Combined DNA-RNA Fluorescent <em>In situ</em> Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Erasmus MC - University Medical Center

In somatic cells of female placental mammals, one X chromosome is inactivated to minimize sex-related dosage differences of X-encoded genes. Random X chromosome inactivation (XCI) in the embryo is a stochastic process, in which each X has an independent probability to initiate XCI, triggered by the nuclear concentration of one or more X-encoded XCI-activators. Here, we identify the E3 ubiquitin ligase RNF12 as an important XCI-activator. Additional copies of mouse Rnf12 or human RNF12 result in initiation of XCI in male mouse ES cells and on both X chromosomes in a substantial percentage of female mouse ES cells. This activity is dependent on an intact open reading frame of Rnf12 and correlates with the transgenic expression level of RNF12. Initiation of XCI is markedly reduced in differentiating female heterozygous Rnf12(+/-) ES cells. These findings provide evidence for a dose-dependent role of RNF12 in the XCI counting and initiation process.

RNF12 is an X-Encoded dose-dependent activator of X chromosome inactivation.

In female somatic cells of mammalian species one X chromosome is inactivated to ensure dosage equality of X-encoded genes between females and males, during development and adulthood. X chromosome inactivation (XCI) involves various epigenetic mechanisms, including RNA mediated gene silencing in cis, DNA methylation, and changes in chromatin modifications and composition. XCI therefore provides an attractive paradigm to study epigenetic gene regulation in a more general context. The XCI process starts with counting of the number of X chromosomes present in a nucleus, and initiation of XCI follows if this number exceeds one per diploid genome. Recently, X-encoded RNF12 has been identified as a dose-dependent activator of XCI. In addition, other factors, including the pluripotency factors OCT4, SOX2 and Nanog, have been implicated to play a role in suppression of initiation of XCI. In this review, we highlight and explain these new and old findings in the context of a stochastic model for X chromosome counting and XCI initiation.

X-changing information on X inactivation.

Murine pluripotent stem cells can exist in two functionally distinct states, LIF-dependent embryonic stem cells (ESCs) and bFGF-dependent epiblast stem cells (EpiSCs). However, human pluripotent cells so far seemed to assume only an epiblast-like state. Here we demonstrate that human iPSC reprogramming in the presence of LIF yields human stem cells that display morphological, molecular, and functional properties of murine ESCs. We termed these hLR5 iPSCs because they require the expression of five ectopic reprogramming factors, Oct4, Sox2, Klf4, cMyc, and Nanog, to maintain this more naive state. The cells are "metastable" and upon ectopic factor withdrawal they revert to standard human iPSCs. Finally, we demonstrate that the hLR5 state facilitates gene targeting, and as such provides a powerful tool for the generation of recombinant human pluripotent stem cell lines.

A murine ESC-like state facilitates transgenesis and homologous recombination in human pluripotent stem cells.

