Mechanisms and Therapeutic Targeting of DNA Damage in Dilated Cardiomyopathy Caused by LMNA Mutations

LMNA 突变引起的扩张型心肌病 DNA 损伤的机制和治疗靶点

• 批准号: 10221032
• 负责人: Ali J Marian
• 金额: $ 48.39万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-03-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10221032
• 关键词: Ablation; Acetylation; Apoptosis; Aspirin; Binding; CCCTC-binding factor; Cardiac Myocytes; Cause of Death; Cell Death; Cessation of life; ChIP-seq; Chromatin; Chromatin Loop; Cleaved cell; Complex; Cytoplasm; DNA; DNA Damage; DNA Double Strand Break; Data; Defect; Dilated Cardiomyopathy; Disease; Fibrosis; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Genomic Instability; Genomic Segment; Heart; Heart failure; Human; IRF3 gene; Immunoprecipitation; Impairment; Incidence; LIG4 gene; Lamin Type A; Lamins; Ligation; Maps; Mediating; Mutation; Myocardial dysfunction; NF-kappa B; Nonhomologous DNA End Joining; Nucleotides; Pathogenesis; Pathway interactions; Pharmacology; Phenotype; Prevention; Prognosis; Proteins; Role; Site; Stimulator of Interferon Genes; Stress; TBK1 gene; TOP2A gene; Techniques; Testing; Therapeutic; Therapeutic Effect; Topoisomerase; Torsion; XRCC4 gene; XRCC5 gene; attenuation; causal variant; citrate carrier; cohesin; cohesion; coronary fibrosis; density; effective therapy; familial dilated cardiomyopathy; gene repair; genome-wide; heart function; mouse model; p53-binding protein 1; premature; recruit; repair enzyme; repaired; response; senescence; sensor; sudden cardiac death; therapeutic target; tyrosyl-DNA phosphodiesterase

Mutations in the LMNA gene, encoding lamin A/C (LMNA) protein, cause a diverse array of diseases referred to as laminopathies. Dilated cardiomyopathy (DCM) is common and the main cause of death in laminopathies. LMNA mutations are also the second most common causes of familial DCM. DCM due to LMNA mutations (hereafter, LMNA-DCM) has a poor prognosis and a high incidence of sudden cardiac death. The underpinning mechanism(s) of LMNA-DCM is unknown. Hence, there is no effective therapy for LMNA-DCM. We have shown that LMNA binds to about 300 genomic regions in human cardiac myocytes, which are referred to as Lamin-Associated Domains (LADs). LADs comprise about 20% of the genome and several hundred genes. We show that LADs are redistributed in LMNA-DCM, resulting in Gain of LADs (GoL) and Loss of LADs (LoL). LoL is associated with active transcription, whereas GoL suppresses gene expression. At the mechanistic level, Preliminary data show that LADs are lost at the binding motifs for CTCF protein. CTCF insulates transcriptionally active chromatin loops and recruits topoisomerase 2B (TOP2B) to cut the compact chromatin and open the loops for active transcription. In parallel with these findings, we show that double stranded DNA breaks (DSBs), induced by TOP2B, are increased. We also show that expression of DSB repair genes is suppressed in LMNA-DCM, partly because of GoL. Consequently, DSBs are released into the cytoplasm and sensed by CGAS protein, which activates DNA damage response (DDR) and expression of genes involved in cell death, senescence, fibrosis, and cardiac dysfunction, phenotypic features of human LMNA-DCM. We propose to study the mechanisms responsible for increased DSBs and impaired DSB repair, and determine therapeutic effects of targeting the DDR pathway in LMNA-DCM. In aim 1, we will test the hypothesis that LoL leads to active transcription, induction of DSBs, and stalled TOP2B, whereas GoL suppresses transcription (hence, no DSBs). To test this hypothesis, we will map genome-wide DSB sites at the nucleotide level by END-Seq technique, compare distribution density of DSBs at LoL, GoL, and non-LAD regions in control and LMNA-DCM hearts, and determine stalling of TOP2B at the DSBs by ChIP-Seq. In aim 2, we will test the hypothesis that DSB repair is impaired in LMNA-DCM partly because GoL suppresses expression of key repair genes and in. part because recruitment of the repair enzymes to the DSB sites is impaired. Recruitment and assembly of the selected repair proteins at the DSBs will be analyzed by ChIP-Seq to map the binding motifs to DSB sites. Interactions between LMNA and the repair proteins will be determined by immunoprecipitation. In aim 3, the DDR pathway will be blocked genetically and pharmacologically by inhibiting CGAS, the key sensor of the cytoplasmic DNA, in two mouse models of LMNA-DCM. The ensuing effects on survival, cardiac function, gene expression, DDR activation, fibrosis, senescence, and apoptosis will be determined. The findings could delineate the mechanisms of increased DSBs and determine therapeutic effects of targeting of the DDR in LMNA-DCM.

