Browsing by Subject "DNA-Binding Proteins"
Now showing 1 - 20 of 24
- Results Per Page
- Sort Options
Item Biochemical and Genetic Studies on CC2D1A, a new NF-κB Activator and a Regulator of Synaptic Functions(2010-05-14) Zhao, Meng; Chen, Zhijian J.CC2D1A is an evolutionarily conserved gene from worm to human. It belongs to a new protein family with four DM14 domains at the NH2 terminus and a C2 domain at the COOH terminus. The function of this protein family remains largely unknown. CC2D1A has been identified as a new NF-κB activator through a large scale screen of human genes by Matsuda et al. Here I show that the conserved DM14 and C2 domains of CC2D1A are important for NF-κB activation. CC2D1A activates the IKK complex and NF-κB target genes through several key components in the canonical pathway including ubiquitin-conjugating enzyme UBC13, a RING domain ubiquitin ligase TRAF2, a protein kinase TAK1, and an essential regulator of IKK complex, NEMO. CYLD, a deubiquitination enzyme specific for Lysine-63 linked polyubiquitin chains, negatively regulates the activity of CC2D1A. These results suggest that CC2D1A activates NF-κB through the canonical IKK pathway. In an attempt to identify the physiological function of CC2D1A, I generated CC2D1A knockout mice. The KO animals die right after birth apparently due to their inability to breathe. Histological analysis identified no significant anatomical defects. In particular, brain, heart and muscle are normal with regard to morphology. In addition, neuromuscular junction at the diaphragm is formed in the absence of Cc2d1a. Human patients with mutations in the gene suffer from mental retardation, implying that Cc2d1a functions in the central nervous system (CNS). Here I show that Cc2d1a expression is enriched in the brain. Deletion of Cc2d1a impairs synapse maturation and function in cortical neurons. Our study may help understand the molecular basis of some human diseases such as mental retardation.Item Characterization of β-glucuronidase for Enzyme Replacement Therapy in DYT6 Dystonia(2023-01-31) Lieu, Linh; Yim, Daron; Pappas, Samuel; Dauer, WilliamBACKGROUND: Dystonia is a debilitating disorder defined by sustained involuntary twisting movements. The current symptomatic treatments for dystonia offer only modest efficacy but numerous side effects. Dominantly inherited, loss of function mutations in the THAP1 transcription factor cause DYT6 dystonia (DYT-THAP1). THAP1 modulates the development of oligodendrocyte progenitor cells (OPC) by regulating the catabolism of glycosaminoglycans (GAGs), a crucial component of the extracellular matrix. The loss of THAP1 within OPCs directly reduces GAG-catabolic lysosomal enzyme β-glucuronidase (GusB) causing the accumulation of GAGs that inhibit their own maturation to myelinating cells. The result is severe dysmyelination during early CNS maturation and impaired neurodevelopment. Genetic overexpression of GusB rescues the maturation deficits and CNS myelination in THAP1 deficient mice raising the critical question of whether β-glucuronidase enzyme replacement could restore myelination in THAP1 null mice. OBJECTIVE: Characterization of β-glucuronidase for enzyme replacement therapy (ERT) in DYT6 dystonia models. METHOD: To establish a symptomatic model of DYT6 dystonia, we utilized a Cre/LoxP based Thap1 conditional knockout mouse model ("THAP1-NCKO") and a GusB transgenic mouse line ("GusB-TG"). We evaluated the enzymatic activity and biodistribution of GusB in the CNS using biochemical and histochemical assays. RESULTS: We determined that THAP1-NCKO mice had lower GusB activity than their control counterparts. Interestingly, the activity of GusB is higher in adult (P60) mice compared to juvenile (P30) mice. Visualization of GusB activity showed that distribution of GusB was highest in white matter tracts. We showed that mice with THAP1- related deficits experienced a significant reduction in GusB activity within white matter tracts but not in other surveyed GusB positive brain areas. CONCLUSIONS: Age differentially affects CNS GusB enzymatic activity in a murine model of DYT6 dystonia. GusB enzyme exhibits a distinct biodistribution that varies regionally. White matter tracts experience more severe defects with THAP1 loss. Our results provide insights into the specific locations where GusB activity is deficient and highlight the importance of a "critical period" in which genetic insults have long lasting neurodevelopmental implications.Item Characterizing New Molecules and Mechanisms of Semaphorin/Plexin/MICAL Signaling(2013-07-16) Yoon, Jimok; Krämer, Helmut; Cobb, Melanie H.; Henkemeyer, Mark; Terman, Jonathan R.The mechanisms that regulate the cellular behaviors including morphology, motility, navigation, and connectivity that are critical for normal human health are still incompletely understood. These types of behaviors are regulated through the ability of guidance cues that are present outside of cells to exert precise effects on the cell’s internal actin cytoskeleton. To help characterize an underlying logic for these cellular changes and proper cellular function I have been characterizing how one of the largest families of extracellular guidance cues, members of the Semaphorin family of proteins, induces actin cytoskeletal changes. Interestingly, Semaphorins have been found to inhibit the movement of cells and their membranous processes but the molecules linking them to these specific behaviors have remained poorly understood. Therefore, during my graduate work, I began further characterizing a new family of proteins, the MICALs that were identified as cytoplasmic binding partners of the Semaphorin receptor Plexin. Combining Drosophila genetics with in vitro biochemical assays, my work revealed that Mical regulates actin organization both in vivo and in vitro and is a novel actin disassembly factor. These results provide a new basis for understanding how extracellular guidance cues regulate the actin cytoskeleton. I then went on to further explore Mical-mediated actin filament (F-actin) disassembly. In one