POSITIONAL CLONING OF THE MEN1 GENE

MEN1 基因的定位克隆

• 批准号: 7594295
• 负责人: settara chandrasekharappa
• 金额: $ 170.87万
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 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/7594295
• 关键词: Affect; Anterior Pituitary Gland; Antisense Oligonucleotides; Antithymoglobulin; Binding; Breast; Cell Differentiation process; Cell Line; Cell Nucleus; Cells; Cellular biology; Colon Carcinoma; Complement; Complex; Development; Diagnostic; Disease; Drosophila genus; Endocrine; Enzymes; Evaluation; Event; Extracellular Matrix Proteins; Fibroblasts; Gene Expression; Gene Mutation; Genes; Genetic Transcription; Genomics; Germ-Line Mutation; Growth; Hematopoiesis; Hematopoietic; Histone H3; Human; Inherited; Injection of therapeutic agent; Islet Cell Tumor; Islets of Langerhans; Knock-out; Knockout Mice; Lead; Light; Liver; Malignant Neoplasms; Maps; Mediating; Menin; Messenger RNA; Methylation; Modeling; Multiple Endocrine Neoplasia Type 1; Mus; Mutation; Neurons; Nuclear Protein; Nuclear Proteins; Numbers; Parathyroid Neoplasms; Parathyroid gland; Pathway interactions; Personal Satisfaction; Phenotype; Phenylalanine-Specific tRNA; Play; Process; Protein Overexpression; Proteins; RNA Splicing; Resolution; Role; Somatic Cell; Specificity; Staging; Syndrome; Techniques; Testing; Tissues; Transcriptional Regulation; Transgenic Organisms; Xenopus; Xenopus laevis; Zebrafish; base; design; embryonic stem cell; gastrointestinal; leukemia; malignant breast neoplasm; mouse model; novel; novel therapeutics; promoter; protein expression; tool; transcription factor; tumor; tumorigenesis; wybutosine

MEN1 is an autosomal dominant cancer syndrome characterized by multiple tumors of the parathyroid, anterior pituitary and GI (gastrointestinal) endocrine tissues. We have shown earlier that mutations in the MEN1 gene are responsible for the disease. We find that the MEN1 encoded nuclear protein, Menin, resides primarily in the nucleus, binds the transcription factors JunD and NFkB, and can repress JunD and NFkB-induced transcription. We find that interaction with menin is required for the growth suppressor function(s) of JunD. Menin was found to interact many other proteins including RPA2, a protein component in a replication complex. We have developed both conventional and conditional mouse knockout models, which yield phenotypes that are remarkably similar to the human MEN1 disease, and have allowed us to delineate the stages in development of the pancreatic endocrine tumors. Tissue specificity of the tumor development was evident from the studies that conditional knockout of menin in liver was well tolerated, a tissue not affected in MEN1 syndrome, whereas similar loss in parathyroid or pancreatic islets resulted in tumors of the respective tissues. In addition, we have developed tissue specific menin-inducible transgenic mouse models. Expression changes associated with menin in cell lines and during tumorigenesis have been evaluated. Mouse fibroblasts lacking menin affected changes in the expression of extracellular matrix proteins, and expression changes for a fraction of these genes was also mediated by TGF-β. Expression changes associated with the loss of menin in ES (embryonic stem) cells suggest that menin may mediate early differentiation of these cells. In order to pursue specific role of menin in differentiation, we are overexpressing menin in mouse P19 cells and evaluating its effect on differentiation into neuronal, meso- and endodermal lineages. Identification of the promoters of the genes with which menin is associated was studied, using ChIP on chip approach. Included among thousands of genes/promoters that menin was associated were hox genes. This is particularly relevant in light of the recent demonstration that menin is a critical component of a huge protein complex that includes MLL (mixed lineage leukemia), which plays key role in transcriptional regulation by methylation of Histone H3, and plays a critical role in hematopoiesis by modulating hox gene expression. Therefore, we tested hematopoietic differentiation of mouse ES cells lacking menin and found that this process was severely affected. We find that not only menin but also hoxa9 can rescue the deficiency in hematopoiesis. This indicates that menin also mediates its role in hematopoiesis via hoxa9, similar to that by MLL. We are exploring the specific role(s) of menin in hematopoiesis in mouse ES cells; the role of menin in hematopoiesis (and other developmental events) in zebrafish and Xenopus laevis, are also being explored. We could bring down the the Men1 mRNA and menin protein levels in zebrafish and Xenopus by injection of antisense oligonucleotides (morpholino derivatives) designed to complement splice junctions and the region around ATG. There was not any apparent effect such a reduction in menin levels in early development in zebrafish. We are evaluating the effect of germline Men1 mutations in zebrafish. In addition, tissue specific transgenic expression and knockout models for MEN1 are developed in Drosophila. These models should help to understand the functional role(s) of menin. Apart from studies associated with menin, we are also evaluation genomic changes associated with breast and colon cancer using high-resolution BAC arrays. The TRMT12 gene encoding an enzyme in the biosynthetic pathway of a modified base, wybutosine (yW) in tRNA Phe was amplified and overexpressed in breast cancer. Application of such high-resolution techniques for evaluation of genomic changes are likely to lead to the identification of novel targets that may serve a s better diagnostic tools or open up novel therapeutic avenues.

