MECHANISTIC STUDIES OF DOT1L FUNCTION IN EMBRYONIC ERYTHROPOIESIS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 精确调节的基因表达对于正常的细胞功能以及各种正常的 哺乳动物的发育过程。组蛋白甲基化状态已成为一个重要的决定因素 基因位点转录活性。DOT1L(端粒沉默干扰物1样)组蛋白H3赖氨酸- 79(H3K79)甲基转移酶与几个不同的生物学过程有关，包括阳性 转录调控。我们从基因陷阱靶向的胚胎干细胞(ES)中产生了DOT1L缺失的小鼠 细胞。与野生型胚胎相比，DOT1L缺失的小鼠发育缓慢，并在E10.5到E13.5之间死亡。 E10.5胚胎表现出明显的贫血，特别是在卵黄囊的小血管，部位 大多数早期的造血术。此外，这些小鼠的造血祖细胞生长有缺陷。 对红系生长因子的反应。GATA2是一种对早期红细胞生成至关重要的转录因子，它是 在DOT1L缺陷的造血祖细胞中显著降低，而转录因子PU.1 抑制红细胞生成和促进骨髓生成的因子显著增加。这些数据 建议一个模型，在该模型中，依赖于DOT1L的H3K79甲基化在 早期造血，调节GATA2和PU1转录的稳定水平，从而控制 适当数量的循环中的红系和髓系细胞。 这项研究的目的是为我们理解这一作用的总体科学目标做出贡献 胚胎红细胞生成中的甲基转移酶。我们已经制定了一个专门的目标来研究这个机制 在此过程中，DOT1L可能调控与细胞命运相关的关键基因的表达 进程。这项研究的完成将为红细胞生成的表观遗传调控提供新的见解 并使我们能够为今后在这一一般调查领域的工作奠定坚实的基础。 虽然拟议工作的范围还远远不够全面，但我们有信心 在分配的时间内提供数据。这些数据将大大有助于我们努力获得长期的、 这项工作的外部资金。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Precisely tuned gene expression is critical for normal cellular functions as well as a variety of normal mammalian developmental processes. Histone methylation status has emerged as an important determinant of gene locus transcriptional activity. The DOT1L (Disruptor of Telomere Silencing 1-Like) histone H3 lysine- 79 (H3K79) methyltransferase has been implicated in several distinct biological processes, including positive regulation of transcription. We generated Dot1L-null mice from gene trap-targeted embryonic stem (ES) cells. Dot1L-null mice developed more slowly than wild-type embryos and died between E10.5 and E13.5. E10.5 embryos displayed a striking anemia, especially apparent in the small vessels of the yolk sac, the site of most early hematopoiesis. Further, hematopoietic progenitors from these mice displayed defective growth in response to erythroid growth factors. GATA2, a transcription factor critical for early erythropoiesis, was significantly reduced in Dot1L-deficient hematopoietic progenitors while expression of PU.1, a transcription factor that inhibits erythropoiesis and promotes myelopoiesis, was significantly increased. These data suggest a model whereby DOT1L-dependent H3K79 methylation serves as a differentiation switch during early hematopoiesis, regulating steady-state levels of GATA2 and PU.1 transcription and thus controlling the appropriate numbers of circulating erythroid and myeloid cells. This study is designed to contribute to our overall scientific goal of understanding the role for this methyltransferase in embryonic erythropoiesis. We have devised a specfic aim to examine the mechanism by which DOT1L might regulate the expression of critical genes involved in cell-fate specification during this process. The completion of this study will provide novel insight into epigenetic regulation of erythropoiesis as well as enable us to produce a substantial foundation for future work in this general area of investigation. Although the scope of the proposed work is far from comprehensive, we are confident that we will obtain data within the time allotted. These data will significantly contribute to our efforts at obtaining long-term, extramural funding for this work.