line of investigation, my work revealed that Mical plays an antagonistic role to F-actin stabilizing/bundling proteins including fascin and espin in regulating the F-actin cytoskeleton in vivo. This work also indicated that Semaphorin/Plexin/Mical activity not only directly disassembles the F-actin cytoskeleton but also triggers other actin regulatory proteins to reorganize a more complex F-actin network, resulting in increased cellular plasticity. In another line of investigation, I found that the Abl non-receptor tyrosine kinase is a new Mical-interacting protein. My functional assays revealed that Abl and Semaphorin/Plexin/Mical work together to regulate F-actin arrangements and these interactions are conserved in many different contexts including bristle cell morphology, axon guidance, and cancer cell survival and invasion. Thus, I have found that MICAL family proteins are novel controllers of the actin cytoskeleton, functioning directly on F-actin and with other actin regulatory proteins to modulate diverse cellular behaviors.Item The Crosstalk Between DNA Mismatch Repair and Replication(2022-05) Zhang, Junqiu; Davis, Anthony John; Castrillon, Diego H.; Erzberger, Jan; Li, Guo-MinDNA replication fidelity relies on DNA mismatch repair (MMR) and the proofreading nuclease activity of DNA polymerases. Normally, biosynthetic errors can be removed by the polymerase's proofreading nuclease activity upon their incorporation, and those errors that have escaped the proofreading nuclease are corrected by MMR. However, this model is challenged by the fact that cells expressing a proofreading-deficient P286R polymerase ɛ (Polɛ-P286R) display a hypermutable phenotype usually seen in MMR-deficient cells, implying the blockage of MMR function by Polɛ-P286R. We show here that consistent with frequent misincorporation by Polɛ-P286R, elevated levels of MMR proteins were found in replicating DNA/chromatin in Polɛ-P286R cells, but this does not result in a reduced mutation frequency, suggesting that cluster binding of MMR proteins at the replication fork inhibits MMR. Instead, the high-level binding of MMR proteins blocks the recruitment of fork protection factors FANCD2 and BRCA1 to replication forks, and promotes MRE11-catalyzed nascent strand degradation. This MMR-dependent degradation causes DNA breaks and chromosome abnormalities, thereby promoting an ultramutator phenotype. Therefore, our findings identify a novel MMR function in triggering replication stress response to promote genome instability when replication forks are filled with biosynthetic errors. The importance of MMR in maintaining genome stability prompts us to further study the mechanism of MMR in vitro, particularly how the MMR initiation complex is formed in response to misincorporation. Using purified recombinant proteins, we assembled MMR initiation complex in vitro and visualized protein-protein and protein-DNA interactions under transmission electron microscopy. These analyses allowed us to gain molecular insights into the mechanism of MMR initiation.Item Delineating the Mechanisms Through Which ZNF165 Supports Growth and Survival of Triple-Negative Breast Cancer(2021-05-01T05:00:00.000Z) Gibbs, Zane Alexander; Cobb, Melanie H.; D'Orso, Iván; Hon, Gary C.; Whitehurst, Angelique WrightCancer/testis (CT) antigens are proteins whose expression is normally restricted to germ cells yet aberrantly activated in tumors, where their functions remain relatively cryptic. The transcription factor ZNF165, a CT antigen frequently expressed in triple-negative breast cancer (TNBC), was previously identified as essential for the growth and survival of TNBC and sustained transforming growth factor beta (TGFβ) signaling. However, the mechanisms through which ZNF165 functions in this context to regulate both TGFβ signaling and survival were poorly understood. To investigate its mechanism of action, I first sought to determine how ZNF165 interfaces with the TGFβ pathway in TNBC using a combination of genomics and biochemical approaches. I found that ZNF165 associates with SMAD3, a key signal transducing factor in the TGFβ pathway, to modulate transcription of TGFβ-dependent genes and thereby promote growth and survival of human TNBC cells. Importantly, my data demonstrate that through functioning as a cofactor for SMAD3, ZNF165 is able to specify a TGFβ-dependent gene expression program that promotes oncogenic phenotypes while repressing tumor-suppressive functions. In addition, I identified the KRAB zinc finger protein, ZNF446, and its associated tripartite motif protein, TRIM27, as obligate components of the ZNF165-SMAD3 complex that also support tumor cell viability. I found that while ZNF446 is bound to chromatin with the ZNF165-SMAD3 complex, TRIM27 alone is necessary for ZNF165 transcriptional activity and is required for TNBC tumor growth in vivo using an orthotopic xenograft model in immunocompromised mice. Moreover, my data also suggest that ZNF165 is SUMOylated by TRIM27 to enhance its protein stability, providing further insight into the mechanisms that regulate ZNF165 activity in TNBC. Together, my findings indicate that aberrant expression of a testis-specific transcription factor is sufficient to co-opt somatic transcriptional machinery to drive a pro-tumorigenic gene expression program in TNBC.Item Development of a T-Loop Assay to Investigate T-Loop Dynamics and Structure(2014-03-27) Mak, Sin Man; Burma, Sandeep; Shay, Jerry W.; Wright, Woodring E.; Yu, HongtaoAn attractive target in cancer therapy development has been the study of telomeres, which are repetitive sequences at the ends of chromosomes critical in maintaining genomic stability. T-loops, formed by the 3’ overhang inserting into the double strand region of the telomere, are thought to protect the ends from being recognized as double-strand breaks. An almost universal marker for cancer, telomerase, is a promising therapeutic target since its inhibition results in critically short telomeres, compromising t-loop structures. The clinical application has yet to be fully realized due to the lag phase between the times in which telomerase is inhibited and the times that telomeres become sufficiently short that the tumor undergoes apoptosis. Thus, improvements are needed including a greater understanding in t-loop dynamics and the cooperative interaction with telomerase to provide the strategy in which the lag phase can be shortened. Unfortunately, the study of t-loops has been challenging due to the difficulty of isolating and visualizing DNA with intact loop structures. Thus, we have developed novel methods to isolate DNA such that biochemical assays and microscopic visualizations for authentic t-loops are now possible. Digestion of proteins that stabilize t-loop and significant melting at the ends of DNA allow the 3’overhang to migrate out of the double strand region, thus unfolding t-loop and exposing the overhang. DNA isolated with typical procedures (Proteinase K at 55°C for 4 hrs, phenol/chloroform extraction) are thus linear and telomeric overhangs are susceptible to digestion by a 3’-5’ exonuclease (ExoI). The “overhangs” in t-loop structures should be resistant to ExoI. If we lower the temperature of Proteinase K digestion to 4°C to reduce the amount of DNA melting that can occur, we find that the ends are in fact resistant to ExoI digestion. A consistent ~2 fold higher overhang signal in isolated t-loops compared to linear telomeres was observed to distinguish between the two samples. Heating these 4°C samples to 37°C and 55°C caused unfolding of t-loops, resulting in sensitivity of the overhangs to ExoI to the same extent as normal DNA preparations. To validate the t-loop assay, transmission electron microscopy (TEM), a method with powerful magnification and extremely high resolution, is used to visualize DNA isolated at 55°C (linear structures) and 4°C (t-loop structures). The assay was then used to investigate t-loop dynamics throughout cell cycle, and we found that t-loops remain in a folded conformation throughout S phase, confirming the hypothesis that t-loops would unfold a second time for late S/G2 C-strand fill in. We also found that an overhang size of ~30 nts is too short to maintain stabilized t-loops compared to ~90 nts in BJ cells. In summary, we have significant evidence that we are able to prepare and analyze t-loops. Using this assay to determine t-loop structure and timing of t-loop repackage following replication and telomerase action will exceedingly add to our understanding of telomere biology. These are the key steps in setting the stage for many additional future studies, such as what factors contribute in generation of t-loops, how t-loop folding varies with telomere length, and what is the timing of t-loop folding and unfolding throughout cell cycle. All of which will provide critical information for the discovery of new improvements in anti-telomerase therapeutics.Item Direct Redox Regulation of F-Actin Assembly and Disassembly by MICAL(2013-04-08) Hung, Ruei-Jiun 1982-; Hiesinger, Peter Robin; Terman, Jonathan R.; Rosen, Michael K.; Yin, Helen L.How guidance cues present outside of cells exert their precise effects on the internal actin cytoskeleton is poorly understood. Such effects are critical for diverse cellular behaviors including polarity, morphology, adhesion, motility, process elongation, navigation, and connectivity. Semaphorins, for example, are one of the largest families of these guidance signals and play critical roles in neurobiology, angiogenesis, immunology, and cancer. One interesting characteristic of the Semaphorins is that they inhibit the movement of cells (and their membranous processes) through their ability to disrupt actin cytoskeletal organization. However, despite considerable progress in the identification of Semaphorin receptors and their signal transduction pathways, the molecules linking them to the precise control of the actin cytoskeleton have remained mysterious. During my graduate studies, I sought to better understand a family of unusual proteins called the MICALs (which includes one Drosophila Mical and three vertebrate MICALs), which associate with the Semaphorin cell-surface receptor Plexin and are important for Semaphorins to exert their effects. Nothing was known, however, regarding the specific role of the MICALs in these Semaphorin-dependent events. Not long after I began my graduate work, my colleagues and I noticed that Mical was necessary for proper actin cytoskeletal organization and sufficient to reorganize the actin cytoskeleton in vivo. Therefore, to better understand the role that Mical plays in actin cytoskeletal rearrangements, I took a biochemical approach, and purified the Mical protein. Utilizing biochemical and imaging approaches with purified proteins, I found that Mical directly binds to actin filaments (F-actin) and is able to induce the rapid disassembly of F-actin. Thus, my results revealed that Mical is a novel F-actin disassembly factor that provides a molecular conduit through which F-actin disassembly can be precisely achieved in response to Semaphorins. So I next wondered how Mical induces F-actin disassembly. Interestingly, the MICALs belong to a class of flavoprotein monooxygenase/hydroxylase enzymes that associate with flavin adenine dinucleotide (FAD) and use the co-enzyme nicotinamide adenine dinucleotide phosphate (NADPH) in oxidationreduction (Redox) reactions. Although MICALs have no known substrate/s, my in vivo and in vitro results revealed that Mical employs its Redox region to bind F-actin and disassembles filaments in an NADPH-dependent manner. Moreover, this Mical-treated actin failed to repolymerize even after removal of Mical, indicating that Mical stably modifies actin to alter polymerization. Mass spectrometric analyses revealed that F-actin subunits were directly modified by Mical on their conserved pointed-end that is critical for filament assembly. Specifically, Mical post-translationally oxidized a conserved amino acid (Methionine 44) within a region of actin that is critical for actin-actin contacts, simultaneously severing filaments and decreasing polymerization. Thus, my thesis observations reveal a novel and specific oxidation dependent signaling mechanism that selectively regulates actin dynamics and cellular behaviors.Item The Effects of Chromatin Remodeling and Pseudokinase Activity on Liver Pathophysiology(August 2021) Moore, Austin Bradley; Agathocleous, Michalis; Hobbs, Helen H.; Hoshida, Yujin; Zhu, HaoThe liver exhibits a remarkable capability to regenerate itself in the face of injury; however, in the setting of sustained damage, this capability can be overwhelmed and eventually lead to chronic liver diseases such as non-alcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma. Although these processes are complex and not completely understood, specific genetic and epigenetic factors that drive aspects of this pathophysiology can shed further light on both how these diseases develop and on how normal, healthy regeneration differs from liver disease. In this body of work, we show that loss of Arid1a, a DNA-binding component of the SWI/SNF chromatin remodeling complex, shifts hepatocyte metabolism to promote lipid accumulation in a manner similar to that seen in non-alcoholic fatty liver disease. We further explore the dynamics of the SWI/SNF complex by examining a mutually exclusive homolog of Arid1a, Arid1b, and provide evidence to suggest that its role is to stabilize the hepatocyte SWI/SNF complex in the absence of Arid1a seen in regeneration and hepatocellular carcinoma. Finally, we harness the power of in vivo CRISPR screening within the liver to identify the pseudokinase STK31 as a positive regulator of hepatocyte proliferation and liver oncogenesis. Our findings underscore the important role that chromatin remodeling has in enforcing hepatocyte identity and functionality as well as allowing for plasticity. Additionally, our work with STK31 highlights the power of in vivo screening within the liver to identify new potential therapeutic targets not only for hepatocellular carcinoma, but other tumor types as well.Item The Functional Roles for SWI/SNF Chromatin Remodeling Complexes in Physiology and Disease(2020-12-01T06:00:00.000Z) Celen, Cemre; Wu, Jiang I.; Zhu, Hao; Olson, Eric N.; Xu, JianSequencing studies have implicated multiple subunits of SWI/SNF complexes in human neurodevelopmental and psychiatric disorders, as well as in cancers. Particularly haploinsufficiency of ARID1B, a SWI/SNF chromatin-remodeling subunit, has been implicated in short stature, autism spectrum disorder, intellectual disability, and corpus callosum agenesis. In addition, ARID1B is the most common cause of Coffin-Siris Syndrome, a developmental delay syndrome characterized by some of the above abnormalities. However, its role in pathologies is not well characterized due to absence of in vivo models. Therefore, in the first part of this thesis, we generated Arid1b heterozygous mice, which showed social behavior impairment, altered vocalization, anxiety-like behavior, neuroanatomical abnormalities, and growth impairment. In the brain, Arid1b haploinsufficiency resulted in changes in the expression of SWI/SNF-regulated genes implicated in neuropsychiatric disorders. A focus on reversible mechanisms identified insulin-like growth factor deficiency with inadequate compensation by Growth Hormone Releasing Hormone and Growth Hormone, underappreciated findings in ARID1B patients. Therapeutically, GH supplementation was able to correct growth retardation and muscle weakness. This model functionally validates the involvement of ARID1B in human disorders and allows mechanistic dissection of neurodevelopmental diseases linked to chromatin-remodeling. ARID1A is a paralogous subunit that is commonly mutated in cancers and plays critical roles in liver regeneration. Chromatin remodeling mechanisms could be generally important for regeneration in other tissues. Since dynamic regulation of β-cell proliferation in pancreatic islets is poorly understood and better understanding could lead to therapeutic approaches for replenishing β-cell mass in type 1 and type 2 diabetes, in the second part of this thesis we focused on the role of ARID1A in β-cells. Arid1a is physiologically suppressed when β-cells proliferate during pregnancy or after pancreas resection. Whole-body Arid1a knockout mice were protected against streptozotocin induced diabetes. Cell-type and temporally specific genetic dissection showed that β-cell specific Arid1a deletion could potentiate β-cell regeneration in multiple contexts. Transcriptomic and epigenomic profiling of mutant islets revealed increased Neuregulin-ERBB-NR4A signaling. Functionally, chemical inhibition of ERBB or NR4A was able to block increased regeneration associated with Arid1a loss. Together, this work defined the role of ARID1A in β-cells and provided new insights into the molecular regulators of β-cell regeneration. Overall, we uncovered important roles of ARID1A and ARID1B-containing SWI/SNF complexes in physiological and disease states.Item Identification of ITGBL1: A Novel Regulator of Adipogenesis(2016-04-01) Spurgin, Stephen B.; Gupta, Rana K.BACKGROUND: Obesity is a global epidemic that increases the risk of chronic metabolic disease. The rising burden of human suffering inflicted by the double-edged sword of obesity and diabetes has increased the urgency of understanding all aspects of adipose tissue biology, including how adipocytes are formed from their progenitor cells (the preadipocyte) when the body has a need to store excess lipid. It has been shown that the pathological expansion of white adipose tissue (WAT) leads to insulin resistance and cardiovascular disease. In spite of this clear observation, the mechanisms driving the formation of new adipocytes in obesity remain unclear. The work done up to this point has shown that the presumptive preadipocytes resemble pericytes: mesenchymal cells embedded in the vascular basement membrane (1). The pericyte is defined by this anatomic location as well as by the expression of the cell-surface marker platelet-derived growth factor receptor β (PDGFRβ) (2). Recently, Gupta et al. have discovered that Zfp423, a key transcriptional regulator of preadipocyte determination, is expressed in a subset of adipose pericytes in the microvasculature of adult adipose tissues (3). Functional analyses of these cells have revealed that Zfp423+ pericytes serve as a subpopulation of committed preadipocytes in the adipocyte lineage. The ability to isolate committed preadipocytes in vivo and study them in vitro is an excellent tool for investigation and discovery of factors that drive adipocyte commitment and differentiation. OBJECTIVE: This research should identify new genes involved in the commitment or differentiation of adipocyte precursors. METHODS: The identification of Zfp423, a transcription factor, as a regulator of preadipocyte determination was a key step forward in understanding adipogenesis (4). Zfp423 regulates the expression of Pparγ, the "master regulator" of adipogenesis. To understand the localization of Zfp423 in vivo, Gupta et al. derived Zfp423GFP BAC transgenic reporter mice, which express GFP under the control of the Zfp423 locus. Interestingly, Zfp423 expression in adipose tissue is found in mature adipocytes and a subset of perivascular cells expressing PDGFRbeta, supporting a long-standing hypothesis of a perivascular location for preadipocytes (3). Having identified a committed preadipocyte in vivo using the Zfp423-GFP mouse, we used a microarray to compare gene expression between the population of non-committed mesenchymal stem cells (PDGFRbeta+; Zfp423-low) and committed adipocyte progenitor cells (PDGFRbeta+; Zfp423-high) in the mouse model. The top differentially expressed genes were identified and their expression was manipulated in preadipocytes in vitro to assess the effect of those genes on the ability of cells to become adipocytes. The most differentially regulated protein was ITGBL1, which was highly expressed in the Zfp423+ mural cells. Given its high expression in these primed early preadipocytes, we investigated the role of ITGBL1 in preadipocyte differentiation in vitro. Here, we show that shRNA or CRISPR-mediated inactivation of ITGBL1 expression increases the propensity of mesenchymal stem cells to undergo adipocyte differentiation. RESULTS: In order to properly assess the potential adipogenic role of the top regulated genes from the microarray analysis, I cloned the genes (see Table 3.1) from cDNA generated from the stromal-vascular fraction of the mouse inguinal fat pad. I then overexpressed the genes in both the 3T3-L1 preadipocyte cell line and the C3H 10T1/2 mesenchymal stem cell line using a retroviral vector (pMSCV-puro), and assessed whether the cells were then more or less likely to differentiate into mature adipocytes when treated with an adipogenic hormonal cocktail. The overexpression did not result in any clear, repeatable phenotypic changes. However, an investigation of knockdown of these genes did yield significant results. I cloned three shRNAs to each gene, as well as three CRISPR gRNAs, and examined the results of mRNA knockdown and genetic deletion of the genes. One gene, ITGBL1, stood out clearly from those I investigated by way of its robust and dramatic impact on adipogenesis. ITGBL1, or "Integrin beta-like protein 1," has not been previously described in any role with regard to the differentiation of mesenchymal stem cells. However, when the C3H 10T1/2 mesenchymal stem cell line was treated with either shITGBL1 or CRISPR-ITGBL1, there was a highly significant increase in adipogenesis in vitro. CONCLUSION: This work has shown that Itgbl1, a previously uncharacterized protein, is a novel repressor of adipogenesis. Through shRNA knockdown of gene expression and CRISPR-cas9 knockout of the gene, I have shown the role of Itgbl1 in preventing the differentiation of an established mesenchymal stem cell line into a mature fat cell in vitro. Further work will focus on the identification of additional genetic factors involved in adipogenesis using murine tools described in this thesis. Additionally, a human model of adipogenesis--the infantile hemangioma--will be utilized to show the relevance of these genes in the human model of disease.Item Intrinsic Specificity of Binding and Regulatory Function of Class II bHLH Transcription Factors(2016-11-28) Casey, Bradford Harris; Krämer, Helmut; Johnson, Jane E.; Konopka, Genevieve; MacDonald, Raymond J.Embryonic development begins with a single cell, and gives rise to the many diverse cells which comprise the complex structures of the adult animal. Distinct cell fates require precise regulation to develop and maintain their functional characteristics. Transcription factors provide a mechanism to select tissue-specific programs of gene expression from the shared genome. ASCL1, ASCL2, and MYOD are class II basic Helix-Loop-Helix (bHLH) transcription factors which play crucial roles in lineage specification in the developing embryo. In vivo, these factors bind to distinct genomic sites, and regulate distinct transcriptional programs. The mechanisms by which they select their cognate binding sites remain poorly defined. Here, we utilize an inducible system to express these master regulatory factors in embryonic stem cells to characterize early events in bHLH factor binding and function in a common cellular context, removed from their role as endogenous master regulators of lineage specification. Using genome-wide sequencing approaches, we demonstrate that these factors maintain distinct binding when ectopically expressed in a common context. We observe that they initiate distinct transcriptional programs, which include key regulators in lineage specification. By comparing chromatin accessibility of bHLH binding sites, we reveal a shared ability for these factors to bind nucleosome-occupied sites, and meet the criteria which define pioneer transcription factors. We further characterize epigenetic features of the empirically observed genome-wide binding sites of these factors, and compare these findings to the conventional understanding of bHLH factor function. This work represents the first comprehensive approach to direct comparison of early events in the binding and transcriptional profiles of ASCL1, ASCL2, and MYOD.Item KU70 Binding Protein 5 (KUB5), A Novel Factor in DNA Double Strand Break Repair and Radio-Resistance in Human Breast Cancer(2011-02-01) Rommel, Amy Ann; Boothman, David A.DNA double strand breaks (DSBs) are considered both mutagenic and carcinogenic if left un-repaired resulting in genomic instability and ultimately cancer. There are two main pathways for DSB repair: homologous recombination (HR) and non-homologous end joining (NHEJ). Defects in DSB repair have already been associated with breast cancer formation and increased breast cancer risk. Breast cancer susceptibility genes, BRCA1 and BRCA2 are largely thought to be involved with HR while LIG4, XRCC4, and Ku70 are linked to NHEJ. Deficiencies in any one of these genes can predispose individuals to breast cancer. In addition to predisposition to breast cancer, altered DNA repair processes can influence chemo- and radiotherapy efficacy by creating resistance to therapy. To study NHEJ further, our laboratory has identified a novel Ku70 binding protein #5 (KUB5) by a yeast two-hybrid screen using Ku70 as bait. Loss of RTT103, a putative yeast homolog of KUB5, resulted in increased sensitivity to IR, similar to that observed in hdf1-deletion yeast, the yeast homolog of Ku70. Results also show that RTT103-deletion yeast are deficient in repairing blunt and non-compatible DNA ends and re-expression of hKub5 can correct the IR-sensitivity and DNA repair deficiency of these deficient yeast demonstrating a strong functional model for human KUB5 function in yeast. Analyses of breast cancer cell lines for their KUB5 protein expression yielded a strong correlation between KUB5 protein level and sensitivity to DNA damage. These data strongly suggests that KUB5 is a novel repair factor involved in NHEJ and endogenous over-expression of KUB5 plays a role in chemotherapeutic and/or radio-therapeutic resistance via increasing the capacity to facilitate NHEJ repair of DSBs in breast cancer cells.Item Mechanisms of Protein Mislocalization in Neurodegenerative Disease(2017-09-06) Pinarbasi, Emile S.; Yu, Gang; Thomas, Philip J.; Chook, Yuh Min; Bezprozvanny, IlyaFronto Temporal Lobar Degeneration (FTLD) and Amyotrophic Lateral Sclerosis (ALS) are two fatal and rapidly progressing neurodegenerative diseases. A unifying characteristic of these diseases is the mislocalization of an RNA-binding protein, TDP-43. In unstressed cells, TDP-43 is predominantly nuclear and constantly shuttling to the cytosol; in ALS/FTLD, TDP-43 is aggregated in the cytosol. Two lines of evidence suggest this shift is a cause, rather than an effect, of disease. First, point mutations in the C-terminus of TDP-43, which enhance its aggregation, are a rare cause of familial ALS. Second, animal models which replicate the disease-linked redistribution of TDP-43 in motor neurons demonstrate the progressive muscle weakness and loss of spinal cord mass seen in patients. However, little is known about the cellular insults that promote TDP-43 mislocalization. My graduate work makes two contributions to this understanding. First, I elucidated a major determinant of normal TDP-43 trafficking. TDP-43 localization is governed by the balance between nuclear import and nuclear export. While a model for TDP-43 nuclear export had been proposed, there was no direct experimental evidence supporting it. I have shown that the proposed model of TDP-43 nuclear export is incorrect; the putative nuclear export signal (NES) does not mediate nuclear export, and TDP-43 nuclear export is XPO1 independent. Additionally, my data suggest no discrete trafficking signal within TDP-43. Rather, I propose that TDP-43 nuclear export is primarily driven by diffusion through the nuclear pore. Second, I focused on an upstream event known to affect TDP-43 localization: progranulin secretion. One genetic cause of FTLD is a single loss-of-function mutation in GRN, which causes progranulin haploinsufficiency. For reasons that are still unclear, a lifetime of progranulin haploinsufficiency in a patient causes FTLD with TDP-43 mislocalization. I focused on how a subset of GRN mutations- the signal sequence mutations- prevent progranulin secretion. I found that the W7R and A9D mutations disrupt co-translational recruitment of the targeting factor SRP (Signal Recognition Particle). This triggers a quality control pathway called RAPP (Regulation of Aberrant Protein Production), which results in degradation of both mutant protein and mutant mRNA. Thus, RAPP mediates progranulin haploinsufficiency in these patients.Item Multiple Approaches to Study of Steroidogenic Factor 1 : Identification of a Novel Regulatory Element and Identification of Novel Target Genes(2005-05-03) Stallings, Nancy Ruth; Parker, Keith L.Steroidogenic Factor 1 (SF-1) is an essential component of the hypothalamic-pituitary-adrenal-gonadal axis. SF-1 knockout (KO) mice lack adrenals, gonads, the ventromedial hypothalamic nucleus (VMH), and pituitary gonadotropes. SF-1 is a transcription factor implicated in the regulation of many genes important in endocrine function. Research into the regulation of SF-1 expression, mostly focused on the proximal promoter, has been unable to fully explain the expression pattern of SF-1. I used DNase hypersensitivity mapping to search for novel regulatory regions of the SF-1 genomic region. One region between the 6th and 7th exons of SF-1 had tissue specific DNase I hypersensitivity. Analysis of this region revealed high conservation with the human genomic sequence and a smaller region that was also highly conserved in the chicken genomic sequence. Transient transfection assays and electrophoretic mobility shift assays have been employed to investigate this conserved element for enhancer activity. Numerous genes are target genes of SF-1, yet loss of known target genes fail to explain why the adrenals, gonads and VMH fail to develop in SF-1 KO mice. I used an SF-1/eGFP transgene as a reporter in both WT and KO E16.5 embryos. eGFP+ cells from the developing VMH of these mice were collected through the use of FACS. Several potential target genes of SF-1 have been identified and analysis of these genes is an ongoing project.Item Polyglutamines & neurodegenerative diseases /(2004-04-29) Cox, Rody P.Item Regulation of Mical Redox Post-Translationally-Driven F-Actin Cytoskeletal Dynamics(2018-04-16) Yesilyurt, Hunkar Gizem; Hibbs, Ryan E.; Terman, Jonathan R.; Rosen, Michael K.; Kavalali, Ege T.The actin cytoskeleton is critical for multiple diverse cellular behaviors, including the ability of an axon to form, extend, navigate, and synapse with its target. Therefore, an important goal is to understand the mechanisms that regulate it. We have been studying one of the largest families of extracellular repulsive guidance cues, the Semaphorins, which were identified in part based on their ability to dramatically dismantle F-actin. More recently, we identified a new actin regulatory protein Mical, which directly associates with both the Semaphorin receptor Plexin and F-actin to post-translationally oxidize actin on its conserved methionine-44 and methionine-47 residues, inducing both F-actin disassembly and altered actin polymerization. Our work has also revealed that this Mical-mediated actin regulatory process is reversible by a specific methionine sulfoxide reductase enzyme called SelR/MsrB. Thus, we have identified an unusual new actin regulatory system - which I sought for my dissertation research to focus on better understanding. I now find that each human MICAL family member, hMICAL-1-3, similar to Drosophila Mical, directly induces F-actin dismantling and controls F-actin-mediated cellular remodeling. Thus, the MICALs are an important phylogenetically-conserved family of catalytically-acting F-actin disassembly factors. I also investigated how this new actin regulatory system fits with classically-studied actin regulatory proteins. Employing a simple biochemical screen, I identified two proteins - cofilin and tropomyosin - that modulate Mical-mediated F-actin disassembly. Further investigation revealed that Mical synergizes with cofilin to rapidly and efficiently dismantle F-actin in a redox regulated manner and that this synergism is also necessary and sufficient for F-actin disassembly in vivo - for remodeling cells, wiring the nervous system, and orchestrating Semaphorin/Plexin repulsion. In contrast, I find that tropomyosin - known to decorate F-actin within specific cellular compartments and at different developmental stages ¬- protects F-actin from Mical-mediated disassembly by stabilizing Mical-oxidized F-actin. Likewise, changing the levels of tropomyosin in vivo results in similar alterations to Mical-mediated F-actin/cellular remodeling suggesting a previously unknown mechanism controlling the plasticity of the actin cytoskeleton with important tissue-specific and developmental/age-related connotations. Thus, my findings provide new insights into the workings of this MICAL-mediated reversible Redox actin regulatory system including its importance to cell, developmental, and neural biology.Item Regulation of the Insulin-like Growth Factor 1-Secretory Clusterin Expression Axis in Genomic Instability and Cell Stress(2009-09-04) Goetz, Eva Marie; Boothman, David A.Secretory clusterin (sCLU) is a pro-survival factor that is up-regulated in human tumors and after exposure to cell stress. Understanding the regulation of sCLU expression in cancer, and after exposure to therapeutic agents, could reveal new therapeutic targets for cancer treatment. A DNA damage induced signaling cascade leading from ATM to sCLU expression mediated by IGF-1/IGF-1R/MAPK activation was uncovered. IGF-1 ligand promoter activity, mRNA, and protein expression induced after exposure to ionizing radiation (IR), hydrogen peroxide, or topoisomerase I and II-alpha poisons matched sCLU expression. Elevated basal IGF-1-sCLU signaling was noted in genomically unstable cells, whether they were deficient in DNA repair factors or telomerase function. ATM function was necessary for induction of sCLU after IR, and for maintaining elevated expression of sCLU in genomically unstable cells. p53 suppressed IGF-1 promoter activity, leading to decreased mRNA and protein expression, and abrogated induction of IGF-1 and sCLU by IR. Loss of p53 by knockdown or knockout enhanced IGF-1 and sCLU induction. Mutations in the p53 DNA binding domain found in cancer did not repress IGF-1 and sCLU. An NF-Y binding site in the IGF-1 promoter was essential for p53 suppression, and both p53 and NF-YA bound to the IGF-1 promoter. Nutlin-3, an Mdm2-p53 inhibitor, stabilized p53 expression, leading to dramatically decreased sCLU expression. Nutlin-3 treatment sensitized wild-type p53 cells to IR exposure. Finally, exogenous IGF-1 exposure led to serine 1981 auto-phosphorylation of ATM, and enhanced DNA damage repair and abrogated cell death after IR exposure. These studies uncovered key molecules important for the regulation of IGF-1-sCLU expression axis after IR exposure, and supported the use of IGF-1 or sCLU expression inhibitors for cancer chemotherapy.Item Regulatory Mechanism of the RNAi Pathway(2011-12-14) Liu, Ying; Liu, QinghuaRNA interference (RNAi) is post-transcriptional gene silencing initiated by Dicer, a RNase III that processes double-stranded RNA (dsRNA) precursors into small interfering RNA (siRNA). In Drosophila, Dicer2 and R2D2 coordinately recruit duplex siRNA to the effector RNA-induced silencing complex (RISC), wherein single-stranded siRNA guides the endoribonuclease Argonaute (Ago) to catalyze sequence-specific cleavage of complementary mRNA. It remains unclear as to what constitutes holo-RISC, how is RISC assembled and how is RISC regulated. Here we took a candidate approach to reconstitute for the first time the long double-stranded RNA- and duplex siRNA-initiated RISC activities with the use of recombinant Drosophila Dicer-2, R2D2, and Ago2 proteins. We further employed this core reconstitution system to purify a RNAi regulator that we named C3PO (component 3 promoter of RISC), a