Browsing by Subject "Repressor Proteins"
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Item Brain-Region-Specific Contributions of FOXP1 to Autism and Intellectual Disability Phenotypes(2017-08-11) Araujo, Daniel John; Eisch, Amelia J.; Konopka, Genevieve; Powell, Craig M.; Volk, Lenora J.; Wu, Jiang I.Mutations and deletions in the transcription factor FOXP1 are causative for severe forms of autism spectrum disorder (ASD) that are often comorbid with intellectual disability (ID). FOXP1 is enriched throughout the brain, with strong expression in the pyramidal neurons of the neocortex, the CA1/CA2 subfields of the hippocampus, and the medium spiny neurons of the striatum. Understanding the role that FOXP1 plays within these brain regions could allow for management of ASD and ID symptoms. This doctoral dissertation leverages multidisciplinary techniques and Foxp1 mutant mouse models to ascertain the role of Foxp1 in the brain and its contribution to specific ASD- and ID-relevant phenotypes. In the first chapter of this dissertation, I review the literature on the characteristics, demographics, and shared genetic underpinnings of ASD and ID and I review the work linking FOXP1 to these disorders. Afterwards, I describe the regional transcriptome regulated by Foxp1 within the brain and I correlate alterations in the gene expression profile of the striatum with deficits in communication (Chapter 2). Briefly, I utilized RNA-sequencing performed on Foxp1 heterozygous knockout animals to uncover the genes regulated by Foxp1 within the neocortex, hippocampus, and striatum. I also recorded the early postnatal ultrasonic vocalizations (USVs) of these animals and I was able to correlate changes in the properties of striatal medium spiny neurons with deficits in USV production. Next, I move onto using a Foxp1 conditional knockout (Foxp1cKO) mouse model to ascertain the contributions of Foxp1 in the neocortex and the hippocampus to ASD and ID-related behaviors (Chapter 3). In brief, I show that total loss of Foxp1 in the pyramidal neurons of the neocortex and the CA1/2 hippocampal subfields results in social communication deficits as well as hyperactivity and anxiety-like behaviors. I also show that Foxp1cKO mice display gross impairments in hippocampal-based spatial-learning tasks and I correlate these deficits with alterations in the expression of genes involved in hippocampal physiology and synaptic plasticity. In my final chapter (Chapter 4), I consider the implications that these data have on our understanding of the role that Foxp1 plays within the brain and I suggest research strategies to answer the new questions that my findings have generated. I also discuss the implications that this research has on our understanding of ASD and ID pathophysiology in general and I recommend future directions for work focused on managing these disorders.Item Cell-Type-Specific Contributions of the Transcription Factor FOXP1 to Striatal Development and Function(2019-08-07) Anderson, Ashley Grace; Kourrich, Said; Konopka, Genevieve; Takahashi, Joseph; Huber, Kimberly M.Mutations in FOXP1, a member of the forkhead box protein (FOXP) family of transcription factors, have been identified as among the most significantly recurring de novo mutations associated with autism spectrum disorder (ASD). ASD is a genetically complex disorder, however, recent studies have identified distinct neuronal cell-types particularly vulnerable in this disorder. These cell-types include deep layer cortical neurons and dopamine receptor 1 (D1) and 2 (D2) expressing striatal spiny projection neurons (SPNs) where FOXP1 is highly expressed. However, the role of Foxp1 within these cell-types was largely unknown. Using a Foxp1 heterozygous mouse model and a human in vitro model system, I reported that FoxP1 regulates conserved pathways within the striatum based on a module preservation analysis between human and rodent gene co-expression networks. I also found a cell-type-specific functional consequence of reduced Foxp1 expression in Foxp1 heterozygous mice, whereby D2 SPNs had increased intrinsic excitability with no significant changes in dSPNs. Together, these data strongly support a conserved, cell-type-specific role for Foxp1 in striatal development and function. The striatum is a critical forebrain structure for integrating cognitive, sensory, and motor information from diverse brain regions into meaningful behavioral output. Therefore, the overarching goal of my project is to investigate the cell-type specific molecular pathways regulated by Foxp1 within distinct striatal SPNs and link these molecular pathways to functional and behavioral outcomes. To do this, I generated mice with deletion of Foxp1 from D1 SPNs, D2 SPNs, or both populations, and used a combination of single-cell RNA-sequencing (scRNA-seq), serial-two-photon tomography, and behavioral assays to delineate the contribution of Foxp1 to striatal development