Characterization of candidate histone methyltransferases

候选组蛋白甲基转移酶的表征

• 批准号: 7592813
• 负责人: David Eric Symer
• 金额: $ 33.87万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7592813
• 关键词: Antibodies; Asthma; B-Lymphocytes; Baculoviruses; Binding; Biochemical; Biological Process; Chimeric Proteins; Chronic Lymphocytic Leukemia; Co-Immunoprecipitations; Defect; Development; Disease; Gene Family; Genes; Genetic Transcription; Goals; Histones; Human; Knock-out; Knockout Mice; Laboratories; Link; Localized; Lysine; Malignant Neoplasms; Methylation; Modeling; Mouse Strains; Mus; Plants; Process; Protein Binding; Proteins; Quantitative Trait Loci; RNA Splicing; Recombinants; Role; Role playing therapy; Structure; Tissues; Transcript; Yeasts; Zinc Fingers; atopy; base; histone methyltransferase; homeodomain; human disease; interest; member; mouse model; research study; yeast two hybrid system

We previously conducted a comprehensive analysis of transcription of a candidate histone methyltransferase, Setdb2, in human and mouse tissues, revealing tissue-specific expression of alternatively spliced and chimeric transcripts. We found in-frame fusion transcripts linking Setdb2 to an adjacent independent gene, Phf11, which encodes a zinc finger-containing plant homeodomain motif. We previously found several protein-binding partners for the putative histone methyltransferases using yeast two hybrid screens; most of these binding partners now have been validated by co-immunoprecipitation experiments. Further functional characterization of interactions between Setdb2 and the identified binding partners, which include an unannotated protein and other biologically interesting proteins, has been conducted. We have generated a viable, hypomorphic knockout mouse model lacking 70-80% of Setdb2 transcripts and probably most or all encoded proteins. Phenotypic characterization of these knockouts on various mouse strain backgrounds has begun. Occasional homozygous hypomorphic mice with a B cell developmental defect have been identified, suggesting they may suffer from a pre-CLL disease process. The knockout mice could comprise a new model for chronic lymphocytic leukemia, for asthma, and/or for other cancers or human diseases. Specific antibodies against mouse and human Setdb2 have been developed. We have expressed recombinant Setdb2 in baculovirus to facilitate characterization of its biochemical activities. On a preliminary basis, we have shown that Setdb2 is an active histone methyltransferase and may also methylate other cellular proteins.

我们以前进行了全面的分析，在人类和小鼠组织中的候选组蛋白甲基转移酶，Setdb 2的转录，揭示了组织特异性表达的选择性剪接和嵌合转录。我们发现框内融合转录连接Setdb 2到一个相邻的独立基因，Phf 11，它编码一个锌指含植物同源结构域基序。我们以前发现了几个蛋白质结合伙伴的假定组蛋白甲基转移酶使用酵母双杂交筛选，这些结合伙伴中的大多数现在已经通过免疫共沉淀实验验证。Setdb 2和所鉴定的结合伴侣之间的相互作用的进一步功能表征已经进行，所述结合伴侣包括未注释的蛋白质和其他生物学上感兴趣的蛋白质。我们已经产生了一个可行的，亚型敲除小鼠模型缺乏70-80%的Setdb 2转录本，可能是大多数或所有编码的蛋白质。已经开始在各种小鼠品系背景上对这些敲除进行表型表征。已经鉴定了偶尔的具有B细胞发育缺陷的纯合子低形态小鼠，表明它们可能患有前CLL疾病过程。基因敲除小鼠可以构成慢性淋巴细胞白血病、哮喘和/或其他癌症或人类疾病的新模型。已经开发了针对小鼠和人Setdb 2的特异性抗体。我们已经表达重组Setdb 2杆状病毒，以方便其生化活性的表征。在初步的基础上，我们已经表明，Setdb 2是一个活跃的组蛋白甲基转移酶，也可能甲基化其他细胞蛋白。