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Browsing by Subject "Molecular, cellular, developmental biology and genetics"

Now showing 1 - 9 of 9
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    Adjacent long non-coding RNA PVT1 and MYC co-operate in breast cancer with gain of 8q24
    (2013-08) Tseng, Yuen-Yi
    Copy number gain of 8q24 is a common structural abnormality in human cancers. Although MYC is usually assumed to be responsible for 8q24 gain cancer, the role of the other genes in the 8q24 region remains mostly unknown. We derived chromosome engineered mice with an extra copy of Myc-Gsdmc region which is syntenic to the common region gained in human 8q24-associated cancer. These mice show aberrant differentiation and loss of proliferation arrest of mammary epithelial cells, excessive branching of mammary ducts, and increased sensitization to mammary tumors. In contrast, mice carrying a duplication of either Myc or Pvt1-Gsdmc were found to be insufficient for neoplasia. We show that the long non-coding RNA Pvt1 and adjacent Myc can co-operate in tumorigenesis. Furthermore, PVT1 can regulate MYC protein stability in human breast cancer cells. Our study reveals a novel mechanism of MYC regulation by a long non-coding RNA in cancer cells and could provide therapeutic targets.
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    A biochemical and molecular analysis of functional differences between dystrophin and utrophin
    (2013-11) Belanto, Joseph John
    The DMD gene encodes the protein dystrophin, a 427kD cytoplasmic protein responsible for linking the actin cytoskeleton to the extracellular matrix via the dystrophin-glycoprotein complex. Mutations in dystrophin that abolish its expression lead to Duchenne muscular dystrophy (DMD). Patients with DMD become wheelchair bound in their early teens and succumb to fatal cardiac and/or respiratory failure in their mid-twenties to early thirties. There is currently no effective or widely available treatment for DMD beyond ventilatory support and the use of corticosteroids. Many therapies for treating dystrophin deficiency aim at upregulating its autosomal homolog utrophin due to its structural similarity and ability to bind an almost identical repertoire of proteins that dystrophin binds. It was previously shown that utrophin cannot bind neuronal nitric oxide synthase (nNOS) even though dystrophin binds nNOS, establishing for the first time a functional difference between dystrophin and utrophin. Here, we show that transgenic overexpression of utrophin on the mdx mouse background (Fiona-mdx) is not sufficient to rescue the disorganized microtubule network of the mdx mouse. Thus, we have elucidated a second functional difference between dystrophin and utrophin. Additionally, Fiona-mdx mice lack full recovery of cage activity after mild exercise. Our results suggest that any deficiency in nNOS binding or microtubule lattice function caused by loss of dystrophin may not be restored by upregulation of utrophin. Previously, our lab reported that dystrophin directly binds to microtubules and organizes them beneath the sarcolemma. Using in vitro microtubule cosedimentation assays, we show that dystrophin binds to microtubules with strong affinity (KD=0.33µM). Through the use of various recombinant constructs tested via in vitro microtubule cosedimentation we show that spectrin-like repeats 20-22 of the dystrophin central rod are responsible for microtubule binding activity. However, we show that these repeats require flanking regions of dystrophin for proper binding activity, making microtubule binding context-dependent. Additionally, we show that recombinant utrophin does not bind microtubules in vitro, corroborating our in vivo findings of the disorganized subsarcolemmal microtubule lattice of the Fiona-mdx mouse. We also provide evidence showing that dystrophin functions as a molecular guidepost to organize microtubules into a rectilinear lattice.
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    Biophysical, cellular, and animal models of dystrophin missense mutations
    (2014-12) Talsness, Dana
    The 427kDa protein dystrophin is expressed in skeletal muscle where it localizes to the costamere and physically links the interior of muscle fibers to the extracellular matrix. Mutations in the DMD gene encoding dystrophin lead to a severe muscular dystrophy known as Duchenne (DMD) or a mild form known as Becker (BMD). Currently, there is no cure for DMD or BMD, but there are several therapies being investigated that target specific types of mutations found in the DMD gene. Nonsense mutations almost always lead to a complete lack of dystrophin protein, with stop codon read-through drugs being studied for personalized treatments. Out-of-frame deletions and insertions also cause nearly a complete lack of dystrophin, for which exon-skipping is currently being investigated. Missense mutations in dystrophin, however, cause a wide range of phenotypic severity in patients, the molecular and cellular consequences of such mutations are not well understood, and there are no therapies currently targeting this genotype. Here, we report on three separate model systems of missense mutations in dystrophin: an in vitro biochemical model, a myoblast cell culture model, and an in vivo animal model. Together, they provide evidence that different missense mutations cause variable degrees of thermal instability, which leads to proportionally decreased dystrophin expression, and subsequently causes dystrophic phenotypes. In addition, our initial studies of small molecule treatments show that it is possible to increase the levels of mutant dystrophin, and may lead to personalized therapeutics for patients with missense mutations.
