Browsing by Subject "Trans-Activators"
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Item Elucidating the Role of Cellular Architecture in the Developing Pancreas(2015-11-30) Marty Santos, Leilani Marie; MacDonald, Raymond J.; Johnson, Jane E.; Carroll, Thomas J.; Cleaver, OndineMany studies have focused on examining the intrinsic factors such as transcriptional regulators that instruct the step-wise acquisition of β-cell fate in the developing pancreas, with the intention of recapitulating the events necessary in order to generate these cells in vitro for replacement therapies. Directed differentiation protocols have improved upon transitioning from 2D to 3D cultures, indicating that the 3D microenvironment in which β cells are born is critical for the acquisition of their cell fate. However, little is known about the mechanisms through which the 3D architecture of the developing pancreas mediates cell fate specification and epithelial organization. In order to address some of the remaining gaps in the field, we proceeded to characterize the Pdx1-/- embryo, a mutant in which pancreatic cell fate and architecture had been reported to fail early in its development, to determine whether the developmental failure was related to defects in the epithelial architecture. After elucidating that Pdx1 is a transcriptional regulator of the cellular adhesion molecule E-cadherin, we then examined the effect that tissue-specific deletion of this molecule has on the developing pancreas. We determined that E-cadherin regulates both endocrine cell fate and isletogenesis, as we observe that there is a reduction in endocrine progenitors and total endocrine volume, in addition to a failure of the endocrine cells to coalesce into islets. Our findings also demonstrate that acinar cells are lost in the post-natal E-cadherinf/f;Pdx1Cre pancreas, due to an increase in cell death, suggesting that E-cadherin is capable of regulating cell survival. This body of work indicates that architectural molecules play a critical role in the regulation of cell fate specification and epithelial morphogenesis in the developing pancreas.Item The Functional Characterization of the Quorum Sensing E. Coli Regulators B and C in EHEC(2005-12-19) Clarke, Marcie B.; Sperandio, VanessaEnterohemorrhagic E. coli (EHEC) is the causative agent of hemorrhagic colitis. During an infection, EHEC can sense and respond to environmental cues, including the cell density of the intestinal normal flora (through the floral-derived AI-3 signal) and the epinephrine/norepinephrine produced naturally by the host. This cell-to-cell signaling may aid in colonization and disease by allowing EHEC to up-regulate its flagella and motility genes to swim closer to the intestinal epithelium. Previously, Sperandio et al. (2002) have shown that the quorum sensing E. coli regulators B and C (QseB&C), a two-component system in EHEC, are responsible for the regulation of the master regulator of flagella and motility genes, flhDC, in response to cell-to-cell signaling [1]. Here, we show that QseC, the membrane-bound sensor kinase, can autophosphorylate itself in response to AI-3 or epinephrine/norepinephrine and then transfer this phosphate to the response regulator, QseB. The autophosphorylation of QseC is not affected by the addition of autoinducer-2 or intestinal hormones, including gastrin, galanin, and secretin. Additionally, autophosphorylation can be antagonized upon the addition of phentolamine, an a-adrenergic receptor antagonist. Given that enterocytes harbor a-adrenergic receptors, it would be consistent for a microbial adrenergic sensor (QseC) to mostly resemble (in an orthologous and not a homologous fashion) an a- and not a ᭡drenergic receptor. Taken together, these results suggest that QseC may be a microbial adrenergic receptor conserved amongst different bacterial and fungal species. After QseC has autophosphorylated and transferred its phosphate to QseB, QseB acts as a transcription factor to activate the expression of flhDC, the master regulator of flagella and motility genes. Nested deletion analyses of the flhDC promoter suggest that QseB may bind to three promoter regions, to either repress or activate transcription. Further transcriptional studies suggest that phosphorylated QseB autoregulates its own transcription in a similar manner. These analyses have identified a QseB consensus binding sequence, which was utilized in an in silico search to identify novel potential targets of QseB. Through the use of both biochemistry and genetics, a comprehensive model of