and function. I show that Foxp1 is crucial for maintaining the cellular composition of the striatum, especially D2 SPN specification, and proper formation of the striosome-matrix compartments at early postnatal and adult timepoints. I uncover downstream targets regulated by Foxp1 within D1 and D2 SPNs and connect these molecular findings to cell-type-specific deficits in motor and limbic system-associated behaviors, including motor-learning, ultrasonic vocalizations, and fear conditioning. Moreover, I identify non-cell autonomous molecular and functional effects produced by disruption of Foxp1 within one SPN subpopulation and the molecular compensation that occurs. Using the scRNA-seq data, I also examined gene expression changes within neuronal and non-neuronal cell-types of the developing striatum. Using my above findings, I attempted to pharmacologically rescue motor-learning deficits in Foxp1 cKO mice by targeting dopaminergic and mTOR-regulated pathways. Finally, I discuss the current challenges and future strategies for therapeutic intervention in cases of FOXP1 mutations. Overall, the findings presented in this thesis provide an important molecular window into striatal development and furthers our understanding of striatal circuits underlying ASD-relevant phenotypes.Item Combinatorial Regulation of Signal-Induced CD45 Exon Repression by hnRNP L 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 Compensation Between Foxp Transcription Factors Maintains Proper Striatal Function(August 2023) Ahmed, Newaz Ibrahim; Tsai, Peter; Chahrour, Maria; Roberts, Todd; Konopka, GenevieveSpiny projection neurons (SPNs) of the striatum are critical in integrating neurochemical information to coordinate motor and reward-based behavior. Mutations in the regulatory transcription factors expressed in SPNs can result in neurodevelopmental disorders (NDDs). Paralogous transcription factors Foxp1 and Foxp2, which are both expressed in the dopamine receptor 1 (D1) expressing SPNs, are known to have variants implicated in NDDs. Paralogous transcription factors are thought to have the ability to compensate for each other and previous work published by the lab supports the hypothesis that Foxp1 and Foxp2 have compensatory roles in D1 SPNs as well. For my dissertation work, I utilized mice with a D1-SPN specific loss of Foxp1, Foxp2, or both and a combination of behavior, electrophysiology, and cell type specific genomic analysis to address if there was compensation occurring. It is only upon the loss of both genes that motor behavior was impaired whereas Foxp1 mediated social behavior impairments were exacerbated upon the further loss of Foxp2 (Chapter Two). I also found that while loss of Foxp1 resulted in KLeak mediated hyperexcitability of D1-SPNs, this too was further impaired with the additional loss of Foxp2 (Chapter Three). Viral mediated re-expression of Foxp1 in the double knockouts was sufficient to restore both behavioral and electrophysiological impairments to baseline. I further studied the contribution of Foxp1 and Foxp2 to regulation of downstream targets genes using single-nuclei RNA-seq and found that in both juvenile and adult D1-SPNs, loss of both transcription factors resulted in differential expression of hundreds of genes (Chapter Four). I was able to use these experiments to also investigate how loss of these transcription factors from the D1-SPNs impacted gene expression in other cell-types (Chapter Five). I also utilized single-nuclei ATAC-Seq and again found that loss of both genes resulted in large scale dysregulation of chromatin state not seen in the single knockouts, including in regions enriched for Fox motifs (Chapter Six). I also began to address the open question of what the direct binding targets of Foxp1 and Foxp2 are using the newly developed CUT&RUN technique (Chapter Seven). The findings from my experiments point towards a form of compensation between Foxp1 and Foxp2 where one transcription factor maintains striatal function upon the loss of the other, which I discuss more in depth (Chapter Eight). I also discuss my involvement in a project where we further study the role of Foxp1 in D1- and D2-SPNs, which I am working on in collaboration with Dr. Nitin Khandelwal (Chapter Nine). I conclude by discussing the implications of my findings and suggest recommendations for further study (Chapter Ten).Item The Functional Characterization of the LysR-Type Transcriptional Regulator QseD and the SorC-Type Transcriptional Regulator LsrR in Enterohemorrhagic Escherichia coli(2010-05-14) 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 EHEC.Item MeCP2 and the Epigenetic Regulation of Excitatory Synaptic Transmission(2007-08-08) Nelson, Erika Dawn; Monteggia, Lisa; Bezprozvanny, Ilya; Kavalali, Ege T.; Lin, WeichunAccurate 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 The Role of Polyploidy in the Liver and Its Implications for Cancer Therapy(2018-04-13) Zhang, Shuyuan; O'Donnell, Kathryn