Chromatin mechanism of molecular motor gene regulation

描述(申请人提供)：病理性应激诱导心肌转录基因重编程，导致肌病和心力衰竭。然而，关于病理性应激如何触发染色质的重组以控制肌病基因的表达，人们知之甚少。这项研究的重点是不同的染色质调节因子如何协同作用，建立抑制染色质的环境，以控制α-肌球蛋白重链(？-MHC)的表达，这是一种分子马达基因，其在应激心脏中的抑制作用导致肌病和心力衰竭。四种不同的染色质调节剂-染色质重构体、组蛋白甲基转移酶、组蛋白去乙酰化酶和DNA甲基转移酶-将被研究它们对MHC的协同抑制。由于化学抑制剂可以抑制这些染色质因子，因此有可能开发DRUS来增强衰竭心脏中的？-MHC，以改善心功能。了解这些染色质因子(药物靶点)是如何相互作用的，将有助于我们设计新药，并了解潜在药物的协同作用/动力学，这些药物抑制不同类别的染色质调节剂。目的1：确定MHC抑制和心力衰竭所需的DNA甲基转移酶的特定亚型。我们将敲除小鼠心肌中的DNA甲基转移酶同工酶，并检测其对应激心脏中MHC抑制和心力衰竭进展的影响。目的2：确定染色质重构体如何与组蛋白和DNA甲基转移酶结合来沉默？-MHC。我们将使用动物模型、手术方法和各种分子生物学方法(染色质免疫沉淀和定量聚合酶链式反应)来确定这些染色质因子之间的相互作用，这些染色质因子对组蛋白和DNA甲基化至关重要。目的3：明确组蛋白脱乙酰酶如何与组蛋白和DNA甲基转移酶协同抑制-MHC启动子。我们将使用动物模型、手术方法和各种分子生物学方法(染色质免疫沉淀、定量PCR和免疫共沉淀)来确定这些组蛋白甲基化和乙酰化所必需的染色质因子之间的相互作用，以及α-MHC启动子的DNA甲基化。

DESCRIPTION (provided by applicant): Pathological stress induces transcriptional gene reprogramming in the heart muscle, leading to myopathy and heart failure. However, little is known about how the pathological stress triggers the restructuring of chromatin to control myopathic gene expression. This research program focuses on how different chromatin-regulating factors act in concert to establish a repressive chromatin environment to control the expression of alpha-myosin heavy chain (¿-MHC), a molecular motor gene whose repression in the stressed hearts contributes to myopathy and heart failure. Four different chromatin regulators-chromatin remodeler, histone methyltransferase, histone deacetylase and DNA methyltransferase-will be examined for their collaborative repression of ¿-MHC. Because chemical inhibitors are available to inhibit these chromatin factors, it is possible to develop drus to boost ¿- MHC in the failing heart to improve cardiac function. Understanding how these chromatin factors (drug targets) interact will help us design new drugs and understand the synergy/dynamics of potential drugs that inhibit different classes of chromatin regulators. Aim 1: Defining specific isoforms of DNA methyltransferase required for ¿-MHC repression and heart failure. We will knock out DNA methyltransferase isoforms in the heart muscle of mice and examine the effects on ¿-MHC repression in the stressed heart and on the progression of heart failure. Aim 2: Determine how chromatin remodeler integrates histone and DNA methyltransferases to silence ¿-MHC. We will use animal models, surgical method, and various molecular biology methods (chromatin immunoprecipitation and quantitative PCR) to define the interactions between these chromatin factors crucial for the histone and DNA methylation of ¿-MHC. Aim 3: Defining how histone deacetylase coordinates with histone and DNA methyltransferases to repress ¿-MHC promoter. We will use animal models, surgical methods, and various molecular biology methods (chromatin immunoprecipitation, quantitative PCR, and co-immunoprecipitation) to define the interactions between these chromatin factors that are essential for histone methylation and acetylation, as well as DNA methylation of ¿-MHC promoter.