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• W2054403078 abstract "Mirk/dyrk1B is a member of the dyrk/minibrain family of serine/threonine kinases that mediate the transition from growth to differentiation in lower eukaryotes and mammals. Depletion of endogenous Mirk from C2C12 myoblasts by RNA interference blocks skeletal muscle differentiation (Deng, X., Ewton, D., Pawlikowski, B., Maimone, M., and Friedman, E. (2003) J. Biol. Chem. 278, 41347-41354). We now demonstrate that knockdown of Mirk blocks transcription of the muscle regulatory factor myogenin. Co-expression of Mirk with MEF2C, but not MyoD or Myf5, enhanced activation of the myogenin promoter in a Mirk kinase-dependent manner. Mirk activated MEF2 not through direct phosphorylation of MEF2 but by phosphorylation of its inhibitors, the class II histone deacetylases (HDACs). MEF2 is sequestered by class II HDACs such as HDAC5 and MEF2-interacting transcriptional repressor (MITR). Mirk antagonized the inhibition of MEF2C by MITR, whereas kinase-inactive Mirk was ineffective. Mirk phosphorylates class II HDACs at a conserved site within the nuclear localization region, reducing their nuclear accumulation in a dose-dependent and kinase-dependent manner. Moreover, less mutant MITR phosphomimetic at the Mirk phosphorylation site localized in the nucleus than wild-type MITR. Regulation of class II HDACs occurs by multiple mechanisms. Others have shown that calcium signaling leads to phosphorylation of HDACs at 14-3-3-binding sites, blocking their association with MEF2 within the nucleus. Mirk provides another level of regulation. Mirk is induced within the initial 24 h of myogenic differentiation and enables MEF2 to transcribe the myogenin gene by decreasing the nuclear accumulation of class II HDACs. Mirk/dyrk1B is a member of the dyrk/minibrain family of serine/threonine kinases that mediate the transition from growth to differentiation in lower eukaryotes and mammals. Depletion of endogenous Mirk from C2C12 myoblasts by RNA interference blocks skeletal muscle differentiation (Deng, X., Ewton, D., Pawlikowski, B., Maimone, M., and Friedman, E. (2003) J. Biol. Chem. 278, 41347-41354). We now demonstrate that knockdown of Mirk blocks transcription of the muscle regulatory factor myogenin. Co-expression of Mirk with MEF2C, but not MyoD or Myf5, enhanced activation of the myogenin promoter in a Mirk kinase-dependent manner. Mirk activated MEF2 not through direct phosphorylation of MEF2 but by phosphorylation of its inhibitors, the class II histone deacetylases (HDACs). MEF2 is sequestered by class II HDACs such as HDAC5 and MEF2-interacting transcriptional repressor (MITR). Mirk antagonized the inhibition of MEF2C by MITR, whereas kinase-inactive Mirk was ineffective. Mirk phosphorylates class II HDACs at a conserved site within the nuclear localization region, reducing their nuclear accumulation in a dose-dependent and kinase-dependent manner. Moreover, less mutant MITR phosphomimetic at the Mirk phosphorylation site localized in the nucleus than wild-type MITR. Regulation of class II HDACs occurs by multiple mechanisms. Others have shown that calcium signaling leads to phosphorylation of HDACs at 14-3-3-binding sites, blocking their association with MEF2 within the nucleus. Mirk provides another level of regulation. Mirk is induced within the initial 24 h of myogenic differentiation and enables MEF2 to transcribe the myogenin gene by decreasing the nuclear accumulation of class II HDACs. The availability of in vitro culture systems that recapitulate the major features of muscle cell differentiation has allowed much progress toward the elucidation of the molecular basis for muscle maturation and regeneration. An improved understanding of these processes might have significant clinical ramifications, such as the development of strategies to ameliorate damage to skeletal or cardiac muscle. A novel gene cloned in our laboratory (1Lee K-M. Friedman E. Proc. Am. Assoc. Cancer Res. 1998; 39: 273Google Scholar, 2Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar), the kinase Mirk, has the unusual properties of mediating cell survival in vitro (2Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar), regulating myoblast differentiation following the commitment