&#x5C0F;&#x9F20;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE;&#x53EF;&#x4EE5;&#x5B58;&#x5728;&#x4E8E;&#x4E24;&#x4E2A;&#x529F;&#x80FD;&#x4E0D;&#x540C;&#x7684;&#x56FD;&#x5BB6;&#x3001; LIF &#x4F9D;&#x8D56;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE; (&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE;) &#x548C; bFGF &#x4F9D;&#x8D56; epiblast &#x5E72;&#x7EC6;&#x80DE; (EpiSCs)&#x3002;&#x7136;&#x800C;&#xFF0C;&#x4EBA;&#x7C7B;&#x591A;&#x80FD;&#x5E72;&#x7EC6;&#x80DE;&#x5230;&#x76EE;&#x524D;&#x4E3A;&#x6B62;&#x4F3C;&#x4E4E;&#x5047;&#x5B9A;&#x53EA;&#x6709;&#x4E00;&#x4E2A;&#x7C7B;&#x4F3C; epiblast &#x7684;&#x56FD;&#x5BB6;&#x3002;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x8868;&#x660E;&#x4EBA;&#x7C7B;&#x673A;&#x4F53;&#x529F;&#x80FD;&#x91CD;&#x65B0;&#x4E0B; LIF &#x6536;&#x76CA;&#x7387;&#x663E;&#x793A;&#x7684;&#x5C0F;&#x9F20;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE;&#x7684;&#x5F62;&#x6001;&#x3001; &#x5206;&#x5B50;&#xFF0C;&#x548C;&#x529F;&#x80FD;&#x5C5E;&#x6027;&#x7684;&#x4EBA;&#x7C7B;&#x5E72;&#x7EC6;&#x80DE;&#x3002;&#x6211;&#x4EEC;&#x79F0;&#x8FD9;&#x4E9B; hLR5 &#x9B54;&#x9B3C;&#xFF0C;&#x56E0;&#x4E3A;&#x5B83;&#x4EEC;&#x9700;&#x8981;&#x8868;&#x8FBE;&#x7684;&#x4E94;&#x4E2A;&#x5F02;&#x4F4D;&#x7F16;&#x7A0B;&#x56E0;&#x7D20;&#x3001; Oct4&#x3001; Sox2&#x3001; Klf4&#x3001; cMyc&#x3001; &#x548C; Nanog&#xFF0C;&#x6765;&#x7EF4;&#x62A4;&#x8FD9;&#x66F4;&#x5929;&#x771F;&#x7684;&#x72B6;&#x6001;&#x3002;&#x5355;&#x5143;&#x683C;&#x662F;&quot;&#x7A33;&quot;&#x548C;&#x5F02;&#x4F4D;&#x56E0;&#x5B50;&#x9000;&#x9009;&#x540E;&#x5C06;&#x5B83;&#x4EEC;&#x8F6C;&#x6362;&#x4E3A;&#x6807;&#x51C6;&#x7684;&#x4EBA;&#x7C7B;&#x9B54;&#x9B3C;&#x3002;&#x6700;&#x540E;&#xFF0C;&#x6211;&#x4EEC;&#x8BC1;&#x660E; hLR5 &#x56FD;&#x5BB6;&#x4FC3;&#x8FDB;&#x57FA;&#x56E0;&#x6253;&#x9776;&#xFF0C;&#x548C;&#x8FD9;&#x79CD;&#x63D0;&#x4F9B;&#x4E00;&#x4E2A;&#x5F3A;&#x5927;&#x7684;&#x5DE5;&#x5177;&#x4E3A;&#x4EE3;&#x7684;&#x91CD;&#x7EC4;&#x4EBA;&#x7C7B;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE;&#x7CFB;&#x3002;

&#x5C0F;&#x9F20; esc &#x952E;&#x7C7B;&#x4F3C;&#x56FD;&#x5BB6;&#x5E2E;&#x52A9;&#x4EBA;&#x7C7B;&#x80DA;&#x80CE;&#x5E72;&#x7EC6;&#x80DE;&#x4E2D;&#x7684;&#x540C;&#x6E90;&#x91CD;&#x7EC4;&#x548C;&#x8F6C;&#x57FA;&#x56E0;&#x6280;&#x672F;&#x3002;

Murins cellules souches pluripotentes peuvent exister dans deux &#xE9;tats distincts, fonctionnellement d&#xE9;pendant de FRV cellules souches embryonnaires (CSE) et bFGF-d&#xE9;pendants des cellules souches &#xE9;piblaste (EpiSCs). Cependant, les cellules pluripotentes humaines jusqu&#x26;#39;&#xE0; pr&#xE9;sent semblait &#xE0; assumer seule un &#xE9;tat &#xE9;piblaste forme. Nous d&#xE9;montrons ici que iPSC humaine reprogrammation dans la pr&#xE9;sence de cellules souches humaines rendements FRV qui affichent des propri&#xE9;t&#xE9;s morphologiques, mol&#xE9;culaires et fonctionnelles des CES murins. Nous appeler ces hLR5 CSPi car ils n&#xE9;cessitent l&#x26;#39;expression de cinq facteurs de reprogrammation ectopiques, Oct4, Sox2, Klf4, cMyc, et Nanog, pour maintenir cet &#xE9;tat plus na&#xEF;f. Les cellules sont &#xAB;m&#xE9;tastable&#xBB; et au moment du retrait facteur extra-ut&#xE9;rine ils reviennent &#xE0; CSPi standards de l&#x26;#39;homme. Enfin, nous d&#xE9;montrons que l&#x26;#39;&#xE9;tat hLR5 facilite le ciblage de g&#xE8;nes, et en tant que telle constitue un outil puissant pour la g&#xE9;n&#xE9;ration de lign&#xE9;es recombinantes cellules souches pluripotentes humaines.

