ultrasound-guided-transthoracic-intramyocardial-injection-in-mice

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

Boston Children's Hospital

Calcium signaling is a central regulator of cardiomyocyte growth and function. Calmodulin is a critical mediator of calcium signals. Because the amount of calmodulin within cardiomyocytes is limiting, the precise control of calmodulin expression is important for the regulation of calcium signaling. In this study, we show for the first time that calmodulin levels are regulated posttranscriptionally in heart failure. The cardiomyocyte-restricted microRNA miR-1 inhibited the translation of calmodulin-encoding mRNAs via highly conserved target sites within their 3' untranslated regions. In keeping with its effect on calmodulin expression, miR-1 downregulated calcium-calmodulin signaling through calcineurin to NFAT. miR-1 also negatively regulated the expression of Mef2a and Gata4, key transcription factors that mediate calcium-dependent changes in gene expression. Consistent with the downregulation of these hypertrophy-associated genes, miR-1 attenuated cardiomyocyte hypertrophy in cultured neonatal rat cardiomyocytes and in the intact adult heart. Our data indicate that miR-1 regulates cardiomyocyte growth responses by negatively regulating the calcium signaling components calmodulin, Mef2a, and Gata4.

MicroRNA-1 negatively regulates expression of the hypertrophy-associated calmodulin and Mef2a genes.

Recent development of methods for genome-wide identification of transcription factor binding sites by chromatin immunoprecipitation (ChIP) has led to novel insights into transcriptional regulation and greater understanding of the function of individual transcription factors. ChIP requires highly specific antibody against the transcriptional regulator of interest, and availability of suitable antibodies is a significant impediment to broader application of this approach. This limitation can be circumvented by tagging the transcriptional regulator of interest with a short bio epitope which is specifically biotinylated by the E. coli enzyme BirA. The biotinylated transcription factor can then be selectively pulled down on streptavidin beads under stringent conditions. This unit provides a detailed protocol for genome-wide location analysis of in vivo biotinylated transcription factors by streptavidin pull-down followed by high-throughput sequencing (bioChIP-seq).

Genome-wide location analysis by pull down of in vivo biotinylated transcription factors.

CompleteMOTIFs (cMOTIFs) is an integrated web tool developed to facilitate systematic discovery of overrepresented transcription factor binding motifs from high-throughput chromatin immunoprecipitation experiments. Comprehensive annotations and Boolean logic operations on multiple peak locations enable users to focus on genomic regions of interest for de novo motif discovery using tools such as MEME, Weeder and ChIPMunk. The pipeline incorporates a scanning tool for known motifs from TRANSFAC and JASPAR databases, and performs an enrichment test using local or precalculated background models that significantly improve the motif scanning result. Furthermore, using the cMOTIFs pipeline, we demonstrated that multiple transcription factors could cooperatively bind to the upstream of important stem cell differentiation regulators.

CompleteMOTIFs: DNA motif discovery platform for transcription factor binding experiments.

Identification of genomic regions that control tissue-specific gene expression is currently problematic. ChIP and high-throughput sequencing (ChIP-seq) of enhancer-associated proteins such as p300 identifies some but not all enhancers active in a tissue. Here we show that co-occupancy of a chromatin region by multiple transcription factors (TFs) identifies a distinct set of enhancers. GATA-binding protein 4 (GATA4), NK2 transcription factor-related, locus 5 (NKX2-5), T-box 5 (TBX5), serum response factor (SRF), and myocyte-enhancer factor 2A (MEF2A), here referred to as "cardiac TFs," have been hypothesized to collaborate to direct cardiac gene expression. Using a modified ChIP-seq procedure, we defined chromatin occupancy by these TFs and p300 genome wide and provided unbiased support for this hypothesis. We used this principle to show that co-occupancy of a chromatin region by multiple TFs can be used to identify cardiac enhancers. Of 13 such regions tested in transient transgenic embryos, seven (54%) drove cardiac gene expression. Among these regions were three cardiac-specific enhancers of Gata4, Srf, and swItch/sucrose nonfermentable-related, matrix-associated, actin-dependent regulator of chromatin, subfamily d, member 3 (Smarcd3), an epigenetic regulator of cardiac gene expression. Multiple cardiac TFs and p300-bound regions were associated with cardiac-enriched genes and with functional annotations related to heart development. Importantly, the large majority (1,375/1,715) of loci bound by multiple cardiac TFs did not overlap loci bound by p300. Our data identify thousands of prospective cardiac regulatory sequences and indicate that multiple TF co-occupancy of a genomic region identifies developmentally relevant enhancers that are largely distinct from p300-associated enhancers.

Co-occupancy by multiple cardiac transcription factors identifies transcriptional enhancers active in heart.

microRNAs (miRNAs) are 21-23-nucleotide non-coding RNAs. It has become more and more evident that this class of small RNAs plays critical roles in the regulation of gene expression at the post-transcriptional level. MEF2A is a member of the MEF2 (myogenic enhancer factor 2) family of transcription factors. Prior report showed that the 3'-untranslated region (3'-UTR) of the Mef2A gene mediated its repression; however, the molecular mechanism underlying this intriguing observation was unknown. Here, we report that MEF2A is repressed by miRNAs. We identify miR-155 as one of the primary miRNAs that significantly represses the expression of MEF2A. We show that knockdown of the Mef2A gene by siRNA impairs myoblast differentiation. Similarly, overexpression of miR-155 leads to the repression of endogenous MEF2A expression and the inhibition of myoblast differentiation. Most importantly, reintroduction of MEF2A in miR-155 overexpressed myoblasts was able to partially rescue the miR-155-induced myoblast differentiation defect. Our data therefore establish miR-155 as an important regulator of MEF2A expression and uncover its function in muscle gene expression and myogenic differentiation.

miR-155 inhibits expression of the MEF2A protein to repress skeletal muscle differentiation.

