The Molecular Mechanisms by which the Paf Oncogene Mediates Hematopoiesis

描述（由申请人提供）：造血干细胞（HSC）的分化是生物体生命中所有血细胞类型的产生和维持所必需的。在 对于人来说，每天大约产生1011 - 1012个新的血细胞以维持体内平衡。造血的异常控制是许多疾病的根本原因，包括免疫缺陷、贫血、自身免疫、白血病和淋巴瘤。过去50多年的基础研究已经允许描述干细胞活性，并促进了一种非常有益的治疗方法-HSC移植的发展。完全理解控制造血的分子机制对于治疗和/或预防人类血液恶性肿瘤和疾病的持续进展至关重要。该领域的进展对于开发更有效的HSC移植策略也至关重要，特别是在成人中。我们最近发现，高度保守的15 kDa增殖细胞核抗原（PCNA）相关因子（Paf）是一种新的和必要的HSC细胞周期调控因子。在没有Paf的情况下，HSC功能受损。特别是，祖细胞发育被破坏，并且中央和外周造血细胞区室的大小减少。当与野生型HSC竞争时，Paf缺陷型HSC的发育被消除。因此，Paf是HSC身份的关键决定因素。在这个R21的应用中，我们描述了实验系统，将确定的分子机制，通过它的PAF介导的HSC的功能和发展。虽然我们已经证明了Paf可以与PCNA相互作用，但尚不清楚这种相互作用是否真的在HSC的发展中起作用。PCNA在DNA复制和修复、细胞周期控制、染色质重塑、表观遗传、姐妹染色单体凝聚和细胞存活等多个过程中发挥重要作用。因此，可能Paf的主要功能是修改一个 或多个PCNA的功能。我们将通过研究表达突变PAF蛋白的小鼠直接测试这种可能性，该突变PAF蛋白不能与PCNA相互作用。我们的研究还表明，从细胞裂解物中纯化的PAF的一个显着的部分是通过添加单和二泛素（Ub）残基K24修改。PAF K24 Ub-Ub连接通过Ub的K63介导，其被理解为修饰蛋白质功能和/或细胞定位。因此，目的2将集中于对表达不能被K24 Ub修饰的PAF突变体的小鼠的分析。这些研究将确定PAF的功能是否依赖于K63连接的Ub介导的与其他蛋白质的相互作用。对HSC功能和发育的全面分析将确定两种突变PAF分子是否纠正了在Paf-/-小鼠中观察到的所有、部分或没有HSC缺陷。从这些实验的数据代表了一个重要的一步，描绘的分子机制，通过它的PAF功能介导造血。

DESCRIPTION (provided by applicant): Differentiation of hematopoietic stem cells (HSCs) is required for the production and maintenance of all blood cell types for the life of an organism. In people, approximately 1011-1012 new blood cells are produced daily in order to maintain the homeostasis. Aberrant control of hematopoiesis is the underlying cause of many diseases including immune deficiency, anemia, autoimmunity, leukemia, and lymphoma. Basic research over the last 50+ years has allowed for the description of stem cell activity and has facilitated the development of an extremely beneficial treatment - HSC transplantation. A complete understanding of the molecular mechanisms that control hematopoiesis is essential for continued progress in the treatment and/or prevention of hematological malignancies and diseases in people. Progress in the field is also essential to develop more efficient strategies fo HSC transplantation, particularly in adults. We recently showed that the highly conserved 15 kDa Proliferating Cell Nuclear Antigen (PCNA) associated factor (Paf) is a novel and essential HSC cell cycle regulator. In the absence of Paf, HSC function is compromised. In particular, progenitor development is disrupted and there is a reduction in the size of central and peripheral hematopoietic cell compartments. When placed in competition with wild type HSCs, Paf deficient HSC development is obliterated. Therefore, Paf is a key determinant for HSC identity. In this R21 application, we describe experimental systems that will determine the molecular mechanisms by which Paf mediates HSC function and development. While we have shown that Paf can interact with PCNA, it is not known if this interaction actually plays a role in HSC development. PCNA has essential roles in multiple processes, such as DNA replication and repair, cell cycle control, chromatin remodeling, epigenetic inheritance, sister chromatid cohesion, and cell survival. Therefore, it is possible that Paf's primary function is to modify one or more of the functions of PCNA. We will directly test this possibility in Aim 1 by studying mice that express a mutant PAF protein that is unable to interact with PCNA. Our studies have also shown that a significant fraction of PAF purified from cell lysates is modified by the addition of mono- and di-ubiquitin (Ub) on residue K24. The PAF K24 Ub-Ub linkage is mediated via K63 of Ub, which is understood to modify protein function and/or the cellular localization. Aim 2, therefore will focus on the analyses of mice that express a PAF mutant that cannot be K24 Ub modified. These studies will determine if the function of PAF is dependent upon K63-linked Ub mediated interactions with other proteins. A comprehensive analysis of HSC function and development will determine if the two mutant PAF molecules correct all, some or none of the observed HSC defects in Paf-/- mice. The data from these experiments represent an essential step towards delineating the molecular mechanisms by which Paf functions to mediate hematopoiesis.