the QseB&C signaling cascade was generated.Item Identification and Characterization of the Multifunctional Epigenetic Regulator CFP1 as an ERK1/2 Substrate(2014-11-21) Klein, Aileen Melanie; Sternweis, Paul C.; Cobb, Melanie H.; Goodman, Joel M.; Conrad, NicholasEpigenetic regulation of gene transcription occurs as an integration of multiple layers of signals at a genetic locus. These signals can include local chromatin structure, covalent modifications to both histone proteins and DNA, the presence of transcription factors, and modification directly to the transcriptional machinery. Our lab is interested in the control of cellular processes by the mitogen activated protein kinases ERK1/2. In a yeast two-hybrid screen with activated ERK2 (extracellular signal-regulated kinase 2) to find novel interacting partners, our lab identified CFP1 (CxxC finger protein 1), a DNA-binding protein that is a vital component of the H3K4 trimethylating Set1A/B complexes to promote gene transcription. CFP1 has also been shown to interact physically and functionally with the major maintenance DNA methyltransferase DNMT1. We are interested in defining how substrate targeting of CFP1 by ERK1/2 regulates downstream transcriptional outcomes. Interaction between ERK2 and CFP1 in cells was validated by co-immunoprecipitation from isolated mononucleosomes. Active ERK2 can phosphorylate CFP1 on multiple sites in vitro, an observation supported by studies in cells. Some of the most likely in vivo ERK1/2 phosphorylation sites include serine 224 and threonine 227. CFP1 is essential for focusing trimethylation of H3K4 at promoters, a histone modification that supports transcription from these loci. We hypothesized that phosphorylation of CFP1 by ERK1/2 during mitogenic signaling may support trimethylation of H3K4 and transcription of ERK1/2-regulated target genes. Introduction of CFP1 containing the mutation T227V into HeLa cells blocked global H3K4 trimethylation to a similar extent as CFP1 depletion. On the other hand, CFP1 S224A shows diminished transactivation capacity against a model transcriptional substrate. Neither of these mutants fail to interact with Set1B in a pulldown, suggesting that these sites may be important for Set1 complex targeting or activity towards chromatin. Consistently, CFP1 knockdown hinders induction of several ERK1/2-regulated immediate early gene targets in response to serum treatment. It will be of interest to test whether this is dependent on stable or inducible H3K4 trimethylation and what impact overexpression of point mutants will play in their transcription. Regulation of H3K4 trimethylation through CFP1 phosphorylation might represent a novel regulatory input to support transcription of ERK1/2-regulated genes.Item Regulation of Skeletal Muscle Development and Disease by an Actin-Dependent Transcriptional Circuit(2018-05-17) Kutluk Cenik, Bercin; Cleaver, Ondine; Olson, Eric N.; MacDonald, Raymond J.; Mangelsdorf, David J.Congenital myopathies are a group of diseases that primarily affect skeletal muscle and cause muscle weakness that manifests at birth. With an incidence of 6 in 100,000 live births every year, myopathies are considered to be one of the top neuromuscular diseases in the world. Among congenital myopathies, nemaline myopathy (NM) is the most common variant. NM patients have generalized muscle weakness and lifelong disability, and its severest forms are neonatal lethal due to respiratory failure. Currently, there is no cure for NM, underscoring the necessity for new insights into the mechanisms of this severe disease. NM results from mutations in the actin thin filament proteins, and is associated with disorganization of myofibrils, reduced contractile force, and consequent failure to thrive. The main goal of my research has been to expand our knowledge of transcriptional networks that regulate sarcomeric actin, and to investigate how perturbations of these networks can lead to muscle disease. The expression of the actin gene, and the stability of the actin protein are tightly regulated during muscle development and maintenance. Myocardin-related transcription factors (MRTFs) play a central role in actin dynamics, by functioning as coactivators of the serum response factor (SRF), a master regulator of actin and other cytoskeletal genes. MRTFs additionally serve as sensors of actin polymerization and are sequestered in the cytoplasm by actin monomers. We explored the role of MRTFs in muscle development in vivo by generating mutant mice harboring a skeletal muscle-specific deletion of MRTF-B