&#x9610;&#x91CA;&#xFF1A; &#x8868;&#x89C2;&#x9057;&#x4F20;&#x6807;&#x8BB0;&#x662F;&#x91CD;&#x8981;&#x5668;&#x5B98;&#x53D1;&#x751F;&#xFF0C;&#x4F46;&#x5728;&#x5FC3;&#x810F;&#x53D1;&#x80B2;&#x4E2D;&#x7684;&#x4F5C;&#x7528;&#x4E86;&#x89E3;&#x751A;&#x5C11;&#x3002;&#x7EC4; Polycomb &#x9547;&#x538B;&#x590D;&#x6742; 2 (PRC2) trimethylates &#x86CB;&#x767D; H3 &#x8D56;&#x6C28;&#x9178; 27&#xFF0C;&#x5728;&#x786E;&#x5B9A;&#x4E86;&#x4FC3;&#x8FDB;&#x7279;&#x5B9A;&#x7EC4;&#x7EC7;&#x7684;&#x5206;&#x5316;&#x7684;&#x6C89;&#x9ED8;&#x5F02;&#x4F4D;&#x57FA;&#x56E0;&#x7A0B;&#x5E8F;&#x7684; H3K27me3 &#x538B;&#x5236;&#x6027;&#x57FA;&#x56E0;&#x6807;&#x8BB0;&#x3002;&#x76EE;&#x6807;&#xFF1A; &#x6211;&#x4EEC;&#x7814;&#x7A76;&#x7684; PRC2 &#x4F7F;&#x7528;&#x7EC4;&#x7EC7;&#x9650;&#x5236;&#x6761;&#x4EF6;&#x706D;&#x6D3B;&#x7B56;&#x7565;&#x7684;&#x5C0F;&#x9F20;&#x5FC3;&#x810F;&#x53D1;&#x80B2;&#x4E2D;&#x7684;&#x51FD;&#x6570;&#x3002;&#x65B9;&#x6CD5;&#x548C;&#x7ED3;&#x679C;&#xFF1A; &#x706D;&#x6D3B; Ezh2 PRC2 &#x4E9A;&#x5355;&#x4F4D;&#x7531; Nkx2-5(Cre) (Ezh2(NK)) &#x9020;&#x6210;&#x81F4;&#x547D;&#x7684;&#x5148;&#x5929;&#x6027;&#x5FC3;&#x810F;&#x7578;&#x5F62;&#xFF0C;&#x5373;&#x5951;&#x7EA6;&#x5FC3;&#x808C;&#x53D1;&#x80B2;&#x4E0D;&#x5168;&#x3001; hypertrabeculation &#x548C;&#x5BA4;&#x95F4;&#x9694;&#x7F3A;&#x635F;&#x3002;&#x786E;&#x5B9A;&#x7684;&#x57FA;&#x56E0;&#x76F4;&#x63A5;&#x538B;&#x5236; EZH2 &#x5FC3;&#x5019;&#x9009;&#x4EBA;&#x548C;&#x5168;&#x57FA;&#x56E0;&#x7EC4; RNA &#x8868;&#x8FBE;&#x5206;&#x6790;&#x548C;&#x67D3;&#x8272;&#x8D28;&#x6C89;&#x6DC0;&#x5206;&#x6790; Ezh2(NK)&#x3002;&#x5176;&#x4E2D;&#x5305;&#x62EC;&#x70C8;&#x6027;&#x7684;&#x7EC6;&#x80DE;&#x5468;&#x671F;&#x6291;&#x5236;&#x5242; Ink4a/b (&#x7EC6;&#x80DE;&#x5468;&#x671F;&#x86CB;&#x767D;&#x4F9D;&#x8D56;&#x6027;&#x6FC0;&#x9176; 4 A &#x548C; B &#x7684;&#x6291;&#x5236;&#x5242;&#xFF09;&#xFF0C;&#x5176;&#x4E2D;&#x4E0A;&#x8C03;&#x662F;&#x4E0E; Ezh2(NK) &#x51CF;&#x5C11;&#x5FC3;&#x808C;&#x7EC6;&#x80DE;&#x589E;&#x6B96;&#x76F8;&#x5173;&#x8054;&#x3002;EZH2 &#x538B;&#x6291;&#x7684;&#x57FA;&#x56E0;&#x88AB;&#x8F6C;&#x5F55;&#x8C03;&#x8282;&#x57FA;&#x56E0;&#x7684; noncardiomyocyte &#x8868;&#x8FBE;&#x7A0B;&#x5E8F;&#x4F8B;&#x5982; Pax6&#x3001; Isl1 &#x548C; Six1 &#x7684;&#x4E30;&#x5BCC;&#x3002;&#x56E0;&#x4E3A; Hcn4&#xFF0C;Mlc2a &#x548C; Bmp10 &#x4E0D;&#x9002;&#x5F53;&#x5730;&#x5728;&#x5FC3;&#x5BA4; RNA &#x4E0A;&#x8C03; EZH2 &#x4E5F;&#x9002;&#x5F53;&#x7684;&#x5FC3;&#x810F;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x7684;&#x65F6;&#x7A7A;&#x76D1;&#x7BA1;&#x6240;&#x5FC5;&#x9700;&#x7684;&#x3002;PRC2 &#x4E5F;&#x9700;&#x8981;&#x7A0D;&#x540E;&#x5728;&#x5FC3;&#x810F;&#x53D1;&#x80B2;&#x4E2D;&#x5FC3;&#x808C;&#x7EC6;&#x80DE;&#x9650;&#x5236; TNT Cre &#x706D;&#x6D3B;&#x7684; PRC2 &#x4E9A;&#x7535;&#x706B;&#x5DE5;&#x54C1;&#x6240;&#x793A;&#x3002;&#x7136;&#x800C;&#xFF0C;&#x7531; TNT Cre Ezh2 &#x706D;&#x6D3B;&#x4E0D;&#x4F1A;&#x9020;&#x6210;&#x53EF;&#x80FD;&#x7684;&#x8D77;&#x56E0;&#x4E0E; Ezh1 &#x529F;&#x80FD;&#x5197;&#x4F59;&#x516C;&#x5F00;&#x8868;&#x578B;&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x7531; Nk2-5(Cre) &#x7684;&#x65E9;&#x671F; Ezh2 &#x706D;&#x6D3B;&#x9020;&#x6210;&#x5FC3;&#x808C;&#x7EC6;&#x80DE;&#x57FA;&#x56E0;&#x8868;&#x8FBE;&#x4E0E;&#x5FC3;&#x810F;&#x53D1;&#x80B2;&#x7684;&#x665A;&#x4E2D;&#x65AD;&#x3002;&#x7ED3;&#x8BBA;&#xFF1A; &#x6211;&#x4EEC;&#x7684;&#x7814;&#x7A76;&#x8868;&#x660E; EZH2 &#x4EE5;&#x524D;&#x53D8;&#x79CD;&#x7684;&#x4F5C;&#x7528;&#x5728;&#x8C03;&#x8282;&#x5FC3;&#x810F;&#x5F62;&#x6210;&#x5E76;&#x663E;&#x793A;&#x65E9;&#x5728;&#x5FC3;&#x810F;&#x53D1;&#x80B2;&#x4E2D;&#x7684;&#x540E;&#x751F;&#x666F;&#x89C2;&#x7684;&#x6444;&#x52A8;&#x5DF2;&#x6301;&#x7EED;&#x53D1;&#x80B2;&#x9636;&#x6BB5;&#x4EE5;&#x540E;&#x7684;&#x7834;&#x574F;&#x6027;&#x5F71;&#x54CD;&#x3002;

Polycomb &#x9547;&#x538B;&#x590D;&#x6742; 2 &#x8C03;&#x8282;&#x5C0F;&#x9F20;&#x5FC3;&#x810F;&#x7684;&#x6B63;&#x5E38;&#x53D1;&#x5C55;&#x3002;

