in-situ-immunofluorescent-staining-of-autophagy-in-muscle-stem-cells

<em>In Situ</em> Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

Italian National Research Council

IRCCS Fondazione Santa Lucia

Terminal differentiation exerts a remarkably tight control on cell proliferation. However, the oncogenic products of DNA tumor viruses, such as adenovirus E1A, can force postmitotic cells to proliferate, thus representing a powerful tool to study progression into S phase. In this study, we identified the gene encoding Np95, a murine nuclear phosphoprotein, as an early target of E1A-induced transcriptional events. In terminally differentiated (TD) cells, the activation of Np95 was specifically induced by E1A, but not by overexpression of E2F-1 or of the cyclin E (cycE)-cyclin-dependent kinase 2 (cdk2) complex. In addition, the concomitant expression of Np95 and of cycE-cdk2 was alone sufficient to induce S phase in TD cells. In NIH-3T3 cells, the expression of Np95 was tightly regulated during the cell cycle, and its functional ablation resulted in abrogation of DNA synthesis. Thus, expression of Np95 is essential for S phase entry. Previous evidence suggested that E1A, in addition to its well characterized effects on the pRb/E2F-1 pathway, activates a parallel and complementary pathway that is also required for the reentry in S phase of TD cells (Tiainen, M., D. Spitkousky, P. Jansen-Dürr, A. Sacchi, and M. Crescenzi. 1996. Mol. Cell. Biol. 16:5302-5312). From our results, Np95 appears to possess all the characteristics to represent the first molecular determinant identified in this pathway.

Np95 is regulated by E1A during mitotic reactivation of terminally differentiated cells and is essential for S phase entry.

Cardiac hypertrophy is induced by a number of stimuli and can lead to cardiomyopathy and heart failure. Present knowledge suggests that cell-cycle regulatory proteins take part in hypertrophy. We have investigated if the D-type cyclins are involved in cardiac hypertrophy.

Involvement of cyclin D activity in left ventricle hypertrophy in vivo and in vitro.

&#x5728;&#x9AA8;&#x9ABC;&#x808C;&#x6210;&#x808C;&#xFF0C;&#x671F;&#x95F4;&#x5411;&#x7EC8;&#x7AEF;&#x5206;&#x5316;&#x57FA;&#x56E0;&#x7EC4;&#x91CD;&#x7F16;&#x7A0B;&#x88AB;&#x901A;&#x8FC7;&#x62DB;&#x8058;&#x67D3;&#x8272;&#x8D28;&#x4FEE;&#x9970;&#x9176;&#x5BF9;&#x808C;&#x8089;&#x7279;&#x5B9A;&#x57FA;&#x56E0;&#x4F4D;&#x70B9;&#x3002;&#x80DE;&#x5916;&#x4FE1;&#x53F7;&#x53F6;&#x6805;&#x4E2D;&#x9488;&#x5BF9;&#x8FD9;&#x4E9B;&#x9176;&#x5BF9;&#x5355;&#x4E2A;&#x57FA;&#x56E0;&#x7684;&#x76F8;&#x5BF9;&#x8D21;&#x732E;&#x662F;&#x672A;&#x77E5;&#x7684;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x663E;&#x793A;&#x5206;&#x5316;&#x6FC0;&#x6D3B; p38 &#x901A;&#x8DEF;&#x76EE;&#x6807; SWI &#x7231;&#x68EE;&#x67D3;&#x8272;&#x8D28;&#x91CD;&#x5851;&#x590D;&#x6742;&#x5230;&#x808C;&#x8F68;&#x8FF9;&#x3002;&#x540E;&#x5206;&#x5316;&#xFF0C;&#x6FC0;&#x9176; p38 &#x53D7;&#x8058;&#x5230;&#x67D3;&#x8272;&#x8D28;&#x808C;&#x8089;&#x8C03;&#x63A7;&#x5143;&#x4EF6;&#x3002;P38 &alpha;/&beta; &#x5C01;&#x9501;&#x9547;&#x538B;&#x808C;&#x8089;&#x57FA;&#x56E0;&#x7684;&#x8F6C;&#x5F55;&#x800C;&#x4E0D;&#x4F1A;&#x5F71;&#x54CD;&#x67D3;&#x8272;&#x8D28;&#x7ED3;&#x5408;&#x7684;&#x808C;&#x8089;&#x8C03;&#x8282;&#x56E0;&#x5B50;&#x548C;&#x4E59;&#x9170;&#x9632;&#x6B62;&#x62DB;&#x52DF;&#x8FD9;&#x4E9B;&#x5143;&#x7D20;&#x5728; SWI &#x7231;&#x68EE;&#x590D;&#x6742;&#x3002;BAF60 &#x53EF;&#x4EE5;&#x78F7;&#x9178;&#x5316; p38 &alpha;-&beta; &#x4F53;&#x5916;&#xFF0C;&#x7531; SWI &#x7231;&#x68EE;&#x4E9A;&#x57FA;&#x548C;&#x5F3A;&#x8FEB;&#x6FC0;&#x6D3B; p38 &alpha;/&beta; &#x6210;&#x808C;&#x7EC6;&#x80DE;&#x4E2D;&#x7684;&#x8868;&#x8FBE;&#x7684;&#x4E00;&#x6D3B;&#x52A8; MKK6 &#xFF08;&#x5F15;&#x7528;&#x3002; 5,6,7&#xFF09; &#x4FC3;&#x8FDB;&#x808C;&#x7EC6;&#x80DE;&#x751F;&#x6210;&#x7D20;&#x542F;&#x52A8;&#x5B50;&#x8BA1;&#x5212;&#x5916;&#x7684; SWI &#x7231;&#x68EE;&#x62DB;&#x52DF;&#x3002;&#x76F8;&#x53CD;&#xFF0C;SWI &#x7231;&#x68EE;&#x9176;&#x4E9A;&#x57FA;&#x7684;&#x706D;&#x6D3B;&#x5E9F;&#x6B62; MKK6 &#x4F9D;&#x8D56;&#x8BF1;&#x5BFC;&#x808C;&#x8089;&#x57FA;&#x56E0;&#x7684;&#x8868;&#x8FBE;&#x3002;&#x8FD9;&#x4E9B;&#x7ED3;&#x679C;&#x786E;&#x5B9A;&#x610F;&#x5916;&#x7684;&#x5206;&#x5316;&#x6FC0;&#x6D3B; p38 &#x5728;&#x5916;&#x90E8;&#x7EBF;&#x7D22;&#x8F6C;&#x5316;&#x4E0A;&#x79BB;&#x6563;&#x7684;&#x57FA;&#x56E0;&#x4F4D;&#x70B9;&#xFF0C;&#x67D3;&#x8272;&#x8D28;&#x4FEE;&#x6539;&#x901A;&#x8FC7;&#x6709;&#x9009;&#x62E9;&#x5730;&#x9488;&#x5BF9; SWI &#x7231;&#x68EE;&#x4E0E;&#x808C;&#x8089;&#x8C03;&#x63A7;&#x5143;&#x4EF6;&#x7684;&#x529F;&#x80FD;&#x3002;

p38 &#x901A;&#x8DEF;&#x76EE;&#x6807; SWI &#x7231;&#x68EE;&#x67D3;&#x8272;&#x8D28;&#x91CD;&#x5851;&#x590D;&#x6742;&#x5230;&#x808C;&#x8089;&#x7279;&#x5B9A;&#x57FA;&#x56E0;&#x4F4D;&#x70B9;&#x3002;