stage of myogenesis (3Deng X. Ewton D. Pawlikowski B. Maimone M. Friedman E. J. Biol. Chem. 2003; 278: 41347-41354Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), and maintaining untransformed cells in G0 arrest (4Deng X. Mercer S. Shah S. Ewton D. Friedman E. J. Biol. Chem. 2004; 279: 22498-22504Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 5Zou Y. Ewton D. Deng D. Mercer S. Friedman E. J. Biol. Chem. 2004; 279: 27790-27798Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In C2C12 myoblasts, and in nontransformed epithelial cells and fibroblasts, Mirk elongates the G0 phase of the cell cycle by stabilizing the CDK 1The abbreviations used are: CDK, cyclin-dependent kinase; HDACs, histone deacetylases; CaMK, calcium/calmodulin-dependent protein kinase; HNF1α, hepatocyte nuclear factor α; RNAi, RNA interference; MITR, MEF2-interacting transcriptional repressor; NLS, nuclear localization sequence; EGFP, enhanced green fluorescent protein; PBS, phosphate-buffered saline; GM, growth medium. inhibitor p27 Kip1 by phosphorylating p27 at serine 10 (4Deng X. Mercer S. Shah S. Ewton D. Friedman E. J. Biol. Chem. 2004; 279: 22498-22504Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) and maintaining p27 in the nucleus where it acts as a CDK2 inhibitor. Mirk also destabilizes the G1 cyclin, cyclin D1, by phosphorylation at threonine 288 (5Zou Y. Ewton D. Deng D. Mercer S. Friedman E. J. Biol. Chem. 2004; 279: 27790-27798Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), adjacent to the GSK3β site of threonine 286. In addition, Mirk is activated through the stress signaling pathway by the mitogen-activated protein kinase kinase MKK3 (6Lim S. Jin K. Friedman E. J. Biol. Chem. 2002; 277: 25040-25046Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) and interacts with p38 MAPK (7Lim S. Zou Y. Friedman E. J. Biol. Chem. 2002; 277: 49438-49445Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Mirk (minibrain-related kinase) is a member of the dyrk/minibrain family of arginine-directed serine/threonine protein kinases (8Kentrup H. Becker W. Heukelbach J. Wilmes A. Schurman A. Huppertz C. Kainulainen H. Joost H-G. J. Biol. Chem. 1996; 271: 3488-3495Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 9Becker W. Weber Y. Wetzel K. Eirmbter K. Tejedor F. Joost H-G. J. Biol. Chem. 1998; 273: 25893-25902Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 10Himpel S. Tegge W. Frank R. Leder S. Joost H. Becker W. J. Biol. Chem. 2000; 275: 2431-2438Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar) and is identical to dyrk1B (11Leder S. Weber Y. Altafaj X. Estivill X. Joost H-G. Becker W. Biochem. Biophys. Res. Commun. 1999; 254: 474-479Crossref PubMed Scopus (57) Google Scholar). Mirk is strongly transcriptionally up-regulated under conditions of mitogen deprivation, such as occurs when myoblasts are transferred from growth to differentiation medium. In particular when IGF-1 is eliminated (2Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar, 3Deng X. Ewton D. Pawlikowski B. Maimone M. Friedman E. J. Biol. Chem. 2003; 278: 41347-41354Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) there is an enrichment of active Mirk kinase in G0, the cell cycle stage in which the initial steps of differentiation occur. Whereas Mirk protein levels are very low in dividing myoblasts, they are increased at least 10-fold when myoblasts commit to terminal differentiation and are maintained at elevated levels in mature muscle cells (3Deng X. Ewton D. Pawlikowski B. Maimone M. Friedman E. J. Biol. Chem. 2003; 278: 41347-41354Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). This unusual cluster of characteristics suggests the following model. Mirk levels increase under conditions favorable for terminal differentiation when myoblasts arrest in G0; Mirk aids in maintaining G0 arrest, and elevated amounts of Mirk protein are further activated by cellular stresses to mediate myoblast differentiation. In this report we have investigated whether Mirk mediates myoblast differentiation solely by maintaining G0 arrest or whether Mirk has an additional role as a transcriptional activator in muscle differentiation. Mirk is also found in normal hepatocytes, and we have shown (6Lim S. Jin K. Friedman E. J. Biol. Chem. 