Rationale: Epigenetic marks are crucial for organogenesis, but their role in heart development is poorly understood. Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27, which establishes H3K27me3 repressive epigenetic marks that promote tissue-specific differentiation by silencing ectopic gene programs. Objective: We studied the function of PRC2 in murine heart development using a tissue-restricted conditional inactivation strategy. Methods and Results: Inactivation of the PRC2 subunit Ezh2 by Nkx2-5(Cre) (Ezh2(NK)) caused lethal congenital heart malformations, namely, compact myocardial hypoplasia, hypertrabeculation, and ventricular septal defect. Candidate and genome-wide RNA expression profiling and chromatin immunoprecipitation analyses of Ezh2(NK) heart identified genes directly repressed by EZH2. Among these were the potent cell cycle inhibitors Ink4a/b (inhibitors of cyclin-dependent kinase 4 A and B), the upregulation of which was associated with decreased cardiomyocyte proliferation in Ezh2(NK). EZH2-repressed genes were enriched for transcriptional regulators of noncardiomyocyte expression programs such as Pax6, Isl1, and Six1. EZH2 was also required for proper spatiotemporal regulation of cardiac gene expression, because Hcn4, Mlc2a, and Bmp10 were inappropriately upregulated in ventricular RNA. PRC2 was also required later in heart development, as indicated by cardiomyocyte-restricted TNT-Cre inactivation of the PRC2 subunit Eed. However, Ezh2 inactivation by TNT-Cre did not cause an overt phenotype, likely because of functional redundancy with Ezh1. Thus, early Ezh2 inactivation by Nk2-5(Cre) caused later disruption of cardiomyocyte gene expression and heart development. Conclusions: Our study reveals a previously undescribed role of EZH2 in regulating heart formation and shows that perturbation of the epigenetic landscape early in cardiogenesis has sustained disruptive effects at later developmental stages.

Polycomb repressive complex 2 regulates normal development of the mouse heart.

Justification: Les marques &#xE9;pig&#xE9;n&#xE9;tiques sont cruciaux pour l&#x26;#39;organogen&#xE8;se, mais leur r&#xF4;le dans le d&#xE9;veloppement du c&#x153;ur est mal comprise. Polycomb complexe r&#xE9;pressive 2 (PRC2) trimethylates histone H3 lysine &#xE0; 27, qui &#xE9;tablit H3K27me3 r&#xE9;pressives marques &#xE9;pig&#xE9;n&#xE9;tiques qui favorisent la diff&#xE9;renciation sp&#xE9;cifique du tissu en faisant taire les programmes de g&#xE8;nes ectopiques. Objectif: Nous avons &#xE9;tudi&#xE9; la fonction de PRC2 dans le d&#xE9;veloppement du c&#x153;ur murin en utilisant une strat&#xE9;gie de tissus restreinte inactivation conditionnelle. M&#xE9;thodes et r&#xE9;sultats: inactivation de la sous unit&#xE9; PRC2 EZH2 par Nkx2-5 (Cre) (EZH2 (NK)) caus&#xE9; l&#xE9;tales malformations cardiaques cong&#xE9;nitales, &#xE0; savoir compacte du myocarde hypoplasie, hypertrabeculation, et le d&#xE9;faut du septum ventriculaire. Candidats et l&#x26;#39;ensemble du g&#xE9;nome profilage de l&#x26;#39;expression de l&#x26;#39;ARN et analyses immunopr&#xE9;cipitation de la chromatine de EZH2 g&#xE8;nes cardiaques (NK) identifi&#xE9;s directement r&#xE9;prim&#xE9;s par EZH2. Parmi ceux-ci &#xE9;taient les puissants inhibiteurs du cycle cellulaire Ink4a / b (inhibiteurs de kinases cycline-d&#xE9;pendantes 4 A et B), la r&#xE9;gulation positive de ce qui a &#xE9;t&#xE9; associ&#xE9;e &#xE0; la prolif&#xE9;ration des cardiomyocytes a diminu&#xE9; dans EZH2 (NK). EZH2-g&#xE8;nes r&#xE9;prim&#xE9;s ont &#xE9;t&#xE9; enrichies pour les r&#xE9;gulateurs de la transcription de programmes d&#x26;#39;expression noncardiomyocyte tels que Pax6, Isl1, et Six1. EZH2 a &#xE9;galement &#xE9;t&#xE9; requise par la r&#xE9;glementation spatio-temporelle ad&#xE9;quate de l&#x26;#39;expression g&#xE9;nique cardiaque, parce HCN4, Mlc2a, et BMP10 ont &#xE9;t&#xE9; mal r&#xE9;gul&#xE9;s &#xE0; la hausse dans l&#x26;#39;ARN ventriculaires. PRC2 a &#xE9;galement &#xE9;t&#xE9; n&#xE9;cessaire plus tard dans le d&#xE9;veloppement du c&#x153;ur, comme indiqu&#xE9; par des cardiomyocytes-restreint TNT-Cre inactivation de la sous-unit&#xE9; PRC2 EED. Cependant, l&#x26;#39;inactivation EZH2 par TNT-Cre ne pas provoquer un ph&#xE9;notype manifeste, probablement en raison de la redondance fonctionnelle avec Ezh1. Ainsi, au d&#xE9;but EZH2 inactivation par NK2-5 (Cre) a provoqu&#xE9; des perturbations plus tard, de l&#x26;#39;expression g&#xE9;nique des cardiomyocytes et le d&#xE9;veloppement du c&#x153;ur. Conclusions: Notre &#xE9;tude r&#xE9;v&#xE8;le un r&#xF4;le pr&#xE9;c&#xE9;demment non d&#xE9;crite de EZH2 dans la r&#xE9;gulation de la formation du c&#x153;ur et montre que la perturbation du paysage &#xE9;pig&#xE9;n&#xE9;tique au d&#xE9;but de cardiogen&#xE8;se a subi des effets perturbateurs &#xE0; des stades de d&#xE9;veloppement plus tard.

2 Polycomb Complexe r&#xE9;pressive r&#xE9;gule le d&#xE9;veloppement normal du c&#x153;ur de souris

Begr&uuml;ndung: Epigenetische Marken sind entscheidend f&uuml;r die Organogenese, aber ihre Rolle in der Entwicklung des Herzens wird oft missverstanden. Polycomb repressiven Komplex 2 (PRC2) Trimethylates Histon H3 an Lysin 27, das H3K27me3 repressive epigenetische Marken herstellt, die Gewebe-spezifischen Differenzierung f&ouml;rdern an, indem ektopische gen Programme. Ziel: Wir studierten die Funktion des PRC2 in murinen Herz-Entwicklung mit einer Gewebe-eingeschr&auml;nkte bedingter Inaktivierung-Strategie. Methoden und Ergebnisse: Inaktivierung von der PRC2-Untereinheit Ezh2 von Nkx2-5(Cre) (Ezh2(NK)) verursacht t&ouml;dliche angeborene Herzfehler, n&auml;mlich kompakt myokardialen Hypoplasie, Hypertrabeculation und Ventrikelseptumdefekt. Kandidaten- und genomweiten RNA Ausdruck Profilerstellung und Chromatin Immunopr&auml;zipitation Analysen von Ezh2(NK) Herz identifizierten Gene direkt von EZH2 unterdr&uuml;ckt. Dazu geh&ouml;rten die potente Zellzyklus-Hemmer Ink4a/b (Inhibitoren cyclin-abh&auml;ngige Kinase 4 A und B), die Hochregulation von denen mit verringerter Cardiomyocyte Proliferation im Ezh2(NK) verbunden war. EZH2 unterdr&uuml;ckten Gene wurden f&uuml;r transkriptionelle Regulatoren der Noncardiomyocyte Ausdruck Programme wie Pax6, Isl1 und Six1 bereichert. EZH2 war auch f&uuml;r die ordnungsgem&auml;&szlig;e raumzeitliche Regulierung der kardialen Genexpression, erforderlich, weil Hcn4, Mlc2a und Bmp10 herraufreguliert in ventrikul&auml;re RNA unangemessen waren. PRC2 wurde auch sp&auml;ter im Herzen Entwicklung, ben&ouml;tigt, wie durch Cardiomyocyte eingeschr&auml;nkten TNT-Cre-Inaktivierung von der PRC2-Untereinheit Eed. Ezh2 Inaktivierung von TNT-Cre waren jedoch keine offene Ph&auml;notyp, wahrscheinlich aufgrund funktionaler Redundanz mit Ezh1 hinzugeben. So verursacht fr&uuml;hen Ezh2 Inaktivierung von Nk2-5(Cre) sp&auml;ter St&ouml;rung Cardiomyocyte Genexpression und Herz-Entwicklung. Schlussfolgerungen: Unsere Studie zeigt eine bisher unbeschriebene Rolle des EZH2 bei der Regulierung von Herzen Bildung und zeigt, dass die St&ouml;rung der epigenetische Landschaft fr&uuml;h im Cardiogenesis zerst&ouml;rerische Auswirkungen auf sp&auml;ter Entwicklungsstadien zugezogen hat.