and a global deletion of MRTF-A and showed that the absence of MRTF-A and MRTF-B in the skeletal muscle leads to sarcomeric disarray and dramatic dysregulation of cytoskeletal genes. These findings highlight the importance of MRTFs in actin cycling and myofibrillogenesis. One of the cytoskeletal genes dysregulated in the MRTF dKO was Leiomodin-3 (Lmod3). This striated muscle-specific gene encodes for a putative actin nucleation factor and is downregulated in the MRTF dKO. Lmod3 is a component of the sarcomere thin filament, and its loss leads to compromised sarcomere integrity and nemaline myopathy (NM), a severe congenital muscle disease. We demonstrated an actin-dependent transcriptional circuit in which SRF cooperates with the myogenic transcription factor MEF2 to sustain the expression of the Lmod3 gene and other components of contractile apparatus. In turn, Lmod3 enhances MRTF-SRF activity by promoting actin polymerization. Together, these factors establish a regulatory loop to maintain skeletal muscle function. Finally, we investigated the Kelch protein family: a group of proteins that function as substrate-specific adaptors for Cullin RING E3 ligases; and are responsible for the balance between protein stability and degradation in many tissues, including striated muscle. Previously we have shown that KLHL40 is required for the stabilization of LMOD3. We further demonstrate that KLHL21, a muscle-enriched Kelch protein, operates in numerous unique pathways that potentially govern muscle and heart development, and the cell cycle. Future studies could pave the pathway to therapeutic approaches that improve heart and muscle regeneration, through our understanding of this gene and protein. Overall, our findings not only provide insight into how actin cycling networks regulate skeletal muscle specific transcripts and/or proteins to contribute to myogenesis, but also pave the way for potential new therapeutic approaches for congenital myopathies through the identification of disease-causing mutations.Item The Role of the WWTR1(TAZ)-CAMTA1 Gene Fusion in Epithelioid Hemangioendothelioma(2021-05-01T05:00:00.000Z) Driskill, Jordan Harrison; Dellinger, Michael T.; Cleaver, Ondine; McFadden, David G.; Pan, DuojiaEpithelioid hemangioendothelioma (EHE) is a devastating and mysterious vascular cancer which has no known definitive treatment. Due to a lack of valid animal or cell-based models of EHE, progress toward understanding and treating this cancer has been severely limited. However, recent studies have determined that 90% of patients exhibit a lone, characteristic in-frame gene fusion, TAZ(WWTR1)-CAMTA1. While expression of the TAZ-CAMTA1 fusion protein has been validated as a biomarker of EHE, it remains unknown whether this genetic abnormality is a passenger or a driver of EHE. In this project, I present the first genetically-engineered mouse model (GEMM) of EHE, showing that the expression of the TAZ-CAMTA1 protein in endothelial cells is sufficient to drive the formation of EHE-like tumors in the lungs of mice. Furthermore, I demonstrate that the cessation of TAZ-CAMTA1 expression leads to the regression of these vascular tumors. I also demonstrate that TAZ-CAMTA1 transforms the MS1 endothelial cell line and that subcutaneous transplantation of these cells into nude mice leads to the formation of solid, progressive EHE-like vascular tumors that have the capacity to metastasize to the lung. Utilizing these two novel models of EHE, I unravel the gene program of TAZ-CAMTA1 and demonstrate that TAZ-CAMTA1 drives a gene signature similar to TAZ, the key effector of the Hippo pathway. Expression of an activated TAZ in endothelial cells is also sufficient to drive EHE-like vascular tumors in mice, and genetic blockade of the transcriptional partners of TAZ, the TEAD family of transcription factors, prevents the formation of TAZ-CAMTA1-induced vascular tumors. Next, I show that TAZ-CAMTA1 induces an angiogenic and regenerative-like gene program in endothelial cells. I validate that TAZ-CAMTA1 exhibits gain-of-function activities by having increased resistance to proteasomal degradation and increased nuclear enrichment over TAZ. Lastly, I show that TAZ-CAMTA1 still maintains its binding to the Hippo pathway proteins which are known to negatively regulate TAZ. In summary, I generate two novel models that pinpoint TAZ-CAMTA1 as the key driver of EHE and utilize these models to suggest several new lines of investigation for the treatment of patients with EHE.Item SMAD3 in Embryonic Patterning, Mesoderm Induction, and