1,046 research outputs found

    REGULATION OF THE L-TYPE PYRUVATE KINASE GENE BY GLUCOSE AND cAMP IN ISLET BETA CELLS

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    Extracellular signals generated during both feeding and fasting coordinately regulate transcription of metabolic enzyme genes that control glucose metabolism in theƒnƒÒ cell. A post-prandial rise in extracellular glucose levels promotes expression of various genes including the gene encoding the glycolytic enzyme L-type pyruvate kinase (L-PK). Conversely, under conditions of fasting, a rise in hormones that stimulate increased intracellular levels of cAMP results in suppression of glucose-activated genes such as L-PK. The L-PK gene is coordinately regulated by these two opposing stimuli. Therefore, we explored the mechanism of induction and repression of the L-PK gene by glucose and cAMP, respectively, using the 832/13 rat insulinoma cell line.Glucose mediates induction of the L-PK gene by stimulating the recruitment of two primary DNA binding transcription factors, the basic helix-loop-helix/leucine zipper protein Carbohydrate Response Element Binding Protein (ChREBP) and the orphan nuclear receptor, Hepatic Nuclear Factor 4ƒÑ (HNF4ƒÑ) to their respective response elements in the proximal L-PK promoter. In addition, glucose stimulates the recruitment of the coactivator CREB binding protein (CBP) to the L-PK gene promoter. Assembly of these three factors on the L-PK gene promoter facilitates alterations in the pattern of acetylation and methylation of histones associated with the promoter and coding region, respectively. These changes in histone modifications correlate with increased occupancy of the RNA Polymerase II (Pol II) holoenzyme on the L-PK promoter. Finally, glucose promotes changes in the phosphorylation state of the carboxyl-terminal domain (CTD) of Pol II at serines 5 and 2, which are necessary for the promoter clearance and elongation phases of transcription.cAMP represses the glucose-mediated induction of the L-PK gene by inhibiting the assembly of the ChREBP, HNF4ƒÑ and CBP-containing complex on the L-PK promoter. The cAMP-dependent decrease in complex assembly on the promoter is associated with alterations in the acetylation and methylation status of histones on both the promoter and coding region. Furthermore, cAMP inhibits the glucose-mediated recruitment and phosphorylation of Pol II CTD, ultimately blocking initiation and elongation of the L-PK gene by Pol II. In summary, these studies provide a detailed insight into the mechanism of regulation of the L-PK gene by glucose and cAMP in islet ƒÒ cells

    Activation of AMP-activated protein kinase by metformin induces protein acetylation in prostate and ovarian cancer cells

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    AMP-activated protein kinase (AMPK) is an energy sensor and master regulator of metabolism. AMPK functions as a fuel gauge monitoring systemic and cellular energy status. Activation of AMPK occurs when the intracellular AMP/ATP ratio increases and leads to a metabolic switch from anabolism to catabolism. AMPK phosphorylates and inhibits acetyl-CoA carboxylase (ACC), which catalyzes carboxylation of acetyl-CoA to malonyl-CoA, the first and rate-limiting reaction in de novo synthesis of fatty acids. AMPK thus regulates homeostasis of acetyl-CoA, a key metabolite at the crossroads of metabolism, signaling, chromatin structure, and transcription. Nucleocytosolic concentration of acetyl-CoA affects histone acetylation and links metabolism and chromatin structure. Here we show that activation of AMPK with the widely used antidiabetic drug metformin or with the AMP mimetic 5-aminoimidazole-4-carboxamide ribonucleotide increases the inhibitory phosphorylation of ACC and decreases the conversion of acetyl-CoA to malonyl-CoA, leading to increased protein acetylation and altered gene expression in prostate and ovarian cancer cells. Direct inhibition of ACC with allosteric inhibitor 5-(tetradecyloxy)-2-furoic acid also increases acetylation of histones and non-histone proteins. Because AMPK activation requires liver kinase B1, metformin does not induce protein acetylation in liver kinase B1-deficient cells. Together, our data indicate that AMPK regulates the availability of nucleocytosolic acetyl-CoA for protein acetylation and that AMPK activators, such as metformin, have the capacity to increase protein acetylation and alter patterns of gene expression, further expanding the plethora of metformin's physiological effects

    Investigating transcriptional regulation of viral and cellular genes by EBV EBNA 2

