Regulation of hematopoietic stem cell maintenance and survival by NKAP

描述（由申请人提供）：我们已经发现，在我们的实验室中首次功能性定义的转录阻遏物NKAP对于维持造血干细胞（HSC）库是绝对必需的。稳态造血通过与HSC的增殖和自我更新平衡的分化来维持。这种平衡的破坏可导致造血功能衰竭，如在没有自我更新的造血分化的情况下，这导致HSC库的损失。对HSC维持的分子调控的理解可能导致治疗干预以加速HSC重建，例如，在骨髓移植中。在成年小鼠中，NKAP的可诱导缺失导致造血衰竭和快速致死。NKAP缺失导致完全的细胞内在损失 所有的HSC，通过减少存活和减少增殖的组合。因此，NKAP对HSC的维持和存活至关重要。为了理解HSC中NKAP需求的分子基础，我们进行了酵母双杂交筛选，并鉴定了NKAP与多梳阻遏复合物-1（PRC 1）E3泛素连接酶Ring 1b相关。PRC 1被募集到H3 K27三甲基化位点，这是一种抑制性表观遗传标记，导致H2 AK 119的单泛素化。PRC 1、Ring 1b、Bmi-1和Rae 28三个组分的突变破坏了HSC功能，表明该途径对维持HSC库的重要性。在离体短期HSC培养物中，NKAP的缺失导致H2 AK 119单重定量的全面和严重下调，表明NKAP是HSC中PRC 1的关键调节剂。本研究旨在阐明NKAP对Ring 1b/PRC 1的调控机制，并揭示NKAP对HSC维持和存活的分子基础。具体目标1：明确NKAP调控Ring 1b/PRC 1功能的机制具体目标2：确定HSC维持和存活所需的NKAP下游基因具体目标3：通过确定NKAP介导的HSC维持和存活调控所需的蛋白质-蛋白质结构域和相关性，确定NKAP在HSC中调控的关键途径。

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.