ATF4 a Novel Regulator of Cardiac Development

项目总结 激活转录因子4(ATF4)，也称为cAMP反应元件结合蛋白2(CREB2)， 属于转录因子ATF/cAMP反应元件结合(CREB)蛋白家族。作为一口井- ATF4是一种典型的应激反应转录因子，在低水平的组成性和普遍表达 但在各种细胞胁迫条件下都能迅速被诱导。此前的研究表明， ATF4在不同的生理和病理条件下，在不同的细胞类型和组织中发挥作用， 包括心脏病。然而，人们对ATF4及其靶基因在肿瘤中的具体作用知之甚少。 哺乳动物心脏发育。为了解决这一知识差距并确定ATF4在 心肌细胞(CMS)，我们已经产生了新的ATF4 CM特异性构成基因敲除(CKO)和他莫昔芬- 可诱导的CM特异性基因敲除(IcKO)小鼠系。我们的初步研究表明，ATF4 CKO小鼠 表现为围产儿死亡和心脏形态缺陷，与CM增殖减少有关。RNA- 胚胎心室组织的SEQ和CHIP-SEQ分析显示一系列细胞周期上调 已知的P53下游的停滞相关基因，以及一系列非P53非依赖性基因的下调 心脏发育/功能和/或细胞周期进展相关基因，其中大多数含有ATF4 结合区域，在ATF4 CKO小鼠中。在CMS发育过程中ATF4的缺失也导致P53蛋白的增加 表达，但不表达TrP53基因。此外，在ATF4 CKO小鼠中，P53消融部分恢复了室壁 E17.5处p53靶细胞周期停滞基因的增厚和上调，但未能挽救 相反，诱导消融成人CMS的ATF4对心脏没有影响。 功能或左心室大小，提示ATF4在CM发育的特定阶段扮演着不同的角色。 综上所述，上述证据使我们得出这样的假设，即ATF4在CM中起着重要作用 心脏特定发育阶段P53依赖和非依赖机制的增殖和功能 发展。因此，我们的具体目标是：1.确定ATF4在心肌细胞中的作用 通过分析ATF4心肌细胞特异性基因敲除(ATF4 CKO)小鼠的增殖和心脏发育，以及 阐明ATF4调节P53依赖和/或靶基因通路的机制 -独立的方式；以及2.确定胚胎和新生儿需要ATF4的时间 通过分析ATF4诱导的心肌细胞增殖和功能(从e7.5到P30) 特异性基因敲除(IcKO)小鼠。我们建议的研究将有助于我们了解ATF4和ATF4的具体作用 P53和其他新的ATF4靶点在CMS体内的关键发育阶段，以及确定一个 ATF4和/或P53抑制剂在心脏病中潜在应用的安全治疗窗口。

PROJECT SUMMARY Activating Transcription Factor 4 (ATF4), also known as cAMP-Response Element Binding Protein 2 (CREB2), belongs to the ATF/cAMP responsive element-binding (CREB) protein family of transcription factors. As a well- characterized stress-response transcription factor, ATF4 is constitutively and ubiquitously expressed at low levels but can be rapidly induced under a variety of cell-stress conditions. Previous studies have shown that ATF4 functions in diverse cell types and tissues under various physiological and pathological conditions, including cardiac diseases. However, little is known as to the specific role of ATF4 and its target genes in mammalian cardiac development. To address this gap in knowledge and to determine the role of ATF4 in cardiomyocytes (CMs), we have generated novel Atf4 CM-specific constitutive knockout (cKO) and tamoxifen- inducible CM-specific knockout (icKO) mouse lines. Our preliminary studies revealed that Atf4 cKO mice exhibited perinatal death and cardiac morphological defects, associated with reduced CM proliferation. RNA- seq and ChIP-seq analyses of embryonic ventricular tissues revealed upregulation of a series of cell cycle arrest-associated genes known to be downstream of p53, and downregulation of a series of p53-independent cardiac development/function and/or cell cycle progression associated genes, most of which harbored ATF4 binding regions, in Atf4 cKO mice. Loss of ATF4 in developing CMs also resulted in increased p53 protein levels but not Trp53 mRNA levels. Moreover, p53 ablation in Atf4 cKO mice partially restored ventricular wall thickness and ameliorated upregulation of p53 target cell cycle arrest genes at E17.5, but failed to rescue lethality beyond postnatal day 1. Conversely, inducible ablation of Atf4 in adult CMs had no effect on cardiac function or left ventricular dimension, suggesting distinct roles for ATF4 at specific stages of CM development. Taken together, the foregoing evidence leads us to the hypothesis that ATF4 plays an essential role in CM proliferation and function via p53-dependent and -independent mechanisms at specific stages of cardiac development. Accordingly, our Specific Aims are: 1. To determine the role of ATF4 in cardiomyocyte proliferation and cardiac development by analyzing Atf4 cardiomyocyte-specific knockout (Atf4 cKO) mice, and to elucidate mechanisms by which ATF4 regulates target gene pathways in a p53-dependent and/or -independent manner; and 2. To determine times at which ATF4 is required for embryonic and neonatal cardiomyocyte proliferation and function (from E7.5 to P30) by analysis of Atf4 inducible cardiomyocyte- specific knockout (icKO) mice. Our proposed studies will help us to understand the specific roles of ATF4 and p53, as well as other novel ATF4 targets in CMs at critical developing stages in vivo, as well as to determine a safe therapeutic window for the potential application of ATF4 and/or p53 inhibitors in cardiac diseases.