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    Epstein-Barr virus (EBV) is linked to the development of several human malignancies. Epstein-Barr Nuclear Antigen 2 (EBNA 2) is required for the immortalisation and continued proliferation of EBV-infected B-cells. EBNA 2 is a transcriptional regulator of both viral and cellular genes. The viral C promoter (Cp), regulated by EBNA 2, drives transcription of an ~120 kb pre-mRNA that is differentially spliced to generate messages encoding all the other EBNAs required for immortalisation. To study the regulation of Cp-transcript elongation, we used a pair of EBV-infected cell-lines to compare the transcriptional complexes associated with the Cp transcriptional unit and two shorter EBNA 2-regulated viral genes, LMP1 and LMP2A. Interestingly, we found an accumulation of RNA Polymerase II (Pol II) in association with the pausing factors DSIF and NELF at Cp, which were absent at the LMP gene locus. Further experiments revealed that C promoter sequences have a much higher propensity to occlude nucleosome formation, promoting TBP recruitment and Pol II accumulation. We also found highlevel recruitment of the Pol II C-terminal domain (CTD) kinase, pTEFb at Cp, increased Pol II Serine 2 CTD phosphorylation and retention at promoter-distal regions. Furthermore pTEFb recruitment at Cp was facilitated by association with the bromodomain protein Brd4 and Pol II pausing. By sustaining a nucleosome-free region and recruiting high levels of pTEFb, Cp elongation appears highly adapted to ensure production of the long EBNA-encoding transcript required to establish and maintain B-cell immortalisation. In additional studies we examined the association of methylated forms of EBNA 2 with viral and cellular genes. EBNA 2 is modified by asymmetric (aDMA) or symmetric (sDMA) arginine di-methylation in the arginine-glycine repeat region. We found that aDMA-modified EBNA 2 preferentially bound promoters to regulate gene expression, implicating this modification as a key regulator of EBNA 2 activity

    The 26S Proteasome and Histone Modifying Enzymes Regulate

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    Major Histocompatibility Complex Class-II (MHC-II) molecules are critical regulators of adaptive immunity that present extracellular antigens required to activate CD4+ T cells. MHC-II are regulated at the level of transcription by master regulator, the Class II Transactivator (CIITA), whose association with the MHC-II promoter is necessary to initiate transcription. Recently, much research focused on novel mechanisms of transcriptional regulation of critical genes like MHC-II and CIITA; findings that the macromolecular complex of the 26S-proteasome is involved in transcription have been perhaps the most exciting as they impart novel functions to a well studied system. Proteasome is a multi-subunit complex composed of a 20S-core particle capped by a 19S-regulatory particle. The 19S contains six ATPases which are required for transcription initiation and elongation. We demonstrate that 19S ATPase-S6a inducibly associates with CIITA promoters. Decreased expression of S6a negatively impacts recruitment of the transcription factors STAT-1 and IRF-1 to the CIITA due to significant loss in histone H3 and H4 acetylation. S6a is robustly recruited to CIITA coding regions, where S6a binding coordinates with that of RNA polymerase II. RNAi mediated S6a knockdown significantly diminishes recruitment of Pol II and P-TEF-b components to CIITA coding regions, indicating S6a plays important roles in transcriptional elongation. Our research is focused on the ways in which accessibility to and transcription of DNA is regulated. While cancers are frequently linked to dysregulated gene expression, contribution of epigenetics to cancers remains unknown. To achieve metastatic ability, tumors alter gene expression to escape host immunosurveilance. MHC-II and CIITA expression are significantly downregulated in highly metastatic MDA-MB-435 breast cancer cells. This suppression correlates with elevated levels of the silencing modification H3K27me3 at CIITA and a significant reduction in Pol II recruitment. We observe elevated binding of the histone methyltransferase to CIITApIV and demonstrate this enzyme is a master regulator of CIITA gene expression. EZH2 knockdown results in significant increases in CIITA and MHC-II transcript levels in metastatic cells. In sum, transcriptional regulation by the 19S-proteasome and histone modifying enzymes represents novel mechanisms of control of mammalian gene expression and present novel therapeutic targets for manipulating MHC expression in disease

    AP-1 Is a Component of the Transcriptional Network Regulated by GSK-3 in Quiescent Cells

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    The protein kinase GSK-3 is constitutively active in quiescent cells in the absence of growth factor signaling. Previously, we identified a set of genes that required GSK-3 to maintain their repression during quiescence. Computational analysis of the upstream sequences of these genes predicted transcription factor binding sites for CREB, NFκB and AP-1. In our previous work, contributions of CREB and NFκB were examined. In the current study, the AP-1 component of the signaling network in quiescent cells was explored.Using chromatin immunoprecipitation analysis, two AP-1 family members, c-Jun and JunD, bound to predicted upstream regulatory sequences in 8 of the 12 GSK-3-regulated genes. c-Jun was phosphorylated on threonine 239 by GSK-3 in quiescent cells, consistent with previous studies demonstrating inhibition of c-Jun by GSK-3. Inhibition of GSK-3 attenuated this phosphorylation, resulting in the stabilization of c-Jun. The association of c-Jun with its target sequences was increased by growth factor stimulation as well as by direct GSK-3 inhibition. The physiological role for c-Jun was also confirmed by siRNA inhibition of gene induction.These results indicate that inhibition of c-Jun by GSK-3 contributes to the repression of growth factor-inducible genes in quiescent cells. Together, AP-1, CREB and NFκB form an integrated transcriptional network that is largely responsible for maintaining repression of target genes downstream of GSK-3 signaling