2002; 277: 25040-25046Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) that Mirk activates the hepatocyte transcription factor HNF1α by phosphorylation. In the myoblast system, depletion of Mirk by RNA interference strongly blocked expression of the myogenic regulatory factor myogenin (3Deng X. Ewton D. Pawlikowski B. Maimone M. Friedman E. J. Biol. Chem. 2003; 278: 41347-41354Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), suggesting that Mirk might control myogenin expression. In the current study, we demonstrate that Mirk functions as a transcriptional activator of MEF2C, a known mediator of myogenin expression, by decreasing the nuclear concentration of class II HDACs that function as MEF2C inhibitors. Mirk phosphorylates these HDACs at a conserved serine within their nuclear localization signal. Small Interfering RNA (RNAi) to Mirk—The RNAi sequence to Mirk, GACCTACAAGCACATCATT, was inserted into the pSilencer plasmid (Ambion) (3Deng X. Ewton D. Pawlikowski B. Maimone M. Friedman E. J. Biol. Chem. 2003; 278: 41347-41354Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). C2C12 cells were transfected with a 10 to 1 ratio (μg/μg) of RNAi plasmid to EGFP selection plasmid in serum-free medium and then isolated by fluorescence-activated cell sorting for EGFP by using a BD Biosciences FACSVantage SE cell sorter. Affymetrix Gene Chips—The MOE430A chip (mouse expression) was utilized. The chips contain 22,690 unique probe identifier oligonucleotide sequence sets. Data were analyzed by using the Microarray Suite 4.0 and, for a more stringent program, the Robust Multi-Chip Analysis program (Gene Traffic, Iobion) to identify genes exhibiting at least a 2-fold reduction, comparing the RNA populations treated with RNAi to Mirk to those treated with the vector control. 12,433 probe sets were identified in the RNAi population and 12,718 in the vector control. 193 transcripts were decreased, and 86 transcripts were increased. Materials—Antibodies to myogenin, MEF2C, MyoD, and β-tubulin were from Santa Cruz Biotechnology. Antibody to the N terminus of HDAC9 from ABGENT was used to detect MITR. Rabbit polyclonal antibody to a unique sequence at the C terminus of Mirk was raised as described (2Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar). Polyvinylidene difluoride transfer paper Immobilon-P was purchased from Millipore. PLUS reagent and Lipofectamine were from Invitrogen; all radioactive materials were purchased from PerkinElmer Life Sciences; ECL reagents were from Amersham Biosciences, and tissue culture reagents were from Mediatech (Fisher). Alexa Fluor 594 (highly cross-adsorbed) secondary antibody conjugates were purchased from Molecular Probes. All other reagents were from Sigma. Cell Culture—C2C12 mouse myoblasts and NIH3T3 cells were obtained from the ATCC and cultured according to their recommendations. C2C12 cells were maintained in growth medium (GM; Dulbecco's modified Eagle's medium, 4 mm l-glutamine, 4.5 g/liter glucose, containing 20% fetal bovine serum) and induced to undergo differentiation by switching to differentiation medium (Dulbecco's modified Eagle's medium containing 2% horse serum). NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium, 4 mm l-glutamine, 4.5g/liter glucose, containing 7% bovine calf serum. Cells were used for experiments only from passages 3 to 10 from our frozen stocks. Plasmids—Plasmids encoding either wild-type or kinase-inactive YF Mirk had been generated previously (2Lee K. Deng X. Friedman E. Cancer Res. 2000; 60: 3631-3637PubMed Google Scholar). The mouse myogenin promoter reporter construct mgnp224-luciferase (-184 to +48 from the start site) and the myogenin expression plasmid in pEMSV were from S. J. Tapscott, Fred Hutchison Cancer Center. Expression plasmids for human MEF2C and MEF2D in pCDNA1 and mouse MyoD in pEMSV, and GST-HDAC5 amino acids 1-283, were the gifts of E. N. Olson, University of Texas Southwestern Medical Center. The MITR expression plasmid was from D. Sparrow, The National Institute for Medical Research, London, UK. The Myf5 expression plasmid in pEMSV was from B. Winter, Technische Universitat Braunschweig. 