Functional examination of H3K4 methyltransferases in myeloid neoplasia

表观遗传调控的扰动与肿瘤的发生有着深刻的联系。组蛋白尾部甲基化的酶，特别是那些参与三胸组和多梳组对立的酶，是表观遗传调控的核心，在许多癌症中经常发生突变。我们已经为所有六种组蛋白3赖氨酸4 （H3K4）三胸基甲基转移酶制作了条件小鼠系，并系统地研究了它们在发育、种系和成年中的作用。这六种包括主要的白血病因子MLL1，其姊妹基因MLL2和SETD1B。在这里，我们提出了基于我们最近发现的工作，即成年小鼠中Setd1b或Mll2的缺失会引起髓系瘤变。我们的发现，加上Mll1在维持造血干细胞（hsc）中的已知作用，以及MLL3和MLL4突变在最近的人类癌症外显子组研究中的突出地位，为研究了解Trithorax-Group在造血中的作用提出了新的迫切需要。此外，我们相信我们的工作将有力地促进和受益于与其他SPP1463合作伙伴的互动，特别是那些致力于MLL1， Polycomb-Group和DNA甲基化的合作伙伴。总的来说，可以获得对髓系谱系中主要表观遗传调控因子的基本综合理解。我们最近的数据表明，在成年小鼠中，Setd1b缺失会产生具有人类慢性髓细胞白血病（CMML）特征的骨髓增生性肿瘤，而Mll2缺失会产生轻度髓系恶性肿瘤。在这些初步数据的基础上，我们计划在条件突变的Setd1b、Mll2和双Setd1b/Mll2小鼠中评估HSC和造血祖细胞室。我们预测造血干细胞的内在缺陷，这导致向髓系谱系分化的倾斜，因此计划进行骨髓移植研究。为了深入了解造血缺陷的分子基础，我们将生成总mRNA表达谱，将Setd1b、Mll2和双Setd1b/Mll2条件敲除与对照HSC进行比较，并辅以来自HSC和祖细胞群体的H3K4me3、-me2和-me1染色质免疫沉淀（ChIPs）。我们将通过比较结构-功能分析来补充小鼠研究，利用BAC转基因来解释Setd1b在Setd1a和Setd1b之间的外显子交换，以解释Setd1b固有的髓细胞转化潜力。通过比较Setd1b和Mll2，我们的目标是将对髓系H3K4甲基转移酶作用的系统理解与其他SPP1463伙伴产生的Polycomb-Group和DNA甲基化知识整合在一起。此外，我们还为该SPP提供了一个关于我们的重组，BAC转基因和通用蛋白质标记方法的研讨会。

Perturbations of epigenetic regulation are profoundly linked with tumorigenesis. The enzymes that methylate histone tails, particularly those involved in the opposition between Trithorax- and Polycomb-Groups, are central to epigenetic regulation and frequently mutated in many cancers.We have made conditional mouse lines for all six histone 3 lysine 4 (H3K4) Trithorax-Group methyltransferases and are systematically examining their roles during development, in the germline and in the adult. These six include the major leukemogenic factor MLL1, its sister gene MLL2, and SETD1B. Here we propose work based on our recent findings that loss of either Setd1b or Mll2 in adult mice provokes myeloid neoplasia. Our findings, together with the known role of Mll1 in the maintenance of hematopoietic stem cells (HSCs) and the prominence of MLL3 and MLL4 mutations in recent human cancer exome studies, raise a new imperative for studies to understand the Trithorax-Group in hematopoiesis. Furthermore, we believe that our work will strongly contribute to and benefit from interactions with other SPP1463 partners, particularly those working on MLL1, Polycomb-Group and DNA methylation. Overall, a substantially integrated understanding of the major epigenetic regulators in the myeloid lineage can be obtained. Our recent data indicate that Setd1b deletion in adult mice produces myeloproliferative neoplasia with features of human chronic myelomonocytic leukemia (CMML), and Mll2 deletion produces a mild myeloid malignancy. On the basis of these preliminary data, we plan to evaluate the HSC and hematopoietic progenitor compartments in conditionally mutated Setd1b, Mll2 and double Setd1b/Mll2 mice. We predict intrinsic defects in HSCs, which results in skewing of differentiation toward the myeloid lineage and therefore plan to perform bone marrow transplantation studies. To gain insight into the molecular basis of the hematopoietic defect, we will generate total mRNA expression profiles comparing Setd1b, Mll2 and double Setd1b/Mll2 conditional knockouts versus control HSCs, complemented by H3K4me3, -me2 and -me1 chromatin immunoprecipitations (ChIPs) from HSC and progenitor cell populations. We will complement the mouse work with comparative structure-function analyses to explain the inherent myeloid transformation potential of Setd1b using BAC transgenesis for exon swaps between Setd1a and Setd1b.By comparison of Setd1b and Mll2, we aim to integrate a systematic understanding of H3K4 methyltransferase action in the myeloid lineage with the Polycomb-Group and DNA methylation knowledge generated by other SPP1463 partners. Furthermore, we offer a workshop on our recombineering, BAC transgenic and generic protein tagging methodologies to this SPP.