The Role of Dot1L in developing and postnatal heart

描述(由申请人提供)：基因转录调控是了解心脏发育、先天性心脏病和开发损伤后心脏再生治疗方法的关键。表观遗传修饰是转录格局的关键调节者，因此，催化这些修饰的酶已经成为一个非常感兴趣的主题。特别是，组蛋白修饰被认为在基因调控的多个方面发挥关键作用，包括介导与染色质重塑因子的相互作用，一般或特定组织特异性转录因子或辅因子的对接，以及可能介导剪接。组蛋白乙酰化和脱乙酰化一直是人们研究的热点。最近，组蛋白甲基化受到了仔细的审查，对特定甲基化标记的作用有了更多的了解。组蛋白甲基化标志物组蛋白H3赖氨酸79(H3K79)是由赖氨酸甲基转移酶DOT1L唯一催化的。多条证据表明DOT1L在心脏发生和出生后心脏中起着关键和特定的作用。DOT1L在整个发育过程中在心脏中高度表达，全局消融DOT1L会导致妊娠中期死亡，并伴有多种心血管异常。在妊娠中期用αMHC-CRE消融心肌细胞中的DOT1L导致成人心肌病，部分原因是dystrophin基因表达的扰动。小鼠胚胎干细胞向心肌细胞分化的证据表明，DOT1L在心肌细胞分化的早期阶段起着关键作用，但还没有活体研究解决这一问题。因此，在初步研究中，我们利用xMLCcre在e7.5处消融了DOT1L，并发现突变体表现出心肌细胞增殖，在出生后早期死亡。在围产期小鼠心肌细胞培养中消融DOT1L的初步研究表明，DOT1L调节心肌细胞周期的持续需求。初步研究表明，在新生儿和成人心肌细胞中，心脏基因座上的H3K79甲基标记丰富。综上所述，这些观察结果导致了我们的假设，即在胚胎、新生儿和成年阶段的心肌细胞中，DOT1L是心肌细胞周期调节和心肌细胞渐进分化所必需的。为了验证这一假设，我们的具体目标是：1)表征早期心肌细胞特异性DOT1L突变体的心脏表型。2)在体内鉴定DOT1L介导的H3K79甲基化直接调控的基因。3)检测出生后和成人心脏对DOT1L的需求量。这些研究的结果将从机制上深入了解DOT1L在不同发育阶段的心肌细胞以及在出生后和成年心脏中的作用。重要的是，这些研究有望深入了解表观遗传修饰调节心肌细胞周期的机制，因此有可能为发育中和成人心脏的心脏修复提供治疗方法。DOT1L抑制剂目前正在测试用于治疗MLL相关白血病，因此我们的研究将有助于了解这些抑制剂的潜在心脏副作用。我们的研究还将探讨DOT1L抑制剂的未来潜力 用于心脏再生治疗。

DESCRIPTION (provided by applicant): Regulation of gene transcription is key to understanding mechanisms underlying heart development, congenital heart disease, and developing therapies for heart regeneration post injury. Epigenetic modifications are key modulators of the transcriptional landscape, and as such, enzymes which catalyze these modifications have become a subject of great interest. In particular, histone modifications are thought to play key roles in multiple aspects of gene regulation, including mediating interactions with chromatin remodeling factors, docking of general or specific tissue specific transcription factors or cofactors, and perhaps mediating splicing. Histone acetylation and deacteylation have been intensely studied. More recently, histone methylation has come under scrutiny, and greater understanding of the role of specific methylation marks has emerged. The histone methylation mark histone H3 lysine 79 (H3K79), is uniquely catalyzed by the lysine methyltransferase, DOT1L. Multiple lines of evidence suggest critical and specific roles for DOT1L during cardiogenesis and in postnatal heart. DOT1L is highly expressed in the heart throughout development, and global ablation of Dot1L results in mid-gestation lethality with multiple cardiovascular abnormalities. Ablation of Dot1L in cardiomyocytes at mid-gestation with αMHC-cre results in cardiomyopathy in adult heart, accounted for in part by perturbation of dystrophin gene expression. Evidence from cardiomyocyte differentiation of mouse embryonic stem cells indicated that DOT1L plays a critical role at earliest stages of cardiomyocyte differentiation, yet no in vivo studies have addressed this issue. Accordingly, in preliminary studies, we have ablated Dot1L at E7.5 utilizing xMLCcre, and found that mutants exhibited cardiomyocyte hyperplasia, dying in the early postnatal period. Preliminary studies ablating Dot1L in perinatal mouse cardiomyocyte cultures demonstrated an ongoing requirement for Dot1L to regulate cardiomyocyte cell cycle. Preliminary studies have demonstrated enrichment of H3K79 methyl marks at cardiac gene loci in neonatal and adult myocytes. Together, these observations have led to our hypothesis that Dot1L is required in cardiomyocytes at embryonic, neonatal and adult stages for cardiomyocyte cell cycle regulation and progressive differentiation of cardiomyocytes. To test this hypothesis, our Specific Aims are: 1) To characterize the cardiac phenotype of early cardiomyocyte-specific Dot1L mutants. 2) To identify genes directly regulated, positively or negatively, by DOT1L mediated-H3K79 methylation in vivo. 3) To examine the requirement for Dot1L in postnatal and adult heart. Results of these studies will give mechanistic insight into the role of DOT1L in cardiomyocytes throughout distinct developmental stages and in postnatal and adult heart. Importantly, these studies promise to give insights into mechanisms by which epigenetic modifications regulate cardiomyocyte cell cycle, and are therefore likely to suggest therapeutic approaches for cardiac repair in both developing and adult heart. DOT1L inhibitors are currently being tested for treatment of MLL-associated leukemias, therefore our studies will be informative as to potential cardiac side effects of these inhibitors. Our studies will also address the future potential of DOT1L inhibitors for cardiac regenerative therapies.