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    Characterization of the function of the C. elegans heterochronic gene lin-42/per during larval development
    (2013-12) McCulloch, Katherine Ann
    The heterochronic pathway of C. elegans ensures the appropriate timing of post-embryonic development. Mutations in heterochronic genes cause skipping or reiteration of larval programs, resulting in severe developmental defects. Many C. elegans heterochronic genes are conserved in both sequence and function; therefore, study of developmental timing in C. elegans contributes to understanding of development in other organisms. The heterochronic gene lin-42 is the C. elegans homolog of period, a component of the circadian clock of Drosophila and mammals. lin-42(lf) results in precocious heterochronic phenotypes, in which later developmental events occur too early. lin-42 also regulates molting, and lin-42 mutants have delayed and prolonged larval molts compared to wild-type animals. In my thesis work, I show that lin-42 confers robustness on developmental and molting pathways against environmental fluctuations, as lin-42(lf) heterochronic and molting phenotypes are very sensitive to changes in environment, which is not observed in wild-type animals. Also, I have found that lin-42 regulates developmental timing by inhibiting expression of let-7-family miRNAs, likely at the level of transcription. Genetic analyses place lin-42 upstream of this miRNAs, showing that regulation of let-7-family miRNAs is a key function of lin-42 in the heterochronic pathway.
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    A conservative mechanism of polarization drives hyphal growth in the opportunistic yeast pathogen Candida albicans
    (2014-08) Pulver, Rebecca
    In eukaryotes, different cell morphologies are generated by fine-tuning the spatiotemporal regulation of the polarized growth machinery. Studying hyphal growth in the multimorphic opportunistic yeast pathogen Candida albicans provides a unique opportunity to understand how highly polarized cell structures are generated and maintained, and has the potential to provide insight into mechanisms of pathogenesis. Hyphal cell morphology requires that polarized growth machinery be held at hyphal tips over extreme distances and through multiple cell cycles. Deletion of the bud-site selection GTPase Rsr1 in C. albicans results in defects in cell size and shape not observed in studies of its ortholog in the related yeast Saccharomyces cerevisiae. This suggests that, in addition to its role in bud site selection, Rsr1 has expanded function in C. albicans, which impacts polarized growth and the generation of the hyphal morphology. Here, I show that loss of Rsr1 results in changes to a hyphal-specific tip structure, the Spitzenkörper, a downstream developmental indicator of Cdc42 signaling, and key regulator of polarized growth. Also, my results show that Rsr1's function impacts the spatiotemporal distribution of Bem1 a marker of the active form of Cdc42. Interestingly, the changes in the distribution of Cdc42 activity are also correlated with reduced expression of the hyphal transcriptional program. In addition, I also show the differential effects of the guanine nucleotide binding states of Rsr1, through the manipulation of the Rsr1 GAP, Bud2, and GEF, Bud5. Through the action of Bud2, Rsr1-GDP acts as a global inhibitor that limits competitive, stochastically-activated clusters of Cdc42, and also as a lateral inhibitor of growth at hyphal tips that strongly influences the overall width of the hypha. In contrast, Rsr1-GTP, through Bud5 activity, is needed to efficiently nucleate single clusters of Cdc42 activity during hyphal emergence, and also contributes to the extremely narrow morphology of the hypha. Altogether, the data presented here suggest that Rsr1 cycling supports a conservative mechanism of polarization that optimizes the efficiency with which polarization occurs, which is required for the maintenance of polarized growth.