During skeletal myogenesis, genomic reprogramming toward terminal differentiation is achieved by recruiting chromatin-modifying enzymes to muscle-specific loci. The relative contribution of extracellular signaling cascades in targeting these enzymes to individual genes is unknown. Here we show that the differentiation-activated p38 pathway targets the SWI-SNF chromatin-remodeling complex to myogenic loci. Upon differentiation, p38 kinases were recruited to the chromatin of muscle-regulatory elements. Blockade of p38 alpha/beta repressed the transcription of muscle genes by preventing recruitment of the SWI-SNF complex at these elements without affecting chromatin binding of muscle-regulatory factors and acetyltransferases. The SWI-SNF subunit BAF60 could be phosphorylated by p38 alpha-beta in vitro, and forced activation of p38 alpha/beta in myoblasts by expression of a constitutively active MKK6 (refs. 5,6,7) promoted unscheduled SWI-SNF recruitment to the myogenin promoter. Conversely, inactivation of SWI-SNF enzymatic subunits abrogated MKK6-dependent induction of muscle gene expression. These results identify an unexpected function of differentiation-activated p38 in converting external cues into chromatin modifications at discrete loci, by selectively targeting SWI-SNF to muscle-regulatory elements.

p38 pathway targets SWI-SNF chromatin-remodeling complex to muscle-specific loci.

Au cours de la myogen&#xE8;se squelettique, reprogrammation g&#xE9;nomique vers la diff&#xE9;renciation terminale est r&#xE9;alis&#xE9;e par le recrutement de la chromatine enzymes qui modifient le muscle-sp&#xE9;cifiques loci. La contribution relative des cascades de signalisation extracellulaire dans le ciblage de ces enzymes &#xE0; des g&#xE8;nes individuels est inconnue. Ici, nous montrons que la voie de diff&#xE9;renciation-p38 activ&#xE9;e cible le SWI-SNF remodelage de la chromatine complexe loci myog&#xE9;niques. Lors de la diff&#xE9;renciation, les kinases p38 ont &#xE9;t&#xE9; recrut&#xE9;s &#xE0; la chromatine du muscle-&#xE9;l&#xE9;ments de r&#xE9;gulation. Blocus de la p38 alpha / b&#xEA;ta r&#xE9;prim&#xE9; la transcription des g&#xE8;nes musculaires par la pr&#xE9;vention du recrutement du complexe SWI-SNF &#xE0; ces &#xE9;l&#xE9;ments sans affecter liaison chromatine du muscle r&#xE9;glementaires facteurs et ac&#xE9;tyltransf&#xE9;rases. La sous-unit&#xE9; SWI-SNF BAF60 pourrait &#xEA;tre phosphoryl&#xE9;e par p38 alpha-b&#xEA;ta in vitro, et forc&#xE9; l&#x26;#39;activation de p38 alpha / beta dans les myoblastes par l&#x26;#39;expression d&#x26;#39;un MKK6 constitutivement active (r&#xE9;f. 5,6,7) promu impr&#xE9;vus SWI-SNF recrutement le promoteur myog&#xE9;nine. A l&#x26;#39;inverse, l&#x26;#39;inactivation de SWI-SNF sous-unit&#xE9;s enzymatiques abrog&#xE9;e MKK6-d&#xE9;pendante l&#x26;#39;induction de l&#x26;#39;expression des g&#xE8;nes musculaires. Ces r&#xE9;sultats mettent en &#xE9;vidence une fonction inattendue de la diff&#xE9;renciation-p38 activ&#xE9;e dans la conversion de signaux externes dans modifications de la chromatine &#xE0; des loci discrets, puisqu&#x26;#39;il cible s&#xE9;lectivement SWI-SNF au muscle-&#xE9;l&#xE9;ments de r&#xE9;gulation.

P38 Pathway Cibles SWI-SNF remodelage de la chromatine Complexe sp&#xE9;cifique du muscle Loci

p38-Signalweg zielt SWI-SNF Chromatin-Remodeling-Komplex, um Muskel-spezifische Loci.

Durante myogenesis scheletrico, riprogrammazione genomica verso la differenziazione terminale &egrave; raggiunto con l&#39;assunzione di enzimi modificanti la cromatina di loci specifici del muscolo. Il contributo relativo di cascate di segnalazione extracellulare in questi enzimi a singoli geni di targeting &egrave; sconosciuto. Qui vi mostriamo che il percorso di differenziazione-attivata p38 obiettivi i loci complessi a miogena rimodellamento della cromatina SWI-SNF. Sulla differenziazione, p38 chinasi sono state reclutate per la cromatina degli elementi muscolo-normativi. Blocco di p38 alfa/beta represse la trascrizione dei geni muscolo impedendo il reclutamento del complesso SWI-SNF a questi elementi senza intaccare il legame della cromatina di fattori muscolo-regolamentazione e acetiltransferasi. La subunit&agrave; SWI-SNF BAF60 potrebbe essere fosforilata da p38 alfa-beta in vitro, e costretto attivazione di p38 alfa/beta in mioblasti dall&#39;espressione di un MKK6 costitutivamente attive (Rif. 5.6.7) promosso non programmata assunzione SWI-SNF al promotore myogenin. Al contrario, inattivazione di subunit&agrave; enzimatiche SWI-SNF abrogata induzione MKK6-dipendente dell&#39;espressione genica di muscolo. Questi risultati identificano un&#39;inaspettata funzione di differenziazione-attivata p38 nella conversione di segnali esterni in modificazioni della cromatina a loci discreti, designando selettivamente SWI-SNF a elementi muscolo-regolatori.

pathway P38 obiettivi loci di complessi muscolo-specifico per SWI-SNF rimodellamento della cromatina.

