• Characterization of Hic-5 in Cancer Associated Fibroblasts: A Role in Extracellular Matrix Deposition and Remodeling

      Turner, Christopher; Goreczny, Gregory (2017)
      Hic-5 (TGFβ1i1) is a focal adhesion scaffold protein that has previously been implicated in many cancer-related processes. However, the contribution of Hic-5 during tumor progression has never been evaluated, in vivo. In Chapter 2 of this thesis, I crossed our Hic-5 knockout mouse with the MMTV-PyMT breast tumor mouse model to assess the role of Hic-5 in breast tumorigenesis. Tumors from the Hic-5 -/-;PyMT mouse exhibited an increased latency and reduced tumor growth. Immunohistochemical analysis of the Hic-5 -/-;PyMT tumors revealed that the tumor cells were less proliferative. However isolated tumor cells exhibit no difference in growth rate. Surprisingly, Hic-5 expression was restricted to the tumor stroma. Further analysis showed that Hic-5 regulates Cancer Associated Fibroblast (CAF) contractility and differentiation which resulted in a reduced ability to deposit and reorganize the extracellular matrix (ECM) in two-and three-dimensions. Furthermore, Hic-5 dependent ECM remodeling supported the ability of tumor cells to metastasize and colonize the lungs.The molecular mechanisms by which CAFs mediate ECM remodeling remains incompletely understood. In Chapter 3 of this thesis, I show that Hic-5 is required to generate fibrillar adhesions, which are specialized structures that are critical for the assembly of fibronectin fibers. Hic-5 was found to promote fibrillar adhesion formation through a newly characterized interaction with tensin1, a scaffold protein that binds to β1 integrin and actin. Furthermore, this interaction was mediated by Src-dependent phosphorylation of Hic-5 in two and three-dimensional matrix environments to prevent β1 integrin internalization and subsequent degradation in the lysosome. This work highlights the importance of the focal adhesion protein, Hic-5 during breast tumorigenesis and provides insight into the molecular machinery driving CAF-mediated ECM remodeling.
    • Characterizing the Role of the Epsilon Subunit in Regulation of the Escherichia coli ATP Synthase.

      Duncan, Thomas; Shah, Naman (2015)
      The F-type ATP synthase is a rotary nanomotor central to cellular energy metabolism in almost all living organisms. In bacteria, the enzyme also plays a role in nutrient uptake and pH regulation underlining its importance. All ATP synthases can be inhibited by ADP, whereas in bacteria, the enzyme is alsoautoinhibitedbyits ε subunit. The inhibition involves a drastic conformationa l change of the C-terminal domain of the ε subunit (εCTD)thatblockscatalytic turnover. Thisregulation by ε is believed to play an important role in maintaining viability of the cell. Recent development in the field of antibiotics has validated ATP synthase as a drug target against pathogenic bacteria. Thus, there is a renewed interest in studying the role of the ε subunit in regulation of the enzyme and exploiting it to develop antimicrobials that can kill pathogenic bacteria. The present work describes advances in our understanding of the regulatory interactions of εCTD in E. coli ATP synthase.In the first approach, we used an optical binding assay to understand the transitions of εCTD between its active and inhibitory conformations.Using different ligands we revealedthe relationship between ADP inhibition and ε inhibition. In the second novel approach, the terminal five amino acids of εCTD were deleted to observe the effects on in vivo and in vitro functions of ATP synthase. The results obtained from these studies advance our understanding of εinhibition inbacteria and also provide a noveltarget within bacterial ATP synthase to obtain antibacterial drugs.
    • Expression and Function of Paxillin Genes in Zebrafish: A Role in Skeletal Muscle Development

      Turner, Chris; Amack, Jeffrey; Jacob, Andrew (2017)
      Paxillin is a key component of the Integrin adhesion complex, which regulates cellular signaling events in response to extracellular matrix interactions. Although the roles for Paxillin in cell migration have been extensively studied, less is understood about its role in vertebrate development. Depletion of Paxillin from mouse embryos results in early lethality due to impaired cardiovascular development and function, necessitating the development of alternative vertebrate genetic models for examining the role of Paxillin during embryogenesis. Zebrafish have emerged as an experimental vertebrate model amenable to genetic manipulation. The work compiled herein first characterizes the expression profiles for Paxillin genes in zebrafish, and then describes the embryonic phenotypes observed upon mutation of these genes. The identification of two Paxillin genes in zebrafish, pxnaand pxnb, provided new insight into the evolution of this gene family in the Teleost lineage. Both overlapping and unique expression profiles for these genes during zebrafish embryogenesis were uncovered. While both genes are expressed in developing skeletal muscle, pxnawas restricted to the notochord during earlier stages of embryogenesis and pxnbwas expressed in the developing heart. Targeted mutation of either gene alone did not impair embryonic development, suggesting partial functional redundancy between each gene during embryogenesis. Accordingly, combined mutations in pxnaand pxnbrevealed defects during the development ofseveral embryonic tissues. In particular, skeletal muscle morphogenesis iiiwas perturbed in these double mutant embryos. Further characterization revealed that Paxillin genes in zebrafish serve to regulate embryonic myotome shape and proper extracellular matrix composition during muscle development. The amount of Laminin was reduced, while the abundance of Fibronectin persisted, during myotome morphogenesis in Paxillin double mutant embryos. In addition, a role for cytoskeletal contractility in regulatingsubcellular localization of Paxillin in developing skeletal muscle was established. Defects in the development of the cardiovascular system were also apparent in Paxillin double mutant embryos, and future work will focus on characterizing these in further detail. Altogether, this work provides a new vertebrate model to use for understanding the role of Paxillin during embryonic development, and uncovers an unrecognized role for Paxillin in establishing the extracellular matrix of skeletal muscle.
    • Role of RPTPzeta/phosphacan in the neural extracellular matrix

