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Srinivasan R.,Waisman Center | Jones E.A.,Program in Cellular and Molecular Biology | Jang S.-W.,Program in Cellular and Molecular Biology | Krueger C.,Waisman Center | And 3 more authors.
Nucleic Acids Research | Year: 2012

Myelin is essential for the rapidity of saltatory nerve conduction, and also provides trophic support for axons to prevent axonal degeneration. Two critical determinants of myelination are SOX10 and EGR2/KROX20. SOX10 is required for specification of Schwann cells from neural crest, and is required at every stage of Schwann cell development. Egr2/Krox20 expression is activated by axonal signals in myelinating Schwann cells, and is required for cell cycle arrest and myelin formation. To elucidate the integrated function of these two transcription factors during peripheral nerve myelination, we performed in vivo ChIP-Seq analysis of myelinating peripheral nerve. Integration of these binding data with loss-of-function array data identified a range of genes regulated by these factors. In addition, although SOX10 itself regulates Egr2/Krox20 expression, leading to coordinate activation of several major myelin genes by the two factors, there is a large subset of genes that are activated independent of EGR2. Finally, the results identify a set of SOX10-dependent genes that are expressed in early Schwann cell development, but become subsequently repressed by EGR2/KROX20. © The Author(s) 2012.


Huebert D.J.,Program in Cellular and Molecular Biology
Nucleus (Austin, Tex.) | Year: 2012

The degree to which nucleosome positioning regulates transcription is an ongoing debate. To address this question, we recently followed dynamic changes in nucleosome occupancy, transcription factor binding and gene expression in yeast cells responding to oxidative stress. Integrating across these dynamic processes revealed new insights into the functions of nucleosome reorganization. Here, we used our data to address the extent to which upstream promoter architecture is a static feature inherent to specific genes vs. a dynamic platform that changes across conditions. Our results argue that, while some aspects of promoter architecture are fixed across environments, the level to which promoters are "open" or "covered" by nucleosomes depends on the conditions investigated.


Boateng L.R.,Program in Cellular and Molecular Biology | Bennin D.,University of Wisconsin - Madison | De Oliveira S.,University of Wisconsin - Madison | Huttenlocher A.,Program in Cellular and Molecular Biology | Huttenlocher A.,University of Wisconsin - Madison
Journal of Biological Chemistry | Year: 2016

Mammalian actin-binding protein-1 (mAbp1) is an adaptor protein that binds actin and modulates scission during endocytosis. Recent studies suggest that mAbp1 impairs cell invasion; however, the mechanism for the inhibitory effects of mAbp1 remain unclear. We performed a yeast two-hybrid screen and identified the adaptor protein, FHL2, as a novel binding partner that interacts with the N-terminal actin depolymerizing factor homology domain (ADFH) domain of mAbp1. Here we report that depletion of mAbp1 or ectopic expression of the ADFH domain of mAbp1 increased Rho GTPase signaling and breast cancer cell invasion. Moreover, cell invasion induced by the ADFH domain of mAbp1 required the expression of FHL2. Taken together, our findings show that mAbp1 and FHL2 are novel binding partners that differentially regulate Rho GTPase signaling and MTLn3 breast cancer cell invasion. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.


Vaughan E.M.,Program in Cellular and Molecular Biology | You J.-S.,Program in Cellular and Molecular Biology | Yu H.-Y.E.,Program in Cellular and Molecular Biology | Vitale N.,University of Wisconsin - Madison | And 3 more authors.
Molecular Biology of the Cell | Year: 2014

After damage, cells reseal their plasma membrane and repair the underlying cortical cytoskeleton. Although many different proteins have been implicated in cell repair, the potential role of specific lipids has not been explored. Here we report that cell damage elicits rapid formation of spatially organized lipid domains around the damage site, with different lipids concentrated in different domains as a result of both de novo synthesis and transport. One of these lipids - diacylglycerol (DAG) - rapidly accumulates in a broad domain that overlaps the zones of active Rho and Cdc42, GTPases that regulate repair of the cortical cytoskeleton. Formation of the DAG domain is required for Cdc42 and Rho activation and healing. Two DAG targets, protein kinase C (PKC) β and η, are recruited to cell wounds and play mutually antagonistic roles in the healing process: PKCβ participates in Rho and Cdc42 activation, whereas PKCη inhibits Rho and Cdc42 activation. The results reveal an unexpected diversity in subcellular lipid domains and the importance of such domains for a basic cellular process. © 2014 et al.


