Characterizing the Novel Protein C15orf65

新型蛋白质 C15orf65 的表征

• 批准号: 9123206
• 负责人: Ruth Ann Howe
• 金额: --
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-05 至 2016-04-06
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9123206
• 关键词: Acute Myelocytic Leukemia; Address; Affinity Chromatography; Animal Model; Animals; Binding; Biochemical; Bioinformatics; Biological Assay; Bone Marrow; Breast; Cell Cycle; Cell Cycle Regulation Pathway; Cell Line; Cell Nucleus; Cell Proliferation; Cells; Cellular biology; ChIP-seq; Chimeric Proteins; Chromatin; Code; Complex; Computer Simulation; Coupled; DNA; Data; Data Set; Development; Disease; Dysmyelopoietic Syndromes; Epigenetic Process; Escherichia coli; Gene Proteins; Genes; Genetic; Genomics; Goals; Hematopoiesis; Hematopoietic; Hematopoietic Neoplasms; Hematopoietic System; Heterochromatin; Histones; Hodgkin Disease; Homologous Gene; Immunohistochemistry; Immunoprecipitation; In Vitro; Investigation; Knock-out; Lead; Lymphoid; MHC Class II Genes; MHC class II transactivator protein; Malignant - descriptor; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Messenger RNA; Microarray Analysis; Modeling; Monoclonal Antibodies; Mus; Myelogenous; Myeloid Cells; Nuclear; Nuclear Magnetic Resonance; Nuclear Protein; Oncogenic; Osteoblasts; PML-RARalpha protein; Pathway Analysis; Pathway interactions; Patients; Peptides; Phenotype; Population; Property; Proteins; RUNX1 gene; Role; S Phase; SAGA; Selenomethionine; Sorting - Cell Movement; Staging; Stem cells; Structure; Techniques; Testing; Tissues; Training; Western Blotting; Work; X-Ray Crystallography; Xenograft procedure; base; cell type; chromatin immunoprecipitation; congeneic transplantation; genome sequencing; in vivo; insight; kidney cell; knock-down; mRNA Expression; member; mouse model; novel; overexpression; pressure; protein function; public health relevance; reconstitution; research study; stem; structural biology; substantia spongiosa; therapeutic development; transcription factor; tumor; whole genome

 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.

 描述（由适用提供）：未表征的基因座C15ORF65，我们以前识别为Hodgkins淋巴瘤中易位的一部分，它编码具有完全未知功能的小型，高度保守的15KDA蛋白。我们生成了针对C15ORF65的C末端的单克隆抗体，并证明该蛋白是以细胞周期依赖性方式表达的，水平在细胞周期的G1/S相峰值。 C15ORF65蛋白的过表达导致细胞循环增加，而敲低降低了细胞循环，并消融了髓样细胞在异种移植后形成肿瘤的能力。在髓样和肾细胞系中，C15ORF65局部局部局部和染色质级分，提高了它可能参与病史结合复合物的考虑。但是，其作用机理仍然出乎意料。对公共微阵列数据集的分析显示了许多组织中的C15orf65 mRNA表达，在乳房，小梁骨成骨细胞以及造血茎和祖细胞中特别高表达。在急性髓样白血病（AML）的PML-RARα亚型中，C15ORF65 mRNA水平得到显着更新，而骨髓增生性综合征（MDS）的RARS亚型表示，表明在恶性和正常造血中具有潜在的作用。 C15ORF65在整个动物界，尤其是脊椎动物谱系中高度保守，并且在动物界之外还具有其他同源物。这种保存程度表示配置函数的选择性压力，我们假设这是由C15orf65的主要域DUF4490介导的。确定结构和功能的DUF4490的最接近的亲戚是SGF29的组蛋白结合帝团域。但是，DUF4490的结构和功能尚未完全均匀化，因此代表了结构确定的潜在信息目标。 C15ORF65的保护和定位与细胞循环表型以及参与多种造血恶性肿瘤的结合表明，C15ORF65可能是先前未描述的表观遗传调节剂，在造血细胞中的细胞循环中具有特殊作用。我们建议1）确定C15orf65的结合伙伴以表征其途径相互作用； 2）确定C15orf65的晶体和NMR结构； 3）使用新生成的有条件敲除模型为C15ORF65的鼠同源物，GM5918描述C15ORF65在造血中的功能作用。我们对C15ORF65的投资将提供对该新基因的生物物理，生化和功能特性的见解，并且也可能导致我们进入新的细胞周期调节途径和新型组蛋白结合结构域的鉴定。