3xMEF2-luciferase and gal4-luciferase were the kind gifts of Dr. Zhenguo Wu, Hong Kong University of Science and Technology. Wild-type HDAC5 (pGEX-HDAC5-1-283) and wild-type MITR were mutated by site-directed mutagenesis (Gene-Editor system, Promega). All mutant constructs were sequenced to confirm the mutated sequence. Transfection and Luciferase Assay—Cells were transfected in 12-well plates for 6 h with Lipofectamine and PLUS reagent (Invitrogen) according to the supplier's manual. Transfected cells were lysed with Cell Culture Lysis Reagent and assayed for luciferase activity (relative luciferase unit) with luciferase assay reagent (Promega) in a Turner Designs Luminometer. The same lysates were diluted and assayed for β-galactosidase activity to normalize transfection efficiency. Data shown are the means of triplicate measurements. Cell Fractionation—5 × 105 C2C12 cells were plated in 60-mm dishes overnight and transfected with the indicated constructs. After 24 h, the cells were trypsinized, washed with ice-cold PBS, and resuspended in 150 μl of Buffer A (10 mm HEPES, pH 7.4, 1 mm KCl, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol) containing 0.5% Nonidet P-40 and protease inhibitor mixture (Sigma P 8340). The cell suspension was kept on ice for 10 min with occasional vortexing and monitored microscopically until >90% of the cells were lysed. The nuclei were pelleted at 800 × g for 10 min. The supernatant containing the cytoplasmic fraction was centrifuged at 16,000 × g for 10 min to remove any unlysed cells. The nuclear pellet was washed with 300 μl of Buffer A without Nonidet P-40 and centrifuged at 800 × g for 10 min. The cytoplasmic and nuclear fractions were solubilized in hot Laemmli sample buffer before analysis by SDS-PAGE. Localization of MITR Constructs—C2C12 myoblasts were plated overnight in Lab-Tek 2-well chamber slides (1 × 105 cells per well) and then transfected with FLAG-MITR (1 μg of plasmid DNA) using Lipofectamine and PLUS reagent in Dulbecco's modified Eagle's medium. After 24 h of expression in GM, cells were washed with PBS, fixed in 4% paraformaldehyde for 5 min, washed with PBS, permeabilized with 0.1% Triton in PBS (PBST) for 5 min, and blocked with 10% normal goat serum in PBST for 30 min. The cells were incubated for 45 min with a 1:1000 dilution of either rabbit antibody to the N terminus of HDAC-9 or murine monoclonal antibody to the FLAG epitope, each diluted in 10% normal goat serum in PBST. After three PBS washes, the cells were incubated for 30 min with a 1:1000 dilution of goat anti-mouse antibody or goat anti-rabbit antibody conjugated to Alexa Fluor 594 (Molecular Probes). Nuclei were detected by incubation for 5 min with a 2 ng/ml solution of 4′,6′-diamidino-2-phenylindole hydrochloride in PBST. Slides were mounted with Biomedia Gel/Mount. Monochrome fluorescence images were obtained at ×400 using a Diagnostic Instruments SPOT RT camera mounted on a Nikon Eclipse E50i fluorescent microscope. SPOT RT software version 4.09 was used to pseudocolor and merge the images. Final figures were arranged using Adobe Photoshop version 7.0. Northern Analysis for Myogenin mRNA—Total RNA was prepared by the RNeasy protocol (Qiagen) and analyzed for mRNA composition by microarray analysis. 4 μg of the remaining total RNA from each cell lysate was electrophoresed in a 1.1% agarose-formaldehyde gel, transferred to nylon membranes by downward capillary transfer, and cross-linked by baking in an oven. The membranes were hybridized to a 32P-labeled 1-kb EcoRI fragment of the myogenin cDNA construct. The probes were labeled with 32P by random priming (Promega). The blot was hybridized overnight at 68 °C with at least 107 cpm of the labeled probe, washed twice at room temperature for 15 min with 1× SSC, 0.1% SDS, and then washed for 20 min at 65 °C in 0.2× SSC, 0.1% SDS and autoradiographed. In Vitro Kinase Assay—GST-Mirk or GST-YF-Mirk preparations were incubated for 5 min at 30 °C with 20 μl of kinase buffer (50 mm Tris-HCl, pH 7.5, 10 mm MgCl2, 0.5 mm dithiothreitol), containing 50 μm cold ATP plus 5 μCi of [γ-32P]ATP and 1 μg of purified recombinant GST-HDAC5 protein as substrate, and then analyzed by PAGE and autoradiography. Glutathione S-Transferase Fusion Proteins—Mirk and YF-Mirk were subcloned into the pGEX-6P1 vector (Amersham Biosciences), and the HDAC5 constructs were subcloned into the pGEX 4T1 vector (Amersham Biosciences), and both were expressed and purified as described. 