Kelly V.R.,Program in Cellular and Molecular Biology | Hammer G.D.,Program in Cellular and Molecular Biology
Molecular and Cellular Endocrinology | Year: 2011

Dax1, an atypical orphan nuclear receptor expressed in steroidogenic tissues, has recently been shown to be expressed in mouse embryonic stem (mES) cells and is required for pluripotency. While the mechanisms of transcriptional regulation of Dax1 in steroidogenic organs have been well characterized, those in mES cells have not. Here we report that 500. bp of the Dax1 gene promoter sequence are sufficient to drive expression in mES cells. In steroidogenic tissues, NR5A1 (Sf1) binds to nuclear receptor binding sites within this sequence to regulate Dax1 expression. In mES cells, while NR5A1 (Sf1) is not expressed, NR5A2 (LRH-1) expression is robust. Luciferase assays, EMSA and overexpression/knockdown studies demonstrate that LRH-1 binds the -128 site and regulates Dax1 in mES cells. Predicated on recent work indicating that Nanog binds to the Dax1 intron, we have used chromatin immunoprecipitation experiments (ChIP) to define an intronic site that is bound by Nanog. Overexpression and knockdown of Nanog in mES cells result in alteration of Dax1 expression, and luciferase assays reveal that this sequence can enhance transcription of a Dax1 reporter construct. These data indicate that LRH-1 and Nanog cooperate to regulate Dax1 expression in mES cells. © 2010 Elsevier Ireland Ltd.


Janssens D.H.,Program in Cellular and Molecular Biology | Lee C.-Y.,Program in Cellular and Molecular Biology | Lee C.-Y.,Life science Institute | Lee C.-Y.,University of Michigan
Seminars in Cell and Developmental Biology | Year: 2014

During malignant transformation the cells of origin give rise to cancer stem cells which possess the capacity to undergo limitless rounds of self-renewing division, regenerating themselves while producing more tumor cells. Within normal tissues, a limitless self-renewal capacity is unique to the stem cells, which divide asymmetrically to produce more restricted progenitors. Accumulating evidence suggests that misregulation of the self-renewal machinery in stem cell progeny can lead to tumorigenesis, but how it influences the properties of the resulting tumors remains unclear. Studies of the type II neural stem cell (neuroblast) lineages in the Drosophila larval brain have identified a regulatory cascade that promotes commitment to a progenitor cell identity by restricting their response to the self-renewal machinery. Brain tumor (Brat) and Numb initiate this cascade by asymmetrically extinguishing the activity of the self-renewal factors. Subsequently, Ea. rmuff (Erm) and the SWI/SNF complex stably restrict the competence of the progenitor cell to respond to reactivation of self-renewal mechanisms. Together, this cascade programs the progenitor cell to undergo limited rounds of division, generating exclusive differentiated progeny. Here we review how defects in this cascade lead to tumor initiation and how inhibiting the self-renewal mechanisms may be an effective strategy to block CSC expansion. © 2014 Elsevier Ltd.


Burnetti A.J.,Program in Cellular and Molecular Biology | Burnetti A.J.,Durham University | Burnetti A.J.,Duke University | Aydin M.,Duke University | Buchler N.E.,Duke University
Molecular Biology of the Cell | Year: 2016

Cells have evolved oscillators with different frequencies to coordinate periodic processes. Here we studied the interaction of two oscillators, the cell division cycle (CDC) and the yeast metabolic cycle (YMC), in budding yeast. Previous work suggested that the CDC and YMC interact to separate high oxygen consumption (HOC) from DNA replication to prevent genetic damage. To test this hypothesis, we grew diverse strains in chemostat and measured DNA replication and oxygen consumption with high temporal resolution at different growth rates. Our data showed that HOC is not strictly separated from DNA replication; rather, cell cycle Start is coupled with the initiation of HOC and catabolism of storage carbohydrates. The logic of this YMC-CDC coupling may be to ensure that DNA replication and cell division occur only when sufficient cellular energy reserves have accumulated. Our results also uncovered a quantitative relationship between CDC period and YMC period across different strains. More generally, our approach shows how studies in genetically diverse strains efficiently identify robust phenotypes and steer the experimentalist away from strain-specific idiosyncrasies. © 2016 Burnetti et al.


Simon C.M.,University of British Columbia | Vaughan E.M.,University of British Columbia | Bement W.M.,Program in Cellular and Molecular Biology | Bement W.M.,University of Wisconsin - Madison | Edelstein-Keshet L.,University of British Columbia
Molecular Biology of the Cell | Year: 2013

The Rho GTPases-Rho, Rac, and Cdc42-control an enormous variety of processes, many of which reflect activation of these GTPases in spatially confined and mutually exclusive zones. By using mathematical models and experimental results to establish model parameters, we analyze the formation and segregation of Rho and Cdc42 zones during Xenopus oocyte wound repair and the role played by Abr, a dual guanine nucleotide exchange factor-GTPase-activating protein, in this process. The Rho and Cdc42 zones are found to be best represented as manifestations of spatially modulated bistability, and local positive feedback between Abr and Rho can account for the maintenance and dynamic properties of the Rho zone. In contrast, the invocation of an Abr-independent positive feedback loop is required to account for Cdc42 spatial bistability. In addition, the model replicates the results of previous in vivo experiments in which Abr activity is manipulated. Further, simulating the model with two closely spaced wounds made nonintuitive predictions about the Rho and Cdc42 patterns; these predictions were confirmed by experiment. We conclude that the model is a useful tool for analysis of Rho GTPase signaling and that the Rho GTPases can be fruitfully considered as components of intracellular pattern formation systems. © 2013 Jo et al.

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