Regulation of hematopoietic stem cell maintenance and survival by NKAP

描述(由申请人提供)：我们发现，我们实验室首次在功能上定义的转录抑制因子NKAP，对于维持造血干细胞库是绝对必要的。稳定状态的造血是通过分化与造血干细胞的增殖和自我更新平衡来维持的。这种平衡的破坏可能会导致造血衰竭，就像没有自我更新的造血分化会导致HSC池的丧失一样。了解HSC维持的分子调控可能导致治疗干预以加速HSC的重建，例如在骨髓移植中。在成年小鼠中，NKAP的可诱导缺失会导致造血衰竭和快速死亡。NKAP缺失会导致完全的细胞固有丢失 在所有的造血干细胞中，由于存活率下降和增殖减少的组合。因此，NKAP对HSC的维持和生存至关重要。为了了解NKAP在HSCs中的分子基础，我们进行了酵母双杂交筛选，发现NKAP与多梳抑制复合体-1(Prc1)E3泛素连接酶Ring1b相关。PRC1被招募到H3K27三甲基化的位点，这是一个抑制性的表观遗传标记，导致H_2AK119的单泛素化。PRC1的三个组成部分Ring1b、Bmi-1和RAE28的突变破坏了HSC的功能，表明这一途径对维持HSC池的重要性。在体外短期培养的HSC中，NKAP的缺失导致H_2AK119单核苷酸的表达下调，表明NKAP是HSC中PRC1的关键调节因子。本研究将致力于阐明NKAP对Ring1b/Prc1的调控机制，揭示NKAP参与HSC维持和存活的分子基础。具体目的1：明确NKAP调控Ring1b/Prc1功能的机制；特异性目标2：确定NKAP下游与HSC维持和存活所需的基因；特异性目标3：通过鉴定NKAP介导的HSC维持和存活调节所需的蛋白-蛋白结构域和结合，确定NKAP在HSC中调控的关键通路。

DESCRIPTION (provided by applicant): We have found that a transcriptional repressor first defined functionally in our laboratory, NKAP, is absolutely required for the maintenance of the hematopoietic stem cell (HSC) pool. Steady state hematopoiesis is sustained through differentiation balanced with proliferation and self-renewal of HSCs. Disruption of this balance can lead to hematopoietic failure, as in the case of hematopoietic differentiation without self-renewal which leads to the loss of the HSC pool. An understanding of the molecular regulation of HSC maintenance could lead to therapeutic interventions to hasten HSC reconstitution, for example, in bone marrow transplants. In adult mice, inducible deletion of NKAP results in hematopoietic failure and rapid lethality. NKAP deletion results in a complete, cell-intrinsic loss of all HSCs, through a combination of decreased survival and decreased proliferation. Therefore, NKAP is critical for HSC maintenance and survival. To understand the molecular basis for the requirement of NKAP in HSCs, we performed a yeast two hybrid screen, and identified that NKAP associates with the polycomb repressive complex-1 (PRC1) E3 ubiquitin ligase Ring1b. PRC1 is recruited to sites of H3K27 trimethylation, an inhibitory epigenetic mark, leading to monoubiquitination of H2AK119. Mutations in three components of PRC1, Ring1b, Bmi-1, and Rae28, disrupt HSC function, indicating the importance of this pathway for maintaining the HSC pool. In ex vivo short term HSC cultures, loss of NKAP leads to a global and severe downregulation of H2AK119 monoubiquitation, indicating that NKAP is a critical regulator of PRC1 in HSCs. This proposal will focus on elucidating the mechanism of Ring1b/PRC1 regulation by NKAP, and to uncover the molecular basis for the contribution of NKAP to HSC maintenance and survival. Specific Aim 1: Defining the mechanism of regulation of Ring1b/PRC1 function by NKAP Specific Aim 2: Identification of genes downstream of NKAP that are required for HSC maintenance and survival Specific Aim 3: Defining the critical pathways regulated by NKAP in HSCs through identification of the protein-protein domains and associations required for NKAP-mediated regulation of HSC maintenance and survival.