2S. Mercer, D. Ewton, S. Lim, T. Mazur, and E. Friedman, submitted for publication. Metabolic Labeling—C2C12 cells were incubated for 6 h in serum-free low phosphate Dulbecco's modified Eagle's medium (9:1 ratio phosphate-free medium/normal medium) with 150 μCi of [32P]orthophosphate/2 ml of medium. Immunodetection—Following treatment as indicated and washing two times with PBS, cells were lysed in hot SDS-PAGE sample buffer (50 mm Tris, pH 6.8, 2% SDS, 10% glycerol), boiled for 5 min, and vortexed vigorously. Protein determinations were made using Coomassie protein assay reagent from Pierce. Depending upon the experiment, 30-50 μg of cell lysate were blotted onto polyvinylidene difluoride membranes after separation on SDS-PAGE. The blots were blocked in 5% milk in TBST (10 mm Tris-HCl, pH 8.0, 150 mm NaCl, and 0.1% Tween 20) for 1 h at room temperature and incubated for 1-2 h at room temperature with primary antibody in TBST buffer, 3% milk, and proteins were subsequently detected by enhanced chemiluminescence. Band Analysis—Immunoblots were scanned using a Lacie Silver-scanner, and densitometry was performed using the IP Lab Gel program (Scanalytics). Use of Small Interfering RNA to Mirk Substantially Decreased the Levels of Myogenin mRNA and Protein in C2C12 Myoblasts—In our recent study (3Deng X. Ewton D. Pawlikowski B. Maimone M. Friedman E. J. Biol. Chem. 2003; 278: 41347-41354Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), we utilized RNAi to demonstrate that Mirk helps to mediate skeletal muscle cell differentiation. Western blotting analysis showed that knockdown of endogenous Mirk led to depletion of other proteins that mediate the myotube phenotype. In the current study we used microarray analysis to identify the genes down-regulated when Mirk levels were reduced by RNA interference. Two RNA populations were compared by microarray analysis as follows: RNA from differentiating C2C12 myoblasts in which Mirk was induced as a natural result of differentiation, and RNA from myoblasts cultured under the same conditions but with endogenous Mirk depleted by RNAi. Cells expressing RNAi were selected by cell sorting for the co-transfected EGFP marker gene, with cells transfected with vector DNA serving as the control. The sorted C2C12 cells were then plated for 1 day and then switched to differentiation medium for 1 day to induce Mirk (vector control, Vc cells) or to allow the RNAi to deplete endogenous Mirk (si cells). Western blotting confirmed that endogenous Mirk levels were decreased 60-fold by RNA interference (Fig. 1A). The MOE430A Affymetrix chip (mouse expression) was utilized for microarray analysis of the RNA populations from Mirk-depleted and control cells. The chips contain 22,690 unique probe identifier oligonucleotide sequence sets. Data were analyzed using the Microarray Suite 4.0 and, a more stringent program, the Robust Multi-Chip Analysis program (Gene Traffic, Iobion) to identify genes exhibiting at least a 2-fold reduction when the mRNA population from the RNAi-treated cells was compared with the mRNA population from vector control-treated cells. 12,433 probe sets were identified in the RNAi population and 12,718 in the vector control. 193 transcripts were decreased, and 86 transcripts were increased. The genes whose expression was most strongly reduced when Mirk was depleted by RNAi are shown in Table I.Table IGenes down-regulated by RNAi to Mirk-Fold changeUniGene name-11.53Collagenous repeat-containing sequence, skeletal development protein-9.99H19 fetal liver mRNA, oct-binding maintenance sequence for unmethylated DNA-8.63Myosin light chain, phosphorylatable, fast skeletal muscle-7.57Myogenin-7.40Myosin light chain, alkali, fast skeletal muscle-6.57Troponin C, fast skeletal-6.24Actin, α1, skeletal muscle-5.59Suppressor of cytokine signaling 2-5.49Actin, α, cardiac-4.87Osteoglycin-4.58Placenta-specific 8-4.43Lymphocyte antigen 6 complex, locus A-4.32Troponin C, cardiac/slow skeletal-4.17Actinin α3-4.04Serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 1a-4.02CD80 antigen-4.00Inhibitor of DNA binding 3-3.96Calcium channel, voltage-dependent, γ subunit 1-3.78Strathmin-like 2-3.68Titin-3.67Myosin, light