Un murin ESC-like &#xC9;tat facilite la transgen&#xE8;se et la recombinaison homologue dans cellules souches pluripotentes humaines

Murine pluripotenten Stammzellen k&ouml;nnen in zwei funktionell unterschiedliche Zust&auml;nde, LIF-abh&auml;ngige embryonale Stammzellen (WSR) und bFGF-abh&auml;ngige Epiblast Stammzellen (EpiSCs) vorhanden sein. Jedoch schien menschliche pluripotenten Zellen bisher nur einen Epiblast-artigen Zustand anzunehmen. Hier zeigen wir, dass die menschlichen iPSC Umprogrammierung in Anwesenheit von LIF menschlichen Stammzellen ergibt, der morphologische, Molekulare und funktionelle Eigenschaften des murinen WSR anzeigen. Wir diese hLR5 iPSCs genannt, weil sie erfordern, dass den Ausdruck der f&uuml;nf ektopische Neuprogrammierung Faktoren, Oct4, Sox2, Klf4 cMyc und Nanog zu erhalten mehr naiven Zustand. Die Zellen sind &quot;metastabil&quot; und nach ektopische Faktor Zur&uuml;cknahme wiederherstellen sie standard menschlichen iPSCs. Schlie&szlig;lich zeigen wir, dass das hLR5 Gene targeting erleichtert, und als solche ein leistungsstarkes Tool f&uuml;r die Generierung von rekombinanten menschlichen pluripotenten Stammzelllinien ist.

Ein muriner ESC-artigen Zustand erleichtert Transgenese und Homologe Rekombination in menschlichen pluripotenten Stammzellen.

Cellule staminali pluripotenti murine possono esistere in due stati distinti funzionalmente, LIF-dipendente le cellule embrionali staminali (CES) e bFGF-dipendente epiblast cellule staminali (EpiSCs). Tuttavia, le cellule pluripotenti umane finora sembravano assumere solo uno stato epiblast-come. Qui dimostriamo che umano iPSC riprogrammazione in presenza di LIF produce cellule staminali umane che visualizzano le propriet&agrave; morfologiche, molecolare e funzionale di CES murine. Abbiamo definito questi iPSCs hLR5 perch&eacute; richiedono l&#39;espressione di cinque fattori di riprogrammazione ectopiche, Oct4, Sox2, Klf4, cMyc e Nanog, per mantenere questo stato pi&ugrave; ingenuo. Le cellule sono &quot;metastabile&quot; e al momento del ritiro del fattore ectopica si ritorna allo standard iPSCs umane. Infine, ci dimostrano che lo stato hLR5 facilita il gene targeting e come tale fornisce un potente strumento per la generazione di recombinant human pluripotent stem cell lines.

Uno stato di ESC murino facilita la transgenesi e ricombinazione omologa in cellule staminali pluripotenti umane.