A.; Zhu, Hao; Brugarolas, James B.; Yu, HongtaoThe description of liver polyploidy dates back to the 1940s, but its functional roles are still largely unknown. Numerous observations and studies have suggested that liver polyploidy may participate in multiple biological processes, including regeneration, stress response, and cancer. However, little evidence has established direct causal links between polyploidy and the observed phenotypes, mainly due to the lack of appropriate tools to specifically manipulate ploidy levels without causing other permanent changes. Specifically, whether polyploidy promotes or inhibits cancer is still under debate. Inspired by a phenomenon we observed in somatically mutated mouse livers, where homozygous Apc deletions were more difficult to obtain due to hepatic polyploidy, we aimed to build inducible tools to manipulate liver ploidy levels in vivo and systematically study the role of polyploidy in liver cancer. By toggling the weaning time and levels of Anln or E2f8 genes to change liver ploidy levels, we found that liver tumorigenesis was inversely correlated with initial polyploidy levels, suggesting a tumor suppressive role for polyploidy. Moreover, the additional alleles in polyploid cells led to a reduced likelihood of loss of heterozygosity (LOH), which largely contributed to the tumor suppressive effect. These results revealed an important function of polyploidy in mammalian livers and also led us to seek related therapeutic strategies for treating liver cancer. Since hepatocyte polyploidization mainly occurs through cytokinesis failure, we hypothesized that inhibiting cytokinesis could be an effective strategy to suppress liver tumorigenesis while preserving normal liver function. Therefore, we inhibited cytokinesis via Anln knockdown in multiple models and found that liver tumor development was significantly suppressed but normal liver function and regeneration capacity were not impaired. These results suggest that cytokinesis inhibition via Anln knockdown is potentially a safe and efficacious strategy for suppressing liver cancer. Overall, we uncovered an important role of polyploidy in the liver and explored its potential applications in liver cancer therapy.Item The Role of Twist2 in Physiologic and Pathologic Myogenesis(2019-05-01) Li, Stephen; Amatruda, James F.; Morrison, Sean J.; Hobbs, Helen H.; Olson, Eric N.Skeletal muscle is a highly regenerative tissue required for vertebrate life. It composes a significant portion of body mass and enables the physiologic processes of movement and breathing. Given its importance, skeletal muscle is also highly susceptible to aging and diseases such as cancer. Aging-related muscle atrophy (sarcopenia) is a process that affects nearly every person, contributing to debilitations and reductions in quality of life. As people age, fast-twitch muscle fibers selectively atrophy resulting in weakness. Normally, muscle regenerates through a population of stem cells called satellite cells, which differentiate and non-selectively fuse to existing myofibers in order to repair the damaged muscle tissue. However, several recent studies have suggested that the contribution of satellite cells to muscle during homeostasis and aging is minimal. Thus, it's possible that loss of an alternative muscle precursor that fuses specifically with fast-twitch fibers may be a mechanism by which aging-related muscle atrophy occurs. Through the technique of lineage-tracing (fate-mapping) of the transcription factor Twist2, we have identified a novel muscle progenitor that fuses specifically to type IIb/x (fast-twitch, glycolytic) muscle fibers during both aging and homeostasis. Additionally, loss of Twist2+ cells result in specific atrophy of fast-twitch myofibers. I show that Twist2 plays a role in regulating fiber-type specificity through upregulation of the membrane receptor Nrp1. Additionally, the Nrp1 chemo-repulsive ligand, Sema3a, is expressed by both type I and IIa fibers. This Sema3a-Nrp1 signaling mechanisms prevents Twist2+ cells from fusing to type I and IIa fibers, and exogenous overexpression of Sema3a in type IIb fibers impairs the contribution of Twist2+ cells to these fibers. I also found that Twist2 is highly amplified in rhabdomyosarcoma (RMS), a pediatric soft tissue sarcoma expressing hallmarks of the skeletal muscle lineage. Twist2 overexpression was capable of reversibly repressing myogenesis and promoting dedifferentiation of myotubes. Through integrated genomic analyses, I show that Twist2 epigenetically remodels the chromatin landscape to redirect MyoD binding from myogenic loci to oncogenic loci, enabling MyoD to adopt novel functions. Our findings identify the previously unknown roles of Twist2 in regulating mammalian muscle biology.Item Transcriptional Regulation of Adult Neurogenesis by NRSF/REST and NeuroD1(2011-08-10) 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.