Browsing by Subject "Repressor Proteins"
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Item Combinatorial Regulation of Signal-Induced CD45 Exon Repression by HNRNPL and PSF(2007-08-08) Melton, Alexis Allyson; Lynch, Kristen W.CD45 is a hematopoetic-specific tyrosine phoshatase. In resting T cells three variable exons are partially repressed, and following antigen challenge, these exons are more highly repressed. Previous work identified the ESS1 silencer element that functions to mediate exon 4 silencing under resting conditions by binding to hnRNP L. ESS1 is also sufficient to confer the activation-induced increase in exon repression, and this document describes two mechanisms responsible for mediating this effect. First, hnRNP L silencing function is slightly increased in activated cells as compared to resting cells. Additionally, PSF binds to the ESS1 complex in a signal-dependent manner and provides a significant increase in repressive activity. Further investigation shows these two mechanisms are largely independent but show some functional crosstalk, and while neither of these mechanisms is sufficient in isolation, the combination of these two effects accounts for an increase in exon silencing that is of similar magnitude to the total observed change in splicing in response to cellular activation.Item The Functional Characterization of the LysR-Type Transcriptional Regulator QseD and the SorC-Type Transcriptional Regulator LsrR in Enterohemorrhagic Escherichia coli(2010-07-12T18:53:55Z) Habdas, Benjamin J.; Sperandio, VanessaEnterohemorrhagic Escherichia coli (EHEC) O157:H7 is a human pathogen responsible for numerous outbreaks of hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) throughout the world. EHEC is able to sense and respond to biotic cues from its environment, such as the human host produced catecholamines epinephrine and norepinephrine, through two two-component systems QseBC and QseEF, and abiotic environmental cues, such as phosphate and sulfate levels through QseEF [1-2]. Additionally, quorum sensing (QS) signaling cascades have evolved to sense microbial population density and diversity through the recognition of bacterially produced autoinducers (AI) AI-2, and 3 by LsrR, and QseBC respectively [1, 3]. Through the interpretation and integration of these multiple regulatory signaling networks that often involve intracellular regulatory proteins, such as the lysine regulator (LysR) type transcriptional (LTTR) family member QseA, EHEC is able to coordinate the expression of its multiple virulence factors [4]. These factors include the production of flagella that confer bacterial motility, the locus of enterocyte effacement (LEE) encoded type three secretion system (TTSS) that facilitates formation of attaching and effacing (AE) lesions on gut epithelium, and is positively regulated by QseA, and Shiga toxin (Stx), which causes cellular damage and HUS. // Here, we show that yjiE, renamed Quorum Sensing E. coli Regulator D (QseD), which was predicted to encode a transcriptional regulator of the LTTR family, functions in a QS-dependent manner to regulate gene expression in both pathogenic and commensal strains of E. coli. LTTRs, the largest known family of prokaryotic DNA binding proteins, contain two functional domains, an N-terminal helix-turn-helix (HTH) and a C-terminal co-factor binding domain which allows for oligomerization [5]. We have demonstrated that QseD indirectly represses transcription of the LEE in EHEC and represses the flagella regulon expression in K-12 E. coli. Additionally QseD regulates the expression of iraD, which has recently been demonstrated to prevent degradation of RpoS by RssB sequestration, leading to an altered bacterial stress-response [6-7]. However, what is most intriguing is that while qseD is prevalent in many enterobacteria it seemingly exists almost exclusively in EHEC O157:H7 isolates as a helix-turn-helix truncated "short" isoform (sQseD). Due to the inability of the sQseD to bind to DNA and the predicted in silico ability of LTTR family members to form hetero-dimers in order to bind DNA, a targeted yeast-two-hybrid (Y2H) approach was used to exclude the known LTTR regulators of LEE transcription QseA and LrhA, as QseD interaction partners. Taken together, these results show that QseD regulates alternate targets in EHEC and K- 12 E. coli, and that EHEC O157:H7 has evolved to encode a truncated form of this protein. // We also studied the role of the LsrR regulon in EHEC pathogenesis and environmental persistence through biofilm formation. LsrR, a negative regulator of lsrK and of the lsrACDBFG operon, has been shown to regulate the uptake and removal of AI-2, the cell-to-cell signaling product of LuxS, from the environment through regulation of the LsrACDB AI-2 uptake pump [8-9]. LsrK, an AI-2 kinase, has been shown to alleviate lsrACDBFG operon repression by generating the inhibitory ligand of LsrR DNA binding, phospho-AI-2 [10]. In E. coli, LsrR has been implicated along with LsrK in AI-2 dependent regulation of biofilm architecture and small-RNA (sRNA) expression [11]. However, while it has been suggested that AI-2 signaling can affect pathogenesis in EHEC, the direct effects of LsrR and LsrK have never been examined [12]. // Here we show that in EHEC both LsrR and LsrK regulate virulence expression, and that this regulation is altered in the absence of a functioning LuxS enzyme. In EHEC, while lsrR and lsrK both positively regulate motility in the presence of luxS, in its absence they both repress motility in a temperature dependent manner. Additionally, in the presence of luxS, lsrR increases biofilm formation. In microarray studies, LsrR was also shown to down-regulate the LEE, and differentially regulate non-LEE effectors (Nle's). Taken together, these results show that both LsrR and LsrK have regulatory roles in the pathogenesis of EHEC and that their effects are altered by the absence of luxS. // These findings have given us a more complete and greater understanding of the genetic regulatory networks and their signaling and integration in EHECItem MeCP2 and the Epigenetic Regulation of Excitatory Synaptic Transmission(2007-08-08) Nelson, Erika Dawn; Bezprozvanny, IlyaAccurate regulation of gene expression is critical for normal brain function. Many human