&#x9AA8;&#x683C;&#x7B4B;&#x5F62;&#x6210;&#x306E;&#x9593;&#x306B;&#x3001;&#x7AEF;&#x672B;&#x306E;&#x5DEE;&#x5225;&#x5316;&#x306B;&#x5411;&#x3051;&#x305F;&#x30B2;&#x30CE;&#x30E0;&#x30EA;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30DF;&#x30F3;&#x30B0;&#x306F;&#x3001;&#x7B4B;&#x7279;&#x7570;&#x7684;&#x907A;&#x4F1D;&#x5B50;&#x306B;&#x30AF;&#x30ED;&#x30DE;&#x30C1;&#x30F3;&#x4FEE;&#x98FE;&#x9175;&#x7D20;&#x3092;&#x52DF;&#x96C6;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x308A;&#x9054;&#x6210;&#x3055;&#x308C;&#x308B;&#x3002;&#x500B;&#x3005;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x306B;&#x3053;&#x308C;&#x3089;&#x306E;&#x9175;&#x7D20;&#x3092;&#x6A19;&#x7684;&#x3068;&#x3059;&#x308B;&#x7D30;&#x80DE;&#x5916;&#x30B7;&#x30B0;&#x30CA;&#x30EB;&#x4F1D;&#x9054;&#x30AB;&#x30B9;&#x30B1;&#x30FC;&#x30C9;&#x306E;&#x76F8;&#x5BFE;&#x7684;&#x306A;&#x5BC4;&#x4E0E;&#x306F;&#x4E0D;&#x660E;&#x3067;&#x3042;&#x308B;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x5206;&#x5316;&#x6D3B;&#x6027;&#x5316;p38&#x306E;&#x7D4C;&#x8DEF;&#x306F;&#x7B4B;&#x539F;&#x6027;&#x907A;&#x4F1D;&#x5B50;&#x5EA7;&#x306B;SWI-SNF&#x30AF;&#x30ED;&#x30DE;&#x30C1;&#x30F3;&#x30EA;&#x30E2;&#x30C7;&#x30EA;&#x30F3;&#x30B0;&#x306F;&#x3001;&#x8907;&#x96D1;&#x306A;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002;&#x5206;&#x5316;&#x3059;&#x308B;&#x3068;&#x3001;p38&#x306E;&#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x306F;&#x7B4B;&#x8089;&#x8ABF;&#x7BC0;&#x30A8;&#x30EC;&#x30E1;&#x30F3;&#x30C8;&#x306E;&#x30AF;&#x30ED;&#x30DE;&#x30C1;&#x30F3;&#x306B;&#x52D5;&#x54E1;&#x3055;&#x308C;&#x307E;&#x3057;&#x305F;&#x3002; p38&#x306E;&#x3B1;/&#x3B2;&#x306E;&#x906E;&#x65AD;&#x306F;&#x3001;&#x7B4B;&#x8089;&#x306E;&#x8ABF;&#x7BC0;&#x56E0;&#x5B50;&#x3068;&#x30A2;&#x30BB;&#x30C1;&#x30EB;&#x30C8;&#x30E9;&#x30F3;&#x30B9;&#x30D5;&#x30A7;&#x30E9;&#x30FC;&#x30BC;&#x306E;&#x30AF;&#x30ED;&#x30DE;&#x30C1;&#x30F3;&#x7D50;&#x5408;&#x306B;&#x5F71;&#x97FF;&#x3092;&#x4E0E;&#x3048;&#x308B;&#x3053;&#x3068;&#x306A;&#x304F;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;&#x8981;&#x7D20;&#x3067;SWI-SNF&#x8907;&#x5408;&#x4F53;&#x306E;&#x52D5;&#x54E1;&#x3092;&#x9632;&#x6B62;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x3088;&#x308A;&#x3001;&#x7B4B;&#x8089;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x306E;&#x8EE2;&#x5199;&#x3092;&#x6291;&#x5236;&#x3002; SWI-SNF&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;BAF60&#x306F;&#x3001;in vitro&#x306B;&#x304A;&#x3051;&#x308B;p38&#x3B1;-&#x3B2;&#x306B;&#x3088;&#x308A;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x3055;&#x308C;&#x3001;&#x6052;&#x5E38;&#x7684;&#x306B;&#x6D3B;&#x6027;MKK6&#xFF08;&#x6587;&#x732E;5,6,7&#xFF09;&#x306E;&#x767A;&#x73FE;&#x306B;&#x3088;&#x3063;&#x3066;&#x7B4B;&#x82BD;&#x7D30;&#x80DE;&#x306B;&#x304A;&#x3051;&#x308B;p38&#x3B1;/&#x3B2;&#x306E;&#x6D3B;&#x6027;&#x5316;&#x3092;&#x5F37;&#x5236;&#x3059;&#x308B;&#x3053;&#x3068;&#x306B;&#x30B9;&#x30B1;&#x30B8;&#x30E5;&#x30FC;&#x30EB;&#x3055;&#x308C;&#x3066;&#x3044;&#x306A;&#x3044;SWI-SNF&#x306E;&#x52DF;&#x96C6;&#x3092;&#x63A8;&#x9032;&#x3057;&#x30DF;&#x30AA;&#x30B2;&#x30CB;&#x30F3;&#x30D7;&#x30ED;&#x30E2;&#x30FC;&#x30BF;&#x30FC;&#x3002;&#x9006;&#x306B;&#x3001;SWI-SNF&#x9175;&#x7D20;&#x306E;&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x306E;&#x6D3B;&#x6027;&#x5316;&#x3001;&#x7B4B;&#x8089;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x306E;MKK6&#x4F9D;&#x5B58;&#x7684;&#x8A98;&#x5C0E;&#x3092;&#x5EC3;&#x6B62;&#x3002;&#x3053;&#x308C;&#x3089;&#x306E;&#x7D50;&#x679C;&#x306F;&#x3001;&#x9078;&#x629E;&#x7684;&#x306B;&#x7B4B;&#x8089;&#x306E;&#x8ABF;&#x7BC0;&#x8981;&#x7D20;&#x306B;SWI-SNF&#x3092;&#x6A19;&#x7684;&#x3068;&#x3059;&#x308B;&#x3053;&#x3068;&#x3067;&#x3001;&#x96E2;&#x6563;&#x7684;&#x907A;&#x4F1D;&#x5B50;&#x5EA7;&#x306E;&#x30AF;&#x30ED;&#x30DE;&#x30C1;&#x30F3;&#x4FEE;&#x98FE;&#x306B;&#x5916;&#x90E8;&#x30AD;&#x30E5;&#x30FC;&#x3092;&#x5909;&#x63DB;&#x3059;&#x308B;&#x969B;&#x306B;&#x5206;&#x5316;&#x6D3B;&#x6027;&#x5316;p38&#x306E;&#x4E88;&#x671F;&#x3057;&#x306A;&#x3044;&#x6A5F;&#x80FD;&#x3092;&#x8B58;&#x5225;&#x3057;&#x307E;&#x3059;&#x3002;

&#x7B4B;&#x7279;&#x7570;&#x7684;&#x907A;&#x4F1D;&#x5B50;&#x5EA7;&#x306B;&#x30B3;&#x30F3;&#x30D7;&#x30EC;&#x30C3;&#x30AF;&#x30B9;P38&#x7D4C;&#x8DEF;&#x306E;&#x30BF;&#x30FC;&#x30B2;&#x30C3;&#x30C8;SWI-SNF&#x30AF;&#x30ED;&#x30DE;&#x30C1;&#x30F3;&#x30EA;&#x30E2;&#x30C7;&#x30EA;&#x30F3;&#x30B0;