    In sickness and in health: the role of methyl-CpG binding protein 2 in the central nervous system

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    The array of specialized neuronal and glial cell types that characterize the adult central nervous system originates from neuroepithelial proliferating precursor cells. The transition from proliferating neuroepithelial precursor cells to neuronal lineages is accompanied by rapid global changes in gene expression in coordination with epigenetic modifications at the level of the chromatin structure. A number of genetic studies have begun to reveal how epigenetic deregulation results in neurodevelopmental disorders such as mental retardation, autism, Rubinstein–Taybi syndrome and Rett syndrome. In this review we focus on the role of the methyl-CpG binding protein 2 (MeCP2) during development of the central nervous system and its involvement in Rett syndrome. First, we present recent findings that indicate a previously unconsidered role of glial cells in the development of Rett syndrome. Next, we discuss evidence of how MeCP2 deficiency or loss of function results in aberrant gene expression leading to Rett syndrome. We also discuss MeCP2's function as a repressor and activator of gene expression and the role of its different target genes, including microRNAs, during neuronal development. Finally, we address different signaling pathways that regulate MeCP2 expression at both the post-transcriptional and post-translational level, and discuss how mutations in MeCP2 may result in lack of responsiveness to environmental signals

    Post-Translational Control of Sp-Family Transcription Factors

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    Sp-family transcription factors are widely expressed in human tissues and involved in the regulation of many cellular processes and response to cellular microenvironment. These responses appear to be mediated by alterations in transcription factor affinity for DNA rather than altered protein level. How might such changes be effected? This review will identify the range of known post-translational modifications (PTMs) of Sp-factors and the sometimes conflicting literature about the roles of PTMs in regulating activity. We will speculate on the interaction between cell environment, chromatin microenvironment and the role of PTM in governing functionality of the proteins and the complexes to which they belong

    Mammalian Mat1 subunit of Transcription Factor IIH in gene-specific and general transcription