MEN1是一种常染色体显性遗传癌症综合征，其特征在于甲状旁腺、垂体前叶和GI（胃肠道）内分泌组织的多发性肿瘤。我们之前已经证明MEN1基因的突变是导致这种疾病的原因。我们发现MEN1编码的核蛋白Menin主要存在于细胞核中，与转录因子JunD和NFkB结合，并能抑制JunD和NFkB诱导的转录。我们发现与menin的相互作用是JunD的生长抑制功能所必需的。Menin被发现与许多其他蛋白质相互作用，包括复制复合物中的蛋白质组分RPA 2。 我们已经开发了常规和条件性小鼠基因敲除模型，其产生的表型与人类MEN1疾病非常相似，并使我们能够描绘胰腺内分泌肿瘤的发展阶段。肿瘤发展的组织特异性从以下研究中显而易见：肝脏中menin的条件性敲除耐受良好，该组织在MEN1综合征中不受影响，而甲状旁腺或胰岛中的类似损失导致相应组织的肿瘤。此外，我们已经开发了组织特异性脑膜炎诱导的转基因小鼠模型。与menin在细胞系和肿瘤发生过程中的表达变化进行了评估。缺乏menin的小鼠成纤维细胞影响细胞外基质蛋白表达的变化，并且这些基因的一部分的表达变化也由TGF-β介导。β与胚胎干细胞中menin缺失相关的表达变化表明，menin可能介导这些细胞的早期分化。 为了研究menin在分化中的特殊作用，我们在小鼠P19细胞中过表达menin，并评估其对分化为神经元、中胚层和内胚层谱系的影响。 使用ChIP芯片方法研究了menin相关基因的启动子的鉴定。在与menin相关的数千个基因/启动子中包括hox基因。这是特别相关的，鉴于最近的证明，menin是一个巨大的蛋白质复合物，其中包括MLL（混合谱系白血病），发挥关键作用，在转录调控组蛋白H3的甲基化，并发挥关键作用，在造血通过调节hox基因表达的关键组成部分。因此，我们测试了缺乏menin的小鼠ES细胞的造血分化，发现这一过程受到严重影响。我们发现不仅menin而且hoxa 9都可以挽救造血缺陷。这表明menin也通过hoxa 9介导其在造血中的作用，类似于MLL。我们正在探索menin在小鼠ES细胞造血中的特定作用;也正在探索menin在斑马鱼和非洲爪蟾造血（和其他发育事件）中的作用。我们可以通过注射反义寡核苷酸（吗啉代衍生物）来降低斑马鱼和非洲爪蟾中Men1 mRNA和menin蛋白的水平，这些反义寡核苷酸被设计用于补充剪接点和ATG周围的区域。在斑马鱼的早期发育中，menin水平的降低没有任何明显的影响。我们正在评估斑马鱼生殖系Men1突变的影响。此外，MEN1的组织特异性转基因表达和敲除模型在果蝇中被开发。这些模型应该有助于理解menin的功能作用。 除了与menin相关的研究，我们还使用高分辨率BAC阵列评估与乳腺癌和结肠癌相关的基因组变化。TRMT12基因编码一种酶的生物合成途径中的一个修饰的碱基，wybutosine（yW）在tRNA苯丙氨酸扩增和过表达的乳腺癌。 应用这种高分辨率技术来评估基因组变化可能会导致识别新的靶点，这些靶点可能会成为更好的诊断工具或开辟新的治疗途径。