&#xACE8;&#xACA9; myogenesis &#xB3D9;&#xC548; &#xD130;&#xBBF8;&#xB110; &#xCC28;&#xBCC4;&#xD654;&#xB97C; &#xD5A5;&#xD574; &#xAC8C;&#xB188; &#xC7AC;&#xD504;&#xB85C;&#xADF8;&#xB798;&#xBC0D; chromatin &#xC218;&#xC815; &#xD6A8;&#xC18C; &#xADFC;&#xC721; &#xAD00;&#xB828; loci &#xBAA8;&#xC9D1; &#xD558; &#xC5EC; &#xC774;&#xB8E8;&#xC5B4;&#xC9D1;&#xB2C8;&#xB2E4;. &#xC774;&#xB7EC;&#xD55C; &#xD6A8;&#xC18C; &#xAC1C;&#xBCC4; &#xC720;&#xC804;&#xC790;&#xB97C; &#xB300;&#xC0C1; &#xC138;&#xD3EC; &#xC678; &#xC2E0;&#xD638; &#xCE90;&#xC2A4;&#xCF00;&#xC774;&#xB4DC;&#xC758; &#xC0C1;&#xB300;&#xC801; &#xAE30;&#xC5EC;&#xB3C4; &#xC54C; &#xC218; &#xC788;&#xB2E4;. &#xC5EC;&#xAE30; &#xC6B0;&#xB9AC;&#xAC00; &#xCC28;&#xBCC4;&#xD654; &#xD65C;&#xC131;&#xD654; p38 &#xD1B5;&#xB85C; SWI SNF &#xAC1C;&#xC7A5; &#xD558;&#xB294; chromatin myogenic &#xBCF5;&#xC7A1; loci &#xB300;&#xC0C1; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. &#xB530;&#xB77C; &#xCC28;&#xBCC4;&#xD654;, p38 kinases &#xADFC;&#xC721; &#xADDC;&#xC81C; &#xC694;&#xC18C; chromatin &#xCC44;&#xC6A9; &#xD588;&#xB2E4;. P38 &#xC54C;&#xD30C;/&#xBCA0;&#xD0C0;&#xC758; &#xBD09;&#xC1C4; chromatin &#xBC14;&#xC778;&#xB529; &#xADFC;&#xC721; &#xADDC;&#xC81C; &#xC694;&#xC778; &#xBC0F; acetyltransferases&#xC758; &#xC601;&#xD5A5;&#xC744; &#xC8FC;&#xC9C0; &#xC54A;&#xACE0; &#xC774;&#xB7EC;&#xD55C; &#xC694;&#xC18C;&#xC5D0; SWI SNF &#xB2E8;&#xC9C0;&#xC758; &#xCC44;&#xC6A9;&#xC744; &#xBC29;&#xC9C0; &#xD558; &#xC5EC; &#xADFC;&#xC721; &#xC720;&#xC804;&#xC790;&#xC758; &#xC804;&#xC0AC;&#xB97C; &#xC5B5;&#xC555;. BAF60 p38 &#xC54C;&#xD30C;-&#xBCA0;&#xD0C0; &#xC2DC;&#xD5D8;&#xAD00;&#xC5D0;&#xC11C; &#xC758;&#xD574; phosphorylated &#xC218; SWI SNF &#xC18C; &#xB2E8;&#xC704; &#xC608;&#xC57D; &#xB418;&#xC9C0; &#xC54A;&#xC740; SWI SNF &#xCC44;&#xC6A9; myogenin &#xBC1C;&#xAE30;&#xC778; p38 &#xC54C;&#xD30C;/&#xBCA0;&#xD0C0; myoblasts&#xC5D0;&#xC11C; &#xD65C;&#xC131;&#xD654; constitutively &#xD65C;&#xC131; MKK6 &#xC2DD;&#xC73C;&#xB85C; &#xAC15;&#xC81C; (&#xCC38;&#xC870;. 5,6,7) &#xC2B9;&#xC9C4;. &#xBC18;&#xB300;&#xB85C;, SWI SNF &#xD6A8;&#xC18C; subunits&#xC758; &#xBE44;&#xD65C;&#xC131;&#xD654; MKK6 &#xC885;&#xC18D; &#xC720;&#xB3C4; &#xADFC;&#xC721; &#xC720;&#xC804;&#xC790; &#xBC1C;&#xD604;&#xC758; &#xD3D0;&#xC9C0;. &#xC774; &#xACB0;&#xACFC; &#xCC28;&#xBCC4;&#xD654; &#xD65C;&#xC131;&#xD654; p38 chromatin &#xC218;&#xC815; &#xC774;&#xC0B0; loci&#xC5D0; &#xC120;&#xD0DD;&#xC801;&#xC73C;&#xB85C; &#xADFC;&#xC721; &#xADDC;&#xC81C; &#xC694;&#xC18C;&#xC5D0; SWI SNF&#xB97C; &#xD0C0;&#xAC9F;&#xD305; &#xD558; &#xC5EC; &#xC678;&#xBD80; &#xC2E0;&#xD638; &#xBCC0;&#xD658;&#xC5D0;&#xC11C; &#xC608;&#xAE30;&#xCE58; &#xC54A;&#xC740; &#xAE30;&#xB2A5;&#xC744; &#xC2DD;&#xBCC4; &#xD569;&#xB2C8;&#xB2E4;.

p38 &#xD1B5;&#xB85C; SWI SNF &#xAC1C;&#xC7A5; &#xD558;&#xB294; chromatin &#xADFC;&#xC721; &#xAD00;&#xB828; &#xBCF5;&#xC7A1; &#xD55C; loci&#xB97C; &#xB300;&#xC0C1;&#xC73C;&#xB85C; &#xD569;&#xB2C8;&#xB2E4;.

Durante miog&#xE9;nesis esquel&#xE9;tico, la reprogramaci&#xF3;n gen&#xF3;mica hacia la diferenciaci&#xF3;n terminal se logra mediante la contrataci&#xF3;n de modificaci&#xF3;n de la cromatina enzimas para m&#xFA;sculos espec&#xED;ficos loci. La contribuci&#xF3;n relativa de las cascadas de se&#xF1;alizaci&#xF3;n extracelulares en la orientaci&#xF3;n de estas enzimas a los genes individuales es desconocida. Aqu&#xED; mostramos que la v&#xED;a de diferenciaci&#xF3;n activada por p38 se dirige a la SWI-SNF remodelaci&#xF3;n de la cromatina-complejo loci miog&#xE9;nicas. Despu&#xE9;s de la diferenciaci&#xF3;n, las quinasas p38 fueron reclutados para la cromatina de los m&#xFA;sculos-elementos reguladores. El bloqueo de p38 alfa / beta reprimida la transcripci&#xF3;n de genes musculares mediante la prevenci&#xF3;n de reclutamiento del complejo SWI-SNF a estos elementos sin que esto afecte de uni&#xF3;n de la cromatina de los reguladores del m&#xFA;sculo-factores y acetiltransferasas. La subunidad SWI-SNF BAF60 podr&#xED;a ser fosforilada por p38 alfa-beta in vitro, y oblig&#xF3; a la activaci&#xF3;n de p38 alfa / beta en mioblastos por la expresi&#xF3;n de un constitutivamente activa MKK6 (ref. 5,6,7) promovido programado SWI-SNF reclutamiento el promotor miogenina. Por el contrario, la inactivaci&#xF3;n de SWI-SNF subunidades enzim&#xE1;ticas derog&#xF3; MKK6 dependiente de la inducci&#xF3;n de la expresi&#xF3;n g&#xE9;nica del m&#xFA;sculo. Estos resultados identifican una funci&#xF3;n inesperada de la diferenciaci&#xF3;n activada por p38 en la conversi&#xF3;n de las se&#xF1;ales externas en modificaciones de la cromatina en los loci discretos, por la orientaci&#xF3;n selectiva de SWI-SNF reguladoras de elementos m&#xFA;sculos-.

