Characterizing the Novel Protein C15orf65

 描述（由申请人提供）：我们先前鉴定为霍奇金斯淋巴瘤易位的一部分的未表征基因座C15 orf 65编码一种功能完全未知的小的高度保守的15 kDa蛋白。我们产生了一个单克隆抗体对C末端的C15 orf 65，并证明该蛋白质的表达在细胞周期依赖的方式，在G1/S期的细胞周期的水平达到峰值。C15 orf 65蛋白的过表达导致细胞周期增加，而敲低则降低细胞周期并消除骨髓细胞在异种移植后形成肿瘤的能力。在髓系和肾细胞系中，C15 orf 65定位于细胞核，特别是染色质部分，这引起了人们对它可能参与组蛋白结合复合物的考虑。然而，其作用机制尚未探索。公共微阵列数据集的分析显示，C15 orf 65 mRNA在许多组织中表达，在乳腺、小梁骨成骨细胞、造血干细胞和祖细胞中表达特别高。C15 orf 65 mRNA水平在急性髓性白血病（AML）的PML-RARα亚型和骨髓增生异常综合征（MDS）的RARS亚型中显著上调，表明在恶性和正常造血中的潜在作用。C15 orf 65在动物界中高度保守，特别是在脊椎动物谱系中，并且在动物界之外具有额外的同源物。这种保守程度表明保守功能的选择压力，我们假设这是由C15 orf 65的主要结构域DUF 4490介导的。结构和功能都已确定的DUF 4490的最近亲属是Sgf 29的组蛋白结合Tudor结构域。然而，DUF 4490的结构和功能尚未完全表征，因此代表了用于结构测定的潜在信息靶点。C15 orf 65的保护和定位与细胞周期表型和参与多种造血系统恶性肿瘤的组合表明，C15 orf 65可能是一个以前未描述的表观遗传调节因子，在造血细胞的细胞周期中具有特定的作用。我们建议：1）确定C15 orf 65的结合伴侣，以表征其途径相互作用; 2）确定C15 orf 65的晶体和NMR结构; 3）使用新生成的C15 orf 65小鼠同源物Gm 5918的条件性敲除模型，描述C15 orf 65在造血中的功能作用。我们对C15 orf 65的研究将深入了解这种新基因的生物物理、生化和功能特性，也可能引导我们找到一种新的细胞周期调控途径和鉴定一种新的组蛋白结合结构域。

 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.