Long Noncoding RNA Control of Cardiac Gene Expression

摘要 心脏发育和病理重塑由转录因子和非转录因子网络控制 编码RNA协调参与心肌细胞增殖，形态发生， 蛋白质合成和收缩能力。作为对损伤或血流动力学应激的反应，成年心肌 经历以心肌细胞大小增加为特征的代偿性肥大生长 以及胎儿心脏基因的重新激活。持续性肥厚是糖尿病发生的主要危险因素 收缩功能障碍与临床心脏病的进展。寻找心脏生长的新调节剂 对于开发治疗心脏病的疗法至关重要，心脏病仍然是 美国的死亡率。 血清反应因子(SRF)是一种广泛表达的转录因子，调节 肌肉特异性基因和生长因子诱导基因。作为对细胞外暗示的反应，SRF与 不同的细胞类型和信号响应的转录辅助激活因子在相反的有丝分裂和 肌源性基因程序，平衡心肌细胞的增殖和分化。Myocardin是一种有效的 SRF的辅激活剂，对心肌分化和肥大是必不可少的。我们最近发现了一种 新的长非编码RNA(LncRNA)转录于myocardin基因的上游，我们将其命名为 肌钙蛋白相关的长非编码RNA，或红衣主教。红衣主教被显著上调了 肌钙蛋白在人类和小鼠心力衰竭中的作用，表明它在该基因中起着重要作用 维持正常心功能和心室重塑所需的表达程序 心脏损伤。在初步研究中，我们发现红衣主教被Myocardin强烈激活，是一种 与染色质有关的核糖核酸。小鼠红衣主教基因突变导致异位 SRF调节的有丝分裂原基因表达和心功能下降。有趣的是，我们发现 Cardinal与SRF形成一个复合体，表明它是第一个描述的lncRNA辅助调节因子。 心脏中依赖SRF的基因网络。 长的非编码RNA是一类新兴的转录辅助调节因子，在很大程度上 尚未探索，部分原因是很难确定它们的结合伙伴和目标基因。在这份提案中， 我们将利用生化、细胞-生物学方法确定红衣主教在直接介导心脏基因表达中的作用。 以独特的动物模型为基础，研究红衣主教在 心。我们开发了一种新的酵母三杂交方法来确定lncRNAs的蛋白质结合伙伴， 我们将利用它来识别和描述Cardinal-SRF监管复合体的组成部分。这些 研究将进一步加深我们对控制心脏基因转录的基本机制的了解，并将 广泛适用于研究其他系统中的lncRNA-蛋白质相互作用。 好了！ 好了！ 好了！ 。 好了！

Abstract Heart development and pathological remodeling are controlled by a network of transcription factors and non- coding RNAs that coordinate the expression of genes involved in cardiomyocyte proliferation, morphogenesis, protein synthesis, and contractility. In response to injury or hemodynamic stress, the adult myocardium undergoes compensatory hypertrophic growth that is characterized by an increase in cardiomyocyte cell size and reactivation of fetal cardiac genes. Sustained hypertrophy is a major risk factor for the development of systolic dysfunction and the progression to clinical heart disease. Identifying novel regulators of cardiac growth is vital to the development of therapeutics for the treatment of heart disease, which remains a leading cause of mortality in the United States. Serum response factor (SRF) is a widely-expressed transcription factor that regulates the expression of both muscle-specific and growth factor-inducible genes. In response to extracellular cues, SRF associates with diverse cell-type and signal-responsive transcriptional coactivators to switch between opposing mitogenic and myogenic gene programs that balance cardiomyocyte proliferation and differentiation. Myocardin is a potent coactivator of SRF that is essential for cardiac muscle differentiation and hypertrophy. We recently identified a novel long noncoding RNA (lncRNA) transcribed upstream of the myocardin locus, that we named the myocardin-associated long noncoding RNA, or CARDINAL. CARDINAL is significantly upregulated with myocardin during heart failure in both humans and mice, suggesting it plays an important role in the gene expression programs required to maintain normal heart function and ventricular remodeling in response to cardiac injury. In preliminary studies, we found that CARDINAL was robustly activated by myocardin and is a nuclear lncRNA that associates with chromatin. Genetic disruption of CARDINAL in mice resulted in ectopic expression of SRF-regulated mitogenic genes and decreased cardiac function. Interestingly, we found that CARDINAL forms a complex with SRF, suggesting it functions as the first described lncRNA coregulator of SRF-dependent gene networks in the heart. Long noncoding RNAs are an emerging class of transcriptional coregulators that remain largely unexplored, in part due to the difficulty in determining their binding partners and target genes. In this proposal, we will determine the role of CARDINAL in directly mediating cardiac gene expression using biochemical, cell- based, and unique animal models to investigate the molecular and biological significance of CARDINAL in the heart. We have developed a novel yeast three hybrid approach to identify protein binding partners for lncRNAs, which we will utilize to identify and characterize components of the CARDINAL-SRF regulatory complex. These studies will further our knowledge of the basic mechanisms controlling cardiac gene transcription, and will be broadly useful for investigating lncRNA-protein interactions in other systems. ! ! ! . !