Polycomb repressiven komplexe 2 regelt die normalen Entwicklung des Herzens Maus.

Spiegazione logica: Marchi epigenetici sono cruciali per organogenesi, ma loro ruolo nello sviluppo del cuore &egrave; capita male. Polycomb repressive complesso 2 (PRC2) trimethylates istone H3 a lisina 27, che stabilisce H3K27me3 repressivo epigenetici che segna promuovere tessuto-specifica differenziazione di silenziamento programmi gene ectopica. Obiettivo: Abbiamo studiato la funzione del PRC2 nello sviluppo del cuore murino mediante una strategia di inattivazione condizionale tessuto-limitato. Metodi e risultati: inattivazione della subunit&agrave; PRC2 Ezh2 da Nkx2-5(Cre) (malformazioni cardiache congenite letali Ezh2(NK)) causato, vale a dire, ipoplasia del miocardio compatto, hypertrabeculation e difetto del setto ventricolare. Candidato e genome-wide RNA expression profiling e cromatina immunoprecipitazione analisi di Ezh2(NK) cuore identificati geni repressi direttamente da EZH2. Tra questi sono stati gli inibitori del ciclo cellulare potente Ink4a/b (inibitori delle chinasi ciclina-dipendente 4 A e B), la sovraregolazione di cui &egrave; stata associata con proliferazione del cardiomyocyte diminuito in Ezh2(NK). Geni repressi EZH2 sono stati arricchiti per regolatori trascrizionali dei programmi di espressione noncardiomyocyte come Pax6, Isl1 e Six1. EZH2 era necessaria per la corretta regolazione spatiotemporal dell&#39;espressione genica cardiaco, anche perch&eacute; Hcn4, Mlc2a e Bmp10 sono stati impropriamente sovraregolati nel RNA ventricolare. PRC2 era necessaria anche pi&ugrave; avanti nello sviluppo del cuore, come indicato dal limitato cardiomyocyte TNT-Cre inattivazione della subunit&agrave; PRC2 Eed. Tuttavia, Ezh2 inattivazione di TNT-Cre non ha causato un fenotipo palese, probabilmente a causa di ridondanza funzionale con Ezh1. Cos&igrave;, primi inattivazione di Ezh2 di Nk2-5(Cre) caus&ograve; danni successivi del cardiomyocyte espressione genica e lo sviluppo del cuore. Conclusioni: Il nostro studio rivela un ruolo precedentemente nuovo di EZH2 nella regolazione della formazione del cuore e mostra che perturbazione del paesaggio epigenetico all&#39;inizio del cardiogenesis ha subito effetti dirompenti a fasi di sviluppo pi&ugrave; tardi.

Polycomb repressive complex 2 regola il normale sviluppo del cuore del mouse.

