Dissecting Regulatory Mechanisms of Cardiac Regeneration Enhancers

项目摘要/摘要 成年哺乳动物在受伤后再生心脏组织的能力有限，例如心肌 脑梗塞。心肌梗死后，多达十亿或更多的心肌细胞死亡，并被 导致心力衰竭和猝死的疤痕组织。相比之下，成年斑马鱼的再生能力非常强 受伤的心脏没有残留的疤痕。尽管斑马鱼和哺乳动物有相同的同源基因， 心脏再生，它们对心脏损伤的转录反应是不同的。然而，这些机制 控制心脏再生过程中的基因表达仍然知之甚少。 我的研究旨在阐明损伤反应基因表达是如何调节的，以促进心脏再生。 我的实验室鉴定出心脏瘦素b连锁再生增强子(Clen)，它能激活基因表达 在心脏损伤时。使用体内转基因检测，我发现需要多个激活元件来 损伤依赖的心脏增强剂活性。令人惊讶的是，我还发现克伦有压抑的成分 这是防止未受损伤心脏中的增强子激活所必需的，提供了第一个抑制的例子 心脏再生促进剂中的元素。基于这些数据，我假设心脏再生 增强子对心脏的直接损伤限制性表达具有激活和抑制双重作用 再生。 在目标1中，我将利用药理学和遗传学的方法来测试损伤诱导的MEK-ERK- AP-1信号通路激活心脏再生促进剂。我还将使用atac-seq、Chip-seq和 用FACS分选未损伤和再生心脏的心内膜细胞进行RNA-SEQ检测AP-1 调节染色质可及性、活性组蛋白标记的沉积和损伤诱导的旁分泌因子 表情。在目标2中，我将使用转基因检测来确定Clen样增强子候选者是否 在斑马鱼、小鼠和人类基因组中发现驱动再生诱导表达。它的功能 候选抑制因素的数量将通过突变分析来确定。我也会利用体内的损失- 功能分析以确定Prdm1a，一种被预测与 Clen中的抑制元件介导未损伤患者的心脏再生和再生促进剂的抑制 红心。 这些研究将利用遗传工具和斑马鱼心脏的内源再生能力来改进 我们对心脏再生潜在的转录机制的理解，建立基因调控网络， 并确定改善心脏修复的潜在目标。

PROJECT SUMMARY/ABSTRACT Adult mammals possess a limited ability to regenerate cardiac tissue after an injury, such as a myocardial infarction. Following a myocardial infarction, up to a billion or more heart muscle cells die and are replaced by scar tissue that contributes to heart failure and sudden death. In contrast, adult zebrafish remarkably regenerate injured hearts with no residual scarring. Although zebrafish and mammals share homologs of genes vital for heart regeneration, their transcriptional responses to cardiac injury are distinct. However, the mechanisms governing gene expression during heart regeneration remain poorly understood. My research aims to elucidate how injury-responsive gene expression is regulated to facilitate heart regeneration. My lab identified the cardiac leptin b-linked regeneration enhancer (cLEN), which activates gene expression upon cardiac injury. Using in vivo transgenic assays, I found that multiple activation elements are required for injury-dependent cardiac enhancer activity. Surprisingly, I also found that cLEN contains a repressive element that is required for preventing enhancer activation in uninjured hearts, providing the first example of an inhibitory element within a cardiac regeneration enhancer. Based on these data, I hypothesize that cardiac regeneration enhancers are dually governed by activation and repression to direct injury-restricted expression for heart regeneration. In Aim 1, I will utilize pharmacological and genetic approaches to test the model that injury-induced MEK–ERK– AP-1 pathway signaling activates cardiac regeneration enhancers. I will also utilize ATAC-seq, ChIP-seq, and RNA-seq using FACS-sorted endocardial cells from uninjured and regenerating hearts to test whether AP-1 regulates chromatin accessibility, deposition of active histone marks, and injury-induced paracrine factor expression. In Aim 2, I will use transgenic assays to determine whether cLEN-like enhancer candidates I identified in the zebrafish, mouse, and human genomes drive regeneration-induced expression. The functionality of candidate repressive elements will be determined via mutational analyses. I will also utilize in vivo loss-of- function assays to determine whether prdm1a, a transcriptional repressor that is predicted to bind to the repressive element in cLEN, mediates heart regeneration and repression of regeneration enhancers in uninjured hearts. These studies will utilize genetic tools and the endogenous regenerative ability of the zebrafish heart to improve our understanding of transcriptional mechanisms underlying heart regeneration, build gene regulatory networks, and identify potential targets for improving heart repair.