ATF4 a Novel Regulator of Cardiac Development

项目摘要 激活转录因子4（ATF 4），也称为cAMP-反应元件结合蛋白2（CREB 2）， 属于转录因子的ATF/cAMP反应元件结合（CREB）蛋白家族。作为一个好- 特征性的应激反应转录因子，ATF 4是组成性的，普遍表达在低水平。 水平，但可以在各种细胞应激条件下快速诱导。以前的研究已经表明 ATF 4在各种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，很少有人知道ATF 4及其靶基因的具体作用， 哺乳动物心脏发育为了解决这一知识差距，并确定ATF 4在以下方面的作用： 心肌细胞（CM），我们已经产生了新的Atf 4 CM特异性组成型敲除（cKO）和他莫昔芬- 诱导型CM特异性敲除（icKO）小鼠系。我们的初步研究表明Atf 4 cKO小鼠 表现出围产期死亡和心脏形态缺陷，与CM增殖减少。核糖核酸 对胚胎心室组织的seq和ChIP-seq分析揭示了一系列细胞周期的上调 已知p53下游的抑制相关基因，以及一系列不依赖于p53的 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ATF 4 在Atf 4 cKO小鼠中，ATF 4在CM中的缺失也导致p53蛋白的增加， 而不是Trp 53 mRNA水平。此外，Atf 4 cKO小鼠中的p53消融部分恢复了心室壁 在E17.5，p53靶细胞周期阻滞基因的上调得到改善，但未能挽救 出生后第1天后的致死率。相反，诱导性消融成人CM中的Atf 4对心脏无影响。 功能或左心室尺寸，表明在CM发展的特定阶段，ATF 4的不同作用。 综上所述，上述证据使我们假设ATF 4在CM中起重要作用 p53依赖性和非依赖性机制在心脏特定阶段的增殖和功能 发展因此，我们的具体目标是：1。为了确定ATF 4在心肌细胞中的作用， 通过分析Atf 4心肌细胞特异性敲除（Atf 4 cKO）小鼠， 阐明ATF 4在p53依赖性和/或 - 独立的方式;及2.为了确定胚胎和新生儿需要ATF 4的时间， 心肌细胞增殖和功能（从E7.5到P30），通过分析Atf 4诱导的心肌细胞- 特异性敲除（icKO）小鼠。我们提出的研究将有助于我们了解ATF 4的具体作用， p53，以及其他新的ATF 4靶点在体内关键发展阶段的CM中，以及确定 安全的治疗窗口的潜在应用的ATF 4和/或p53抑制剂在心脏疾病。

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.