P38 Camino Objetivos SWI-SNF remodelaci&#xF3;n de la cromatina-complejo de m&#xFA;sculos espec&#xED;ficos Loci

DNA damage induces cell cycle arrest and DNA repair or apoptosis in proliferating cells. Terminally differentiated cells are permanently withdrawn from the cell cycle and partly resistant to apoptosis. To investigate the effects of genotoxic agents in postmitotic cells, we compared DNA damage-activated responses in mouse and human proliferating myoblasts and their differentiated counterparts, the myotubes. DNA double-strand breaks caused by ionizing radiation (IR) induced rapid activating autophosphorylation of ataxia-teleangiectasia-mutated (ATM), phosphorylation of histone H2AX, recruitment of repair-associated proteins MRE11 and Nbs1, and activation of Chk2 in both myoblasts and myotubes. However, IR-activated, ATM-mediated phosphorylation of p53 at serine 15 (human) or 18 (mouse) [Ser15(h)/18(m)], and apoptosis occurred in myoblasts but was impaired in myotubes. This phosphorylation could be enforced in myotubes by the anthracycline derivative doxorubicin, leading to selective activation of proapoptotic genes. Unexpectedly, the abundance of autophosphorylated ATM was indistinguishable after exposure of myotubes to IR (10 Gy) or doxorubicin (1 microM/24 h) despite efficient phosphorylation of p53 Ser15(h)/18(m), and apoptosis occurred only in response to doxorubicin. These results suggest that radioresistance in myotubes might reflect a differentiation-associated, pathway-selective blockade of DNA damage signaling downstream of ATM. This mechanism appears to preserve IR-induced activation of the ATM-H2AX-MRE11/Rad50/Nbs1 lesion processing and repair pathway yet restrain ATM-p53-mediated apoptosis, thereby contributing to life-long maintenance of differentiated muscle tissues.

Differentiation-induced radioresistance in muscle cells.

&#x805A;&#x9170;&#x80FA;&#x82EF;&#x4E01;&#x9178;&#x6C2E;&#x82A5;&#x5171;&#x8F6D; (1R Chl) &#x902E;&#x6355;&#x5404;&#x79CD;&#x5404;&#x6837;&#x7684;&#x80BF;&#x7624;&#x7EC6;&#x80DE; G2/M &#x9636;&#x6BB5;&#x7684;&#x7EC6;&#x80DE;&#x5468;&#x671F;&#x53CA;&#x75C5;&#x6BD2;&#x7EC4; H4c &#x86CB;&#x767D;&#x57FA;&#x56E0;&#x7684;&#x8868;&#x8FBE;&#x3002;&#x4F46;&#x662F;&#xFF0C;&#x9488;&#x5BF9; H4c mRNA siRNA &#x5BFC;&#x81F4; G1/S &#x902E;&#x6355;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#xFF0C;&#x6211;&#x4EEC;&#x62A5;&#x544A; G2/M &#x88AB;&#x6355;&#x524D;&#x7684;&#xFF0C;1R Chl &#x75C5;&#x6BD2; H4c&#x3002;G2/M &#x902E;&#x6355;&#x662F;&#x5BFC;&#x81F4;&#x78F7;&#x9178;&#x5316; H2A 1R-&#x667A;&#x5229;&#x3001; &#x5E7F;&#x6CDB; DNA &#x635F;&#x4F24;&#x7684;&#x7ED3;&#x679C;&#x3002;X &#x4E0A;&#x4E1D;&#x6C28;&#x9178; 139&#x3001; Nbs1 &#x4FEE;&#x590D;&#x86CB;&#x767D;&#x8D28;&#x3001; &#x62DB;&#x8058;&#x548C;&#x7EA7;&#x8054;&#x7684;&#x672A;&#x77E5;&#x4E8B;&#x4EF6;&#x6700;&#x7EC8;&#x4E0E; cdc2 &#x78F7;&#x9178;&#x5316;&#x916A;&#x6C28;&#x9178; 15 &#x548C; cdc2 &#x6FC0;&#x9176;&#x6D3B;&#x6027;&#x88C1;&#x64A4;&#x3002;&#x63A7;&#x5236;&#x805A;&#x9170;&#x80FA; Chl &#x5171;&#x8F6D;&#xFF0C;&#x5176;&#x4E2D;&#x65E2;&#x4E0D;&#x7ED1;&#x5B9A;&#x5230; H4c &#x57FA;&#x56E0;&#x4E5F;&#x4E0D;&#x7531;&#x672C;&#x8EAB;&#x5177;&#x6709;&#x6297;&#x589E;&#x6B96;&#x3001; G2/M &#x902E;&#x6355;&#x65F6;&#x5BFC;&#x81F4;&#x7EC6;&#x80DE;&#x88AB;&#x89C6;&#x4E3A;&#x4E0E;&#x7279;&#x5B9A;&#x7684; siRNAs H3 &#x6216; H4c&#x3002;

Pre-mitotic &#x902E;&#x6355;&#x7684;&#x80BF;&#x7624;&#x7EC6;&#x80DE;&#x589E;&#x6B96;&#x7684;&#x805A;&#x9170;&#x80FA;-alkylator &#x5171;&#x8F6D;&#x4E8C;&#x6253;&#x51FB;&#x673A;&#x5236;&#x3002;

A polyamide-chlorambucil conjugate (1R-Chl) arrests a wide range of human cancer cell lines at the G2/M phase of the cell cycle and downregulates histone H4c gene expression. However, an siRNA against H4c mRNA causes G1/S arrest. Here, we report that 1R-Chl downregulates H4c prior to G2/M arrest. G2/M arrest is the result of extensive DNA damage by 1R-Chl, which leads to phosphorylation of H2A.X at serine 139, recruitment of the Nbs1 repair protein, and a cascade of unknown events culminating with cdc2 phosphorylation at tyrosine 15 and abolishment of cdc2 kinase activity. A control polyamide-Chl conjugate, which neither binds to the H4c gene nor has an anti-proliferative effect by itself, causes G2/M arrest when cells are treated with siRNAs specific for H3 or H4c.

A two-hit mechanism for pre-mitotic arrest of cancer cell proliferation by a polyamide-alkylator conjugate.

Un polyamide-chlorambucil conjugu&eacute; arrestations (1R-Chl) une vaste gamme de lign&eacute;es cellulaires canc&eacute;reuses humaines lors de la phase G2/M de l&#39;expression de g&egrave;nes d&#39;histone H4c cycle cellulaire et att&eacute;nue. Toutefois, un siARN contre H4c ARNm provoque une arrestation G1/S. Nous rapportons ici, cette action 1R-Chl H4c avant l&#39;arrestation de G2/M. G2/M arrestation est le r&eacute;sultat de vastes dommages &agrave; l&#39;ADN par 1R-Chl, qui m&egrave;ne &agrave; la phosphorylation de H2A.X &agrave; s&eacute;rine 139, recrutement de la prot&eacute;ine de r&eacute;paration Nbs1 et une cascade d&#39;&eacute;v&eacute;nements inconnus, culminant avec cdc2 phosphorylation de tyrosine 15 et suppression de l&#39;activit&eacute; kinase cdc2. Un contr&ocirc;le polyamide-Chl conjugu&eacute;, ni se lie au g&egrave;ne H4c ni a un effet anti-prolif&eacute;rante par lui-m&ecirc;me, entra&icirc;ne G2/M arrestation quand les cellules sont trait&eacute;es avec le siARN sp&eacute;cifique pour H3 ou H4c.

