PRDM16 in cardiac development

项目摘要 心脏形态发生需要复杂和精心策划的转录程序。PRDI-BF 1和RIZ 含有同源性（PR）结构域的16（PRDM 16）是保守的PRDM家族的成员，其功能为 转录调节因子和甲基转移酶在不同的细胞类型。PRDM 16在两种小鼠中表达， 和人心肌细胞（CM）。PRDM 16中的突变与先天性心脏病（CHD）相关， 强调了它对心脏发育的重要性。此外，以前的一项研究报告说，全球 Prdm 16缺陷小鼠（gKO）围产期死亡，并显示心脏异常。然而，很少有人知道 PRDM 16在CM中的特定作用，或PRDM 16缺失导致CHD的分子机制。此外，本发明还 PRDM 16的DNA结合功能和/或组蛋白甲基转移酶（HMT）活性的要求， CM尚未确定。为了检查PRDM 16在CM中的作用，我们生成了一个PRDM 16 CM特定的 敲除（cKO）小鼠模型。我们的初步数据显示Prdm 16 cKO小鼠在出生前死亡， 7，表明gKO小鼠致死的主要原因是由于CM中PRDM 16的丢失。Prdm16 cKO 小鼠表现出显著的左心室扩张，这在胚胎第15.5天（E）首次观察到。总的来说， 上述观察表明PRDM 16在发展CM中起关键作用。以确定目标 Prdm 16基因在发展中的CM中，我们对心室组织进行了RNA和ChIP测序分析， 在E13.5从Prdm 16 cKO和对照心脏分离。结果表明，基因的显着改变， 表达，69.3%的失调基因具有PRDM 16结合峰，表明PRDM 16起作用。 一个关键的作用，在转录程序的发展厘米。因此，我们的假设是PRDM 16 在CMs中发挥重要作用，表现出独特的功能活动，形成转录调控中的关键事件。 调节心脏形态发生和突变，以消除其DNA结合或HMT活性将损害 PRDM 16的特定方面起导致心脏发育缺陷的作用。研究的具体作用 PRDM 16 DNA结合和HMT活性，我们已经产生了两种新的小鼠模型， 其DNA结合或HMT活性已分别通过突变功能关键氨基酸而被消除， 酸。在“DB”突变体中，关键的“DNA结合”精氨酸（R1000）突变为谷氨酰胺（Q），导致 PRDM 16的DNA结合活性的丧失。在“HMT”突变体中，酪氨酸（Y113）和缬氨酸（V115）在“HMT”突变体中的表达降低。 PR结构域分别突变为苯丙氨酸（F）和甘氨酸（G），导致HMT丧失 活动我们的具体目标是：（1）确定PRDM 16所需的作用和机制， Prdm 16 cKO小鼠心脏发育的组织学、生理学、生物化学和分子分析; 和（2）通过以下步骤确定PRDM 16的DNA结合和/或HMT活性在CM发展中的特定作用： 分析“DB”和“HMT”突变小鼠，其中DNA结合或HMT活性已被消除。

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.