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: 10455102
• 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/10455102
• 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; 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.

编码核纤层蛋白 A/C (LMNA) 蛋白的 LMNA 基因突变会导致多种疾病 称为核纤层蛋白病。扩张型心肌病 (DCM) 很常见，是导致死亡的主要原因 核纤层蛋白病。 LMNA 突变也是家族性 DCM 的第二常见原因。 LMNA 引起的 DCM 突变（以下简称LMNA-DCM）预后较差，心源性猝死发生率较高。这 LMNA-DCM 的基础机制尚不清楚。因此，LMNA-DCM 没有有效的治疗方法。 我们已经证明 LMNA 与人类心肌细胞中约 300 个基因组区域结合，这些区域是 称为核纤层蛋白相关结构域 (LAD)。 LAD 约占基因组的 20%，并且有几个 一百个基因。我们证明 LAD 在 LMNA-DCM 中重新分布，导致 LAD 增益 (GoL) 和损失 LAD（笑）。 LoL 与活跃转录相关，而 GoL 则抑制基因表达。 在机制层面，初步数据显示，LAD 在 CTCF 蛋白的结合基序处丢失。 CTCF 隔离转录活性染色质环并招募拓扑异构酶 2B (TOP2B) 来切割 压缩染色质并打开环以进行主动转录。与这些发现同时，我们表明 TOP2B 诱导的双链 DNA 断裂 (DSB) 增加。我们还表明 DSB 的表达 修复基因在 LMNA-DCM 中受到抑制，部分原因是 GoL。因此，DSB 被释放到 细胞质并被 CGAS 蛋白感知，激活 DNA 损伤反应 (DDR) 和基因表达 参与细胞死亡、衰老、纤维化和心脏功能障碍，是人类 LMNA-DCM 的表型特征。 我们建议研究 DSB 增加和 DSB 修复受损的机制，以及 确定针对 LMNA-DCM 中 DDR 通路的治疗效果。在目标 1 中，我们将检验假设 LoL 会导致活跃的转录、DSB 的诱导以及 TOP2B 的停滞，而 GoL 会抑制 转录（因此，没有 DSB）。为了验证这一假设，我们将绘制核苷酸上的全基因组 DSB 位点图谱。 通过 END-Seq 技术水平，比较对照中 LoL、GoL 和非 LAD 区域的 DSB 分布密度 和 LMNA-DCM 心脏，并通过 ChIP-Seq 确定 DSB 处 TOP2B 的停滞。在目标 2 中，我们将测试 假设 DSB 修复在 LMNA-DCM 中受损，部分原因是 GoL 抑制关键修复的表达 基因，部分是因为修复酶向 DSB 位点的募集受到损害。招聘及 DSB 处所选修复蛋白的组装将通过 ChIP-Seq 进行分析，以将结合基序映射到 DSB 网站。 LMNA 和修复蛋白之间的相互作用将通过免疫沉淀来确定。瞄准目标 3、通过抑制CGAS，DDR途径将从基因和药理上被阻断，CGAS是细胞的关键传感器。 两种 LMNA-DCM 小鼠模型中的细胞质 DNA。对生存、心脏功能、基因的后续影响 将测定表达、DDR 激活、纤维化、衰老和细胞凋亡。研究结果可以描绘 DSB 增加的机制并确定 LMNA-DCM 中 DDR 靶向的治疗效果。