Cardiac lineage determination and nuclear architecture

此R35应用程序提出了一个概念框架，其中PI在过去产生的工作主体 20年被用作启动创新研究的基础， 了解核结构（染色质如何在细胞核中以三维方式组织）以产生 谱系决定和细胞命运同一性的新范例。我们的目标是更好地了解 表征细胞身份的基因表达程序受到调控，以告知再生能力。 心血管疾病的治疗方法。初步数据表明，基因组中定位的区域 到核外围（称为“核纤层蛋白相关结构域”或LAD）被特定的组蛋白标记沉默， 包括H3K9me2，并且这些区域在谱系确定后从外周释放， 以允许同时激活整个基因程序。数据显示H3K9me2标记 LAD的特征是通过有丝分裂“记忆”，提供了谱系表观遗传记忆的机制 身份所提出的模型表明，定义LAD的表观遗传标记如H3K9me2， 这些组蛋白表位可以被介导空间定位的“LAD系链”识别，并且这些组蛋白表位可以被“屏蔽”， 通过组蛋白尾部的相邻氨基酸残基的磷酸化（包括H3S10和H3S12的磷酸化）， H3T11）。我们建议测试，在有丝分裂过程中，极光B激酶磷酸化H3S10， 通过屏蔽H3K9me2表位，允许释放LAD，随后细胞核的破坏， 膜和DNA复制，然后去除S10磷酸化并重建LAD， 在暴露的组蛋白标记周围形成子核膜。因此，如果全基因组模式 给定细胞中的LAD通过表示沉默的替代谱系程序的“代码”来定义其身份， 细胞身份可以通过有丝分裂记忆并在子细胞中有效地重建。影响 用于重编程、转分化、不对称细胞分裂和谱系身份的稳定性（因此 癌症易感性）将被探索。信号转导级联调节细胞内 代谢和功能（如葡萄糖和脂肪酸代谢特征之间的转换） 发展和生病的心肌细胞和癌细胞）可能会影响核结构和LAD动力学 通过集中研究包括H3T11在内的组蛋白残基的磷酸化。这一观点得到了出版物的支持。 数据表明，与代谢转变有关的丙酮酸激酶的核形式可以磷酸化 H3T11可以与Hdac3相互作用，我们已经证明Hdac3是LAD系链，导致表观遗传变化， 激活特定基因位点。因此，该提案提供了为以下方面提供实验支持的机会： 一种基因调控和细胞特性的模型，包括染色质的三维调控 细胞核内的包装扩展了我们对细胞身份的理解，并提供了一种新的机制 来理解整个基因程序是如何协调调节的。

This R35 application proposes a conceptual framework in which a body of work produced by the PI over the past 20 years is utilized as the foundation for launching innovative studies that seek to incorporate cutting edge understanding of nuclear architecture (how chromatin is organized in three dimensions in the nucleus) to produce a novel paradigm of lineage determination and cell fate identity. The goal is to better understand how broad gene expression programs that characterize cell identity are regulated in order to inform regenerative approaches to cardiovascular disease. Preliminary data suggest that regions of the genome that are localized to the nuclear periphery (called “lamin associated domains” or LADs) are silenced by specific histone marks, including H3K9me2, and that these regions are released from the periphery upon lineage determination in order to allow for simultaneous activation of entire gene programs. Data suggests that the H3K9me2 mark characteristic of LADs is “remembered” through mitosis providing a mechanism for epigenetic memory of lineage identity. The proposed model suggests that epigenetic marks such as H3K9me2 that define LADs are recognized by “LAD-tethers” that mediate spatial localization, and that these histone epitopes can be “shielded” by phosphorylation of adjacent amino acid residues of the histone tails (including phosphorylation of H3S10 and H3T11). We propose to test that during mitosis, aurora B kinase which phosphorylates H3S10, acts to un-tether LADs by shielding the H3K9me2 epitope, allowing for the release of LADs, subsequent breakdown of the nuclear membrane and DNA replication, followed by removal of S10 phosphorylation and re-establishment of LADs as the daughter nuclear membranes form around the exposed histone mark. Thus, if the genome-wide pattern of LADs in a given cell defines its identity by representing a “code” of silenced alternative lineage programs, then cellular identity can be remembered through mitosis and efficiently re-established in daughter cells. Implications for reprogramming, trans-differentiation, asymmetric cell division, and stability of lineage identity (and thus cancer susceptibility) will be explored. Signal transduction cascades that regulate dramatic changes in cellular metabolism and function (such as the switch between glucose and fatty acid metabolism characteristic of developing and ailing cardiac myocytes and of cancer cells) may impact nuclear architecture and LAD dynamics by converging on phosphorylation of histone residues including H3T11. This notion is supported by published data indicating that a nuclear form of pyruvate kinase that is implicated in metabolic shifts can phosphorylate H3T11 and can interact with Hdac3 which we have shown is a LAD tether, resulting in epigenetic changes and activation of specific gene loci. Thus, this proposal provides the opportunity to provide experimental support for a model of gene regulation and cellular identity that incorporates three dimensional regulation of chromatin packaging within the nucleus extending our understanding of cellular identity and providing a novel mechanism to understand the way in which entire gene programs are coordinately regulated.