Un m&eacute;canisme &agrave; deux frappes pour arr&ecirc;ter la prolif&eacute;ration des cellules canc&eacute;reuses par un conjugu&eacute; de polyamide-alkylator pre-mitotic.

Ein Polyamid-Chlorambucil Konjugat (1R-Chl) Verhaftungen eine Vielzahl von menschlichen Krebszelllinien bei der G2/M-Phase des Zellzyklus und Downregulates-Histon H4c-Genexpression. Ein SiRNA gegen H4c mRNA bewirkt jedoch, dass G1/S Verhaftung. Hier berichten wir, dass 1R-Chl Downregulates H4c vor der Verhaftung der G2/M. G2/M-Festnahme ist das Ergebnis einer umfangreichen DNA-Sch&auml;den durch 1R-Chl, Phosphorylierung von H2A f&uuml;hrt.X Serin 139, Rekrutierung von Nbs1-Reparatur-Protein und eine Kaskade von unbekannte Ereignisse, die ihren H&ouml;hepunkt mit cdc2 Phosphorylierung an Tyrosin 15 und Abschaffung von cdc2-Kinase-Aktivit&auml;t. Ein Steuerelement-Polyamid-Chl-Konjugat, die weder an das H4c-gen bindet noch eine antiproliferative Wirkung selbst, G2/M-Verhaftung verursacht, wenn Zellen behandelt werden mit spezifischen SiRNAs f&uuml;r H3 oder H4c.

Eine zweifache Mechanismus pre-mitotic Verhaftung von Krebs Zellproliferation von einem Polyamid-Alkylator-Konjugat.

Un poliammide-clorambucil coniugato (1R-Chl) arresti una vasta gamma di linee di cellule tumorali umane nella fase G2/M del ciclo cellulare e sottoregola istone H4c espressione di gene. Tuttavia, un siRNA contro H4c mRNA provoca arresto G1/S. Qui, segnaliamo che sottoregola 1R-Chl H4c prima dell&#39;arresto di G2/M. Arresto di G2/M &egrave; il risultato di danni del DNA da 1R-Chl, che conduce alla fosforilazione di H2A.X di serina 139, reclutamento della proteina di riparazione Nbs1 e una cascata di eventi sconosciuti che si conclude con cdc2 fosforilazione in tirosina 15 e abolizione di attivit&agrave; della chinasi cdc2. Un controllo di poliammide-Chl coniugato che n&eacute; si lega al gene H4c n&eacute; ha un effetto anti-proliferativo di per s&eacute;, causa arresto G2/M quando le cellule sono trattate con specifici siRNA per H3 o H4c.

Un meccanismo del due-colpo per pre-mitotic arresto della proliferazione di cellule cancerogene di un coniugato di poliammide-alkylator.

&#x30DD;&#x30EA;&#x30A2;&#x30DF;&#x30C9; &#x30A2;&#x30DF;&#x30CE;&#x5171;&#x5F79; (1 r, &#x30AF;&#x30ED;&#x30ED;&#x30D5;&#x30A3;&#x30EB;&#xFF09; &#x902E;&#x6355;&#x69D8;&#x3005; &#x306A;&#x3072;&#x3068;&#x764C;&#x7D30;&#x80DE;&#x306E;&#x7D30;&#x80DE;&#x5468;&#x671F;&#x3068;&#x306F;&#x30D2;&#x30B9;&#x30C8;&#x30F3; H4c &#x907A;&#x4F1D;&#x5B50;&#x306E;&#x767A;&#x73FE;&#x306E; G2/M &#x76F8;&#x3067;&#x3002;&#x3057;&#x304B;&#x3057;&#x306B; H4c mRNA &#x306B;&#x5BFE;&#x3059;&#x308B; siRNA G1/S &#x902E;&#x6355;&#x304C;&#x767A;&#x751F;&#x3057;&#x307E;&#x3059;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x305D;&#x306E; 1 r Chl &#x306F; H4c G2/M &#x306E;&#x902E;&#x6355;&#x306E;&#x524D;&#x306B;&#x5831;&#x544A;&#x3057;&#x307E;&#x3059;&#x3002;G2/M &#x902E;&#x6355; H2A &#x306E;&#x30EA;&#x30F3;&#x9178;&#x5316;&#x306B;&#x3064;&#x306A;&#x304C;&#x308B; 1R-&#x30AF;&#x30ED;&#x30ED;&#x30D5;&#x30A3;&#x30EB;&#x306B;&#x3088;&#x308B;&#x5E83;&#x7BC4;&#x306A; DNA &#x640D;&#x50B7;&#x306E;&#x7D50;&#x679C;&#x3067;&#x3059;&#x3002;&#x30BB;&#x30EA;&#x30F3; 139&#x3001;Nbs1 &#x4FEE;&#x5FA9;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x306E;&#x52DF;&#x96C6;&#x304A;&#x3088;&#x3073; cdc2 &#x30EA;&#x30F3;&#x9178;&#x5316;&#x30C1;&#x30ED;&#x30B7;&#x30F3; 15 &#x3068; cdc2 &#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x6D3B;&#x6027;&#x306E;&#x5EC3;&#x6B62;&#x3067;&#x6700;&#x9AD8;&#x6F6E;&#x306B;&#x9054;&#x3059;&#x308B;&#x672A;&#x77E5;&#x30A4;&#x30D9;&#x30F3;&#x30C8;&#x306E;&#x30AB;&#x30B9;&#x30B1;&#x30FC;&#x30C9; X&#x3002;&#x30B3;&#x30F3;&#x30C8;&#x30ED;&#x30FC;&#x30EB; &#x30DD;&#x30EA;&#x30A2;&#x30DF;&#x30C9; &#x30AF;&#x30ED;&#x30ED;&#x30D5;&#x30A3;&#x30EB;&#x306E;&#x5171;&#x5F79;&#x3001;&#x3069;&#x3061;&#x3089;&#x3082; H4c &#x907A;&#x4F1D;&#x5B50;&#x306B;&#x7D50;&#x5408;&#x3059;&#x308B;&#x3001;&#x307E;&#x305F;&#x6297;&#x5897;&#x6B96;&#x52B9;&#x679C;&#x81EA;&#x4F53;&#x306B;&#x3088;&#x3063;&#x3066;&#x6301;&#x3063;&#x3066;&#x3044;&#x308B;&#x3001;&#x30BB;&#x30EB;&#x6271;&#x308F;&#x308C;&#x308B;&#x3068;&#x304D; G2/M &#x902E;&#x6355; siRNAs &#x3068;&#x7279;&#x5B9A; H3 &#x307E;&#x305F;&#x306F; H4c &#x306E;&#x539F;&#x56E0;&#x3057;&#x307E;&#x3059;&#x3002;