&#x30DE;&#x30A6;&#x30B9;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x306F;&#x3001;2&#x3064;&#x306E;&#x6A5F;&#x80FD;&#x7684;&#x306B;&#x7570;&#x306A;&#x308B;&#x72B6;&#x614B;&#x3001;LIF-&#x4F9D;&#x5B58;&#x6027;&#x306E;&#x80DA;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#xFF08;ESC&#x306F;&#xFF09;&#x3068;bFGF&#x306B;&#x4F9D;&#x5B58;&#x3059;&#x308B;&#x80DA;&#x76E4;&#x8449;&#x4E0A;&#x5C64;&#x5E79;&#x7D30;&#x80DE;&#xFF08;EpiSCs&#xFF09;&#x306B;&#x5B58;&#x5728;&#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059;&#x3002;&#x3057;&#x304B;&#x3057;&#x3001;&#x30D2;&#x30C8;&#x591A;&#x80FD;&#x6027;&#x7D30;&#x80DE;&#x306F;&#x3001;&#x3053;&#x308C;&#x307E;&#x3067;&#x306E;&#x3068;&#x3053;&#x308D;&#x552F;&#x4E00;&#x306E;&#x80DA;&#x76E4;&#x8449;&#x4E0A;&#x5C64;&#x306E;&#x3088;&#x3046;&#x306A;&#x72B6;&#x614B;&#x3092;&#x60F3;&#x5B9A;&#x3057;&#x3066;&#x3044;&#x308B;&#x3088;&#x3046;&#x3060;&#x3063;&#x305F;&#x3002;&#x3053;&#x3053;&#x3067;&#x306F;&#x3001;&#x305D;&#x306E;&#x4EBA;&#x9593;&#x306E;IPSC&#x306F;&#x3001;&#x30DE;&#x30A6;&#x30B9;ESC&#x306E;&#x3001;&#x5F62;&#x614B;&#x5B66;&#x7684;&#x5206;&#x5B50;&#x3001;&#x6A5F;&#x80FD;&#x306E;&#x30D7;&#x30ED;&#x30D1;&#x30C6;&#x30A3;&#x3092;&#x8868;&#x793A;LIF&#x5229;&#x56DE;&#x308A;&#x30D2;&#x30C8;&#x5E79;&#x7D30;&#x80DE;&#x306E;&#x5B58;&#x5728;&#x4E0B;&#x3067;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30DF;&#x30F3;&#x30B0;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x5F7C;&#x3089;&#x306F;&#x3053;&#x306E;&#x3088;&#x308A;&#x5358;&#x7D14;&#x306A;&#x72B6;&#x614B;&#x3092;&#x7DAD;&#x6301;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x3001;5&#x7570;&#x6240;&#x6027;&#x518D;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30DF;&#x30F3;&#x30B0;&#x56E0;&#x5B50;&#x306E;&#x767A;&#x73FE;&#x3001;OCT4&#x3001;&#x30EC;&#x30C8;&#x30ED;&#x30A6;&#x30A4;&#x30EB;&#x30B9;&#x30D9;&#x30AF;&#x30BF;&#x30FC;&#x3001;KLF4&#x3001;cMyc&#x3001;&#x304A;&#x3088;&#x3073;Nanog&#x3092;&#x5FC5;&#x8981;&#x3068;&#x3059;&#x308B;&#x305F;&#x3081;&#x3001;&#x6211;&#x3005;&#x306F;&#x3001;&#x3053;&#x308C;&#x3089;&#x306E;hLR5&#x6027;IPSC&#x3068;&#x547C;&#x3070;&#x308C;&#x308B;&#x3002;&#x7D30;&#x80DE;&#x306F; &#x26;quot;&#x5B89;&#x5B9A;&#x26;quot;&#x3067;&#x3042;&#x308A;&#x3001;&#x7570;&#x6240;&#x6027;&#x56E0;&#x5B50;&#x306E;&#x64A4;&#x9000;&#x306B;&#x3001;&#x5F7C;&#x3089;&#x306F;&#x6A19;&#x6E96;&#x7684;&#x306A;&#x4EBA;&#x9593;&#x6027;IPSC&#x306B;&#x623B;&#x308A;&#x307E;&#x3059;&#x3002;&#x6700;&#x5F8C;&#x306B;&#x3001;&#x6211;&#x3005;&#x306F;hLR5&#x72B6;&#x614B;&#x304C;&#x6A19;&#x7684;&#x3068;&#x3057;&#x3001;&#x305D;&#x306E;&#x3088;&#x3046;&#x306A;&#x7D44;&#x63DB;&#x3048;&#x30D2;&#x30C8;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x682A;&#x3092;&#x751F;&#x6210;&#x3059;&#x308B;&#x305F;&#x3081;&#x306E;&#x5F37;&#x529B;&#x306A;&#x30C4;&#x30FC;&#x30EB;&#x3092;&#x63D0;&#x4F9B;&#x3059;&#x308B;&#x3088;&#x3046;&#x306B;&#x907A;&#x4F1D;&#x5B50;&#x3092;&#x5BB9;&#x6613;&#x306B;&#x3059;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x308B;&#x3002;