polypeptide 9, regulatory-3.62Biglycan-3.47IGF-BP7-3.43Early growth response 1 (EGR1)-3.38Enolase 3, β muscle-3.36Dynein, cytoplasmic, intermediate chain I-3.35SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin d-3.31Granzyme E-3.29Cadherin 15-2.96Myomesin 2-2.93Troponin T1, skeletal slow-2.82Thioredoxin-interacting protein-2.81IGF-BP5-2.76Sarcoglycan, β (43-kDa dystrophin-associated glycoprotein) Open table in a new tab Most of the genes reduced by knockdown of endogenous Mirk by RNA interference were skeletal muscle differentiation genes. None of the Id family of differentiation inhibitor genes was increased when Mirk was depleted (Table II). One of the genes reduced most in expression was myogenin, whose expression was reduced 7.5-fold. Northern blotting confirmed the microarray analysis. Myogenin mRNA was undetectable by Northern blotting, although similar levels of 18 S and 28 S rRNAs were found in the RNA preparations (Fig. 1B). As an additional control, cyclin D1 mRNA levels were measured in the Northern blots and were identical in control and RNA interference samples (data not shown). Therefore, Mirk controls the transcription of myogenin. Our prior study supports the hypothesis that Mirk mediates expression of myogenin. Myogenin and Mirk are induced at the protein level with similar kinetics during the initial differentiation of primary cultured murine hindlimb muscle satellite cells, C2C12 cells, and L6 rat myoblasts placed in differentiation medium (3Deng X. Ewton D. Pawlikowski B. Maimone M. Friedman E. J. Biol. Chem. 2003; 278: 41347-41354Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Furthermore, depletion of Mirk by RNAi led to a 13-fold reduction in myogenin protein levels (Fig. 1A), and overexpression of Mirk induced a more rapid appearance of myogenin (3Deng X. Ewton D. Pawlikowski B. Maimone M. Friedman E. J. Biol. Chem. 2003; 278: 41347-41354Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Thus myogenin down-regulation following loss of Mirk is mediated on the transcriptional level.Table IIGenes up-regulated by RNAi to Mirk-Fold changeUniGene name7.29Cubilin (intrinsic factor-cobalamin receptor)6.14Niban protein5.93Serine (or cysteine) proteinase inhibitor, clade B, member 25.66Pyruvate dehydrogenase kinase, isoenzyme 44.85CD53 antigen4.65RIKEN cDNA 1190002H23 gene3.77Serine protease inhibitor 133.55Calcitonin-related polypeptide, β3.40Neurofilament, light polypeptide3.29Activated leukocyte cell adhesion molecule3.27ESTs3.11Histamine N-methyltransferase2.89Mannosidase 1, α2.89Mus musculus, similar to protein phosphatase 1, regulatory subunit 10,clone IMAGE:5012018, mRNA2.83src-like adaptor2.77Serum/glucocorticoid-regulated kinase2.76Hedgehog-interacting protein2.75Occludin2.73Methylenetetrahydrofolate dehydrogenase (NAD+-dependent), Methenyltetrahydrofolate cyclohydrolase2.67RIKEN cDNA 1300019I21 gene2.66Asparagine synthetase2.63RAR-related orphan receptor β2.60Branched chain aminotransferase 1, cytosolic Open table in a new tab RNAi to Mirk Blocks Activation of the Myogenin Promoter— Myogenesis is regulated by the following four transcription factors called muscle regulatory factors: Myf5, MyoD, myogenin, and MRF-4, which function in a sequential program, and a second group of transcription factors termed myocyte enhancer-2 (MEF2A through 2D). Myf5 and MyoD are essential for establishing the myogenic lineage, before Mirk is induced through RhoA action at the onset of differentiation (3Deng X. Ewton D. Pawlikowski B. Maimone M. Friedman E. J. Biol. Chem. 2003; 278: 41347-41354Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Myogenin is up-regulated later during the process of terminal differentiation. Both an E box (CANNTG) and an MEF2-binding site within the myogenin promoter are essential for myogenin transcription. The transcription factors MyoD, MEF-2, Myf5, and the ubiquitous E box proteins have been implicated in myogenin regulation (13Edmondson D. Cheng T. Cserjesi P. Chakraborty T. Olson E. Mol. Cell. Biol. 1992; 12: 3665-3677Crossref PubMed Scopus (257) Google Scholar, 14Malik S. Huang C. Schmidt J. Eur. J. Biochem. 1995; 230: 88-96Crossref PubMed Scopus (25) Google Scholar, 15Friday B. Mitchell P. Kegley K. Pavlath G. Differentiation. 