      Matthews, Rick; Eill, Geoffrey (2020)
      The mammalian brain is the most structurally and functionally complex system in biology. In order to carry out diverse functions such as thought and cognition, neurons in the brain must properly differentiate, make millions of functional interconnections, and incredibly be able to maintain those interconnections while retaining plasticity and the ability to learn throughout its lifetime. In order for neurons to carry out these complex functions, they must have an intricate relationshipwith their extracellular environment, which provides numerous molecules, such as growth factors and neurotransmitters, and physical cues to initiate critical downstream signaling cascades. In the central nervous system (CNS), the neural extracellular matrix (ECM) largely organizes this extracellular environment, and as such, is implicated in a multitude of neuronal functions. Not only can it serve as a physical barrier, the neural ECM is shown to regulate neuronal cell differentiation, migration, synaptogenesis, and maintain the mature state by restricting plasticity and neurite outgrowth. Of particular import, a subset of the neural ECM, the perineuronal net (PNN), is implicated to regulate neuronal plasticity in the CNS. PNNs were historically believed to be critical for restricting experience dependent plasticity in the brain but more recently shown to regulate several forms of learning and memory, in addition to multiple neurological diseases. However, despite these implicated functions, PNNs comprise only a fraction of the total ECM in the CNS. A majority of the neural ECM is derived from the diffuse ECM, a structure that is ubiquitously expressed throughout the CNS. As the diffuse ECM is very similar in molecular composition and structure to PNNs, it is difficult to specifically target PNNs for functional and mechanistic studies.As such, a better toolset is needed to differentiate the contributions ivbetween PNNs and the diffuse ECM in regulating plasticity and learning and memory. To develop thistoolset, there is a great need to better understand PNN molecular composition and structure. Therefore, the purpose of this work was to provide critical insight into the molecular composition and structure of PNNs soas to better understand its function in the CNS. In Chapter 2, using multiple ECM genetic knockout models, we show compelling evidence of an additional PNN anchor that importantlylays the groundworkforfuture functional studies. Specifically, we suggestchondroitin sulfate proteoglycan (CSPG) phosphacan, secreted isoform of receptor protein tyrosine phosphatase zeta (RPTPζ), iscritical for PNN structure as it partially anchors PNNs to the neuronal surface through cooperation with tenascin-R. Additionally, as the neural ECM is involved in numerous neurological diseases, in Chapter 3, we investigated the function of major ECM component, RPTPζ, in a group of O-mannosyl related congenital muscular dystrophy with associated brain abnormalities (CMD). Our data suggest a possible role of RPTPζ in proper cortical lamination in a CMD mouse model. Interestingly, we found evidence of a novel O-mannosyl substrate in the developing brain that could critically contribute to the underlying deficits of CMD. In conclusion, the neural ECM, once previously disregarded in the field, is becoming a novel source to understand mature mammalian brain function and disease, but more work is needed to better differentiate the specific roles of its substructures.
    • The Role of TET Methylcytosine Dioxygenase 2 in Myeloid Malignancies

