Role of MLL1 and MLL1 leukemogenic fusions in maintaining transcriptional memory

描述（由申请人提供）：正常的发育需要细胞建立特定的基因表达模式，并通过细胞分裂稳定地传递。在有丝分裂期间，转录停止，因为大多数转录调节因子从靶基因中移除。新形成的子细胞重组转录装置以重新激活适当的基因表达程序的机制被认为与核因子有关，这些核因子仍然与有丝分裂染色体结合，称为有丝分裂书签。然而，是什么使这些因子在有丝分裂中保留，以及它们在谱系承诺和/或谱系稳定性中的具体作用的理解仍然不完整。本提案的目的是确定书签因子MLL1（混合谱系白血病1）占据其有丝分裂靶点的机制，并确定MLL1突变是否以干扰造血的方式改变有丝分裂功能。我们实验室最近的研究表明，正常胚胎和成人造血所需的组蛋白甲基转移酶MLL1在有丝分裂染色体上全局保留。MLL1染色质占用模式在有丝分裂中重组，向最高表达基因转移。然而，控制MLL1有丝分裂再分配的物理相互作用及其与正常造血的相关性尚不清楚。MLL1经常通过染色体易位重排，产生白血病融合蛋白，维持异常的基因表达程序并阻断造血分化。初步结果表明，MLL1融合蛋白MLL-AF9仍然与有丝分裂染色体结合，然而，这是否是MLL-AF9维持导致白血病的不适当转录模式所必需的尚不清楚。在本提案中，我将研究指导MLL1有丝分裂占用的相互作用，并检查MLL1和MLL-AF9在正常和恶性造血中的有丝分裂功能。在Aim 1中，我将系统地定义MLL1结合有丝分裂染色质所必需和充分的最小区域，并评估MLL1在造血祖细胞中的有丝分裂特异性功能。在Aim 2中，我将研究MLL-AF9和野生型MLL1在白血病细胞间期和有丝分裂中的整体占用模式，并定义其与正常和白血病特异性转录程序的关系。我将通过设计在有丝分裂期间选择性降解的MLL-AF9融合蛋白来确定白血病转化是否需要有丝分裂特异性MLL-AF9功能。总之，这些实验有望深入了解MLL1执行其有丝分裂功能的机制，以及致癌MLL融合是否表现出干扰有丝分裂书签特性，从而阻碍正常的分化程序。更一般地说，据我们所知，提出的研究是第一次直接测试表观遗传有丝分裂记忆在细胞转化中的作用。

DESCRIPTION (provided by applicant): Normal development necessitates that cells establish specific gene expression patterns and transmit them stably through cell division. During mitosis, transcription ceases as most transcriptional regulators are removed from their target genes. The mechanism by which newly formed daughter cells reassemble the transcriptional apparatus to reactivate appropriate gene expression programs is thought to involve nuclear factors that remain bound to mitotic chromosomes, termed mitotic bookmarks. However, an understanding of what enables mitotic retention of these factors, as well as their specific role(s) in lineage commitment and/or lineage stability remains incomplete. The goal of this proposal is to define the mechanism by which the bookmarking factor MLL1 (mixed lineage leukemia 1) occupies its mitotic targets, and to determine whether mutations in MLL1 alter mitotic functions in such a way as to perturb hematopoiesis. Recent studies from our lab demonstrate that MLL1, a histone methyltransferase that is required for normal embryonic and adult hematopoiesis, is globally retained on mitotic chromosomes. MLL1 chromatin occupancy patterns are reorganized in mitosis, shifting towards the most highly expressed genes. Yet the physical interactions that control MLL1 mitotic redistribution, as well as its relevance to normal hematopoiesis are unknown. MLL1 is frequently rearranged via chromosomal translocation to generate leukemogenic fusion proteins that sustain aberrant gene expression programs and block hematopoietic differentiation. Preliminary results suggest that the MLL1 fusion protein, MLL-AF9, remains bound to mitotic chromosomes, however, whether this is required for MLL-AF9 to sustain inappropriate transcriptional patterns that result in leukemia is not known. In this proposal I will investigate the interactions that direct MLL1 mitotic occupancy, and examine the mitotic functions of MLL1 and MLL-AF9 in normal and malignant hematopoiesis. In Aim 1, I will systematically define the minimal region(s) of MLL1 that are both necessary and sufficient to bind mitotic chromatin, and assess mitosis-specific functions of MLL1 in hematopoietic progenitor cells. In Aim 2, I will examine the global occupancy patterns of MLL-AF9 and wild-type MLL1 in interphase and mitosis in leukemia cells, and define relationships to both normal and leukemia-specific transcriptional programs. I will determine if mitosis-specific MLL-AF9 function is required for leukemic transformation by engineering MLL-AF9 fusion proteins that are degraded selectively during mitosis. Together these experiments are expected to provide insight into the mechanisms by which MLL1 carries out its mitotic functions, and whether oncogenic MLL fusions exhibit perturbed mitotic bookmarking properties that impede normal programs of differentiation. More generally, the proposed studies are, to our knowledge, the first to directly test a role for epigenetic mitotic memory in cellular transformation.