neurodevelopmental and neurodegenerative disorders are associated with mutations in genes important for controlling transcription. Mutations in one such gene, the transcriptional repressor methyl-CpG-binding protein 2 (MeCP2), lead to a form of mental retardation called Rett Syndrome (RTT). Though the MeCP2 protein is expressed ubiquitously, symptoms of RTT patients are primarily neurological, which include reduced mental capacity, autistic-like behavior and autonomic dysfunction. In addition, a mouse model with reduced MeCP2 expression specifically in postnatal, forebrain neurons recapitulates many of the phenotypes seen in human patients. These findings, among others, lead to interest in MeCP2's function in the brain. Our research has focused on the transcriptional repression activity of MeCP2 and its role in the regulation of synapse function. Using mainly electrophysiological techniques, we found that the loss of MeCP2 in hippocampal neurons results in deficits in both spontaneous and evoked excitatory synaptic transmission. Using pharmacological manipulations, we were able to attribute these deficits to the loss of transcriptional repression by MeCP2. By utilizing a conditional knockout approach, we found that these effects were not due to the loss of MeCP2 during neurodevelopment and that they were primarily due to a deficiency in presynaptic vesicle release. We further extended these findings by looking at two mechanisms for controlling the repression of gene expression, DNA methylation and histone deacetylation, both of which are important for MeCP2's function as a transcriptional repressor. Using inhibitors of DNA methyltransferases, we discovered that synaptic activity-dependent decreases in DNA methylation occur in post-mitotic neurons, and that these changes in DNA methylation can regulate spontaneous synaptic transmission. We were also able to rescue the MeCP2-dependent decrease in spontaneous activity by treating neurons with the methyl donor, S-adenosyl-L-methionine. Finally, we addressed the role of histone deacetylation in synapse function by conditionally deleting histone deacetylases (HDACs) 1 and 2 from mature hippocampal neurons. HDAC1 and 2 are present in the transcriptional repressor complex containing MeCP2. After acute knockdown of HDAC1 or HDAC2, we found deficits in excitatory synaptic transmission that mimicked the defects seen after the constitutive loss of MeCP2. In summary, we have discovered a role for the transcriptional repressor, MeCP2, and two components of its repressor complex, DNA methylation and HDACs, in the control of excitatory synaptic transmission between hippocampal neurons.Item Muscle-Specific Regulation of Serum Response Factor by Differential DNA Binding Affinity and Cofactor Interactions(2003-04-01) Chang, Priscilla Shin-Ming; MacDonald, Raymond J.Serum response factor (SRF) is a MADS-box transcription factor that regulates muscle-specific and growth factor-inducible genes by binding the CArG box consensus sequence CC(A/T)6GG. Because SRF expression is not muscle-restricted, its expression alone cannot account for the muscle-specificity of some of its target genes. To further understand the role of SRF in muscle-specific transcription, two distinct approaches were taken. First, tandem multimers of different CArG boxes with flanking sequences were analyzed in transgenic mice. CArG elements from the SM22 and skeletal a-actin promoters directed highly restricted expression in developing smooth, cardiac, and skeletal muscle cells during early embryogenesis. In contrast, the CArG box and flanking sequences from the cfos promoter directed expression throughout the embryo, with no preference for muscle cells. Systematic swapping of the core and flanking sequences of the SM22 and c-fos CArG boxes revealed that cell type-specificity was dictated in large part by sequences immediately flanking the CArG box core. Sequences that directed widespread expression bound SRF more strongly than those that directed muscle-restricted expression. Therefore, sequence variations among CArG boxes influence cell type-specificity of expression and account, at least in part, for the ability of SRF to distinguish between growth factor-inducible and muscle-specific genes in vivo. Second, a novel transcriptional cofactor for SRF called Myocardin was characterized. Myocardin belongs to the SAP domain family of nuclear proteins, is expressed specifically in cardiac and smooth muscle cells, and is a potent activator of cardiac and smooth muscle genes, including SM22. Myocardin activates through CArG boxes, and its activation is dependent on its interaction with the MADS box domain of SRF. Myocardin is the founding member of a new class of muscle-specific transcription factors and provides another mechanism whereby SRF can convey myogenic activity to muscle-specific genes. These results describe two mechanisms for muscle-specific activation of target genes by SRF. Muscle-specific genes contain CArG boxes with relatively low affinities for SRF, and thus are only able to respond to the high levels of SRF found in muscle. Also, Myocardin, a muscle-specific transcription factor, is able to associate with SRF and cooperatively activate transcription of muscle genes.Item Transcriptional Regulation of Adult Neurogenesis by NRSF/REST and NeuroD1(2011-08-26T17:35:21Z) Ure, Kerstin Maria; Hsieh, JennyNeurogenesis in the adult brain is a complex and lifelong process that is regulated by multiple pathways and is sensitive to many external stimuli. Two critical regulatory factors in this process are NRSF/REST and NeuroD1. NRSF/REST, a transcriptional repressor that binds a specific NRSE site and recruits corepressors and chromatin remodeling machinery to repress its target genes, is critical for maintenance of the neural stem cell pool and for proper pacing of neuronal differentiation. NeuroD1, a bHLH transcription factor, is necessary for the terminal differentiation, maturation, and survival of newborn neurons. In addition, both factors are necessary for the neurogenic response to both physiological and pathological stimuli, which may induce neurogenesis through different pathways. Thus, NRSF/REST and NeuroD1 are necessary for neurogenesis to occur correctly, to persist throughout the organism’s lifespan, and to respond to external stimuli.