2003; 71: 217-227Crossref PubMed Scopus (106) Google Scholar) and could serve as potential Mirk substrates. The muscle regulatory factor Myf-5 is co-expressed with MyoD only in dividing myoblasts but is lost within 12 h of the start of differentiation and is not seen in cells expressing myogenin, indicating that MyoD, not Myf-5, mediates myogenin transcription (16Kitzmann M. Carnac G. Vandromme M. Primig M. Lamb N. Fernandez A. J. Cell. Biol. 1998; 142: 1447-1459Crossref PubMed Scopus (248) Google Scholar). We first determined whether endogenous Mirk could up-regulate myogenin expression by transfecting a myogenin promoter reporter construct into growing C2C12 myoblasts. The cells were then switched to differentiation medium for 24 h to induce the up-regulation of Mirk (3Deng X. Ewton D. Pawlikowski B. Maimone M. Friedman E. J. Biol. Chem. 2003; 278: 41347-41354Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The cells were not sorted in this experiment, and the transfection efficiency was at best 30%, so some background was detected due to the residual endogenous Mirk activity. However, a clear difference was observed. The myogenin promoter reporter construct was activated 4-fold in differentiating C2C12 cells that had up-regulated endogenous Mirk expression as part of their differentiation program (Fig. 2). However, when the myogenin reporter construct was co-transfected with an expression plasmid for RNAi to Mirk to knockdown endogenous Mirk levels and cells were then cultured in differentiation medium, little increase in myogenin reporter activity was observed. Therefore, reduction of endogenous Mirk levels blocked the activation of the myogenin promoter. Mirk Activates a Myogenin Reporter Construct in a Kinase-dependent Manner in Concert with MEF2C but Not MyoD or Myf5—Mirk activates the transcription factor HNF1α and phosphorylates HNF1α at a residue within its CBP-binding domain (6Lim S. Jin K. Friedman E. J. Biol. Chem. 2002; 277: 25040-25046Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). We postulated that Mirk would also phosphorylate, and thus activate, one of the transcription factors that mediate transcription of the myogenin gene. We co-transfected a myogenin promoter reporter construct together with expression plasmids for either Mirk, kinase-inactive YF-Mirk, or the vector, and we then performed parallel experiments with the Mirk constructs in the presence of expression constructs for MEF2C, MEF2D, MyoD, Myf5, or combinations. Mirk activated the myogenin promoter reporter construct about 2-fold, whereas YF-Mirk had no effect (Fig. 3A, vector lanes). When either MEF2C or MEF2D were co-expressed with wild-type Mirk, there was a further 2-fold increase in reporter activity. MEF2D, and to a lesser extent MEF2C, increased myogenin reporter expression when co-expressed with kinase-inactive YF-Mirk, probably because each factor complexed with endogenous transcription factors. However, MEF2C clearly activated the myogenin reporter in a Mirk kinase-dependent manner. In contrast, MyoD increased the activity of the myogenin reporter construct about 10-fold, but expression of Mirk did not alter this activation (Fig. 3B). When MyoD and MEF2C were co-expressed, the stimulatory effect of Mirk was seen again (Fig. 3C). As in the case with MyoD, Myf5 increased the activity of the myogenin reporter in a Mirk-independent manner (Fig. 3D). However, when MEF2C was co-expressed with Myf5, the large activation of the myogenin reporter that was seen was further enhanced by expression of wild-type Mirk but not kinase-inactive Mirk. These data, taken together, demonstrated that Mirk functioned by activating MEF2C. Mirk Does Not Directly Activate MEF2C by Phosphorylation—In an earlier study, we had found that Mirk activate" @default.
• W2054403078 created "2016-06-24" @default.
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• W2054403078 creator A5031238366 @default.
• W2054403078 creator A5032939076 @default.
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• W2054403078 date "2005-02-01" @default.
• W2054403078 modified "2023-10-02" @default.
• W2054403078 title "Mirk/dyrk1B Decreases the Nuclear Accumulation of Class II Histone Deacetylases during Skeletal Muscle Differentiation" @default.
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