Long Noncoding RNA Control of Cardiac Gene Expression

摘要 心脏发育和病理性重塑是由转录因子和非转录因子网络控制的。 编码RNA，其协调参与心肌细胞增殖、形态发生 蛋白质合成和收缩性。在对损伤或血流动力学应激的反应中，成人心肌 经历以心肌细胞大小增加为特征的代偿性肥大生长 和胎儿心脏基因的重新激活。持续性肥大是发生 收缩功能障碍和临床心脏病的进展。鉴定新的心脏生长调节因子 对于治疗心脏病的疗法的发展至关重要，心脏病仍然是导致心脏病的主要原因。 死亡率在美国。 血清反应因子（SRF）是一种广泛表达的转录因子，它调节以下因子的表达： 肌肉特异性基因和生长因子诱导型基因。响应细胞外信号，SRF与 不同的细胞类型和信号应答转录辅激活因子在相反的促有丝分裂和 平衡心肌细胞增殖和分化的生肌基因程序。Myocardin是一种有效的 SRF的辅激活因子，其对于心肌分化和肥大是必需的。我们最近发现了一个 一种新的长链非编码RNA（lncRNA）在心肌蛋白基因座的上游转录，我们将其命名为 心肌素相关长非编码RNA，或CARDINAL。CARDINAL在以下情况下显著上调： 在人类和小鼠的心力衰竭过程中，心肌蛋白，这表明它在基因中起着重要作用。 表达程序所需的维持正常的心脏功能和心室重塑， 心脏损伤在初步研究中，我们发现CARDINAL被myocardin强烈激活，并且是一种 与染色质结合的核lncRNA。小鼠中CARDINAL的遗传破坏导致异位 SRF调节的促有丝分裂基因的表达和心功能下降。有趣的是，我们发现， CARDINAL与SRF形成复合物，表明它作为第一个描述的lncRNA辅助调节因子起作用。 心脏中的SRF依赖基因网络。 长链非编码RNA是一类新兴的转录辅调节因子， 这些基因尚未被探索，部分原因是难以确定它们的结合伴侣和靶基因。在这一提议中， 我们将使用生物化学、细胞生物学和生物化学方法， 基于独特的动物模型，以研究CARDINAL在 心我们已经开发了一种新的酵母三杂交方法来鉴定lncRNA的蛋白质结合伴侣， 我们将利用它来鉴定和表征CARDINAL-SRF调节复合物的组分。这些 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认识， 广泛用于研究其他系统中的lncRNA-蛋白质相互作用。 ! ! ! . !

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. ! ! ! . !