The Molecular Mechanisms by which the Paf Oncogene Mediates Hematopoiesis

描述(由申请人提供)：造血干细胞(HSCs)的分化是生物体生命中生产和维持所有血细胞类型所必需的。在……里面 人，每天大约产生1011-1012个新的血细胞，以维持体内平衡。异常的造血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。在过去的50多年里，基础研究使得干细胞活性的描述成为可能，并促进了一种极其有益的治疗方法--造血干细胞移植的发展。对控制造血的分子机制的全面了解对于继续在治疗和/或预防人类血液系统恶性肿瘤和疾病方面取得进展是至关重要的。这一领域的进展对于制定更有效的造血干细胞移植策略也是至关重要的，特别是在成人中。我们最近发现高度保守的15 kDa增殖细胞核抗原相关因子(PAF)是一种新的、必不可少的HSC细胞周期调节因子。在没有PAF的情况下，HSC的功能受到损害。特别是，祖细胞发育受到干扰，中心和外周造血细胞室的大小缩小。当与野生型HSC竞争时，PAF缺乏的HSC发育被抹杀。因此，PAF是HSC同一性的关键决定因素。在R21的应用中，我们描述了将确定PAF介导HSC功能和发育的分子机制的实验系统。虽然我们已经证明PAF可以与增殖细胞核抗原相互作用，但这种相互作用是否真的在HSC的发育中发挥作用尚不清楚。增殖细胞核抗原在DNA复制和修复、细胞周期控制、染色质重塑、表观遗传、姐妹染色单体聚集和细胞存活等多个过程中发挥重要作用。因此，PAF的主要功能可能是修改一个 或更多的增殖细胞核抗原的功能。我们将通过研究表达不能与增殖细胞核抗原相互作用的突变PAF蛋白的小鼠，直接在AIM 1中测试这种可能性。我们的研究还表明，从细胞裂解物中纯化的PAF的很大一部分是通过在残基K24上添加单泛素和双泛素(Ub)来修饰的。PAF K24Ub-Ub的连锁是通过Ub的K63介导的，它被认为是改变蛋白质功能和/或细胞定位的。因此，目标2将侧重于对表达不能被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功能和发育的综合分析将确定这两个突变的PAF分子是否纠正了PAF-/-小鼠观察到的全部、部分或全部HSC缺陷。这些实验的数据代表了描绘PAF介导造血的分子机制的重要一步。

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.