PRDM16 in cardiac development

项目总结 心脏形态发生需要复杂和精心编排的转录程序。PRDI-BF1和RIZ 同源(PR)结构域包含16(PRDM16)是保守的PRDM家族的成员，其功能是 不同细胞类型中的转录调节因子和甲基转移酶。PRDM16在两种小鼠中均有表达 和人心肌细胞(CMS)。PRDM16突变与先天性心脏病(CHD)有关， 强调了它对心脏发育的重要性。此外，之前的一项研究报告称，全球 PRDM16基因缺陷小鼠(GKO)在围产期死亡，并表现出心脏异常。然而，人们对此知之甚少 PRDM16在CMS中的特殊作用，或PRDM16缺失导致CHD的分子机制。此外， 中国人PRDM16DNA结合功能(S)和/或组蛋白甲基转移酶活性的要求 细胞质雄性不育症尚未确定。为了研究PRDM16在CMS中的作用，我们生成了一个特定于Prdm16 CM的 基因敲除(CKO)小鼠模型。我们的初步数据显示Prdm16 CKO小鼠在出生后一天前死亡 7，提示GKO小鼠致死的主要原因是CMS中PRDM16的缺失。PRDM16 CKO 小鼠表现出戏剧性的左心室扩张，首次观察到是在胚胎发育的15.5天。总而言之， 上述观察结果表明，PRDM16在细胞质雄性不育的发生中起着关键作用。要确定目标， Prdm16基因在CMS的发生发展过程中，我们对心脏组织进行了RNA和芯片测序分析 从Prdm16 CKO分离出来，并在E13.5控制心脏。结果显示基因发生了显著的变化 表达，69.3%的异常基因有PRDM16结合峰，表明PRDM16发挥作用 在发育CMS的转录程序中起着关键作用。因此，我们的假设是PRDM16 通过展示独特的功能活动来塑造转录中的关键事件，从而在CMS中发挥重要作用 调节心脏形态发生，并突变以取消其DNA结合或HMT活性将损害 PRDM16功能的特定方面会导致心脏发育缺陷。研究……的具体作用 PRDM16DNA结合和HMT活性在CMS发育过程中，我们产生了两个新的小鼠模型，在 其DNA结合活性或HMT活性分别通过突变功能关键氨基而被取消 酸(S)。在“DB”突变体中，一个关键的“DNA结合”精氨酸(R1000)被突变为谷氨酰胺(Q)，导致 PRDM16的DNA结合活性丧失。在“HMT”突变体中，酪氨酸(Y113)和Valine(V115)在 PR结构域已分别突变为苯丙氨酸(F)和甘氨酸(G)，导致HMT丢失 活动。我们的具体目标是(1)确定PRDM16在以下方面的作用和机制 Prdm16 CKO小鼠心脏发育的组织学、生理学、生化和分子分析； 和(2)通过以下方式确定PRDM16的DNA结合和/或HMT活性在CMS形成中的特定作用 分析“DB”和“HMT”突变小鼠，在这些突变小鼠中，DNA结合或HMT活性已经取消。

PROJECT SUMMARY Cardiac morphogenesis requires complex and well-orchestrated transcriptional programs. PRDI-BF1 and RIZ homology (PR) domain-containing 16 (PRDM16) is a member of the conserved PRDM family that function as transcriptional regulators and methyltransferases in diverse cell types. PRDM16 is expressed in both murine and human cardiomyocytes (CMs). Mutations in PRDM16 are associated with congenital heart disease (CHD), highlighting its importance for cardiac development. Furthermore, a previous study has reported that global Prdm16-deficient mice (gKO) die perinatally and display heart abnormalities. However, little is known as to the specific role of PRDM16 in CMs, or molecular mechanisms by which loss of PRDM16 results in CHD. Further, requirements for the DNA-binding function(s) and/or histone methyltransferase (HMT) activity of PRDM16 in CMs are yet to be determined. To examine the role of PRDM16 in CMs, we generated a Prdm16 CM-specific knockout (cKO) mouse model. Our preliminary data revealed that Prdm16 cKO mice die before postnatal day 7, suggesting that the primary cause of lethality in gKO mice is due to loss of PRDM16 in CMs. Prdm16 cKO mice exhibited dramatic left ventricular dilation, first observed at embryonic day (E)15.5. Taken together, the foregoing observations suggest that PRDM16 plays a critical role in developing CMs. To determine the target genes of Prdm16 in developing CMs, we performed RNA- and ChIP-sequencing analysis of ventricular tissue isolated from Prdm16 cKO and control hearts at E13.5. Results demonstrated significant alterations in gene expression, with 69.3% of dysregulated genes having PRDM16 binding peaks, suggesting that PRDM16 plays a critical role in the transcriptional program of developing CMs. Accordingly, our hypothesis is that PRDM16 plays an essential role in CMs by exhibiting unique functional activities that shape key events in transcriptional regulation of cardiac morphogenesis, and mutation to abolish its DNA-binding or HMT activity will impair specific aspects of PRDM16 function to lead to cardiac developmental defects. To study the specific roles of PRDM16 DNA-binding and HMT activity in developing CMs, we have generated two novel mouse models, in which the DNA-binding or HMT activity has been abolished, respectively, by mutating functionally critical amino acid(s). In the “DB” mutant, a critical “DNA-binding” Arginine (R1000) is mutated to Glutamine (Q), resulting in the loss of DNA-binding activity of PRDM16. In the “HMT” mutant, the Tyrosine (Y113) and Valine (V115) in the PR domain have been mutated to Phenylalanine (F) and Glycine (G), respectively, resulting in loss of HMT activity. Our specific aims are to (1) determine the role and mechanisms by which PRDM16 is required in cardiac development by histological, physiological, biochemical, and molecular analyses of Prdm16 cKO mice; and (2) determine specific roles of DNA-binding and/or HMT activities of PRDM16 in developing CMs by analyzing “DB” and “HMT” mutant mice, in which either the DNA-binding or HMT activity has been abolished.