      Mohi, Golam; Nath, Dipmoy (2017)
      TET methylcytosine dioxygenase 2 (TET2) catalyzes the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), an intermediate stage in the DNA demethylation processthat controls the transcription of genes. TET2 is highly expressed in the hematopoietic system and is suggestedto regulate the maintenance and differentiation of hematopoietic stem/progenitors. Mutations in TET2 has been found in different hematological malignancies including Acute Myeloid Leukemia (AML), Chronic Myelomonocytic Leukemia (CMML), Myeloproliferative Neoplasms (MPN), Myelodysplastic Syndrome (MDS), etc. However, the mechanisms by which TET2 controls hematopoiesis and contributes to myeloidmalignancies remainunknown. The direct targets of TET2 have not been elucidated yet. In order to identify the direct targets of TET2, we have performed chromatin immunoprecipitation using TET2 specific antibody followed by genome-wide sequencing. We have found enrichment in binding of TET2 in the promoters of SHP1, SOCS3 and PLZF among other targets. Interestingly, the expression of these genes is also significantly downregulated in the hematopoietic progenitors of TET2 deficient mice. Furthermore, we have found that CMML patients with TET2 mutations also have decreased expression of these genes. Knockdown of TET2 resulted in downregulation of these genes in leukemic Molm14 and murine Ba/F3 cells. Conversely, overexpression of TET2 in monocytic U937 cells increased the expression of these genes. Using methylation specific PCR, we also have observed increased methylation in the promoters of SHP1, SOCS3 and PLZF in TET2 deficient Molm14 and Ba/F3 cells. Also, using methyl/hydroxymethyl-DNA immunoprecipitation, we observed an increase in the 5mC level and decrease in the 5hmC level in the promoter region of these genes suggesting that TET2 directly regulates the expression of these genes by regulating the methyl and hydroxymethyl level of the promoter of these genes. Although loss of function of TET2 has been associated with multiple hematopoietic malignancies, TET2 is most commonly mutated in CMML with almost 50% patients bearing TET2 mutations. Interestingly, TET2 mutations are frequently associated with CBL mutations in CMML. In order to assess the concurrent effects of TET2 and CBL deficiencies, we generated TET2 CBL double knockout mice. We observed that simultaneous deletion of TET2 and CBL resulted in increased leukocytes and neutrophil and enhanced splenomegaly compared to control mice. The double knockout mice showed increase in the granulocyte macrophage progenitors and a significant expansion of the stem progenitor cell population. The overall survival of these mice also reduced substantially. It suggests that concurrent deletion of TET2 and CBL increased the severity of the CMML like disease in mice and thus TET2 and CBL deletion may cooperate in the pathogenesis of CMML. Although most studies suggest a tumor suppressor function of TET2, we also have found a tumor promoter function, especially in MLL rearranged leukemia. We have found that knockdown of TET2 resulted in decreased proliferation in MLL-AF9 positive Molm14 leukemia cells and murine Ba/F3 MLL-AF9 expressing cells. Conversely overexpression of TET2 significantly increased the proliferation level of Molm14 cells. In orderto understand the in vivo role of TET2 in MLL-AF9 mediated leukemia, we performed a retroviral BMT experiment. Whereas the expression of MLL-AF9 in wild type BM resulted in marked increase in WBC and NE and splenomegaly, the deletion of TET2 reduced the white blood cell and neutrophil count and also caused reduction in the spleen size. MLL-AF9 overexpression resulted in the increase in hematopoietic stem/progenitor cells and granulocyte macrophage progenitors and granulocyte/monocyte precursors which was significantly reduced in TET2 KO MLL-AF9 mice. There was a significant reduction of the hematopoietic colony formation ability mediated by MLL-AF9 in TET2 KO mice. The overall survival of the knockout mice was markedly improved compared to the control MLL-AF9 mice. Together these results suggest a tumor promoter role of TET2. Taken together, all the results indicate a dual role of TET2 in myeloid leukemia.
    • Role of the SMC5/6 complex in DNA replication and DNA damage repair

      Feng, Wenyi; Peng, Jie (2017)
      The structural maintenance of chromosome (SMC) proteins form the core of Cohesin, Condensin and the Smc5/6 complex, which are essential for organization and metabolism of chromosomes during the cell cycle. The Smc5/6 complex is implicated a role in non-essential homologous recombination-mediated (HR) DNA repair. Inactivating Smc5/6 (via temperature-sensitivity mutant) in the S phase, but not in the G2 phase, causes mitotic failure. Hence, we hypothesize that the Smc5/6 complex has an major role in DNA replication. We analyzed the genome-wide DNA replication temporal program in different genetic backgrounds. Mapping replication fork-associated ssDNA in WT and rad53 cells in the presence of replication stress allowed us to separate early and late replication origins. Using this method, we revealed a strain background difference of origin usage in A364a and W303. We then studied the genome-wide replication dynamics in a hypomorphic mutant, smc6-P4, using a density transfer coupled with microarray method. Overall replication dynamics of the mutant are similar to that of the WT cells with exceptions in the early S phase. However, we captured a difference in the replication profile of an early S phase sample in the mutant, prompting the hypothesis that the mutant incorporates ribonucleotides and/or accumulates single-stranded DNA gaps during replication. We then tested whether we can exacerbate the DNA replication stress hypersensitivity of the smc6 mutant by inhibiting ribonucleotides excision repair (RER) pathway. Contrary to our expectation, impairment of ribonucleotide excision repair, as well as virtually all other DNA repair pathways, alleviated smc6 mutant’s hypersensitivity to induced replication stress. We propose that nucleotide incision with impaired Smc5/6 complex has a more disastrous outcome than the damage itself. We found that the smc6 mutant has a significant increase in ssDNA level under replication stress but inhibiting RER pathway decreases it. This result suggests that Smc5/6 complex prevents unsupervised ssDNA formation during DNA replication as a result of damage incision repair. We reason that excess ssDNA in the Smc5/6 defective cells may subsequently engage in toxic recombination. Our study thus provides novel perspectives for the role of the Smc5/ 6 complex during DNA replication.