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癌综合征，其特征是甲状旁腺，垂体前和GI（胃肠道）内分泌组织的多种肿瘤。我们之前已经表明，Men1基因中的突变是疾病的原因。我们发现MEN1编码的核蛋白主要驻留在细胞核中，结合了JUND和NFKB的转录因子，并且可以抑制JUND和NFKB诱导的转录。我们发现，JUND的生长抑制功能需要与Menin的相互作用。发现Menin与许多其他蛋白质相互作用，包括RPA2，RPA2是复制复合复合络合物中的蛋白质成分。 我们已经开发了常规的小鼠敲除模型，它们产生了与人类Men1疾病非常相似的表型，并使我们能够描绘胰腺内分泌肿瘤发展的阶段。从研究中可以明显看出，肿瘤发育的组织特异性是肝脏中梅宁的条件敲除良好的耐受性，这是MEN1综合征不影响的组织，而在甲状旁腺或胰腺胰岛中类似的损失会导致各自组织的肿瘤。此外，我们开发了特定于组织特异性的Menin诱导的转基因小鼠模型。已经评估了与细胞系和肿瘤发生过程中梅宁相关的表达变化。缺乏MENIN的小鼠成纤维细胞影响细胞外基质蛋白的表达变化，而这些基因的一部分的表达变化也由TGF-β介导。与ES（胚胎干）细胞中Menin丢失相关的表达变化表明，Menin可能会介导这些细胞的早期分化。 为了探讨梅宁在分化中的特定作用，我们在小鼠p19细胞中过度表达了梅宁，并评估了其对神经元，中胚层和内胚层谱系分化的影响。 使用芯片方法上的芯片研究了与梅宁相关的基因的启动子的鉴定。梅宁与数千种基因/启动子中有相关的基因。鉴于最近的证明是Menin是包括MLL（混合谱系白血病）的巨大蛋白质复合物的关键组成部分，这一点尤其重要，该蛋白质复合物（混合谱系白血病）通过组蛋白H3的甲基化在转录调节中起关键作用，并且通过调节HOX基因表达来调节造血中的甲基化，在造血中起关键作用。因此，我们测试了缺乏Menin的小鼠ES细胞的造血分化，发现该过程受到严重影响。我们发现，不仅梅宁，而且Hoxa9也可以挽救造血的缺乏。这表明Menin还通过HOXA9介导了其在造血中的作用，类似于MLL。我们正在探索梅宁在小鼠ES细胞中造血中的特定作用。也正在探索Menin在肿瘤和Xenopus laevis中造血（以及其他发育事件）中的作用。我们可以通过注射反义寡核苷酸（morpholino衍生物）来降低斑马鱼和Xenopus中的MEN1 mRNA和MENIN蛋白水平，旨在补充旨在补充剪接连接及ATG区域的ATG。在斑马鱼的早期发育中，没有任何明显的效果降低了梅宁水平。我们正在评估斑马鱼中种系MEN1突变的作用。此外，果蝇中开发了MEN1的组织特异性转基因表达和敲除模型。这些模型应有助于了解Menin的功能作用。 除了与Menin相关的研究外，我们还使用高分辨率BAC阵列进行了评估与乳腺癌和结肠癌相关的基因组变化。在乳腺癌中放大并过表达了修饰碱基Wybutosine（YW）的生物合成途径中编码酶的TRMT12基因。 这种高分辨率技术在评估基因组变化时的应用可能会导致鉴定出可以提供更好诊断工具或开放新型治疗途径的新型目标。