&#x6839;&#x62E0;&#xFF1A;&#x30A8;&#x30D4;&#x30B8;&#x30A7;&#x30CD;&#x30C6;&#x30A3;&#x30C3;&#x30AF;&#x30DE;&#x30FC;&#x30AF;&#x304C;&#x5668;&#x5B98;&#x5F62;&#x6210;&#x306B;&#x91CD;&#x8981;&#x3067;&#x3042;&#x308B;&#x304C;&#x3001;&#x5FC3;&#x81D3;&#x306E;&#x958B;&#x767A;&#x306B;&#x304A;&#x3051;&#x308B;&#x5F7C;&#x3089;&#x306E;&#x5F79;&#x5272;&#x306F;&#x3042;&#x307E;&#x308A;&#x7406;&#x89E3;&#x3055;&#x308C;&#x3066;&#x3044;&#x306A;&#x3044;&#x3002;&#x30DD;&#x30EA;&#x30B3;&#x30FC;&#x30E0;&#x6291;&#x5727;&#x7684;&#x306A;&#x8907;&#x96D1;&#x306A;2&#xFF08;PRC2&#xFF09;&#x306F;&#x7570;&#x6240;&#x6027;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30E0;&#x3092;&#x30B5;&#x30A4;&#x30EC;&#x30F3;&#x30B7;&#x30F3;&#x30B0;&#x306B;&#x3088;&#x3063;&#x3066;&#x7D44;&#x7E54;&#x7279;&#x7570;&#x7684;&#x5206;&#x5316;&#x3092;&#x4FC3;&#x9032;&#x3059;&#x308B;H3K27me3&#x6291;&#x5727;&#x7684;&#x306A;&#x30A8;&#x30D4;&#x30B8;&#x30A7;&#x30CD;&#x30C6;&#x30A3;&#x30C3;&#x30AF;&#x30DE;&#x30FC;&#x30AF;&#x3092;&#x78BA;&#x7ACB;&#x3057;&#x3001;&#x30EA;&#x30B8;&#x30F3;27&#x3067;&#x30D2;&#x30B9;&#x30C8;&#x30F3;H3&#x3092;&#x3001;trimethylates&#x3002;&#x76EE;&#x7684;&#xFF1A;&#x6211;&#x3005;&#x306F;&#x7D44;&#x7E54;&#x306E;&#x5236;&#x9650;&#x6761;&#x4EF6;&#x306E;&#x4E0D;&#x6D3B;&#x5316;&#x6226;&#x7565;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x30DE;&#x30A6;&#x30B9;&#x306E;&#x5FC3;&#x81D3;&#x306E;&#x958B;&#x767A;&#x306B;PRC2&#x306E;&#x6A5F;&#x80FD;&#x3092;&#x8ABF;&#x3079;&#x305F;&#x3002;&#x65B9;&#x6CD5;&#x3068;&#x7D50;&#x679C;&#xFF1A;NKX2-5&#xFF08;CRE&#xFF09;&#xFF08;EZH2&#xFF08;NK&#xFF09;&#xFF09;&#x306B;&#x3088;&#x308B;PRC2&#x30E6;&#x30CB;&#x30C3;&#x30C8;EZH2&#x306E;&#x4E0D;&#x6D3B;&#x6027;&#x5316;&#x3001;&#x3059;&#x306A;&#x308F;&#x3061;&#x81F4;&#x547D;&#x7684;&#x306A;&#x5148;&#x5929;&#x6027;&#x5FC3;&#x5947;&#x5F62;&#x3001;&#x30B3;&#x30F3;&#x30D1;&#x30AF;&#x30C8;&#x306A;&#x5FC3;&#x7B4B;&#x5F62;&#x6210;&#x4E0D;&#x5168;&#x3001;hypertrabeculation&#x3068;&#x3001;&#x5FC3;&#x5BA4;&#x4E2D;&#x9694;&#x6B20;&#x640D;&#x304C;&#x767A;&#x751F;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x5019;&#x88DC;&#x8005;&#x3068;EZH2&#x306E;&#x30B2;&#x30CE;&#x30E0;&#x30EF;&#x30A4;&#x30C9;&#x306A;RNA&#x767A;&#x73FE;&#x30D7;&#x30ED;&#x30D5;&#x30A1;&#x30A4;&#x30EA;&#x30F3;&#x30B0;&#x3068;&#x30AF;&#x30ED;&#x30DE;&#x30C1;&#x30F3;&#x514D;&#x75AB;&#x6C88;&#x964D;&#x89E3;&#x6790;&#xFF08;NK&#xFF09;&#x5FC3;&#x81D3;&#x306B;&#x76F4;&#x63A5;EZH2&#x306B;&#x3088;&#x3063;&#x3066;&#x6291;&#x5236;&#x907A;&#x4F1D;&#x5B50;&#x3092;&#x540C;&#x5B9A;&#x3057;&#x305F;&#x3002;&#x4E2D;&#x3067;&#x3082;&#x5F37;&#x529B;&#x306A;&#x7D30;&#x80DE;&#x5468;&#x671F;&#x963B;&#x5BB3;&#x5264;INK4A / B&#xFF08;&#x30B5;&#x30A4;&#x30AF;&#x30EA;&#x30F3;&#x4F9D;&#x5B58;&#x6027;&#x30AD;&#x30CA;&#x30FC;&#x30BC;&#x306E;&#x963B;&#x5BB3;&#x5264;4&#x304A;&#x3088;&#x3073;B&#xFF09;&#x3001;EZH2&#xFF08;NK&#xFF09;&#x306E;&#x6E1B;&#x5C11;&#x5FC3;&#x7B4B;&#x7D30;&#x80DE;&#x5897;&#x6B96;&#x306B;&#x95A2;&#x9023;&#x4ED8;&#x3051;&#x3089;&#x308C;&#x3066;&#x3044;&#x305F;&#x305D;&#x306E;&#x3046;&#x3061;&#x306E;&#x4E0A;&#x65B9;&#x3067;&#x3042;&#x3063;&#x305F;&#x3002; EZH2&#xB7;&#x6291;&#x5727;&#x907A;&#x4F1D;&#x5B50;&#x306F;&#x3001;Pax6&#x306E;&#x3001;ISL1&#x3001;&#x3068;Six1&#x3068;&#x3057;&#x3066;noncardiomyocyte&#x767A;&#x73FE;&#x30D7;&#x30ED;&#x30B0;&#x30E9;&#x30E0;&#x306E;&#x8EE2;&#x5199;&#x8ABF;&#x7BC0;&#x306E;&#x305F;&#x3081;&#x306B;&#x6FC3;&#x7E2E;&#x3057;&#x305F;&#x3002; HCN4&#x3001;Mlc2a&#x3001;&#x3068;Bmp10&#x304C;&#x4E0D;&#x9069;&#x5207;&#x5FC3;&#x5BA4;RNA&#x306B;&#x30A2;&#x30C3;&#x30D7;&#x30EC;&#x30AE;&#x30E5;&#x30EC;&#x30FC;&#x30C8;&#x3055;&#x308C;&#x305F;&#x306E;&#x3067;&#x3001;EZH2&#x306F;&#x307E;&#x305F;&#x3001;&#x5FC3;&#x81D3;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x306E;&#x9069;&#x5207;&#x306A;&#x6642;&#x7A7A;&#x9593;&#x7684;&#x8ABF;&#x7BC0;&#x306E;&#x305F;&#x3081;&#x306B;&#x5FC5;&#x8981;&#x3067;&#x3042;&#x3063;&#x305F;&#x3002; EED PRC2&#x30B5;&#x30D6;&#x30E6;&#x30CB;&#x30C3;&#x30C8;&#x306E;&#x5FC3;&#x7B4B;&#x7D30;&#x80DE;&#x304C;&#x5236;&#x9650;&#x3055;TNT-Cre&#x30EA;&#x30B3;&#x30F3;&#x30D3;&#x30CA;&#x30FC;&#x30BC;&#x306E;&#x6D3B;&#x6027;&#x5316;&#x306B;&#x3088;&#x3063;&#x3066;&#x793A;&#x3055;&#x308C;&#x308B;&#x3088;&#x3046;&#x306B;PRC2&#x306F;&#x307E;&#x305F;&#x3001;&#x5F8C;&#x306B;&#x5FC3;&#x81D3;&#x306E;&#x958B;&#x767A;&#x306B;&#x5FC5;&#x8981;&#x3068;&#x3055;&#x308C;&#x305F;&#x3002;&#x3057;&#x304B;&#x3057;&#x3001;TNT-Cre&#x30EA;&#x30B3;&#x30F3;&#x30D3;&#x30CA;&#x30FC;&#x30BC;&#x306B;&#x3088;&#x3063;&#x3066;EZH2&#x4E0D;&#x6D3B;&#x6027;&#x5316;&#x306E;&#x305F;&#x3081;Ezh1&#x3068;&#x6A5F;&#x80FD;&#x5197;&#x9577;&#x6027;&#x306E;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x660E;&#x767D;&#x306A;&#x8868;&#x73FE;&#x578B;&#x3092;&#x5F15;&#x304D;&#x8D77;&#x3053;&#x3059;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x305B;&#x3093;&#x3067;&#x3057;&#x305F;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;NK2-5&#xFF08;CRE&#xFF09;&#x306B;&#x3088;&#x308B;&#x65E9;&#x671F;EZH2&#x4E0D;&#x6D3B;&#x5316;&#x5F8C;&#x306B;&#x5FC3;&#x7B4B;&#x7D30;&#x80DE;&#x306E;&#x907A;&#x4F1D;&#x5B50;&#x767A;&#x73FE;&#x3068;&#x5FC3;&#x81D3;&#x306E;&#x958B;&#x767A;&#x306E;&#x4E2D;&#x65AD;&#x3092;&#x5F15;&#x304D;&#x8D77;&#x3053;&#x3057;&#x305F;&#x3002;&#x7D50;&#x8AD6;&#xFF1A;&#x6211;&#x3005;&#x306E;&#x7814;&#x7A76;&#x306F;&#x3001;&#x5FC3;&#x81D3;&#x5F62;&#x6210;&#x306E;&#x8ABF;&#x7BC0;&#x306B;&#x304A;&#x3044;&#x3066;EZH2&#x306E;&#x4EE5;&#x524D;&#x306B;&#x672A;&#x8A18;&#x8F09;&#x306E;&#x5F79;&#x5272;&#x3092;&#x660E;&#x3089;&#x304B;&#x306B;&#x3057;&#x3001;&#x5FC3;&#x81D3;&#x767A;&#x751F;&#x306E;&#x521D;&#x671F;&#x306E;&#x30A8;&#x30D4;&#x30B8;&#x30A7;&#x30CD;&#x30C6;&#x30A3;&#x30C3;&#x30AF;&#x306A;&#x98A8;&#x666F;&#x3001;&#x305D;&#x306E;&#x6442;&#x52D5;&#x306F;&#x5F8C;&#x3067;&#x767A;&#x9054;&#x6BB5;&#x968E;&#x3067;&#x7834;&#x58CA;&#x7684;&#x306A;&#x52B9;&#x679C;&#x3092;&#x6301;&#x7D9A;&#x3057;&#x3066;&#x3044;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;