LMNA基因的突变，编码层粘连蛋白A/C（LMNA）蛋白，引起多种疾病 称为椎板病。扩张的心肌病（DCM）很常见，是死亡的主要原因 椎板病。 LMNA突变也是家族性DCM的第二大最常见原因。 DCM由于LMNA 突变（以下简称LMNA-DCM）的预后较差，心脏猝死的发病率很高。这 LMNA-DCM的基础机制尚不清楚。因此，没有有效的LMNA-DCM疗法。 我们已经表明，LMNA与人类心肌细胞中约300个基因组区域结合，这是 称为层固定相关域（LAD）。小伙子约占基因组的20％和几个 一百个基因。我们表明，在LMNA-DCM中重新分配了LAD，导致LAD（GOL）和损失获得 小伙子（大声笑）。 HOL与主动转录有关，而GOL抑制基因表达。 在机械水平上，初步数据表明，在CTCF蛋白的结合基序中，小伙子丢失了。 CTCF绝缘具有转录活性的染色质环路，并招募拓扑异构酶2B（TOP2B）切割 紧凑型染色质并打开环的主动转录。与这些发现并行，我们证明 由TOP2B诱导的双链DNA断裂（DSB）增加。我们还表明了DSB的表达 修复基因在LMNA-DCM中被抑制，部分原因是GOL。因此，DSB被释放到 细胞质并通过CGAS蛋白感测，该蛋白激活DNA损伤反应（DDR）和基因的表达 参与细胞死亡，衰老，纤维化和心脏功能障碍，人LMNA-DCM的表型特征。 我们建议研究负责增加DSB和DSB维修受损的机制，以及 确定靶向LMNA-DCM中DDR途径的治疗效应。在AIM 1中，我们将检验假设 大声笑会导致主动转录，诱导DSB和停滞的top2b，而GOL抑制 转录（因此，无DSB）。为了检验该假设，我们将在核苷酸的全基因组DSB位点绘制 按末端技术水平，比较在控制中，在LOL，GOL和非LAD区域的DSB的分布密度 和LMNA-DCM心脏，并确定TOP2B在DSB处通过chip-seq停滞。在AIM 2中，我们将测试 LMNA-DCM中DSB修复受损的假设部分是因为GOL抑制了钥匙修复的表达 基因及其。部分是因为将修复酶募集到DSB位点受到损害。招募和 Chip-Seq将分析选定的修复蛋白在DSBS上的组装，以将结合基序映射到 DSB站点。 LMNA与修复蛋白之间的相互作用将由免疫沉淀确定。目标 3，DDR途径将通过抑制CGA，遗传和药理阻塞，CGA是该途径的关键传感器 在LMNA-DCM的两个小鼠模型中，细胞质DNA。随后对生存，心脏功能，基因的影响 将确定表达，DDR激活，纤维化，衰老和凋亡。这些发现可以描述 DSB的增加的机制并确定DDR靶向LMNA-DCM中的治疗作用。