&#x30DE;&#x30A6;&#x30B9;ESC&#x306E;&#x3088;&#x3046;&#x306A;&#x72B6;&#x614B;&#x306F;&#x3001;&#x30D2;&#x30C8;&#x591A;&#x80FD;&#x6027;&#x5E79;&#x7D30;&#x80DE;&#x306B;&#x304A;&#x3051;&#x308B;&#x907A;&#x4F1D;&#x5B50;&#x7D44;&#x63DB;&#x3048;&#x3068;&#x76F8;&#x540C;&#x7D44;&#x63DB;&#x3048;&#x3092;&#x5BB9;&#x6613;&#x306B;

Murine pluripotent &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xC904;&#xAE30; &#xC138;&#xD3EC; (EpiSCs) &#xB450; &#xAC00;&#xC9C0; &#xAE30;&#xB2A5; &#xC0C1;&#xD0DC;, LIF &#xC885;&#xC18D; &#xBC30;&#xC544; &#xC904;&#xAE30; &#xC138;&#xD3EC; (ESCs)&#xC640; bFGF &#xC885;&#xC18D; epiblast &#xC874;&#xC7AC;&#xD560; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, &#xC778;&#xAC04;&#xC758; &#xB9CC;&#xB2A5; &#xC138;&#xD3EC;&#xB294; &#xC9C0;&#xAE08;&#xAE4C;&#xC9C0; epiblast &#xAC19;&#xC740; &#xC0C1;&#xD0DC;&#xB97C; &#xAC00;&#xC815; &#xD558;&#xB294; &#xAC83; &#xAC19;&#xC558;&#xB2E4;. &#xC5EC;&#xAE30; &#xC6B0;&#xB9AC;&#xB294; &#xC778;&#xAC04;&#xC758; iPSC LIF &#xC874;&#xC7AC; &#xC7AC;&#xD504;&#xB85C;&#xADF8;&#xB798;&#xBC0D; murine Escs&#xC758; &#xD615;&#xD0DC;&#xD559;, &#xBD84;&#xC790;, &#xADF8;&#xB9AC;&#xACE0; &#xAE30;&#xB2A5;&#xC801; &#xC18D;&#xC131;&#xC744; &#xD45C;&#xC2DC; &#xD558;&#xB294; &#xC778;&#xAC04;&#xC758; &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xB97C; &#xC0DD;&#xC131; &#xD558;&#xB294; &#xBC29;&#xBC95;&#xC744; &#xBCF4;&#xC5EC; &#xC90D;&#xB2C8;&#xB2E4;. 5 &#xC18C;&#xC131; &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC744; &#xB2E4;&#xC2DC; &#xB9CC;&#xB4E4; &#xC694;&#xC778;, Oct4, Sox2, Klf4, cMyc, &#xBC0F; Nanog, &#xC774;&#xAC83;&#xC744; &#xB354; &#xC720;&#xC9C0; &#xD558;&#xAE30; &#xC704;&#xD574;&#xC758; &#xC2DD;&#xC774; &#xD544;&#xC694;&#xB85C; &#xD558;&#xAE30; &#xB54C;&#xBB38;&#xC5D0; &#xC6B0;&#xB9AC;&#xAC00; &#xC774;&#xB7EC;&#xD55C; hLR5 iPSCs &#xB418; &#xB098; &#xC21C;&#xC9C4;&#xD55C; &#xC0C1;&#xD0DC;. &#xC140;&#xC740; &quot;&#xD655;&#xC815;&quot; &#xD558; &#xACE0; &#xC18C;&#xC131; &#xC694;&#xC18C; &#xCCA0;&#xC218;&#xC5D0; &#xB530;&#xB77C; &#xADF8;&#xB4E4;&#xC740; &#xD45C;&#xC900; &#xC778;&#xAC04;&#xC758; iPSCs &#xB418;&#xB3CC;&#xB9AC;&#xB824;&#xBA74;. &#xB9C8;&#xC9C0;&#xB9C9;&#xC73C;&#xB85C; &#xC6B0;&#xB9AC;&#xAC00; &#xC124;&#xBA85; hLR5 &#xC0C1;&#xD0DC; &#xC720;&#xC804;&#xC790;&#xB97C; &#xB300;&#xC0C1;&#xC73C;&#xB85C;, &#xC6A9;&#xC774; &#xD558; &#xAC8C; &#xD558; &#xACE0; &#xAC19;&#xC740; &#xC7AC;&#xC870;&#xD569; &#xC778;&#xAC04; &#xB9CC;&#xB2A5;&#xC758; &#xC138;&#xB300;&#xB97C; &#xC704;&#xD55C; &#xC904;&#xAE30; &#xC138;&#xD3EC; &#xAC15;&#xB825;&#xD55C; &#xB3C4;&#xAD6C;&#xB97C; &#xC81C;&#xACF5; &#xD569;&#xB2C8;&#xB2E4;.