&#x30DD;&#x30EA;&#x30B3;&#x30FC;&#x30E0;&#x6291;&#x5727;&#x7684;&#x306A;&#x8907;&#x5408;&#x4F53;2&#x306F;&#x3001;&#x30DE;&#x30A6;&#x30B9;&#x5FC3;&#x81D3;&#x306E;&#x6B63;&#x5E38;&#x306A;&#x767A;&#x9054;&#x3092;&#x8ABF;&#x7BC0;&#x3059;&#x308B;

&#xC124;&#xBA85;: Epigenetic &#xB9C8;&#xD06C; organogenesis&#xC5D0; &#xB300; &#xD55C; &#xB9E4;&#xC6B0; &#xC911;&#xC694; &#xD558;&#xC9C0;&#xB9CC; &#xC2EC;&#xC7A5; &#xBC1C;&#xB2EC;&#xC5D0; &#xC788;&#xB294; &#xADF8;&#xB4E4;&#xC758; &#xC5ED;&#xD560;&#xC740; &#xC81C;&#xB300;&#xB85C; &#xC774;&#xD574;. Polycomb &#xC5B5;&#xC555; &#xC801;&#xC778; &#xBCF5;&#xC7A1; &#xD55C; 2 (PRC2) trimethylates &#xD788;&#xC2A4;&#xD1A4; H3 lysine 27 &#xC77C;&#xC5D0; H3K27me3 &#xC5B5;&#xC555; &#xC801;&#xC778; epigenetic &#xD45C;&#xC2DC;&#xB97C; &#xC124;&#xC815; &#xD558;&#xB294; &#xC18C;&#xC131; &#xC9C4; &#xD504;&#xB85C;&#xADF8;&#xB7A8; &#xC785;&#xC744; &#xC870;&#xC9C1; &#xAD00;&#xB828; &#xCC28;&#xBCC4;&#xD654; &#xD64D;&#xBCF4;. &#xBAA9;&#xD45C;: &#xC6B0;&#xB9AC; murine &#xC2EC;&#xC7A5; &#xAC1C;&#xBC1C; &#xC870;&#xC9C1; &#xC81C;&#xD55C; &#xC870;&#xAC74;&#xBD80; &#xBE44;&#xD65C;&#xC131;&#xD654; &#xC804;&#xB7B5;&#xC744; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC; PRC2 &#xD568;&#xC218;&#xB97C; &#xACF5;&#xBD80; &#xD588;&#xB2E4;. &#xBC29;&#xBC95; &#xBC0F; &#xACB0;&#xACFC;: PRC2 &#xC18C; &#xB2E8;&#xC704; Ezh2 Nkx2-5(Cre) (Ezh2(NK)) &#xC778; &#xD55C; &#xCE58;&#xBA85;&#xC801;&#xC778; &#xC120; &#xCC9C; &#xC131; &#xC2EC;&#xC7A5; &#xAE30;&#xD615;, &#xC989;, &#xC18C;&#xD615; &#xC2EC;&#xADFC; hypoplasia, hypertrabeculation, &#xBC0F; &#xC2EC; &#xC2E4; septal &#xACB0;&#xD568; &#xC5EC;&#xC758; &#xBE44;&#xD65C;&#xC131;&#xD654;. &#xD6C4;&#xBCF4; &#xBC0F; &#xAC8C;&#xB188; &#xC804;&#xCCB4; RNA &#xC2DD; &#xD504;&#xB85C; &#xD30C;&#xC77C;&#xB9C1; &#xD558; &#xACE0; chromatin immunoprecipitation &#xBD84;&#xC11D; ezh2(nk)&#xC758; &#xB9C8;&#xC74C;&#xC744; &#xC9C1;&#xC811; ezh2&#xC5D0; &#xC758;&#xD574; &#xC5B5;&#xC555; &#xD558;&#xB294; &#xD655;&#xC778; &#xB41C; &#xC720;&#xC804;&#xC790;. &#xC774;&#xB7EC;&#xD55C; &#xAC00;&#xC6B4;&#xB370;&#xC758; upregulation &#xAC10;&#xC18C; cardiomyocyte &#xD655;&#xC0B0; ezh2(nk)&#xC640; &#xC5F0;&#xACB0; &#xB41C; &#xAC15;&#xB825;&#xD55C; &#xC138;&#xD3EC; &#xC8FC;&#xAE30; &#xC5B5;&#xC81C;&#xC81C; Ink4a/b (&#xF06C; &#xC885;&#xC18D; &#xD0A4; &#xB2C8; &#xC544; &#xC81C; 4 A&#xC640; B&#xC758; &#xC800; &#xD574;&#xC81C;) &#xD588;&#xB2E4;. EZH2 &#xC5B5;&#xC555; &#xC720;&#xC804;&#xC790; Pax6, Isl1, Six1 &#xB4F1; noncardiomyocyte &#xC2DD; &#xD504;&#xB85C;&#xADF8;&#xB7A8;&#xC758; transcriptional &#xB808; &#xADE4; &#xB808;&#xC774; &#xD130;&#xC5D0; &#xB300; &#xD55C; &#xD48D;&#xBD80;&#xD55C; &#xD588;&#xB2E4;. Ezh2&#xB294; Hcn4, Mlc2a, &#xBC0F; Bmp10 upregulated &#xC2EC; &#xC2E4; RNA&#xC5D0; &#xBD80;&#xC801;&#xC808; &#xD558; &#xAC8C; &#xD588;&#xB2E4; &#xB54C;&#xBB38;&#xC5D0; &#xB610;&#xD55C; &#xC2EC;&#xC7A5; &#xC720;&#xC804;&#xC790; &#xD45C;&#xD604;&#xC758; &#xC801;&#xC808; &#xD55C; spatiotemporal &#xADDC;&#xC81C;&#xC5D0; &#xB300; &#xD55C; &#xD544;&#xC694; &#xD588;&#xC2B5;&#xB2C8;&#xB2E4;. PRC2 &#xC18C; &#xB2E8;&#xC704; Eed&#xC758; TNT Cre &#xBE44;&#xD65C;&#xC131;&#xD654; cardiomyocyte &#xC81C;&#xD55C; &#xD558; &#xC5EC; &#xD45C;&#xC2DC; &#xB41C; &#xB300;&#xB85C; PRC2 &#xC2EC;&#xC7A5; &#xAC1C;&#xBC1C; &#xB4B7;&#xBD80;&#xBD84;&#xC5D0;&#xC11C;&#xB3C4; &#xD544;&#xC694; &#xD588;&#xB2E4;. &#xADF8;&#xB7EC;&#xB098;, TNT Cre&#xC5D0; &#xC758;&#xD574; Ezh2 &#xBE44;&#xD65C;&#xC131;&#xD654; &#xBA85;&#xBC31;&#xD55C; &#xD615;, ezh1&#xC640; &#xAE30;&#xB2A5; &#xC911;&#xBCF5; &#xB54C;&#xBB38;&#xC5D0; &#xAC00;&#xB2A5;&#xC131;&#xC774; &#xBC1C;&#xC0DD; &#xD558;&#xC9C0; &#xC54A;&#xC558;&#xB2E4;. &#xB530;&#xB77C;&#xC11C;, nk2-5(cre)&#xC5D0; &#xC758;&#xD574; &#xCD08;&#xAE30; Ezh2 &#xBE44;&#xD65C;&#xC131;&#xD654; cardiomyocyte &#xC720;&#xC804;&#xC790; &#xBC1C;&#xD604; &#xBC0F; &#xC2EC;&#xC7A5; &#xAC1C;&#xBC1C; &#xC774;&#xD6C4; &#xC911;&#xB2E8;&#xC744; &#xBC1C;&#xC0DD;&#xD569;&#xB2C8;&#xB2E4;. &#xACB0;&#xB860;: &#xC6B0;&#xB9AC;&#xC758; &#xC5F0;&#xAD6C; &#xC2EC;&#xC7A5; &#xD615;&#xC131; &#xC870;&#xC808;&#xC5D0; ezh2&#xC758; &#xC774;&#xC804;&#xC5D0; undescribed &#xC5ED;&#xD560;&#xC744; &#xACC4;&#xC2DC; &#xD558; &#xACE0; cardiogenesis&#xC5D0; &#xC77C;&#xCC0D; epigenetic &#xD504;&#xB9AC;&#xC758; &#xC12D; &#xB3D9; &#xBC1C;&#xB2EC; &#xB2E8;&#xACC4; &#xB098;&#xC911;&#xC5D0; &#xD30C;&#xAD34;&#xC801;&#xC778; &#xD6A8;&#xACFC; &#xC9C0;&#xC18D; &#xD558;&#xACE0;&#xC788;&#xB2E4; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;.

