Characterizing the Novel Protein C15orf65

 描述(由申请人提供)：C15orf65这一未鉴定的基因座，是我们之前发现的霍奇金淋巴瘤易位的一部分，它编码一种功能完全未知的小的、高度保守的15 kDa蛋白。我们制备了一种针对C15orf65 C末端的单抗，并证明该蛋白的表达依赖于细胞周期，其表达水平在细胞周期的G1/S阶段达到峰值。C15orf65蛋白的过度表达导致细胞周期增加，而基因敲除则降低了细胞周期，并在异种移植时消除了髓系细胞形成肿瘤的能力。在髓系和肾细胞系中，C15orf65定位于细胞核，尤其是染色质部分，这增加了它可能参与组织结合复合体的考虑。然而，其作用机制尚不清楚。对公共微阵列数据的分析表明，C15orf65在许多组织中都有表达，尤其是在乳房、骨小梁成骨细胞和造血干/祖细胞中高表达。C15orf65mRNA在急性髓系白血病的α亚型和骨髓增生异常综合征的RARS亚型中显著上调，提示C15orf65mRNA在恶性和正常造血中都有潜在的作用。C15orf65在动物界中高度保守，特别是在脊椎动物谱系中，并且在动物界之外还有额外的同源物。这种保守程度表明对保守功能的选择压力，我们假设这是由C15orf65的S主结构域DUF4490介导的。DUF4490的结构和功能都已确定的最近的近亲是Sgf29的组蛋白结合的Tudor结构域。然而，DUF4490的结构和功能迄今完全未知，因此是一个潜在的结构确定的信息靶点。C15orf65‘S的保守和定位与细胞周期表型和多种血液系统恶性肿瘤的参与相结合，提示C15orf65可能是一种未知的表观遗传调节因子，在调节造血细胞的细胞周期中具有特殊的作用。我们建议1)寻找C15orf65的结合伙伴以表征其途径相互作用；2)确定C15orf65的晶体和核磁共振结构；以及3)使用新产生的C15orf65小鼠同源基因Gm5918的条件基因敲除模型来描述C15orf65在造血中的功能作用。我们对C15orf65的研究将为我们深入了解这一新基因的生物物理、生化和功能特性提供线索，并可能为细胞周期调控和新的组蛋白结合域的鉴定提供一条新的途径。

 DESCRIPTION (provided by applicant): The uncharacterized locus C15orf65, which we previously identified as part of a translocation in Hodgkins lymphoma, encodes a small, highly conserved, 15kDa protein of completely unknown function. We generated a monoclonal antibody against the C terminus of C15orf65 and demonstrated that the protein is expressed in a cell-cycle-dependent fashion, with levels peaking in the G1/S phase of the cell cycle. Overexpression of the C15orf65 protein resulted in increased cell cycling, whereas knockdown decreased cell cycling and ablated the ability of myeloid cells to form tumors upon xenotransplantation. In myeloid and kidney cell lines, C15orf65 localized to the nucleus and to the chromatin fraction in particular, raising the consideration that it may participate in a histon-binding complex. However, its mechanism of action remains unexplored. Analysis of public microarray datasets showed C15orf65 mRNA expression in many tissues, with particularly high expression in the breast, trabecular bone osteoblasts, and hematopoietic stem and progenitor cells. C15orf65 mRNA levels are significantly upregulated in the PML-RARα subtype of acute myeloid leukemia (AML) and the RARS subtype of myelodysplastic syndrome (MDS), indicating a potential role in malignant as well as normal hematopoiesis. C15orf65 is highly conserved across the animal kingdom, especially within the vertebrate lineage, and has additional homologs outside of the animal kingdom. This degree of conservation indicates selective pressure for a conserved function, which we postulate is mediated by C15orf65's principal domain, DUF4490. The closest relative of DUF4490 for which both structure and function have been determined is the histone-binding Tudor domain of Sgf29. However, the structure and function of DUF4490 are as yet completely uncharacterized and thus represent a potentially informative target for structural determination. The combination of C15orf65's conservation and localization with a cell cycling phenotype and involvement in multiple hematopoietic malignancies suggests that C15orf65 may be a previously undescribed epigenetic regulator with a particular role in governing cell cycling in hematopoietic cells. We propose to 1) Identify binding partners for C15orf65 to characterize its pathway interactions; 2) Determine crystal and NMR structures for C15orf65; and 3) Delineate the functional role of C15orf65 in hematopoiesis using a newly generated conditional knockout model for the murine homolog of C15orf65, Gm5918. Our investigation of C15orf65 will provide insight into the biophysical, biochemical, and functional properties of this novel gene, and may also lead us to a new pathway for cell cycle regulation and identification of a novel histone binding domain.