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    All protein-encoding genes in eukaryotes are transcribed into messenger RNA (mRNA) by RNA Polymerase II (RNAP II), whose activity therefore needs to be tightly controlled. An important and only partially understood level of regulation is the multiple phosphorylations of RNAP II large subunit C-terminal domain (CTD). Sequential phosphorylations regulate transcription initiation and elongation, and recruit factors involved in co-transcriptional processing of mRNA. Based largely on studies in yeast models and in vitro, the kinase activity responsible for the phosphorylation of the serine-5 (Ser5) residues of RNAP II CTD has been attributed to the Mat1/Cdk7/CycH trimer as part of Transcription Factor IIH. However, due to the lack of good mammalian genetic models, the roles of both RNAP II Ser5 phosphorylation as well as TFIIH kinase in transcription have provided ambiguous results and the in vivo kinase of Ser5 has remained elusive. The primary objective of this study was to elucidate the role of mammalian TFIIH, and specifically the Mat1 subunit in CTD phosphorylation and general RNAP II-mediated transcription. The approach utilized the Cre-LoxP system to conditionally delete murine Mat1 in cardiomyocytes and hepatocytes in vivo and and in cell culture models. The results identify the TFIIH kinase as the major mammalian Ser5 kinase and demonstrate its requirement for general transcription, noted by the use of nascent mRNA labeling. Also a role for Mat1 in regulating general mRNA turnover was identified, providing a possible rationale for earlier negative findings. A secondary objective was to identify potential gene- and tissue-specific roles of Mat1 and the TFIIH kinase through the use of tissue-specific Mat1 deletion. Mat1 was found to be required for the transcriptional function of PGC-1 in cardiomyocytes. Transriptional activation of lipogenic SREBP1 target genes following Mat1 deletion in hepatocytes revealed a repressive role for Mat1apparently mediated via co-repressor DMAP1 and the DNA methyltransferase Dnmt1. Finally, Mat1 and Cdk7 were also identified as a negative regulators of adipocyte differentiation through the inhibitory phosphorylation of Peroxisome proliferator-activated receptor (PPAR) γ. Together, these results demonstrate gene- and tissue-specific roles for the Mat1 subunit of TFIIH and open up new therapeutic possibilities in the treatment of diseases such as type II diabetes, hepatosteatosis and obesity.Transkriptio on prosessi, jolla eukaryoottisolu kääntää proteiineja koodaavat geenit lähetti-RNA:ksi RNA Polymeraasi II:sen välityksellä. Koska lähes kaikki eliön solut sisältävät saman perimätiedon geeneissä, RNA Polymeraasi II:n säätely on elintärkeää, jotta erilaiset solut voivat ilmentää juuri niille tarvittavia proteiineja juuri oikeasssa tilanteessa. Yksi tärkeä säätelymekanismi on RNA Polymeraasi II:n C-terminuksen seriinitähteiden fosforylaatio, joka aktivoi transkription sekä rekrytoi paikalle muita transkriptiota sääteleviä proteiineja kuten kromatiinia muokkaavia tekijöitä sekä lähetti-RNA:n muokkaamiseen tarvittavia entsyymejä. Vaikka hiivatutkimuksien perusteella on oletettu, että Mat1/Cdk7/CycH kinaasikompleksi osana TFIIH transkriptiotekijää fosforyloi RNA Polymeraasi II:sta, nisäkässoluissa sekä TFIIH kinaasin että RNA Polymeraasi II:n fosforylaation rooli ja merkitys transkriptiossa on epäselvä. Tämän väitöskirjatutkimuksen tavoitteena oli selvittää TFIIH kinaasikompleksin, etenkin Mat1 alayksikön, roolia RNA Polymeraasi II:n fosforylaatiossa sekä transkription säätelyssä. Hyödyntämällä Mat1 poistogeenisiä soluja maljalla, ensimmäinen osatyö identifioi Mat1-proteiinin tarpeelliseksi RNA Polymeraasi II:n fosforylaatioon sekä proteiineja koodaavien geenien transkriptioon nisäkässoluissa. Lisäksi kyseinen osatyö paljasti Mat1-proteiinille roolin lähetti-RNA:n puoliajan säätelyssä. Toinen väitöskirjan tavoite oli tutkia TFIIH kinaasikompleksin ja Mat1:sen mahdollisia geeni- ja kudosspesifisiä rooleja hyödyntämällä kudosspesifisiä Mat1-poistogeenisiä hiiriä sekä erilaistuvia solumalleja. Toisessa osatyössä osoitettiin, että TFIIH kinaasi säätelee rasvasolujen muodostumista fosforyloimalla PPARg transkriptiotekijää, täten estämällä solua erilaistumasta rasvasoluksi. Kolmas osatyö osoitti, että sydänlihassoluissa Mat1:stä tarvitaan rasvahappojen hapettamiseen tarpeellisten geenien säätelyyn. Neljännessa osatyössä löydettiin uusi, negatiivisesti rasvametaboliageenien transkriptiota säätelevä rooli Mat1:selle maksasoluissa. Mat1 säätelee rasvametabolia geenejä rekrytoimalla geeneille kromatiinia muokkaavia entsyymejä, jotka vuorostaan vähentävät geenien ilmentämistä ja täten ylläpitävät maksan tervettä rasva-aineenvaihduntaa. Mat1:sen poisto maksasta aiheuttaa kyseisten geenien yli-ilmentämisen ja rasvan kertymistä maksasoluihin, johtaen rasvamaksan muodostumiseen. Väitöskirjatyö paljastaa TFIIH kinaasille yleisen tehtävän kaikkien proteiineja koodaavien geenien säätelyssä RNA Polymeraasi II:n fosforylaation kautta, ja lisäksi geeni- ja kudosspesifisiä rooleja sekä negatiivisena että positiivisena transkription säätelijänä, ja antaa viitteitä terapeuttisista mahdollisuuksista aineenvaihduntatautien, kuten tyypin II diabeteksen tai liikalihavuuden hoidossa

    Thr435 phosphorylation regulates RelA (p65) NF-κB subunit transactivation

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    Phosphorylation of the RelA (p65) NF-κB (nuclear factor κB) subunit has been previously shown to modulate its ability to induce or repress transcription. In the present study we have investigated the consequences of Thr435 phosphorylation within the C-terminal transactivation domain of RelA. We confirm that Thr435 is phosphorylated in cells and is induced by TNFα (tumour necrosis factor α) treatment. Mutational analysis of this site revealed gene-specific effects on transcription, with a T435D phosphomimetic mutant significantly enhancing Cxcl2 (CXC chemokine ligand 2) mRNA levels in reconstituted Rela−/− mouse embryonic fibroblasts. Chromatin immunoprecipitation analysis revealed that this mutation results in enhanced levels of histone acetylation associated with decreased recruitment of HDAC1 (histone deacetylase 1). Moreover, mutation of this site disrupted RelA interaction with HDAC1 in vitro. Thr435 phosphorylation of promoter-bound RelA was also detected at NF-κB target genes following TNFα treatment in wild-type mouse embryonic fibroblasts. Phosphorylation at this site therefore provides an additional mechanism through which the specificity of NF-κB transcriptional activity can be modulated in cells
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