Polycomb &#xC5B5;&#xC555; &#xC801;&#xC778; &#xBCF5;&#xC7A1; &#xD55C; 2 &#xB9C8;&#xC6B0;&#xC2A4; &#xC2EC;&#xC7A5;&#xC758; &#xC815;&#xC0C1;&#xC801;&#xC778; &#xBC1C;&#xC804;&#xC744; &#xADDC;&#xC81C; &#xD55C;&#xB2E4;.

Justificaci&#xF3;n: Las marcas epigen&#xE9;ticas son cruciales para la organog&#xE9;nesis, pero su papel en el desarrollo del coraz&#xF3;n es dif&#xED;cil de entender. Complejo Polycomb represivas 2 (PRC2) trimethylates histona H3 en la lisina 27, que establece H3K27me3 marcas epigen&#xE9;ticas represivas que promueven la diferenciaci&#xF3;n espec&#xED;fica de tejido por el silenciamiento de genes programas ect&#xF3;picos. Objetivo: Se estudi&#xF3; la funci&#xF3;n de PRC2 en el desarrollo del coraz&#xF3;n de rat&#xF3;n utilizando una estrategia de tejidos restringido inactivaci&#xF3;n condicional. M&#xE9;todos y resultados: la inactivaci&#xF3;n de la subunidad PRC2 EZH2 por Nkx2-5 (Cre) (EZH2 (NK)) caus&#xF3; malformaciones cardiacas cong&#xE9;nitas letales, es decir, compacta hipoplasia del miocardio, hipertrabeculaci&#xF3;n, y el defecto septal ventricular. Candidatos y en todo el genoma de perfiles de expresi&#xF3;n de RNA y an&#xE1;lisis de inmunoprecipitaci&#xF3;n de cromatina de EZH2 (NK), los genes del coraz&#xF3;n identificados directamente reprimida por EZH2. Entre ellos, los inhibidores potentes del ciclo celular Ink4A / b (inhibidores de la quinasa dependiente de ciclina 4 A y B), la regulaci&#xF3;n al alza de los cuales se asoci&#xF3; con disminuci&#xF3;n de la proliferaci&#xF3;n de los cardiomiocitos en EZH2 (NK). EZH2 reprimidos los genes fueron enriquecidos por los reguladores de la transcripci&#xF3;n de los programas de expresi&#xF3;n noncardiomyocyte como Pax6, ISL1 y Six1. EZH2 se requiere tambi&#xE9;n para una adecuada regulaci&#xF3;n espacio-temporal de la expresi&#xF3;n de genes cardiacos, porque HCN4, MLC2a y Bmp10 se upregulated indebidamente en el ARN del ventr&#xED;culo. PRC2 se requiere tambi&#xE9;n m&#xE1;s adelante en el desarrollo del coraz&#xF3;n, como se indica por cardiomiocito restringida en TNT-Cre inactivaci&#xF3;n de la subunidad PRC2 EED. Sin embargo, EZH2 inactivaci&#xF3;n por TNT-Cre no caus&#xF3; un fenotipo manifiesto, probablemente a causa de la redundancia funcional con Ezh1. As&#xED;, a principios EZH2 inactivaci&#xF3;n por NK2-5 (Cre) caus&#xF3; m&#xE1;s trastornos de la expresi&#xF3;n g&#xE9;nica y el desarrollo de los cardiomiocitos del coraz&#xF3;n. Conclusiones: Nuestro estudio pone de manifiesto un papel no descrita con anterioridad en la regulaci&#xF3;n de la EZH2 la formaci&#xF3;n del coraz&#xF3;n y muestra que la perturbaci&#xF3;n del paisaje epigen&#xE9;tico a principios de cardiog&#xE9;nesis ha sufrido los efectos perturbadores en las etapas posteriores del desarrollo.

2 Polycomb complejo represivo regula el desarrollo normal del coraz&#xF3;n del rat&#xF3;n

Polycomb-repressive complex 2 (PRC2) promotes tissue-specific differentiation by depositing trimethylated histone H3 Lys 27 (H3K27me3) epigenetic marks to silence ectopic gene expression programs. Here, we show that EZH2, the catalytic subunit of PRC2, is required for cardiac morphogenesis. Both in vitro and in fetal hearts, EZH2 interacted with cardiac transcription factor GATA4 and directly methylated it at Lys 299. PRC2 methylation of GATA4 attenuated its transcriptional activity by reducing its interaction with and acetylation by p300. Our results reveal a new mechanism of PRC2-mediated transcriptional repression in which PRC2 methylates a transcription factor to inhibit its transcriptional activity.

PRC2 directly methylates GATA4 and represses its transcriptional activity.