Murine esc &#xD0A4;&#xC640; &#xAC19;&#xC740; &#xC0C1;&#xD0DC; transgenesis &#xBC0F; &#xB3D9;&#xC885; &#xC7AC;&#xC870;&#xD569; &#xC778;&#xAC04; pluripotent &#xC904;&#xAE30; &#xC138;&#xD3EC;&#xC5D0;&#xC11C;&#xB97C; &#xC6A9;&#xC774; &#xD558; &#xAC8C;.

Murino c&#xE9;lulas madre pluripotentes pueden existir en dos estados funcionales distintos, LIF-dependientes de las c&#xE9;lulas madre embrionarias (CME) y bFGF dependientes de las c&#xE9;lulas madre del epiblasto (EpiSCs). Sin embargo, las c&#xE9;lulas humanas pluripotentes hasta ahora parec&#xED;a asumir s&#xF3;lo un estado similar al epiblasto. Aqu&#xED; demostramos que la reprogramaci&#xF3;n de IPSC humana en la presencia de rendimientos LIF c&#xE9;lulas madre humanas que muestran las propiedades morfol&#xF3;gicas, moleculares y funcionales de los CES de rat&#xF3;n. Hemos llamado estos hLR5 iPSCs debido a que requieren la expresi&#xF3;n de los cinco factores de reprogramaci&#xF3;n ect&#xF3;picos, Oct4, Sox2, Klf4, cMYC, y Nanog, para mantener este estado m&#xE1;s ingenuo. Las c&#xE9;lulas son &#x26;quot;metaestable&#x26;quot;, y tras la retirada factor de embarazo ect&#xF3;pico vuelven a est&#xE1;ndares iPSCs humanos. Por &#xFA;ltimo, demostrar que el estado hLR5 facilita la orientaci&#xF3;n de genes, y como tal, proporciona una poderosa herramienta para la generaci&#xF3;n de l&#xED;neas recombinantes humanos de c&#xE9;lulas madre pluripotentes.

Un murino CES-como Estado Facilita La transg&#xE9;nesis y la recombinaci&#xF3;n hom&#xF3;loga en c&#xE9;lulas madre pluripotentes

X chromosome inactivation (XCI) is a process required to equalize the dosage of X-encoded genes between female and male cells. XCI is initiated very early during female embryonic development or upon differentiation of female embryonic stem (ES) cells and results in inactivation of one X chromosome in every female somatic cell. The regulation of XCI involves factors that also play a crucial role in ES cell maintenance and differentiation and the XCI process therefore provides a beautiful paradigm to study ES cell biology. In this chapter we describe the important cis and trans acting regulators of XCI and introduce the models that have been postulated to explain initiation of XCI in female cells only. We also discuss the proteins involved in the establishment of the inactive X chromosome and describe the different chromatin modifications associated with the inactivation process. Finally, we describe the potential of mouse and human ES and induced pluripotent stem (iPS) cells as model systems to study the XCI process.

X chromosome inactivation and embryonic stem cells.

In somatic cells of female placental mammals, one of the two X chromosomes is transcriptionally silenced to accomplish an equal dose of X-encoded gene products in males and females. Initiation of random X chromosome inactivation (XCI) is thought to be regulated by X-encoded activators and autosomally encoded suppressors controlling Xist. Spreading of Xist RNA leads to silencing of the X chromosome in cis. Here, we demonstrate that the dose dependent X-encoded XCI activator RNF12/RLIM acts in trans and activates Xist. We did not find evidence for RNF12-mediated regulation of XCI through Tsix or the Xist intron 1 region, which are both known to be involved in inhibition of Xist. In addition, we found that Xist intron 1, which contains a pluripotency factor binding site, is not required for suppression of Xist in undifferentiated ES cells. Analysis of female Rnf12⁻/⁻ knockout ES cells showed that RNF12 is essential for initiation of XCI and is mainly involved in the regulation of Xist. We conclude that RNF12 is an indispensable factor in up-regulation of Xist transcription, thereby leading to initiation of random XCI.