Colorectal Cancer in the Mouse(2005-04-29) Wieduwilt, Matthew J.; Wakeland, Edward K.Smad3 transduces TGF-β/nodal/activin signals. We ventured to define the roles of Smad3 in development and colorectal cancer in the mouse. Embryos deficient in Smad3 and a related molecule, Smad2, developed defects in anterior morphogenesis, left-right determination, and anterior primitive streak induction indicating that these molecules function redundantly in transducing nodal signals in the mouse embryo. In addition, loss of Smad2 and Smad3 in embryonic stem cells led to impaired mesoderm formation by these cells. Smad3 mutant mice develop colon cancer (Zhu et al., 1998). We show that Smad3 loss in the mouse promotes the transition of benign Apc deficient intestinal polyps to invasive cancers. Using a conditional Smad3 mutant, nullizygosity of Smad3 in the colonic epithelium is demonstrated to be non-essential for the development of colorectal cancer in Smad3 mutant mice. We show that Smad3 mutant mice have gene expression changes in the colon consistent with bowel inflammation, potentially in response to intestinal flora antigens. Suppression of intestinal bacterial flora in Smad3 mutant mice with neomycin plus metronidazole led to nearly complete suppression of colorectal tumorigenesis. These data indicate that Smad3 has important immunosuppressive and tumor suppressive functions in vivo consistent with its role in TGF-β signaling. Lastly, we investigated the role of cyclooxygenase-2 (Cox-2) in modifying tumorigensis in Smad3 mutant mice. Contrary to previous reports in Apc mutant mice, Cox-2 heterozygosity did not effect tumorigenesis in Smad3 mutant mice. In carcinogen treated and ApcMin mice, Cox-2 heterozygosity had no effect on tumorigenesis whereas nullizygosity for Cox-2 suppressed colonic tumor number. Small intestinal polyp number, a sensitive indicator of tumor initiation, was unchanged in Cox-2 deficient ApcMin mice. Treatment of Cox-2 wild type or heterozygous ApcMin mice with the NSAID sulindac suppressed intestinal tumor number and size to a greater extent than Cox-2 nullizygosity suggesting that NSAIDs target Cox-2 independent pathways in vivo. To test this, we treated Cox-2 null ApcMin mice with sulindac and found significant, large magnitude decreases in polyp number and polyp size. These data suggest that using selective Cox-2 inhibitors as chemotherapeutic agents for colorectal cancer may not yield the maximal anti-neoplastic effects of NSAIDs.Item Structural and Mechanistic Roles of Novel Chemical Ligands on Quorum Sensing Transcription Regulator SdiA in Enterohemorrhagic Escherichia coli(2014-11-18) Nguyen, Y. Nhu; Alto, Neal; Sperandio, Vanessa; Hooper, Lora V.; Winter, Sebastian E.Microorganisms and their eukaryotic hosts have co-evolved for millions of years. How bacteria sense and adapt to different environments is still unclear. Most Gram-negative bacteria use the LuxR family of transcription factors to regulate gene expression to coordinate population behavior by sensing endogenously produced chemical signaling molecules, acyl-homoserine lactones (AHLs) [1]. However, some bacteria such as Escherichia coli (E. coli) do not produce AHLs and, therefore, their quorum sensing LuxR-type proteins are thought to be regulated by AHLs from other bacteria [2-4]. These sub-family of LuxR proteins known as LuxR solos can also regulate and detect non-AHL signals to regulate gene expression independently of AHLs [5,6]. This AHL-dependent and -independent regulation of transcription is still unknown. Here we present several structures of one such solo LuxR-type protein, SdiA, from E. coli, in the presence and absence of AHL. Our study demonstrated that without AHL, SdiA is actually not in an apo-state, but regulated by a previously unknown endogenous ligand, 1-octanoyl-rac-glycerol (OCL), which is ubiquitously found throughout the tree of life, and serve as energy sources, signaling molecules, and substrates for membrane biogenesis. While exogenous AHL renders SdiA much higher stability and DNA binding affinity, we propose that OCL may function as a chemical chaperone placeholder in the absence of AHL and stabilizes SdiA as a dimer, allowing for some basal activity. Structural comparison between SdiA-AHL and SdiA-OCL complexes provides some crucial mechanistic insights into the ligand regulation of SdiA transcription activity. Understanding the role of ligand binding on the function SdiA is important for elucidating how SdiA regulates expression of virulence genes in the human pathogen enterohemorrhagic E. coli (EHEC) O157:H7. Although EHEC causes foodborne infections worldwide that result in bloody diarrhea and hemolytic uremic syndrome (HUS), cattle is the major reservoir of EHEC. In cattle, EHEC colonizes predominately at the recto-anal junction (RAJ). Colonization at the RAJ poses a serious risk for fecal shedding and contamination of the environment. We previously demonstrated that EHEC senses AHLs produced by the microbiota in the rumen to activate the gad acid resistance genes necessary for survival through the acidic stomachs in cattle and to repress the locus of enterocyte effacement (LEE) genes important for colonization of the RAJ, but unnecessary in the rumen. Devoid of AHLs, the RAJ is the prominent site of colonization of EHEC in cattle. To determine whether the presence of AHLs in the RAJ could repress colonization at this site, we engineered EHEC to express the Yersinia enterocolitica (Y. enterocolitica) AHL synthase gene yenI, which constitutively produces AHLs, to mimic a constant exposure of AHLs in the environment. The yenI⁺ EHEC produces endogenous AHLs, and has a significant reduction in LEE expression, effector protein secretion, and attaching and effacing (A/E) lesion formation in vitro compared to the wild type (WT). The yenI⁺ EHEC also activated expression of the gad genes. To assess whether AHL production, which decreases LEE expression, would decrease RAJ colonization by EHEC, cattle were challenged at the RAJ with WT or yenI⁺ EHEC. Although the yenI⁺ EHEC colonized the RAJ with equal efficiency to that of the WT, there was a trend for the cattle to shed the WT strain longer than the yenI⁺ EHEC. The findings demonstrate that the regulation of EHEC in cattle is complex. Other factors such as fimbriae [161] may also contribute the colonization of EHEC in cattle. Identifying new factors and mechanisms of EHEC regulation is crucial for developing a better preventive approach against EHEC survival and colonization in cattle and subsequent EHEC contamination in the environment.Item Structural Characterization and Chemical Inhibition of the ARNT/TACC3 Complex(2015-04-13) Guo, Yirui; Rosenbaum, Daniel M.; Roth, Michael G.; Rice, Luke M.; Gardner, Kevin H.My research project focuses on mechanistic studies of a new group of transcriptional coactivators (coiled-coil coactivators: TACC3, TRIP230, CoCoA), involved in cancer development and progression. Normally, these coactivators play an essential role in the HIF hypoxia response, directly interacting with the ARNT subunit of HIF in a novel and promoter-specific way. However, misregulation by overexpression or activating fusions (for example, FGFR-TACC3) is sufficient for transformation and associated with the development of glioblastoma, renal cell carcinoma and other cancers. In light of this connection between coiled-coil coactivators (CCCs) and HIF signaling, tools that inhibit HIF/CCC complex formation might present opportunities to interrogate the linkage between different CCC-containing pathways and may offer a novel route to blocking cancer formation and progression. As a member of a new group of transcription coactivators, knowing how TACC3 interacts with ARNT is critical in understanding the general role of CCCs in HIF-dependent transcription machinery. In the first half of my study, I characterized the ARNT/TACC3 complex with various biophysical and biochemical methods including solution NMR, X-ray crystallography, circular dichroism, luminescence proximity and numerous cell-based assays. A 3.15 Å ARNT/TACC3 crystal structure was solved by molecular replacement, revealing details of this protein complex and providing a structural funcation for coactivator recruitment in HIF signaling pathway. The second half of this study focuses on the search for ARNT/TACC3 inhibitors with in vitro screens to regulate ARNT/CCCs activity in a rapid and flexible way. From a fragment-based NMR screen, I identified small molecules that specifically bound within the second PER-ARNT-SIM (PAS) domain of ARNT and perturb its interaction with TACC3. However, these small molecules have drawbacks, such as low potency or unclear modes of action. To identify higher potency small molecules targeting ARNT/TACC3 complexes, I developed an AlphaScreen-based high throughput screen. Hopefully the discovery of artificial ligands with known mode-of-action that inhibit this typically "undruggable" protein complex will provide new perspectives in small molecule inhibitor development, and also serve as a very useful tool in cell biology for studying pathways utilizing ARNT/TACC3.