Functional dissection of the regulatory network that governs cardiomyocyte maturation.

摘要 从出生到成年，心肌细胞（CM）在大小，超微结构， CM成熟是指细胞的生长、代谢和基因表达的过程，这一过程统称为CM成熟。虽然高度 协调，控制这一过程的转录网络还不清楚。这种缺乏理解 是心脏再生医学的一个障碍，我们目前无法使CM成熟， 肌细胞限制了它们用于疾病建模或替代疗法。此外，成熟的破坏， 接受先天性心脏病矫正手术的新生儿的异常血流动力学负荷 可能导致了他们成年后心力衰竭的高发病率。对监管的充分理解 管理CM成熟的网络将激发假设驱动的尝试，以克服这些挑战。 在小鼠中，CM成熟的一个关键标志是肌节同种型转换，包括有据可查的 新生儿肌球蛋白重链7（Myh 7）转换为肌球蛋白重链6（Myh 6）。我们进行了 验证了CM成熟的转录调节因子的体内高通量CRISPR筛选，使用 Myh 7/6同种型开关作为读数。来自该屏幕的两个顶级候选物Rnf 20和Rnf 40形成复合物 它控制着人们知之甚少的染色质修饰组蛋白2B单泛素化的沉积 （H2Bub1）。H2 Bub 1在心脏中的功能尚未探索，与先天性心脏病突变相关 在人类病人身上。因此，在目标1中，我们将详细描述Rnf 20/40对 CM成熟的遗传调控。 控制CM成熟的转录网络知之甚少的主要原因是 以前没有合适的工具。在本提案中，我们将利用一套新颖的体内工具， 开始绘制控制CM成熟的转录调控网络。在目标2中，我们将寻求 通过剖析两个Myh 7顺式调节元件来实现这一目标。此外，我们将比较基因 通过发现和描述与病理性肥大相关的关键调节因子， Myh 7的机制，其在成熟过程中失活，并响应于病理性炎症而重新激活。 应力这些实验将产生关于基因表达如何协调控制的机制线索 在成熟和疾病期间，从而能够改进CM生产方案和靶向治疗。 总的来说，这些实验将开始绘制控制成熟的转录调控网络 通过定义选择的反式作用调节子和顺式调节元件的功能。

ABSTRACT Between birth and adulthood, cardiomyocytes (CMs) undergo profound changes in size, ultrastructure, metabolism, and gene expression, a process collectively referred to as CM maturation. Although highly coordinated, the transcriptional network that governs this process is not understood. This lack of understanding is a barrier to cardiac regenerative medicine, where our current inability to mature CMs differentiated from non- myocytes limits their use for disease modeling or replacement therapy. In addition, disruption of maturation by abnormal hemodynamic loads in neonates who have undergone surgery to correct congenital heart defects likely contributes to their high incidence of heart failure in adulthood. A sound understanding of the regulatory network governing CM maturation will inspire hypothesis driven attempts to surmount these challenges. In mice, a key hallmark of CM maturation is sarcomere isoform switching, including the well documented neonatal switch from Myosin Heavy Chain 7 (Myh7) to Myosin Heavy Chain 6 (Myh6). We have conducted and validated an in vivo high throughput CRISPR screen for transcriptional regulators of CM maturation, using the Myh7/6 isoform switch as the readout. Two top candidates from this screen, Rnf20 and Rnf40, form a complex which governs deposition of the poorly understood chromatin modification histone-2B mono-ubiquitination (H2Bub1). H2Bub1 function is unexplored in the heart, and associated with congenital heart disease mutations in human patients. Therefore, In Aim 1 we will perform a detailed characterization of the impact of Rnf20/40 on genetic regulation of CM maturation. A major reason why the transcriptional networks controlling CM maturation are poorly understood is that appropriate tools were previously unavailable. In this proposal we will utilize a suite of novel in vivo tools to begin mapping the transcriptional regulatory networks that govern CM maturation. In Aim 2 we will seek to accomplish this goal by dissecting two Myh7 cis-regulatory elements. Furthermore, we will contrast the genetic regulation of CM maturation with pathological hypertrophy by discovering and describing the key regulatory mechanisms of Myh7, which is deactivated during maturation and re-activated in response to pathological stress. These experiments will yield mechanistic clues as to how gene expression is coordinately controlled during maturation and disease, thus enabling improved CM production protocols and targeted therapies. Collectively these experiments will begin to map the transcriptional regulatory network that governs maturation by defining the functions of select trans-acting regulators and cis-regulatory elements.