POSITIONAL CLONING OF THE MEN1 GENE

我们早些时候已经证明，MEN1基因的突变与多发性内分泌肿瘤1型(MEN1)综合征有关，该综合征的特征是甲状旁腺、垂体前叶和胃肠道内分泌组织的多发性肿瘤。MEN1编码核蛋白Menin，与转录因子Jund和NFkB结合，并能抑制Jund和NFkB诱导的转录。通过在MAND和JUND阴性的小鼠成纤维细胞系中表达WT或突变的JUND，我们发现JUND的生长抑制功能(S)需要与MENIN相互作用。 我们已经开发了常规和有条件的小鼠基因敲除模型，这些模型产生的表型与人类MEN1疾病非常相似，并使我们能够描绘肿瘤发展的阶段。肝脏脑膜素的条件性敲除耐受性良好，MEN1综合征患者的脑膜组织不受影响，而甲状旁腺或胰岛的类似缺失会导致相应组织的肿瘤。此外，我们还建立了组织特异性薄荷素诱导的转基因小鼠模型。目前正在利用芯片技术研究脑膜素在细胞系和肿瘤发生过程中的表达变化，特别是与脑膜素相关的基因启动子的鉴定，以了解脑膜素的生物学特性。鉴于最近的研究表明，脑膜素是包括MLL(混合血统白血病)在内的巨大蛋白质复合体的关键成分，这一点尤其相关，MLL在组蛋白H3甲基化的转录调控中发挥关键作用。MLL在造血中起着至关重要的作用，因此我们对缺乏脑膜素的小鼠胚胎干细胞进行了造血分化试验，发现这一过程受到了严重影响。我们正在探索薄荷素在斑马鱼和非洲爪哇的造血和其他早期发育过程中的作用，这两种动物是这些研究的优秀模型。注射反义寡核苷酸(吗啉衍生物)来补充MEN1到1-2细胞期胚胎的剪接连接，导致MEN1 mRNA的剪接改变和Menin蛋白水平的降低。在斑马鱼和非洲爪哇的早期发育中，薄荷素水平的这种降低的影响将被跟踪。此外，还在果蝇中建立了MEN1的组织特异性转基因表达和敲除模型。这些模型应该有助于理解薄荷素的功能作用(S)。

We have shown earlier that mutations in the MEN1 gene are responsible for the Multiple endocrine neoplasia type 1 (MEN1) syndrome, characterized by multiple tumors of the parathyroid, anterior pituitary and GI endocrine tissues. The MEN1 encoded nuclear protein, Menin, binds the transcription factors JunD and NFkB, and can repress JunD and NFkB-induced transcription. By expressing WT or mutant JunD in mouse fibroblast cell lines that are null for menin and JunD, we find that interaction with menin is required for the growth suppressor function(s) of JunD. 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 tumor development. 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, and specifically identification of the promoters of the genes with which menin is associated are being studied, using ChIP on chip approach, to understand the biology of menin. 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. MLL plays a critical role in hematopoiesis, and therefore we tested hematopoietic differentiation of mouse ES (embryonic stem) cells lacking menin and found that this process was severely affected. We are exploring the role of menin in hematopoiesis and other early developmental processes in zebrafish and Xenopus laevis, which are excellent models for these studies. Injection of antisense oligonucleotides (morpholino derivatives) designed to complement splice junctions of Men1 to 1-2 cell stage embryos resulted in altered splicing of the Men1 mRNA and reduction in the levels of menin protein. The effect of such a reduction in menin levels in early development of zebrafish and Xenopus will be followed. 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.