Pre-mitotic &#x902E;&#x6355;&#x30DD;&#x30EA;&#x30A2;&#x30DF;&#x30C9; alkylator &#x5171;&#x5F79;&#x306B;&#x3088;&#x308B;&#x764C;&#x7D30;&#x80DE;&#x306E;&#x5897;&#x6B96;&#x306E;&#x305F;&#x3081;&#x306E; 2 &#x30D2;&#x30C3;&#x30C8; &#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3002;

&#xD3F4; &#xB9AC;&#xC544; &#xBBF8;&#xB4DC; chlorambucil &#xC138;&#xD3EC; &#xC8FC;&#xAE30;&#xC640; downregulates &#xD788;&#xC2A4;&#xD1A4; H4c &#xC720;&#xC804;&#xC790; &#xBC1C;&#xD604;&#xC758; G2/M &#xB2E8;&#xACC4;&#xC5D0;&#xC11C; &#xB2E4;&#xC591; &#xD55C; &#xC778;&#xAC04; &#xC554; &#xC138;&#xD3EC; &#xB77C;&#xC778; (1R Chl) &#xCCB4;&#xD3EC; &#xCF24;&#xB808;. &#xADF8;&#xB7EC;&#xB098; H4c Mrna&#xC5D0; &#xB300; &#xD55C; siRNA G1/S &#xCCB4;&#xD3EC;&#xB97C; &#xBC1C;&#xC0DD;&#xD569;&#xB2C8;&#xB2E4;. &#xC5EC;&#xAE30;, &#xC6B0;&#xB9AC;&#xB294; 1 &#xB77C;&#xC6B4;&#xB4DC; Chl downregulates H4c G2/M &#xCCB4;&#xD3EC; &#xC774;&#xC804;&#xC5D0; &#xBCF4;&#xACE0;&#xD569;&#xB2C8;&#xB2E4;. G2/M &#xCCB4;&#xD3EC; 1 &#xB77C;&#xC6B4;&#xB4DC;-Chl h2a&#xC758; &#xC778; &#xC0B0;&#xD654;&#xC5D0; &#xC774;&#xB974;&#xAC8C; &#xD558; &#xC5EC; &#xAD11;&#xBC94;&#xC704; &#xD55C; DNA &#xC190;&#xC0C1;&#xC758; &#xACB0;&#xACFC; &#xC774;&#xB2E4;.&#xB5A0;&#xB4E4;&#xACE0; 139, Nbs1 &#xC218;&#xB9AC; &#xB2E8;&#xBC31;&#xC9C8;&#xC758; &#xBAA8;&#xC9D1; &#xBC0F; &#xC54C; &#xC218; &#xC5C6;&#xB294; &#xC774;&#xBCA4;&#xD2B8; cdc2 &#xC778; &#xC0B0;&#xD654; &#xD2F0;&#xB85C;&#xC2E0; 15&#xC640; cdc2 kinase &#xD65C;&#xB3D9;&#xC758; &#xD3D0;&#xC9C0;&#xC640; culminating&#xC758; &#xD3ED;&#xD3EC;&#xC5D0;&#xC11C; X. &#xCEE8;&#xD2B8;&#xB864; &#xD3F4; &#xB9AC;&#xC544; &#xBBF8;&#xB4DC; Chl &#xACF5;&#xC561;-&#xC5B4;&#xB290; H4c &#xC720;&#xC804;&#xC790;&#xC5D0; &#xBC14;&#xC778;&#xB529;&#xD569;&#xB2C8;&#xB2E4;&#xB3C4; &#xC790;&#xCCB4; &#xC548;&#xD2F0; &#xC99D;&#xC2DD; &#xD6A8;&#xACFC;&#xAC00;, &#xC6D0;&#xC778;&#xC774; G2/M &#xCCB4;&#xD3EC; &#xC140; &#xCE58;&#xB8CC;&#xB294; siRNAs &#xD2B9;&#xC815; H3 &#xB610;&#xB294; H4c.

&#xD3F4; &#xB9AC;&#xC544; &#xBBF8;&#xB4DC; alkylator &#xACF5;&#xC561;&#xC5D0; &#xC758;&#xD574; &#xC554; &#xC138;&#xD3EC; &#xC99D;&#xC2DD;&#xC758; pre-mitotic &#xCCB4;&#xD3EC;&#xC5D0; &#xB300; &#xD55C; &#xB450; &#xD788;&#xD2B8; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;.

Un poliamida-clorambucilo conjugado detenciones (1R-Chl) una amplia gama de l&iacute;neas celulares de c&aacute;ncer humano en la fase G2/M de la expresi&oacute;n del gen histona H4c ciclo celular y algo. Sin embargo, un siRNA contra H4c mRNA causa detenci&oacute;n G1/S. Aqu&iacute;, divulgamos regula 1R-Chl H4c antes de G2/M detenci&oacute;n. Detenci&oacute;n de G2/M es el resultado de da&ntilde;os de ADN por 1R-Chl, que conduce a la fosforilaci&oacute;n de H2A.X en serina 139, reclutamiento de la prote&iacute;na de reparaci&oacute;n de Nbs1 y una cascada de eventos desconocidos culminando en cdc2 fosforilaci&oacute;n en tirosina de 15 y la abolici&oacute;n de la actividad de la quinasa cdc2. Un conjugado de poliamida-Chl control, que ni se une al gen H4c ni tiene un efecto anti proliferativo por s&iacute; misma, causa detenci&oacute;n de G2/M cuando las c&eacute;lulas se tratan con siRNAs espec&iacute;ficos para H3 o H4c.

Un mecanismo de dos hits para pre-mitotic detenci&oacute;n de la proliferaci&oacute;n celular de c&aacute;ncer por un conjugado de poliamida-un f&aacute;rmaco alquilante.

Studies on DNA damage responses in proliferating cells have revealed the relationship between sensing and repair of the DNA lesions and the regulation of the cell cycle, leading to the discovery and molecular characterization of the DNA damage-activated cell cycle checkpoints. Much less is known about the DNA damage response in progenitors of differentiated cells, in which cell cycle arrest is a critical signal to trigger the differentiation program, and in terminally differentiated cells, which are typically post-mitotic. How DNA lesions are detected, processed and repaired in these cells, the functional impact of DNA damage on transcription of differentiation-specific genes, how these events are coordinated at the molecular level, the consequence of defective DNA damage response on tissue-specific functions and its potential relationship with age-related diseases are currently open questions. In particular the biological complexity inherent to the global genome reprogramming of tissue progenitors, such as embryonic or adult stem cells, suggests the importance of an accurate DNA damage response at the transcription level in these cells to ensure the genomic integrity of regenerating tissues.

DNA damage and cellular differentiation: more questions than responses.

