of transcriptional dysregulation as a likely contributing factor to hnRNP U-related disease, possibly through the dysfunction of subiculum-derived excitatory neurons. Future studies evaluating the relationship between reduced activity within the ventral subiculum and hnRNP U disease phenotypes are an important next step, and may serve as the basis for targeted therapeutic discovery.

No description available.

published_or_final_version / abstract / toc / Molecular Biology / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy