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.

In Pyrococcus furiosus (Pfu), a hyperthermophile archaeon, two transcription factor Bs, TFB1 and TFB2 are encoded in the genomic DNA. TFB1 is the primary TFB in Pfu, and is homologous to transcription factor IIB (TFIIB) in eukaryotes. TFB2 is proposed to be a secondary TFB that is compared to TFB1, TFB2 lacks the conserved B-finger / B-reader / B-linker regions which assist RNA polymerase in transcription start site selection and promoter opening functions respectively. P. furiosus, like all Archaea, encodes a single transcription factor E (TFE), that is homologous to the N-terminus of transcription factor II E (TFIIE) α subunit in eukaryotes. TFE stabilizes the transcription bubble when present, although it is not required for in vitro transcription. In this study, in vitro transcription is used to reveal how TFB2 responds to different temperature (65 °C, 70 °C, 75 °C, 80 °C, and 85 °C) at promoters for three different kinds of gene: non-temperature responsive, heat-shock induced, and cold-shock induced in the absence or presence of TFE. The activity of transcription complexes formed by TFB2 is always lower than by TFB1 in all temperatures and promoters. However, with heat-shock gene promoters, the activity of transcription complexes formed by TFB2 increases more than those formed with TFB1 with increasing temperatures. The temperature-dependent activities of TFB1 and TFB2 are similar with the non-temperature responsive gene promoter. With the cold-shock gene promoter, the activity of transcription complexes formed by both TFB1 and TFB2 has the highest activity in lower temperatures. When TFE is present, the activity of transcription complexes formed by TFB2 is enhanced with heat-shock gene promoters particularly at lower temperatures, and makes TFB2 behave more similarly to TFB1. With the non-temperature responsive gene promoter, TFB2 still behaves similarly to TFB1 when TFE is present. However, with the cold-shock gene promoter, most of the activity of transcription complexes formed by TFB1 and TFB2 remain the same, but only the activity of TFB1 decreases at 75 °C. The results suggest that TFB2 may play a role in heat-shock response through its increased sensitivity to temperature, and that TFE can modulate this temperature response.

The ABF-1 gene encodes for a protein that belongs to the basic helix-loop-helix family of transcription factors. ABF-1 mRNA molecules have been detected in the lymphoid tissues, which include the bone marrow, lymph nodes, and appendix, as well as transformed B cells lines infected with Epstein-Barr virus. This study investigates the role of ABF-1 in regulating downstream target genes in the human mature B cell line RAJI, as well as the plasma cell line, ARH-77. Quantitative real-time polymerase chain reaction and DNA microarray technology was used to investigate target genes that are subjected to transcriptional regulation by ABF-1. Using ABF-1 inducible cell lines or B cell lines that overexpress ABF -1 by transient transfection experiments, we discovered many cellular genes that change in their transcriptional profiles in response to ABF -1 expression. Based upon the analysis of genes being affected following ABF-1 induction, our results support the hypothesis that ABF-1 primarily functions as a transcriptional repressor in vivo. Many genes that regulate the cellular processes of apoptosis, as well as the cell cycle, were repressed following ABF-1 expression. Because EBV has been reported to control ABF-1 gene expression, the identification of downstream target genes regulated by ABF-1 may provide insight into the molecular events that follow after EBV infection.

No description available.

The termini of the autonomous parvovirus LuIII, which encapsulates plus and minus DNA strands equally, were cloned and sequenced. The left and right termini of LuIII differ in nucleotide sequence and these termini can assume T- and U-shaped intra-strand base-paired structures, respectively. The LuIII termini are virtually identical in nucleotide sequence and secondary structure to those of the rodent parvoviruses MVM and H-1. The presence of non-identical LuIII termini demonstrated that identical ends are not required for the encapsidation of both DNA strands with equal frequency, as suggested for parvoviruses B19 and AAV. An infectious genomic clone of LuIII was constructed and sequenced. The LuIII genome is 5135 bases and it shares over 80% sequence identity with the sequence of the genomes of MVM and H-1. The genome organization of LuIII is virtually identical to that of the rodent parvoviruses of known sequence. The major ORFs, the left and right ORFs, are restricted to the plus strand. Promoter-like sequences are present at map units 4 and 38. The transactivation responsive element (TAR), characterized in H-1, upstream of P38, is also present in LuIII. Regulatory sequences and splice donor-acceptor consensus sequences, characterized in MVM and H-1, are also present in LuIII. This suggests that both LuIII promoters are functional, and that the transcription map for LuIII could be very similar to that of MVM. The LuIII sequence has only a single copy of a repeat present in tandem at the right end of the MVMp genome. Downstream of this sequence, an A-T rich region of 47 nt is present in LuIII. Since this A-T rich region is absent from the genomes of MVM and H-1, we propose that it represents a putative encapsidation signal responsible for the encapsidation pattern observed for LuIII. Northern analysis of BPV RNAs suggests that, like the human parvovirus B19, most, if not all, BPV transcripts initiate at promoter sequences localized at map unit 4. Amplification of BPV cDNA ends by the polymerase chain reaction resulted in a number of BPV-specific fragments. Four of these fragments were cloned and sequenced. Sequencing revealed two splices, one of which is very likely a major splice for several BPV transcripts. cDNA fragments were assigned to transcripts possibly coding for three BPV non-structural proteins. Amplification of BPV transcripts with primers specific to the mid-ORF suggests that the amino terminus of the capsid protein VP1 is not coded for by the mid-ORF as suggested by earlier studies, but instead results from one or both of the two small ORFs present upstream of the right ORF, in the same reading frame. / Ph. D.

The specific goal of this study was to construct and test transcriptional fusions of zein promoters and a yeast reporter gene that will serve as part of a two plasmid system that will allow for the identification of maize transcriptional regulators of zein genes. Zein genes are expressed coordinately and tempO~ly during endosperm development and are controlled at the transcriptional level (Pedersen et aL, 1980; Kodryzcki et al. t 1989). The accumulation of zein proteins in the endosperm presents an ideal model system to study plant gene regulation. These proteins are synthesized only in the endosperm tissue, and their concentration in the endosperm determine the nutritional quality of the seed. Because of the coordinate and temporal regulation of zein gene transcription, there is a strong likelihood that there exists positive regulatory elements of zein gene expression during early endosperm development. We know that the control of storage protein gene expression is mediated by regulatory elements in the endosperm of maize seeds. It has been shown that the recessive mutation opaque-2 (02) specifically reduces the 22,000 zein polypeptide .. Schmidt et at (1990) and Aukennan et at (1991) show that the wild-type 02 encodes a protein containing a basic leucine zipper domain / Master of Science

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 1977 / Vita. / Includes bibliographical references. / Ph. D. / Ph. D. Massachusetts Institute of Technology, Department of Biology

The process of specific gene transcription by RNA polymerase II (Pol II) is initiated by the<p>binding of specific transcription factors to DNA. A global understanding of the mechanisms of gene<p>transcriptional regulation of Saccharomyces cerevisiae goes through the description of the targets and<p>the behavior of those transcription factors.<p>The GATA factors are specific transcription factors intervening in the regulation of Nitrogen<p>Catabolite Repression (NCR)-sensitive genes, a mechanism encompassing the transcriptional<p>regulations leading to the preferential use of good nitrogen sources of the growth medium of yeast in<p>the presence of less good nitrogen sources. Those 4 GATA factors involved in NCR comprise 2<p>activators (Gat1 and Gln3) and 2 repressors (Gzf3 and Dal80).<p>Generally speaking, the promoters of genes have always been described like the main place for<p>the integration of the transcription regulation signals relayed by the general and specific transcription<p>factors and the chromatin remodeling factors. Furthermore, the GATA factors have been described as<p>integrating the external signals of nitrogen availability thanks to their specific DNA binding to<p>consensus GATA sequences in the promoter of NCR-sensitive genes. The results presented here<p>introduce many nuances to the model, notably implying new proteins but also new regions in the<p>regulation process of the NCR-sensitive gene regulation. Indeed, the first goal of this work is to<p>discover and understand the mechanisms of NCR-sensitive gene regulation that will explain the<p>variations in their expression levels in the presence of various nitrogen sources and their dependency<p>towards the GATA factors.<p>Strikingly, it appeared that GATA factor positioning was not limited to the promoter, but<p>occurred also in the transcribed region. It seems that the transcription factors may have been driven<p>by the general transcription machinery (Pol II). The binding of a chromatin remodeling complex, RSC,<p>has also been demonstrated in the coding region of NCR-sensitive genes. Moreover, the binding of the<p>histone acetyltransferase complex, SAGA, recruited by the GATA activators, was highlighted along<p>NCR-sensitive genes. The SAGA complex was also implied in their transcriptional regulation.<p>Finally, a ChIP-sequencing experiment revealed an unsuspected number and diversification of<p>targets of the GATA factors in yeast, which were not limited to NCR-sensitive genes.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished