RSS 1.0Upstate Graduate Student Dissertations & Theses
Recent Submissions
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Investigating the mechanism of interaction of R-loops and the Fragile X protein, FMRP: an entanglement of disordered tails and multivalencyFragile X syndrome (FXS) is one of the most prevalent forms of inherited intellectual disability and is the leading monogenetic cause of autism spectral disorder. FXS is caused by lack of expression or mutations of the FMR1 gene which encodes fragile X messenger ribonucleoprotein, FMRP. Recently, FMRP has been shown to undergo liquid-liquid phase separation (LLPS) in vitro and to localize different isoforms in distinct membrane-less organelles in cellulo. Despite three decades of research, the molecular mechanisms by which FMRP functions are still not fully understood. FMRP is best known as a cytoplasmic mRNA-binding translational regulator. Although the presence of a small fraction of FMRP in the nucleus has long been realized, it was only recently that studies are beginning to uncover its role in influencing genomic function and stability [1]. The Feng lab recently discovered a novel genome protective role for FMRP. FXS patient-derived cells undergo higher level of DNA double-strand breaks (DSBs) than normal cells, especially during DNA replication stress. These DSBs occur at sequences prone to forming R-loops, which are co-transcriptional RNA:DNA hybrids associated with multiple functions including genome instability. Exogenously expressed WT FMRP, but not an I304N disease-causing mutant abates R-loop-induced DSBs. This unexpected finding suggests that FMRP promotes genome integrity by preventing R-loop accumulation and chromosome breakage. However, the mechanism by which FMRP performs this critical function, and how disease-causing mutations affect this process is not fully understood. Here, we set out to elucidate the mechanism underlying FMRP's role in maintaining genome stability. First, we demonstrate that FMRP directly binds R-loops primarily through its C-terminal Intrinsically Disordered Region (C-IDR). In FMR1 CRISPR knock-out HEK293T cells, we observed dynamic condensates in WT FMRP but not in I304N mutant, suggesting that this mutation, located in the central RNA binding KH2 domain, disrupted the ability of I304N to assemble into higher order condensates. Furthermore, unlike the I304N FMRP, WT FMRP show increase in nuclear condensates that overlap with R-loops under replication stress. While we found that WT and I304N mutant can co-phase separate with R-loops in vitro, WT FMRP tends to form hollow droplets with R-loop substrates localized at the periphery, but the vast majority of I304N droplets are filled with dispersed R-loops substrates. Taken together, these data support the hypothesis that the ability of FMRP to form higher order assemblies with R-loops is critical to maintaining genome stability. Our study sets the stage to test the proposed phase separation-function paradigm in other FXS disease mutants.
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Skewed Distribution Models: Data Analysis, Identification, and Applications in Biomolecular Systems and R-loop Biology of CancerModeling and computational analysis can be used to crystallize, integrate, and extract knowledge from large datasets generated by biology, medicine, and next-generation sequencing. The use of probability models, multifactor hypothesis testing, and computational analyses is crucial to studies in systems biology. These studies provide insights into understanding large and diverse molecular biology data sets. It is no longer enough to study individual molecules, their properties, and their interactions with other molecules in cells and organisms. In addition to generating numerous case studies with unique data, such studies provide a limited understanding of the underlying complexity and dynamics of the leading mechanisms determining the states and behaviors of a whole biological system. Sequencing and multi-omics experiments generate big data needed to model processes, organization and behavior of biological systems in a more comprehensive, less biased manner. Analysis of such enormously heterogeneous and complex information requires mathematical models and computational algorithms. It is the motivation and challenge of current systems biology and medicine. Applied to cancer systems biology, we will consider basic probabilistic aspects of big data. We study skewed frequency distributions commonly observed in diverse omics experiments. We focus on modeling and developing computational algorithms to quantify big data's statistical characteristics, aiming for accurate and unbiased characterization of the systems variation. In several applications, we focus on the identification of the skewed distributions for quantification and differentiation of the of R-loop formation patterns in non-cancer, pre-malignant states and cancer genomes. Current studies involving R-loops rely on the S9.6 antibody which generates noisy signals. We show that using R-loop forming sequences for filtering specific S9.6 signals selects biologically meaningful signals. R-loops have been shown to play a role in tumorigenesis. Using our R-loop forming sequence enrichment method, we investigate the roles of R-loops in tumorigenesis across different detection modalities primarily in breast cancer.
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Loss of Nicotinamide Nucleotide Transhydrogenase Potentiates Autoimmunity in the C57BL/6J Mouse StrainIn Chapter I, we will discuss recent studies showing that mTOR pathway activation plays a critical role in the pathogenesis of autoimmune diseases. The mTOR pathway is a central regulator of growth and survival signals, integrating environmental cues to control cell proliferation and differentiation. Activation of mTOR underlies inflammatory lineage specification, and mTOR blockade-based therapies show promising efficacy in several autoimmune diseases. In Chapter II, we will discuss nicotinamide nucleotide transhydrogenase, NNT, an enzyme localized to the inner mitochondrial membrane which contributes to mitochondrial NADPH production. In C57BL/6J mice, the spontaneous loss of NNT creates a natural model for researching oxidative stress and its ability to potentiate autoimmune disease via the mTOR/AKT pathway. We identify the loss of NNT as a driver of autoimmune pathogenesis, including in multiple sclerosis and ulcerative colitis models. In sum, we highlight the link between upstream pathways of mTOR activation, particularly oxidative stress, and the downstream pathological shift in autoimmune disease due to mTOR activation. We show the novel finding that loss of NNT in the C57BL/6J mouse potentiates autoimmune pathogenesis, and that restoration of wild-type NNT reduces disease burden in select autoimmune models via restoration of redox balance.
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The neuroinflammatory basis of schizophrenia and bipolar disorder: spotlight on brain macrophages, cytokines, and the blood-brain barrierSchizophrenia is a severe mental disorder that has been associated with dysregulation of the immune system. Using inflammatory cytokine transcript levels, we found approximately 40% of individuals with schizophrenia are classified as having elevated levels of inflammation with worse neuropathology. The aim of this study was to investigate the extent to which neuroinflammation is associated with schizophrenia in the dorsolateral prefrontal cortex (DLPFC) and midbrain. We used postmortem human brain tissue to investigate multiple inflammation-related molecular mechanisms. First, in the DLPFC, we found macrophages and astrocytes, rather than microglia, contributed more to neuroinflammation in schizophrenia, by showing unchanged or decreased microglial markers and elevated macrophage markers. Macrophage marker expression was more related to pro-inflammatory marker and macrophage recruitment chemokine expression. In addition, we classified "high" and "low" inflammatory (HI and LI) subgroups using inflammatory cytokine and macrophage marker protein levels as discriminators in the DLPFC for the first time. 30% of controls (CTRL) and 56% schizophrenia (SCZ) cases were classified as high inflammation individuals. We found higher CD163+ macrophage density in the DLPFC of SCZ-HI subgroup mainly around small blood vessels of both grey and white matter. In the midbrain, we characterized a substantial proportion of individuals in schizophrenia (46%) and bipolar disorder (29%) expressing elevated inflammatory mRNA (IL6, IL1, TNF and SERPINA3), which were termed the "high" inflammatory (HI) subgroups, and we confirmed increases in IL6 and IL1 at the protein level in these subgroups. Furthermore, we showed elevated macrophage and chemokine marker expression in schizophrenia and bipolar disorder HI subgroups which were associated with changed blood-brain barrier markers, indicating potential macrophage transmigration, supported by altered mRNA and protein levels in the adhesion molecules, tight junction proteins, basement membrane proteins, and angiogenic factors related to blood vessel regulation. Importantly, we observed a novel but unknown neuropathology of more frequent claudin-5 "cell bursts" between blood vessel fragments in schizophrenia and bipolar high inflammatory subgroups. These findings have implications for new immune-related treatments, therapy development, and potential targets for measuring disease progression or early detection of schizophrenia and bipolar disorder.
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Analysis of phosphatidylinositol 3-phosphate binding to the erlin complexThe Endoplasmic reticulum (ER) membrane lipid raft-associated proteins erlin 1 (E1) and erlin2 (E2) are ~40 kDa proteins, and they are members of a superfamily of stomatin/prohibitin/ flotillin/HflK/C (SPFH) domain-containing proteins. E1 and E2 form a massive (~2 MDa) hetero-oligomeric complex that is an essential mediator of inositol 1,4,5-trisphosphate receptor (IP3R) ubiquitination and degradation. Mutations of E1 and E2 are involved in many pathological processes in neurological disorders, such as Hereditary Spastic Paraplegia (HSP), with unknown molecular mechanisms. The Wojcikiewicz laboratory has previously provided evidence that the erlin complex, immunopurified from mammalian cells binds to phosphatidylinositol 3-phosphate (PI(3)P), a key player in membrane dynamics and trafficking regulation in endocytosis and autophagy. In addition, the erlin complex may be involved in different cellular processes beyond IP3Rs degradation, but the exact nature of these roles has remained elusive. My research described in this thesis has uncovered intriguing new insights into the erlin complex and its role in previously unknown aspects of cellular biology. Through the application of diverse biochemical and molecular biology assays, I successfully identified specific regions on E2 that are essential for its binding to PI(3)P. Additionally, my research revealed that the erlin complex plays an important role in regulating cellular PI(3)P levels through its interaction and stabilization with this lipid. This binding and stabilization of PI(3)P are crucial for the regulation of autophagy and lysosome function. These findings contribute to our understanding of the erlin complex's importance in cellular biology and provide valuable knowledge about related processes that have implications for human health and disease.
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Distinct Interaction Modes for the Eukaryotic RNA Polymerase Alpha-like Subunits and Implications for Disease ModelingEukaryotic DNA-dependent RNA Polymerases (Pols I-III) encode two distinct ⍺- like heterodimers where one is shared between Pols I and III, and the other is unique to Pol II. Human alpha-like subunit mutations are associated with several diseases including Treacher Collins Syndrome (TCS), 4H Leukodystrophy, and Primary Ovarian Sufficiency. Yeast is commonly used to model human disease mutations, yet it remains unclear whether the alpha-like subunit interactions are functionally similar between yeast and human homologs. To examine this, we mutated several regions of the yeast and human small alpha-like subunits and used biochemical and genetic assays to establish the regions and residues required for heterodimerization with their corresponding large alpha-like subunits. Here we show that different regions of the small alpha-like subunits serve differential roles in heterodimerization, in a polymerase- and species-specific manner. We found that the small human alpha-like subunits are more sensitive to mutations, including a "humanized" yeast that we used to characterize the molecular consequence of the TCS-causing POLR1D G52E mutation. These findings help explain why some alpha subunit associated disease mutations have little to no effect when made in their yeast orthologs and offer a better yeast model to assess the molecular basis of POLR1D associated disease mutations.
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Disorder in the Loop: Identification of a Role for Intrinsic Disorder and Liquid-Liquid Phase Separation in R-Loop BiologyR-loops are non-canonical nucleic acid structures composed of a DNA:RNA hybrid, a displaced single-stranded (ss)DNA, and a trailing ssRNA overhang. R-loops perform critical biological functions under normal and disease conditions. To elucidate their cellular functions, we need to understand the mechanisms underlying R-loop formation, recognition, signaling, and resolution. Previous high-throughput screens identified multiple proteins that bind R-loops, including Enzymes and non-enzymatic Readers. However, the precise mechanisms by which these proteins modulate R-loop functions are not fully known. The Fragile X Protein (FMRP) has been recently shown to prevent R-loop-mediated DNA double strand breaks (DSBs), but the mechanism was unknown. FMRP has been previously shown to undergo Liquid-Liquid Phase Separation (LLPS) by itself and with another non-canonical nucleic acid structure, RNA G-quadruplexes, via its C-terminal intrinsically disordered region (C-IDR). Here, we identified the same C-IDR as the predominant R-loop binding site. This unexpected discovery prompted us to explore the hypothesis that disordered regions of other R-loop binding proteins are also utilized to recognize R-loops. Our analysis of the R-loop interactome revealed that low-complexity IDRs are prevalent in this interactome, and that Readers and Enzyme IDRs are distinct (Gly, Ser, Arg, and Pro-rich vs. Glu, Lys, Arg, and Ser-rich, respectively). Furthermore, like FMRP, both R-loop Readers and Enzymes are not only modular, (i.e., contain folded domain(s) interspersed with IDRs), but are also predicted to undergo LLPS. Next, we demonstrated that the IDRs from the R-loop binding protein RBM3 and the R-loop helicase DDX21 also bind to R-loops, providing additional examples of IDR-mediated R-loop binding from an R-loop Reader and Enzyme, respectively. Finally, we demonstrated that FMRP C-IDR and DDX21 N-IDR can undergo co-LLPS with R-loops suggesting that IDR-based R-loop binding and co-LLPS is a universal mechanism shared by all members of the interactome. Therefore, we propose that IDRs can provide a functional link between R-loop recognition and downstream signaling through the assembly of LLPS-mediated membrane-less R-loop foci, where the activities of the folded domains are coordinated to regulate the biological functions of R-loops. Mutations or dysregulation of the function of IDR-enriched R-loop interactors can potentially lead to severe genomic defects, such as the R-loop-mediated DSBs observed in Fragile X patient-derived cells.
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Effects of the hepatic glucocorticoid receptor in the setting of sepsis, infection, and inflammationEach year hundreds of thousands of people develop life-threatening sepsis, defined by a combination of infection and organ dysfunction. Although many affected biological pathways are typically regulated by the glucocorticoid receptor (GR), during sepsis this is deficient and supplementation with exogenous glucocorticoids is often ineffective in reducing mortality. The GR has different effects in different organs. In liver many effects are beneficial, whereas in immune or muscular systems many effects are deleterious. A sampling of this literature is reviewed in chapter 1. With the hypothesis that liver-specific glucocorticoid therapy will be more clinically beneficial than systemic therapy, we studied liver-specific GR effects in infection, inflammation, or sepsis. Chapter 2 describes in-vitro chemokine alterations from GR activation in primary human hepatocytes, with inflammation or infection modeled by TNFα and/or lipopolysaccharide. Comparisons were made in primary human hepatocytes, the human hepatoma cell line HEPG2, and the non-liver cell line HEK293t. Chapter 3 outlines outcomes of liver-specific GR deficiency using mice with inducible liver-specific GR knockout, modeling sepsis with cecal ligation and puncture. Results of these two models show mRNA and/or protein changes induced by GR in chemokines; transcription factors; genes related to protein degradation, metabolism, metal management, inflammation, liver regeneration, and hemodynamic stability. Results of these 2 models demonstrate a significant role of the hepatic GR in many pathways dysregulated during sepsis. Therefore, targeting GR therapy to the liver instead of systemic treatment may prove more clinically beneficial to reducing the morbidity and mortality of sepsis.
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Defining the mechanism of STAT3 regulation by ABI1 in prostate cancerProstate cancer, driven by hormones and the androgen receptor (AR), initially responds to AR pathway-targeted treatments. However, tumor relapse arises from a process called the prostate cancer cell lineage switch. This switch involves transcriptional and epigenetic reprogramming, allowing cancer cells to acquire a new identity and bypass the stress caused by anti-AR treatments, resulting in increased proliferation and metastasis. Our study delves into the regulatory mechanism of STAT3, a key modulator, by the tumor suppressor ABI1 during the process of lineage switch. We observed an inverse correlation between ABI1 expression and the progression of the lineage switch. Using tumor models, we demonstrated that ABI1 modulates the phosphorylation of STAT3 by regulating kinase activities. Additionally, we discovered that ABI1 interacts with DNA through unique intrinsic disordered DNA binding regions. Notably, during prostate cancer lineage switch, a specific ABI1 EXON4 undergoes abnormal splicing, enhancing the ABI1-DNA interaction and influencing epigenetic remodeling by modulating chromatin accessibility. Our findings highlight the role of ABI1 in regulating STAT3 activities through its DNA interaction and reveal a reciprocal regulation between ABI1 and STAT3 in terms of nuclear localization, thereby influencing the lineage switch driven by STAT3. Overall, we propose that ABI1 acts as a master regulator of the lineage switch by maintaining the homeostasis of epigenetic and transcriptional processes.
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Pharmacological inhibition of protein phosphatase-5 and induction of the extrinsic apoptotic pathway in kidney cancerSerine/threonine protein phosphatase-5 (PP5) is involved in tumor progression and survival, making it an attractive therapeutic target. Specific inhibition of protein phosphatases has remained challenging because of their conserved catalytic sites. PP5 contains its regulatory domains within a single polypeptide chain, making it a more desirable target. Here we used an in silico approach to screen and develop a selective inhibitor of PP5. Compound P053 is a competitive inhibitor of PP5 that binds to its catalytic-domain and causes apoptosis in renal cancer. We further demonstrated that PP5 interacts with FADD, RIPK1 and caspase 8, components of the extrinsic apoptotic pathway complex II. Specifically, PP5 dephosphorylates and inactivates the death effector protein FADD, preserving complex II integrity and regulating extrinsic apoptosis. Our data suggests that PP5 promotes renal cancer survival by suppressing the extrinsic apoptotic pathway. Pharmacological inhibition of PP5 activates this pathway, presenting a viable therapeutic strategy for renal cancer.
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Specific Structural Features of the RNA Polymerase I Core Promoter Element Targeted by Core FactorIn yeast, Core Factor (CF) is a critical and essential RNA Polymerase I (Pol I) transcription factor that plays fundamental roles in the transcription process by recruiting Pol I and opening Pol I promoter DNA before initiation. CF binds to a ~24 bp region in the rDNA promoter called the Core Element (CE) prior to Pol I recruitment. Pol I transcribes the rDNA gene into the 35S precursor rRNA (pre-rRNA) which serves both catalytic and structural roles in the ribosome. Up-regulation of Pol I transcription has been linked to a variety of human cancers, as increased protein production can facilitate the rapid growth of cancer cells. Thus, Pol I transcription is a promising target for therapeutic development. Previous studies from our lab suggest that CF and its human orthologue, Selectivity Factor 1 (SL1), use an evolutionarily conserved mechanism to target DNA, governed by the structural features of their respective promoters. Eukaryotic rDNA promoters also exhibit conserved structural features, such as intrinsic curvature and kinks but show a distinct lack of sequence conservation. These sequence independent structurally conserved features of rDNA promoters might explain how they are being recognized by CF and its orthologues. Our findings here revealed that CF is capable of tolerating mutations at some positions of the CE while mutation in the rigid “A” patch being particularly sensitive to mutations changing structural properties. Along with conditional tolerance for sequence mutations, our results show that CF prefers a variety of structural features such as overall increased bendability and decreased curvature as well as specific profiles of bendability. Furthermore, we describe the preferences of CF for the parameters of helix twist, propeller twist, roll, and minor groove width.
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Uncovering a new phase: the interactions that mediate MBD2 and MBD3 LLPSChromatin structure and organization controls DNA's accessibility to regulatory factors and influences gene regulation. Heterochromatin, or condensed chromatin containing mostly silenced genes, self-assembles through weak, multivalent interactions with its associated proteins that contain intrinsically disordered regions (IDRs) and undergoes liquid-liquid phase separation (LLPS). However, the details of the intricate molecular interactions that drive heterochromatin LLPS are not fully understood. It is crucial that we uncover the molecular mechanisms involved as it regulates vital nuclear functions, and dysregulation is implicated in neurological disorders and cancer. Here, we focus on two members of the methyl-CpG-binding domain (MBD) family of proteins, MBD2 and MBD3, that recognize and interpret methylated residues on heterochromatin's underlying DNA. We use an integrated approach to explore the driving forces that allow them to undergo LLPS and how known interactors influence this process. Using computational approaches that assess amino acid sequence features, we found that MBD2 and MBD3 are highly disordered proteins predicted to undergo LLPS. Although they are highly similar in sequence, they have distinct clustering patterns of certain residue types that suggest the molecular basis of how they phase separate differs between them. We have tested these predictions in vitro and in cellulo and have demonstrated their ability to phase separate individually, together and with methylated DNA using UV-Vis spectroscopy and microscopy. Through truncations of MBD2 and MBD3, we have found that their ability to undergo LLPS is dictated by a balance between hydrophobic interactions, likely arising from their associative domains, and electrostatic interactions, arising from their highly charged termini, occurring within or between the proteins and DNA. Finally, using scattering techniques such as small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS), we have demonstrated that MBD2 and MBD3 are self-interacting proteins that form large assemblies. We propose that MBD2 and MBD3, through their ability to self-interact via hydrophobic and electrostatic forces, undergo LLPS and foster a biochemically unique environment to sequester binding partners and perform their functions as transcriptional repressors and heterochromatin organizers. Uncovering the driving forces that assemble MBD protein-based droplets will give us insight into the higher-order, LLPS-mediated organization of heterochromatin and how it functions within this structure. Additionally, understanding how disease-related aberrations influence biomolecular condensate dynamics will provide novel therapeutic targets.
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Characterization of the Role of Myosin 1e in the Progression of Breast Cancer and Focal Segmental GlomerulosclerosisMyosin 1e (myo1e) is a long-tailed class I myosin implicated in breast cancer progression and the development of focal segmental glomerulosclerosis (FSGS). This dissertation characterizes how myosin functions in these distinct pathologies. In chapter 2, I dissect the role that myo1e plays in the metastasis of breast cancer cells. Using the highly invasive 4T1 cell line, I demonstrate that cells deficient in myo1e exhibit altered morphologies and slower migration rates. Dissection of the migration defects in myo1e KO cells led us to investigate the role of myo1e in organelle trafficking, integrin endocytosis and the assembly and disassembly of focal adhesions. Our preliminary results suggest that cells deficient in myo1e exhibit reduced rate of focal adhesion disassembly. In chapter 3, I characterize three novel mutations in myosin 1e that were isolated from patients with FSGS: A92E, H506D, and G562R. Using in silico and comparative sequence analyses, I demonstrate that these mutations are likely pathogenic and highly evolutionarily conserved. Expressing these mutants in Madin-Darby Canine Kidney (MDCK) cells, I demonstrate that mutants myo1eH506D and myo1eG562R exhibit proper membrane enrichment, while the myo1eA92E mutant mislocalizes to the cytoplasm. Additional characterization of the properly localizing myo1eG562R mutant demonstrated that its junctional dynamics were not different from the junctional dynamics of myo1eWT. Taken together, our findings in chapter 3 demonstrate functional differences among myo1eA92E and myo1eH506D and myo1eG562R mutants and how these differences may contribute to their pathogenicity.
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Investigating how FHOD-family formin promotes Z-line organization and striation formation in C. elegans striated musclesStriated muscles are composed of basic structural and functional units called sarcomeres. The assembly of these sarcomeres is a well-studied process among vertebrates and multiple lines of evidence suggest formins as regulators of sarcomere assembly. Formins are regulators of unbranched actin networks and thus were ideal candidates to test for the initiators of thin filament assembly. We examined how Caenorhabditis elegans formins, FHOD-1 and CYK-1 regulate striated body wall muscle (BWM) growth. We found that FHOD family-related, FHOD-1 was the only formin that promoted BWM growth in a cell autonomous manner. However, the DIAPH-family related CYK-1 effect on BWMs was rather indirect. Interestingly, both these formins did not function as thin filament initiators. Our focus was to investigate the mechanisms of how FHOD-1 regulates striated muscle development. Loss of FHOD-1 however caused disorganized Z-lines in BWMs. Dense bodies (DBs) are analogous to Z-lines and are also similar to integrin-based focal adhesions. They are often arranged in rows that appear parallel in wild-type animals. We investigated how the loss of FHOD-1 affects the distribution, arrangement and morphology of the DBs. We found that loss of FHOD-1 led to the accumulation of non-parallel striations and FHOD-1 was enriched at the sites of new DB assembly as well as at sites where non-parallel striations would intersect. FHOD-1 supports the orientation of new striations. We also found that DBs from worms that lack FHOD-1 were fragile and were not able to withstand prolonged contractions. We interpret that FHOD-1 could regulate the actin dynamics or act as a linker to bundle actin filaments that are a part of this unique DB-associated cytoskeletal system, which provides structural integrity to the DBs.
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Unraveling differential origin firing and replication stress mediated genome instability using S. cerevisiae as a model organismYeasts have been used as model organisms because of their compact genome size, simple growth requirements, and homology to higher eukaryotes. The sites for origin initiation are well defined in the S. cerevisiae genome, and several metabolism and resistance genes are well conserved to higher eukaryotes, making it an ideal model system to study origin dynamics and replication inhibitor mediated genome instability. In this thesis, using S. cerevisiae as a model we answered two big questions. First, we studied factors regulating differential origin firing between two commonly used laboratory strains. Second, we identified the general mechanism used by replication inhibitors to induce genome-wide DSBs. To identify the factors regulating differential origin firing we compared, 1) S-phase progression, 2) the relative level of checkpoint protein, and 3) the binding of Rad53 and Cdc45 at a select few origins. Amongst the three, we found that difference in binding of Rad53 at the origins was responsible for differential firing between the two strains. In a new origin class, "Rad53 dependent" we looked the role of active transcription in regulating origin firing. Our results show that active transcription during S-phase does not wipe out the origin firing activity. To study replication inhibitor-induced genome instability, we used a broad range of inhibitors that induce replication stress differently. Our results suggested that replication-transcription collision is a common mechanism used by these inhibitors to induce DSBs. Associating each DSB with an upregulated gene, we found an enrichment in the head-on orientations. As these inhibitors are regularly used in the clinic as anticancer therapeutics, studying the gene expression helped us identify their mechanism of action to specifically target cancer cells. We found that all of these individually upregulate the oxidation-reduction pathway, and downregulate glycolysis. Finally, at one locus on chromosome II which induces DSB specifically in CPT, we were able to demonstrate replication fork pause and correlation of the DSB with active transcription-deposited histone marks.
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Bridging the Gaps: Investigating Molecular Mechanisms That Coordinate Actin Filament AssemblyCell division, migration, and maintaining cell morphology are all essential and dynamic cell processes that require precise coordination of the actin cytoskeleton. Actin monomers assemble into polar actin filaments that have a faster-growing (barbed) end and a slower-growing (minus) end. Here we examine the role of IQ-motif containing GTPase Activating Protein 1 (IQGAP1) in regulating actin filament assembly. This 189 kDa actin- binding protein slows actin filament assembly by interacting with the barbed end. Using extensive truncation analysis and single molecule microscopy techniques, we determined that IQGAP1 interacts with actin filament ends via residues within its IQ motifs. The barbed ends of actin filaments are intricately and competitively regulated by specific proteins and complexes of proteins that promote (formins) or inhibit (capping proteins) actin filament growth. We next examined the role of IQGAP1 in competitive interactions with the prominent barbed end regulators including formin and capping protein. Using fluorescently tagged proteins, IQGAP1 can be directly visualized on filament ends with individual formins and capping proteins and with formin-capping protein complexes. Interactions between IQGAP1 and formin on the ends of filaments slows formin-mediated actin assembly from 22.68 ± 2.9 subunits s-1μM-1 to 6.13 ± 0.7 subunits s-1μM-1. Further, IQGAP1 interacts with decision complexes on filament ends, creating a more complex decision complex, which decreases the dwell time on the end by 18-fold. We next examined the relevance of IQGAP1-mediated capping in cells using readouts of actin assembly: cell morphology, actin filament structure, and cell migration. Cells lacking IQGAP1 displayed significant changes to cell morphology and actin filament structures Cells expressing IQGAP1 or a capping deficient IQGAP1(CD), unable to bind filament ends, on a plasmid did not display significant changes to cell morphology or actin filament structure compared to wildtype cells. However, cells expressing IQGAP1(CD) displayed significantly slower wound closure compared to cells with endogenous IQGAP1. These results suggest that IQGAP1-mediated capping is a physiologically relevant mechanism of regulating actin filament assembly. This study reveals a role for IQGAP1 as a transient capper that promotes protein exchange on filament ends, which may have implications in the regulation of actin filament lengths in cells.
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Phosphoinositide-Specific binding by Human V-ATPase a-subunit isoformsThe individual organelles in a eukaryotic cell have tightly regulated pH, essential for their function and viability. This distinct pH defines organelle identity and is maintained principally by vacuolar H+-ATPases (V-ATPases). V-ATPases are highly conserved, ATP driven proton pumps comprised of a peripheral, cytosolic V1 domain, and an integral membrane bound Vo domain. The cytosolic N-terminal domain of the Vo a-subunit (aNT), positioned at the interface of V1 and Vo, modulates organelle specific regulation and targeting of V-ATPases. The a-subunit is encoded by several tissue and organelle-specific isoforms that help target V-ATPases to various organelles and confer distinct functional properties. Importantly, loss of V-ATPase a-subunit isoform function is associated with human diseases, making V-ATPases potential drug targets. However, the mechanisms for targeting V-ATPases to distinct membranes and achieving organelle-specific regulation are incompletely understood. Phosphatidylinositol phosphates (PIP) are low abundance lipids localized in the outer leaflets of organelle membranes and implicated in V-ATPase regulation and organelle pH maintenance. Studies have shown that the yeast a-subunit isoforms, Vph1NT and Stv1NT, interact with distinct PIPs in their resident organelle and affect activity, regulation, and localization of V-ATPases accommodating these isoforms. Higher organisms, including humans express four a-subunit isoforms. We hypothesize that V-ATPases and PIP lipids interact with the NT domains of human Vo a-subunit isoforms. The Hua1 and Hua2 isoforms function in endolysosomes and Golgi respectively. Our data shows that bacterially expressed HuaNTs bind specific PIP lipids, Hua1NT binds endosome/lysosome enriched PI(3)P and PI(3,5)P2 and Hua2NT bind Golgi-enriched PI(4)P. Cryo-EM structures from yeast and mammals show that aNT is dumbbell shaped, with globular proximal and distal ends supporting specific interactions with V1 and Vo subunits with poorly conserved loops facing the membrane. Modeling on existing structures has identified potential PIP binding sites in the HuaNT domains, which were mutagenized and tested for PIP specificity. In both the isoforms, binding sites were identified in the distal domain loops, highlighting their importance in PIP specificity of the a-subunit. Defining PIP binding codes on V-ATPase will improve our understanding of organelle specific pH control and provide new avenues for controlling V-ATPase subpopulations.
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Modelling prenatal stress in human neural progenitor cellsPsychiatric disorders are a leading cause of disability, premature mortality, and economic burden globally, with a 48.1% increase in cases between 1990 and 2019. Stress in early life has been linked to hippocampal damage and the development of psychiatric disorders later in life. Personal stressors have also been found to be significantly associated with psychological distress, anxiety, and depression. Prenatal stress, which occurs when the fetus is exposed to excess glucocorticoids from maternal stress or synthetic glucocorticoids, has been linked to cognitive and behavioral outcomes later in life, possibly due to its impact on fetal development. In this project, I aimed to study the effect of cortisol on neural progenitor cells (NPCs) differentiated from human induced pluripotent stem cells (iPSCs). Cell type was confirmed using iPSC and NPC marker gene and protein expression by qPCR and immunofluorescence. The loss of undifferentiation marker SSEA4 in NPCs suggests that they are differentiated. The NPCs expressed SOX2, Nestin, SOX1, and PAX6, as confirmed by qPCR and immunofluorescence analyses. The percentage of SOX2, SOX1, PAX6, and Nestin positive cells was quantified based on colocalized signals with the nuclear marker DAPI. Novel Nestin isoforms that may be present in NPCs but not in iPSCs were identified. Currently, only one Nestin isoform is known to us. Novel, unpublished data from collaborators was used to confirm that there are Nestin isoforms present in fetal and adult human isoform sequencing study. Glucocorticoid (GC) response genes were upregulated after cortisol treatment in NPCs, as confirmed by qPCR. I measured NPC proliferation in response to cortisol and identified that cortisol did not significantly increase NPC proliferation. However, RNA sequencing showed differential expression of proliferation-related genes in cortisol-treated NPCs. RNA sequencing analysis revealed that 59 genes were differentially expressed in cortisol treated NPCs (including ZBTB16 and TSC22D3 measured previously using qPCR) compared to controls, out of which nine genes are associated with psychiatric traits based on GWAS studies. The study showed that cortisol can alter gene expression in NPCs, which may have implications for psychiatric disorders. Finally, the study emphasizes the novelty of exploring the role of cortisol in iPSC derived NPCs and highlights the need for further research in this area.
