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

编码核纤层蛋白A/C（LMNA）蛋白的LMNA基因突变导致多种疾病 称为核纤层蛋白病。扩张型心肌病（DCM）是常见的，也是死亡的主要原因。 核纤层蛋白病LMNA突变也是家族性DCM的第二大常见原因。由于LMNA导致DCM 突变型心肌病（以下称为LMNA-DCM）预后差，心脏性猝死发生率高。的 LMNA-DCM的基础机制未知。因此，对于LMNA-DCM没有有效的治疗。 我们已经证明，LMNA与人心肌细胞中约300个基因组区域结合， 称为层相关结构域（LAD）。LAD占基因组的约20%， 百个基因我们表明，LAD在LMNA-DCM中重新分布，导致LAD增益（GoL）和损失 （注：LoL）。LoL与活跃的转录相关，而GoL抑制基因表达。 在机制水平上，初步数据显示，LADs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（TOP 2B）来切割转录活性染色质环。 压缩染色质并打开环以进行活跃的转录。与这些发现同时，我们表明， 由TOP 2B诱导的双链DNA断裂（DSB）增加。我们还表明，表达DSB 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 CGAS蛋白可激活DNA损伤反应（DDR）和基因表达 参与细胞死亡、衰老、纤维化和心功能障碍，人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， 确定靶向DDR途径在LMNA-DCM中的治疗效果。在目标1中，我们将检验假设 LoL导致活跃的转录，DSB的诱导和停滞的TOP 2B，而GoL抑制 转录（因此，没有DSB）。为了验证这一假设，我们将绘制核苷酸序列中的全基因组DSB位点， 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测定T0 P2B在DSB处的停滞。在目标2中，我们将测试 假设LMNA-DCM中DSB修复受损部分是因为GoL抑制了关键修复的表达， 基因与内部分原因是修复酶在DSB位点的募集受损。招募和 将通过ChIP-Seq分析所选修复蛋白在DSB处的组装，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。在aim中 3.通过抑制CGAS，DDR通路将被遗传阻断和阻断，CGAS是DDR通路的关键传感器。 细胞质DNA，在两个小鼠模型的LMNA-DCM。对存活率心脏功能基因 将测定表达、DDR活化、纤维化、衰老和凋亡。这些发现可以描述 增加DSB的机制，并确定靶向DDR在LMNA-DCM中的治疗效果。

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.