RNF12 activates Xist and is essential for X chromosome inactivation.

The use of bacterial artificial chromosomes (BACs) provides a consistent and high targeting efficiency of homologous recombination in embryonic stem (ES) cells, facilitated by long stretches of sequence homology. Here, we introduce a BAC targeting method which employs restriction fragment length polymorphisms (RFLPs) in targeted polymorphic C57BL/6/Cast/Ei F1 mouse ES cell lines to identify properly targeted ES cell clones. We demonstrate that knockout alleles can be generated either by targeting of an RFLP located in the open reading frame thereby disrupting the RFLP and ablating gene function, or by introduction of a transcription stop cassette that prematurely stops transcription of an RFLP located downstream of the stop cassette. With both methods we have generated Rnf12 heterozygous knockout ES cells, which were identified by allele specific PCR using genomic DNA or cDNA as a template. Our results indicate that this novel strategy is efficient and precise, by combining a high targeting efficiency with a convenient PCR based readout and reliable detection of correct targeting events.

Precise BAC targeting of genetically polymorphic mouse ES cells.

TGF-β members are of key importance during embryogenesis and tissue homeostasis. Smad7 is a potent antagonist of TGF-β family/Smad-mediated responses, but the regulation of Smad7 activity is not well understood. We identified the RING domain-containing E3 ligase RNF12 as a critical component of TGF-β signaling. Depletion of RNF12 dramatically reduced TGF-β/Smad-induced effects in mammalian cells, whereas ectopic expression of RNF12 strongly enhanced these responses. RNF12 specifically binds to Smad7 and induces its polyubiquitination and degradation. Smad7 levels were increased in RNF12-deficient mouse embryonic stem cells, resulting in mitigation of both BMP-mediated repression of neural induction and activin-induced anterior mesoderm formation. RNF12 also antagonized Smad7 during Nodal-dependent and BMP-dependent signaling and morphogenic events in early zebrafish embryos. The gastrulation defects induced by ectopic and depleted Smad7 were rescued in part by RNF12 gain and loss of function, respectively. These findings demonstrate that RNF12 plays a critical role in TGF-β family signaling.

RNF12 controls embryonic stem cell fate and morphogenesis in zebrafish embryos by targeting Smad7 for degradation.

Evolution of the mammalian sex chromosomes has resulted in a heterologous X and Y pair, where the Y chromosome has lost most of its genes. Hence, there is a need for X-linked gene dosage compensation between XY males and XX females. In placental mammals, this is achieved by random inactivation of one X chromosome in all female somatic cells. Upregulation of Xist transcription on the future inactive X chromosome acts against Tsix antisense transcription, and spreading of Xist RNA in cis triggers epigenetic changes leading to X-chromosome inactivation. Previously, we have shown that the X-encoded E3 ubiquitin ligase RNF12 is upregulated in differentiating mouse embryonic stem cells and activates Xist transcription and X-chromosome inactivation. Here we identify the pluripotency factor REX1 as a key target of RNF12 in the mechanism of X-chromosome inactivation. RNF12 causes ubiquitination and proteasomal degradation of REX1, and Rnf12 knockout embryonic stem cells show an increased level of REX1. Using chromatin immunoprecipitation sequencing, REX1 binding sites were detected in Xist and Tsix regulatory regions. Overexpression of REX1 in female embryonic stem cells was found to inhibit Xist transcription and X-chromosome inactivation, whereas male Rex1(+/-) embryonic stem cells showed ectopic X-chromosome inactivation. From this, we propose that RNF12 causes REX1 breakdown through dose-dependent catalysis, thereby representing an important pathway to initiate X-chromosome inactivation. Rex1 and Xist are present only in placental mammals, which points to co-evolution of these two genes and X-chromosome inactivation.

RNF12 initiates X-chromosome inactivation by targeting REX1 for degradation.