complex of Translin and Trax. C3PO is a novel Mg2+ -dependent endoribonuclease that promotes RISC activation by removing the siRNA passenger strand cleavage products. Similar as Drosophila C3PO, human C3PO also degrades passenger strand fragments and facilitates RISC activation. RISC is a multiple-turnover enzyme, wherein single-stranded (ss)-siRNA guides Ago2 to catalyze sequence-specific cleavage of the target mRNA at the effector step. We employed human minimal RISC reconstitution system to purify antoantigen La as a novel activator of the RISC effector step. Biochemical studies indicated that La promotes the multiple-turnover of RISC catalysis by facilitating the release of RISC cleaved products. Moreover, we demonstrated that La is required for efficient RNAi, antiviral defense, and transposon silencing in mammalian and Drosophila cells. Taken together, our findings of C3PO and La reveal a general concept that regulatory factors are required to remove Ago2-cleaved products to assemble or restore active RISC. The robust reconstitution system establishes a powerful platform for in-depth studies of the assembly, function, and regulation of RISC. Similar to the discovery of C3PO and La, it can be used to identify novel regulators and study post-translational regulations of RNAi, therefore, connecting RNAi to other cellular signaling pathways. As such, these biomedical studies could have a major and lasting impact on the biological understanding and therapeutic application of RNAi.Item Role of c-Cbl in Invasion of Mammalian Cells by Rickettsia Conorii(2007-08-08) Ravikumar, Sai P.; Seemann, Joachim[SPECIFIC AIMS] Rickettsia conorii is an intracellular bacterium that causes Mediterranean spotted fever. R. conorii is transmitted from ticks to humans and invades vascular endothelial cells. A recent publication (Martinez et al., 2005) has identified Ku70 as a host cell receptor that binds to rOmpB, an R. conorii surface ligand. The engagement of Ku70 by rOmpB leads to a rapid ubiquitination of Ku70 by c-Cbl, followed by the entry of R. conorii into the host cell. However, the role of c-Cbl in ubiquitinating Ku70 and communicating with the invading bacterium remain to be clarified. Based on the report of Martinez et al and other groups on the roles played by R. conorii surface ligands rOmpA and rOmpB, we propose the following hypothesis: [HYPOTHESIS] c-Cbl ubiquitinates Ku70 in response to signals from a Rickettsia conorii cell surface protein called rOmpA. This ubiquitination event enables Ku70 and the attached bacteria to be endocytosed into the cell. [SPECIFIC AIMS] 1. To analyse the effect of c-Cbl - mediated ubiquitination on Ku70 localisation. We will investigate if ubiquitination of Ku70 by c-Cbl is necessary for Ku70 to localize to the bacterial entry foci. We will culture c-Cbl deficient cells and observe the effects of R. conorii infection on them. 2. To map the putative signal(s) involved in the communication between R. conorii rOmpA outside the cell and c-Cbl inside the cell. We will identify the putative rOmpA receptor on host cells and protein(s) that may act as bridges of communication between rOmpA and c-Cbl. 3. To investigate the mechanism of how ubiquitination of Ku70 enables R. conorii to enter a host cell. We will determine if any c-Cbl interacting proteins and/or endocytic proteins are recruited to the cell surface by ubiquitinated Ku70 to form a phagosome. If endocytic proteins are recruited, we will proceed.Item Steroidogenic Factor 1 and Beta-Catenin: Two Critical Regulators of Endocrine Organ Development(2009-06-18) Reuter, Anne Louise; Repa, Joyce J.The endocrine system comprises a diverse array of organs and hormones that regulate many aspects of development and homeostasis. Steroidogenic hormones - secreted by the adrenal cortex, testis, and ovary - are required for electrolyte balance, maintenance of intermediary metabolism, and reproduction. The nuclear receptor steroidogenic factor 1 (SF-1, officially designated NR5A1) originally was identified as a transcriptional regulator of steroidogenic synthetic enzymes. In addition to expression in the adrenal cortex and somatic cells of the gonads, however, SF-1 is expressed in the ventromedial hypothalamic nucleus (VMH) and pituitary gonadotropes, suggesting a broader role in endocrine physiology. Global knockout of SF-1 in mice confirmed this possibility, as it resulted in complete adrenal and gonadal agenesis and XY sex reversal, causing postnatal death due to adrenal insufficiency. Humans with mutations in SF-1 exhibit a spectrum of phenotypes ranging from 46, XY sex reversal and adrenal insufficiency to patients with normal adrenal function and mild gonadal dysgenesis. Herein is reported a patient with compound heterozygosity for a previously described SF-1 polymorphism and a novel mutation, p.R84C, which impairs DNA binding and transactivation activity. SF-1 interacts with numerous coactivators, including beta-catenin, a central mediator of the canonical Wnt signaling pathway. Upon activation by Wnt, beta-catenin translocates to the nucleus where it enhances the transcription of Wnt target genes, and - as recently appreciated - a subset of SF-1 gene targets. Due to the reported functional synergy between SF-1 and beta-catenin, and as Wnt4 deficiency in mice resulted in adrenal and gonadal defects, we investigated the consequences of beta-catenin disruption in certain SF-1-expressing tissues, specifically the adrenal and pituitary glands, and the VMH. SF-1/Cre-mediated beta-catenin knockout mice died immediately after birth and lacked adrenal glands. In contrast, the VMH and pituitary gland were largely unaffected from a structural viewpoint. Analysis of adrenal development revealed that though the adrenal primordium forms, adrenocortical cell numbers quickly decline due to impaired proliferation. The potential for beta-catenin/SF-1 synergy on a fetal adrenal specific enhancer was investigated. These results implicate beta-catenin - presumably as part of the Wnt signaling pathway - as a required factor in adrenocortical development.