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    FGFR1-induced soluble factors promote mammary tumorigenesis and chemoresistance
    (2013-08) Bade, Lindsey Kay
    The fibroblast growth factor receptor (FGFR) family consists of four receptor tyrosine kinases that are known regulators of cellular processes such as proliferation, migration, survival, and angiogenesis. Anomalous expression or uncontrolled activation of these receptors or their ligands has been correlated with progression of various types of cancer, including breast cancer. Specifically, the chromosomal locus of FGFR1, 8p11-12, is found to be aberrantly amplified in approximately 10% of patients diagnosed with breast cancer. Patients who harbor the FGFR1 amplification do not respond well to current therapies and develop resistance to hormone-based therapies. Therefore, understanding the molecular mechanisms of how FGFR1 overexpression promotes tumorigenesis may provide insights into better targets for novel, more effective therapies. The work presented here shows that FGFR1 activation significantly upregulates expression of the ligands AREG and EREG at the transcript and protein levels both in vitro and in vivo, which then activate EGFR signaling. AREG is critical for normal ductal morphogenesis in the mammary gland and has also been linked to breast cancer progression. Studies examining AREG expression in human breast cancers have found AREG expression to significantly correlate with regional lymph node metastases, large tumor size, and high-grade tumors. While EREG promotes proliferation of several normal and cancerous cell types, the role of EREG has not been extensively characterized in the mammary gland. However, recent studies have demonstrated that EREG is a potent mediator of metastasis of breast cancer cells to the lung and that overexpression of EREG is an indicator of poor prognosis for inflammatory breast cancer patients. EGFR, a member of the ErbB receptor tyrosine kinase family, has been well studied in the mammary gland, and it is known that EGFR is required for normal mammary gland ductal morphogenesis. Alternatively, overexpression or constitutive activation of EGFR in the mammary gland has been linked to mammary tumorigenesis. Additionally, overexpression of EGFR in the breast is associated with recurrence of earlier stage breast cancers and decreased disease-free and overall survival in later stage breast cancer patients. Notably, we demonstrate that EGFR activation is at least in part required for FGFR1-induced proliferation and migration and ERK1/2 activation, as inhibition of EGFR with the small molecule kinase inhibitor erlotinib significantly blocks these processes. Moreover, we show that FGFR1 and EGFR are co-expressed in TNBC cell lines and that both FGFR1 and EGFR can mediate Doxorubicin chemoresistance. We further show that FGFR1 upregulates expression of the cytokine LIF, which then signals through gp130/JAK to activate STAT3 in vitro. Directly inhibiting either FGFR1 or STAT3 significantly reduces chemoresistance and increases apoptosis in vitro. Furthermore, inhibition of FGFR1 with the small molecule inhibitor PD173074 results in increased chemosensitivity and apoptosis in a mouse model of mammary tumorigenesis. These results are significant because they are the first to show that FGFR1 signals through EGFR and that FGFR1 mediates chemoresistance through activation of STAT3. This study furthers our understanding of FGFR1-amplified mammary tumorigenesis and presents alternative factors for targeted therapies for patients with FGFR1-amplified breast cancers.
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    Identification of novel signatures of murine definitive hematopoiesis
    (2014-01) Webber, Beau
    Pluripotent stem cells (PSC) are a tantalizing prospect for a renewable source of patient-specific hematopoietic stem cells (HSC), however efforts to obtain PSC derived HSC capable of long-term engraftment have largely failed. We set out with the primary aim of identifying novel molecular signatures of definitive hematopoiesis, so that these signatures could be applied to improve generation and isolation of HSC in vitro. Toward this end we pursued both discovery and application based strategies centered on Runx1; a transcription factor that is critical for the development of definitive HSC. The discovery arm identified epigenetic modifications at Runx1 cis-regulatory elements that temporally associate with the transition from primitive to definitive hematopoiesis in vivo. We replicated these signatures in vitro by overexpressing HOXB4 in hematopoietic progenitors derived from murine embryonic stem cells (ESC), and found that HOXB4 directly interacts with the definitive-specific distal Runx1 promoter and mediates increased transcription, loss of DNA methylation, and acquisition of active histone modifications at this locus. We next applied our understanding of Runx1 regulation to generate a panel of clonal mESC lines harboring targeted, single-copy fluorescent reporters under the transcriptional control of Runx1 cis-regulatory elements. These lines were used to interrogate the hematopoietic activity of each element independent of copy number and chromosomal position, allowing us to identify combinations that provided optimal activity and fidelity. Building upon this, we established mESC lines harboring synthetic fluorescent and bioluminescent mini genes replicating the structure of the endogenous Runx1 locus and demonstrated that these lines reflect the dynamic promoter switching that occurs at Runx1 during hematogenesis. Sub-fractionation of embryoid body cells based on promoter activity revealed that nearly all colony forming cells (CFC) reside in the distal promoter expressing fraction. With this information we identified specific conditions that could further mature and expand distal positive cells. Collectively, this work identified a previously undescribed molecular signature of definitive hematopoiesis and the mechanism by which it is established. In addition, we applied this knowledge to generate tools with which to interrogate hematopoietic development in vitro, and have demonstrated their utility in optimizing strategies for obtaining definitive hematopoietic progenitors from PSC.