Female mammalian cells silence one of their two X chromosomes, resulting in equal expression levels of X-encoded genes in female XX and male XY cells. In mice, the X chromosomes in female cells go through sequential steps of inactivation and reactivation. Depending on the developmental time window, imprinted or random X chromosome inactivation (XCI) is initiated, and both processes lead to an inactive X chromosome that is clonally inherited. Here, we review new insights into the life cycle of XCI and provide an overview of the mechanisms regulating X inactivation and reactivation.

X chromosome inactivation in the cycle of life.

X chromosome inactivation (XCI) is a dynamically regulated developmental process with inactivation and reactivation accompanying the loss and gain of pluripotency, respectively. A functional relationship between pluripotency and lack of XCI has been suggested, whereby pluripotency transcription factors repress the master regulator of XCI, the noncoding transcript Xist, by binding to its first intron (intron 1). To test this model, we have generated intron 1 mutant embryonic stem cells (ESCs) and two independent mouse models. We found that Xist's repression in ESCs, its transcriptional upregulation upon differentiation, and its silencing upon reprogramming to pluripotency are not dependent on intron 1. Although we observed subtle effects of intron 1 deletion on the randomness of XCI and in the absence of the antisense transcript Tsix in differentiating ESCs, these have little relevance in vivo because mutant mice do not deviate from Mendelian ratios of allele transmission. Altogether, our findings demonstrate that intron 1 is dispensable for the developmental dynamics of Xist expression.

The pluripotency factor-bound intron 1 of Xist is dispensable for X chromosome inactivation and reactivation in vitro and in vivo.

Esophageal atresia with or without tracheoesophageal fistula (EA/TEF) is a relatively common birth defect often associated with additional congenital anomalies such as vertebral, anal, cardiovascular, renal and limb defects, the so-called VACTERL association. Yet, little is known about the causal genetic factors. Rare case reports of gastrointestinal anomalies in children with triple X syndrome prompted us to survey the incidence of structural and numerical changes of chromosome X in patients with EA/TEF. All available (n=269) karyotypes of our large (321) EA/TEF patient cohort were evaluated for X-chromosome anomalies. If sufficient DNA material was available, we determined genome-wide copy number profiles with SNP array and identified subtelomeric aberrations on the difficult to profile PAR1 region using telomere-multiplex ligation-dependent probe amplification. In addition, we investigated X-chromosome inactivation (XCI) patterns and mode of inheritance of detected aberrations in selected patients. Three EA/TEF patients had an additional maternally inherited X chromosome. These three female patients had normal random XCI patterns. Two male EA/TEF patients had small inherited duplications of the XY-linked SHOX (Short stature HOmeoboX-containing) locus. Patients were small for gestational age at birth (<P5) and had additional, mostly VACTERL associated, anomalies. Triple X syndrome is rarely described in patients with EA/TEF and no duplications of the SHOX gene were reported so far in these patients. As normal patterns of XCI were seen, overexpression of X-linked genes that escape XCI, such as the SHOX gene, could be pathogenic by disturbing developmental pathways.European Journal of Human Genetics advance online publication, 8 January 2014; doi:10.1038/ejhg.2013.295.

Structural and numerical changes of chromosome X in patients with esophageal atresia.

X chromosome inactivation (XCI) in female placental mammals is a vital mechanism for dosage compensation between X-linked and autosomal genes. XCI starts with activation of Xist and silencing of the negative regulator Tsix, followed by cis spreading of Xist RNA over the future inactive X chromosome (Xi). Here, we show that XCI does not require physical contact between the two X chromosomes (X-pairing) but is regulated by trans-acting diffusible factors. We found that the X-encoded trans-acting and dose-dependent XCI-activator RNF12 acts in concert with the cis-regulatory region containing Jpx, Ftx, and Xpr to activate Xist and to overcome repression by Tsix. RNF12 acts at two subsequent steps; two active copies of Rnf12 drive initiation of XCI, and one copy needs to remain active to maintain XCI toward establishment of the Xi. This two-step mechanism ensures that XCI is very robust and fine-tuned, preventing XCI of both X chromosomes.

Tahsin Stefan Barakat

Department of Reproduction and Development

Erasmus MC - University Medical Center

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Tahsin Stefan Barakat

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Reproduction and Development

Erasmus MC - University Medical Center

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Department of Reproduction and Development