Ms1/STARS is a novel muscle-specific actin-binding protein that specifically modulates the myocardin-related transcription factor (MRTF)-serum response factor (SRF) regulatory axis within striated muscle. This ms1/STARS-dependent regulatory axis is of central importance within the cardiac gene regulatory network and has been implicated in cardiac development and postnatal cardiac function/homeostasis. The dysregulation of ms1/STARS is associated with and causative of pathological cardiac phenotypes, including cardiac hypertrophy and cardiomyopathy. In order to gain an understanding of the mechanisms governing ms1/STARS expression in the heart, we have coupled a comparative genomic in silico analysis with reporter, gain-of-function, and loss-of-function approaches. Through this integrated analysis, we have identified three evolutionarily conserved regions (ECRs), α, SINA, and DINA, that act as cis-regulatory modules and confer differential cardiac cell-specific activity. Two of these ECRs, α and DINA, displayed distinct regulatory sensitivity to the core cardiac transcription factor GATA4. Overall, our results demonstrate that within embryonic, neonatal, and adult hearts, GATA4 represses ms1/STARS expression with the pathologically associated depletion of GATA4 (type 1/type 2 diabetic models), resulting in ms1/STARS upregulation. This GATA4-dependent repression of ms1/STARS expression has major implications for MRTF-SRF signaling in the context of cardiac development and disease.

Cardiac expression of ms1/STARS, a novel gene involved in cardiac development and disease, is regulated by GATA4.

Transcription factors regulate formation and function of the heart, and perturbation of transcription factor expression and regulation disrupts normal heart structure and function. Multiple mechanisms regulate the level and locus-specific activity of transcription factors, including transcription, translation, subcellular localization, posttranslational modifications, and context-dependent interactions with other transcription factors, chromatin remodeling enzymes, and epigenetic regulators. The zinc finger transcription factor GATA4 is among the best-studied cardiac transcriptional factors. This review focuses on molecular mechanisms that regulate GATA4 transcriptional activity in the cardiovascular system, providing a framework to investigate and understand the molecular regulation of cardiac gene transcription by other transcription factors.

Regulation of GATA4 transcriptional activity in cardiovascular development and disease.

Mature cardiomyocytes recall their progenitor experience via polycomb repressive complex 2.

Transcriptional profiling is a useful strategy to study development and disease. Approaches to isolate RNA from specific cell types, or from specific cellular compartments, would extend the power of this strategy. Previous work has shown that isolation of genetically tagged ribosomes (translating ribosome affinity purification; TRAP) is an effective means to isolate ribosome-bound RNA selectively from transgene-expressing cells. However, widespread application of this technology has been limited by available transgenic mouse lines. Here we characterize a TRAP allele (Rosa26(fsTRAP)) that makes this approach more widely accessible. We show that endothelium-specific activation of Rosa26(fsTRAP) identifies endothelial cell-enriched transcripts, and that cardiomyocyte-restricted TRAP is a useful means to identify genes that are differentially expressed in cardiomyocytes in a disease model. Furthermore, we show that TRAP is an effective means for studying translational regulation, and that several nuclear-encoded mitochondrial genes are under strong translational control. Our analysis of ribosome-bound transcripts also shows that a subset of long intergenic noncoding RNAs are weakly ribosome-bound, but that the majority of noncoding RNAs, including most long intergenic noncoding RNAs, are ribosome-bound to the same extent as coding transcripts. Together, these data show that the TRAP strategy and the Rosa26(fsTRAP) allele will be useful tools to probe cell type-specific transcriptomes, study translational regulation, and probe ribosome binding of noncoding RNAs.

Interrogating translational efficiency and lineage-specific transcriptomes using ribosome affinity purification.

Transcriptional networks orchestrate complex developmental processes, and such networks are commonly instigated by master regulators for development. By now, considerable progress has been made in elucidating GATA factor-dependent genetic networks that control red blood cell development. Here we reported that GATA-1 and GATA-2 co-regulated the expression of two microRNA genes, microRNA-27a and microRNA-24, with critical roles in regulating erythroid differentiation. In general, GATA-2 occupied the miR-27a≈24 promoter and repressed their transcription in immature erythroid progenitor cells. As erythropoiesis proceeded, GATA-1 directly activated miR-27a≈24 transcription, and this involved a GATA-1-mediated displacement of GATA-2 from chromatin, a process termed 'GATA switch'. Furthermore, the mature miR-27a and miR-24 cooperatively inhibited GATA-2 translation and favoured the occupancy switch from GATA-2 to GATA-1, thus completing a positive feedback loop to promote erythroid maturation. In line with the essential role of GATA factors, ectopic expression of miR-27a or miR-24 promoted erythropoiesis in human primary CD34+ haematopoietic progenitor cells and mice, whereas attenuated miR-27 or miR-24 level led to impaired erythroid phenotypes in haematopoietic progenitor cells and zebrafish. Taken together, these data integrated micro RNA expression and function into GATA factor coordinated networks and provided mechanistic insight into a regulatory circuit that comprised GATA1/2 switch and miR-27a/24 in erythropoiesis.

A regulatory circuit comprising GATA1/2 switch and microRNA-27a/24 promotes erythropoiesis.

Engraftment of IL-6 deficient donor into wild-type recipient could significantly improve allograft survival through T cell lineage particularly regulatory T cells (Tregs) in non-sensitized transplant host. However, its effect on innate immune responses remains uncertain. Our data revealed that donor IL-6 deficiency significantly increased infiltration of two subsets of MDSCs (CD11b+Gr1+myeloid-derived suppressor cells), CD11b+Gr1(-low) and CD11b+Gr1(-int) with strong immunosuppression activity in the transplanted graft. It resulted in a dramatic increase of CD11b+Gr1(-low) frequency and a significant decrease of the frequency of CD11b+Gr1(-high) and CD4-CD8-NK1.1+ cells in the recipient's spleen. Unexpectedly, donor IL-6 deficiency could not significantly reduce macrophage frequency irrespective of in the host's spleen or graft. Taken together, suppression of innate immune effector cells and enhanced activity of regulatory MDSCs contributed to tolerance induction by blockade of IL-6 signaling pathway. The unveiled novel mechanism of targeting IL-6 might shed light on clinical therapeutic application in preventing accelerated allograft rejection for those pre-sensitized transplant recipients.

Alteration of innate immunity by donor IL-6 deficiency in a presensitized heart transplant model.

Study of monogenic mitochondrial cardiomyopathies may yield insights into mitochondrial roles in cardiac development and disease. Here, we combined patient-derived and genetically engineered induced pluripotent stem cells (iPSCs) with tissue engineering to elucidate the pathophysiology underlying the cardiomyopathy of Barth syndrome (BTHS), a mitochondrial disorder caused by mutation of the gene encoding tafazzin (TAZ). Using BTHS iPSC-derived cardiomyocytes (iPSC-CMs), we defined metabolic, structural and functional abnormalities associated with TAZ mutation. BTHS iPSC-CMs assembled sparse and irregular sarcomeres, and engineered BTHS 'heart-on-chip' tissues contracted weakly. Gene replacement and genome editing demonstrated that TAZ mutation is necessary and sufficient for these phenotypes. Sarcomere assembly and myocardial contraction abnormalities occurred in the context of normal whole-cell ATP levels. Excess levels of reactive oxygen species mechanistically linked TAZ mutation to impaired cardiomyocyte function. Our study provides new insights into the pathogenesis of Barth syndrome, suggests new treatment strategies and advances iPSC-based in vitro modeling of cardiomyopathy.

Aibin He

Department of Cardiology

Boston Children's Hospital

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Aibin He

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Boston Children's Hospital

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Department of Cardiology