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The Role of Paxillin in Regulating Rab5 through Modulating Microtubule AcetylationCell migration is vital to many cellular processes such as development, immune surveillance and wound healing. Cells interact with and move along the extracellular matrix by utilizing transmembrane integrin receptors and large macromolecular structures, called focal adhesions. Focal adhesions are comprised of many signaling and scaffolding proteins, and these complexes constantly undergo formation and turnover. The trafficking of focal adhesion components is mediated by RabGTPases, which are essential in regulating cell migration. Defects in Rab-associated vesicle trafficking have been implicated in diseases such as cancer cell migration. Understanding the mechanisms contributing to vesicle trafficking is vital to the development of therapeutics to treat diseases. The focal adhesion adaptor protein, paxillin, plays a central role in regulating focal adhesion dynamics and cell migration. Recent studies have implicated paxillin in regulating vesicle trafficking. Paxillin, through regulation of microtubule acetylation via modulation of HDAC6 activity, regulates anterograde trafficking of ts-VSVG to the plasma membrane. Additionally, paxillin interacts with components of endocytic machinery, suggesting it may also regulate retrograde trafficking. Further studies are needed to characterize paxillin's role in regulating vesicle trafficking. Herein, this thesis identifies a role for paxillin in regulating the RabGTPase, Rab5, in a HDAC6-dependent manner. Through modulation of HDAC6 activity, paxillin regulates Rab5 vesicle size and distribution, Rab5 activity and dynein-mediated retrograde Rab5 vesicle motility in MDA-MB-231 cells and paxillin (-/-) fibroblasts. In addition to its role in regulating Rab5, paxillin was also shown to promote Rab7 vesicle size and co-localization with Rab5-positive vesicles. Co-localization analysis revealed that the distribution of active β1 integrin at zyxin-positive focal adhesions was disrupted upon paxillin depletion but was rescued upon inhibition of HDAC6 activity. Altogether, this work expands on a recently identified role of paxillin regulating vesicle trafficking in both normal and transformed cancer cells.
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Isoform-Specific Regulation of the Yeast V-ATPase a-subunitV-ATPase primarily acidifies organelles of the endocytic and secretory pathway. The lumen of each of these organelles maintains a distinct pH, but how this diverse pH range is established is not well understood. Different modes of regulation of the V ATPase may help fine-tune their activity. The a-subunit from the membrane bound Vo subcomplex is a regulatory hub of V-ATPase which harbors many regulatory interactions in its cytosolic N-terminal (aNT) domain. The aNT domains of all isoforms consist of a proximal and a distal globular domain connected by a coiled-coil. Vo a-subunit exhibits organelle- and tissue-specific isoforms. Vph1 and Stv1 are the two organelle-specific a subunit isoforms in yeast. V-ATPase containing the Vph1 isoform reside in the lysosome-like yeast vacuole whereas V-ATPase containing the Stv1 isoform reside predominantly in the Golgi apparatus. The RAVE (Regulator of H+ -ATPase of vacuoles and endosomes) complex and phosphoinositide phospholipids (PIP lipids) are two important factors previously implicated in isoform-specific V-ATPase regulation. The a subunit isoforms vary in their dependence on the RAVE and PIP lipid. But where the information of RAVE and PIP lipid recognition reside in the aNT domain and how these two regulatory inputs are integrated to control V-ATPase function are not well understood. We hypothesize that the aNT domain contains distinct sequences for RAVE and PIP lipid recognition, and that the differential interactions of the a-subunit isoforms with RAVE and PIP lipids result in isoform-specific function and regulation of the V ATPase. To better understand the regulatory information present in the aNT domain, we generated chimeric aNT constructs by combining parts of Vph1NT and Stv1NT. Vph1NT was previously shown to bind to a RAVE subunit and Vph1 V-ATPases require the RAVE complex for their assembly. Stv1-containing V-ATPases on the other hand assemble independent of RAVE and Stv1NT does not bind to RAVE subunits in vitro. In chapter 2, we have shown that replacing the proximal domain of Vph1NT with the proximal end of Stv1NT fully restores the RAVE interaction, implicating this region of Vph1 in RAVE-dependent assembly. The two isoforms also exhibit preferences for distinct PIP lipids enriched in their organelle of residence; Stv1NT binds tightly to Golgi PI(4)P and Vph1NT binds to vacuolar PI(3,5)P2. A PI4P binding site in the proximal domain of Stv1NT was previously reported. Our analysis with chimeric constructs suggests that a 6-amino acid sequence containing this site is sufficient to transfer PI(4)P binding to Vph1NT. Our results also suggest that both the proximal and the distal ends of Stv1NT contain sequences that promote PI4P binding. Interestingly, when expressed in yeast as a full-length a-subunit, the chimera containing both PI4P and PI(3,5)P2 binding sites has wild-type level activity and assembly in isolated vacuoles even though it lacks a RAVE binding site. Although V-ATPases in this chimeric strain is fully functional there are consequences of their altered regulatory properties. V-ATPases in this chimera disassemble during glucose deprivation but do not reassemble efficiently after glucose re addition, consistent with a lack of RAVE binding. We also observed a delayed growth in media containing raffinose suggesting they cannot readily adjust during a transition to a less preferred carbon source. While the lack of RAVE binding gives rise to the growth defect of this chimera in a poor carbon source, increased PI(3,5)P2 binding of this mutant, on the other hand, contributes to growth benefit in alkaline pH stress. Together these data reveal the interplay between two mechanisms of V-ATPase regulation and suggest that aNT domains can functionally integrate multiple regulatory inputs.