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    Purification of bacterially-expressed chick NSD3-SET that is active in an in vitro methyltransferase assay
    (2014-12) Kretzschmar, Daniel Alan
    The lysine methyltransferase NSD3, which belongs to a family of histone H3 lysine 36 methyltransferases that are conserved among vertebrates and overexpressed in many cancers, is independently required for both neural crest cell specification and migration in the developing chick embryo. Since cytoplasmic protein lysine methylation is also required for neural crest cell migration in chick, one possibility is that NSD3 methylates non-histone substrates. To test this hypothesis, it is necessary to purify NSD3 protein that is active in a methyltransferase assay, in order to evaluate candidate substrates individually and on protein arrays. In this thesis, I describe a detailed method for high-yield purification of a chick NSD3 catalytic fragment, followed by a chick NSD3 methyltransferase assay with which to test for activity on polynucleosomes and/or candidate non-histone substrates.
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    Reprogramming of different cell types into pancreatic beta cells by using transcription factor genes Pdx1, Ngn3 and MafA
    (2012-08) Akinci, Ersin
    In this dissertation we studied the effect of the pancreatic transcription factor genes Pdx1, Ngn3 and MafA, all of which were cloned into single adenoviral construct. First, we investigated the reprogramming competency to Pdx1, Ngn3 and MafA of different cell types at different developmental stages from mouse and rat. Second, we looked at the effect of these three genes on a pancreatic rat exocrine cell line AR42J-B13 to see if this gene combination provides a true beta cell reprogramming event. Next, we investigated the effect of some small molecules to see if they increased the reprogramming efficiency of this gene combination. Finally we tried to sort the small fraction of insulin-positive cells formed after Pdx1, Ngn3, MafA transduction to enrich the insulin-positive cell population hence getting more accurate data from them.Transduction of different cell types with Ad-PNM showed that the Pdx1, Ngn3 and MafA gene combination is enough to stimulate the expression of Insulin genes with varying intensities depending on cell type. Rat cell lines responded to Ad-PNM in a better way than mouse cell lines by activating more beta cell genes. The number of responding cells to Ad-PNM was also higher for the rat cell lines than the mouse cell lines. Moreover progenitor-like cells as well as the cells developmentally related to beta cells are prone to be reprogrammed into beta-like state. Supporting this final statement, embryonic mouse heptoblasts from Pdx1-GFP transgenic mice gave the most promising result by activating the endogenous Pdx1 as well as many other important beta cell genes upon transduction with Ad-PNM.Further characterization of the effect of Ad-PNM on the most resposive cell type AR42J-B13 cells showed that after Ad-PNM the cells became post-mitotic, began to express Insulin genes together with many other beta cell genes, produced mature insulin hormone, changed the epigenetic state of their chromatin, and rescued diabetic mice if transplanted into the kidney capture. However, some important beta cell genes were not activated thereby making these cells unresponsive to glucose which is an indispensable beta cell quality. It is obvious that even though the changes are dramatic, the combination of Pdx1, Ngn3 and MafA did not provide a true beta cell reprogramming in vitro at least for this cell system. Among some of the small molecules which had been reported to favor beta cell formation, regeneration and/or survival, three of them including DAPT, BIX-01294 and NECA increased the effect of Ad-PNM on mouse hepatocyte-derived small cells. Moreover when all three were used together they showed a better efficiency. Even though there was a significant increase in the number of insulin positive cells, the fraction of the Ad-PNM-responding cells was still low after the small molecules. For that reason the effect of small molecules on reprogramming efficiency of Ad-PNM was compared by counting the fraction of insulin-positive cells out of Ad-PNM-bearing cells between control and experimental cell groups instead of performing qRT-PCR.Of the attempts that we performed to increase the low fraction of insulin-positive cells via different sorting techniques to get more accurate and reliable results from Ad-PNM responding cells, only fluorescence-activated cell sorting of insulin-immunostained cells worked. Even though these cells were enriched through the FACS, because the cells were nonviable not alive we could not perform any downstream application that necessitate living cells. This system allows us to do qRT-PCR only.