Despite having distinct expression patterns and phenotypes in mutant mice, the myogenic regulatory factors Myf5 and MyoD have been considered to be functionally equivalent. Here, we report that these factors have a different response to DNA damage, due to the presence in MyoD and absence in Myf5 of a consensus site for Abl-mediated tyrosine phosphorylation that inhibits MyoD activity in response to DNA damage. Genotoxins failed to repress skeletal myogenesis in MyoD-null embryos; reintroduction of wild-type MyoD, but not mutant Abl phosphorylation-resistant MyoD, restored the DNA-damage-dependent inhibition of muscle differentiation. Conversely, introduction of the Abl-responsive phosphorylation motif converts Myf5 into a DNA-damage-sensitive transcription factor. Gene-dosage-dependent reduction of Abl kinase activity in MyoD-expressing cells attenuated the DNA-damage-dependent inhibition of myogenesis. The presence of a DNA-damage-responsive phosphorylation motif in vertebrate, but not in invertebrate MyoD suggests an evolved response to environmental stress, originated from basic helix-loop-helix gene duplication in vertebrate myogenesis.

An evolutionarily acquired genotoxic response discriminates MyoD from Myf5, and differentially regulates hypaxial and epaxial myogenesis.

Upon exposure to genotoxic stress, skeletal muscle progenitors coordinate DNA repair and the activation of the differentiation program through the DNA damage-activated differentiation checkpoint, which holds the transcription of differentiation genes while the DNA is repaired. A conceptual hurdle intrinsic to this process relates to the coordination of DNA repair and muscle-specific gene transcription within specific cell cycle boundaries (cell cycle checkpoints) activated by different types of genotoxins. Here, we show that, in proliferating myoblasts, the inhibition of muscle gene transcription occurs by either a G 1- or G 2-specific differentiation checkpoint. In response to genotoxins that induce G 1 arrest, MyoD binds target genes but is functionally inactivated by a c-Abl-dependent phosphorylation. In contrast, DNA damage-activated G 2 checkpoint relies on the inability of MyoD to bind the chromatin at the G 2 phase of the cell cycle. These results indicate an intimate relationship between DNA damage-activated cell cycle checkpoints and the control of tissue-specific gene expression to allow DNA repair in myoblasts prior to the activation of the differentiation program.

Coordination of cell cycle, DNA repair and muscle gene expression in myoblasts exposed to genotoxic stress.

Epigenetic control of reprogramming and cellular differentiation.

Reporting in this issue of Developmental Cell, L'honoré et al. (2014) show that a network composed of Pitx2/Pitx3 and downstream antioxidant enzymes protects differentiating skeletal muscle from excessive reactive oxygen species production during fetal myogenesis. Genetic deficiency of Pitx2/Pitx3 results in irreversible oxidative DNA damage and apoptosis, impairing skeletal muscle development.

Redox or death: checking on fetal myogenesis.

The progressive loss of muscle regenerative capacity with age or disease results in part from a decline in the number and function of satellite cells, the direct cellular contributors to muscle repair. However, little is known about the molecular effectors underlying satellite cell impairment and depletion. Elevated levels of inflammatory cytokines, including interleukin-6 (IL-6), are associated with both age-related and muscle-wasting conditions. The levels of STAT3, a downstream effector of IL-6, are also elevated with muscle wasting, and STAT3 has been implicated in the regulation of self-renewal and stem cell fate in several tissues. Here we show that IL-6-activated Stat3 signaling regulates satellite cell behavior, promoting myogenic lineage progression through myogenic differentiation 1 (Myod1) regulation. Conditional ablation of Stat3 in Pax7-expressing satellite cells resulted in their increased expansion during regeneration, but compromised myogenic differentiation prevented the contribution of these cells to regenerating myofibers. In contrast, transient Stat3 inhibition promoted satellite cell expansion and enhanced tissue repair in both aged and dystrophic muscle. The effects of STAT3 inhibition on cell fate and proliferation were conserved in human myoblasts. The results of this study indicate that pharmacological manipulation of STAT3 activity can be used to counteract the functional exhaustion of satellite cells in pathological conditions, thereby maintaining the endogenous regenerative response and ameliorating muscle-wasting diseases.

STAT3 signaling controls satellite cell expansion and skeletal muscle repair.

A hallmark of aging is alteration of organismal homeostasis and progressive decline of tissue functions. Alterations of both cell intrinsic functions and regenerative environmental cues contribute to the compromised stem cell activity and reduced regenerative capability occurring in aged muscles. In this perspective, we discuss the new evidence supporting the hypothesis that skeletal muscle stem cells (MuSCs) are intrinsically defective in elderly muscles. In particular, we review three recent papers leading to identify fibroblast growth factor receptor-1, p38 mitogen-activated protein kinase, and p16INK4a as altered signaling in satellite cells from aged mice. These pathways contribute to age-related loss of MuSCs asymmetric polarization, compromised self-renewal capacity, and acquisition of pre-senescent state. The pharmacological manipulation of those networks can open novel strategies to rejuvenate MuSCs and counteract the functional decline of skeletal muscle during aging.

Forever young: rejuvenating muscle satellite cells.

Epigenetic Regulation Shapes the Stem Cells State.

Mitochondrial dysfunction occurs in many muscle degenerative disorders. Here, we demonstrate that mitochondrial biogenesis was impaired in limb-girdle muscular dystrophy (LGMD) 2D patients and mice and was associated with impaired OxPhos capacity. Two distinct approaches that modulated histones or peroxisome proliferator-activated receptor-gamma coactivator 1 α (PGC-1α) acetylation exerted equivalent functional effects by targeting different mitochondrial pathways (mitochondrial biogenesis or fatty acid oxidation[FAO]). The histone deacetylase inhibitor Trichostatin A (TSA) changed chromatin assembly at the PGC-1α promoter, restored mitochondrial biogenesis, and enhanced muscle oxidative capacity. Conversely, nitric oxide (NO) triggered post translation modifications of PGC-1α and induced FAO, recovering the bioenergetics impairment of muscles but shunting the defective mitochondrial biogenesis. In conclusion, a transcriptional blockade of mitochondrial biogenesis occurred in LGMD-2D and could be recovered by TSA changing chromatin conformation, or it could be overcome by NO activating a mitochondrial salvage pathway.

Reversal of Defective Mitochondrial Biogenesis in Limb-Girdle Muscular Dystrophy 2D by Independent Modulation of Histone and PGC-1α Acetylation.

The molecular determinants of muscle progenitor impairment to regenerate aged muscles are currently unclear. We show that, in a mouse model of replicative senescence, decline in muscle satellite cell-mediated regeneration coincides with activation of DNA damage response (DDR) and impaired ability to differentiate into myotubes. Inhibition of DDR restored satellite cell differentiation ability. Moreover, in replicative human senescent fibroblasts, DDR precluded MYOD-mediated activation of the myogenic program. A DDR-resistant MYOD mutant could overcome this barrier by resuming cell cycle progression. Likewise, DDR inhibition could also restore MYOD's ability to activate the myogenic program in human senescent fibroblasts. Of note, we found that cell cycle progression is necessary for the DDR-resistant MYOD mutant to reverse senescence-mediated inhibition of the myogenic program. These data provide the first evidence of DDR-mediated functional antagonism between senescence and MYOD-activated gene expression and indicate a previously unrecognized requirement of cell cycle progression for the activation of the myogenic program.

Lucia Latella

Department of Medicine,
Institute of Translational Pharmacology,
Department of Medicine, Institute of Translational Pharmacology

Italian National Research Council

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Lucia Latella

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Italian National Research Council

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Department of Medicine,
Institute of Translational Pharmacology,
Department of Medicine, Institute of Translational Pharmacology