POSITIONAL CLONING OF THE MEN1 GENE

我们之前已经证明MEN1基因的突变是家族性癌症综合征，多发性内分泌肿瘤1型（MEN1）的原因。MEN1以甲状旁腺、垂体前叶和胃肠道内分泌组织多发肿瘤为特征。我们发现MEN1编码的蛋白menin主要存在于细胞核中。我们发现menin与转录因子JunD结合，这种相互作用是JunD生长抑制功能所必需的。Menin与另一个转录因子NFkb相互作用，在这两种情况下，相互作用都导致由JunD或NFkb驱动的转录抑制。Menin已被发现是包括MLL（混合谱系白血病）在内的巨大蛋白复合物的一个组成部分，MLL在组蛋白H3甲基化的转录调节中起关键作用。Menin是该复合体的关键成分，因为它是MLL最佳甲基化酶活性所必需的。在menin与转录调控因子相互作用的提示下，利用ChIP芯片方法，我们已经确定了与menin相关的数千个基因启动子。这项研究使我们能够确定与MLL单独相关的启动子，以及与MLL和menin都相关的启动子。MLL在造血过程中起着至关重要的作用，因此我们测试了menin是否需要这种分化过程。我们制备了缺乏menin的小鼠胚胎干细胞，并诱导其进行造血分化。我们发现，在缺乏menin的细胞中，产生造血集落的能力受到严重影响。MLL通过调节Hox家族成员如Hoxa9和Hoxc6的表达来调节其在造血中的作用，我们发现这些基因在menin-null ES细胞中的表达降低。通过在胚胎中注射反义寡核苷酸（morpholino衍生物）减少menin，使我们能够评估menin在斑马鱼造血中的作用；这种减少似乎不会对胚胎的血液发育产生不利影响，也不会改变这一过程中关键基因（如Hoxa9和Gata1）的表达。现在我们已经分离出一种斑马鱼menin错义突变体，由化学诱变诱导，我们正在努力评估menin在斑马鱼生长和发育中的作用，如果有的话。我们继续利用爪蟾和果蝇等其他模型系统探索menin的生物学功能：果蝇MEN1的转基因表达和敲除模型帮助我们确定了menin和Jun/Fos之间的遗传相互作用。我们已经开发了常规和条件小鼠敲除模型，其产生的表型与人类MEN1疾病非常相似，并使我们能够描述肿瘤发展的阶段。肝脏中menin的条件敲除耐受良好，MEN1综合征不影响组织，而甲状旁腺或胰岛的类似缺失导致相应组织的肿瘤。我们开发了高密度BAC阵列，并通过鉴定乳腺癌中几个新的区域扩增和缺失，证明了它们在评估肿瘤基因组变化方面的有用性：我们打算使用这些阵列来研究与MEN1肿瘤相关的基因组改变。除了在肿瘤发生中发挥作用外，menin似乎在早期发育中发挥关键作用，因为小鼠Men1等位基因的纯合缺失会导致胚胎致死（E11.5-E13.5），并导致包括心脏在内的几个器官发育缺陷。我们建立了缺乏menin的小鼠胚胎成纤维细胞（MEF）系，并对其表达模式等特性进行了评价。Menin-null mef显示编码细胞外基质蛋白的几个基因的表达改变。这些由TGF-b调节的基因被认为是心脏早期发育所必需的。我们还发现缺乏menin的MEF细胞系对TGF-b的反应较差。menin在menin-null mef中的重新表达，无论是短暂的还是稳定的，都导致menin调控的鉴定基因。评估与menin相关的表达变化，以及各种动物模型的有用性，可能会揭示menin的基本生物学功能。

We have shown earlier that mutations in the MEN1 gene are responsible for the familial cancer syndrome, Multiple endocrine neoplasia type 1 (MEN1). MEN1 is characterized by multiple tumors of the parathyroid, anterior pituitary and GI endocrine tissues. We found that the MEN1 encoded protein, menin, resides primarily in the nucleus. We discovered that menin binds the transcription factor JunD, and this interaction is required for the growth suppressor function(s) of JunD. Menin interacts with another transcription factor NFkb, and in both instances, the interaction results in the repression of transcription driven by JunD or NFkB. Menin has been found to be a component of a huge protein complex that includes MLL (mixed lineage leukemia), which plays key role in transcriptional regulation by methylation of Histone H3. Menin is a critical component of this complex as it is required for the optimal methylase activity of MLL. Prompted by the interaction of menin with transcriptional regulators, using ChIP on chip approach, we have identified thousands of promoters of the genes with which menin is associated. This study has allowed us to identify the promoters that are also associated with MLL alone, and with both MLL and menin. MLL plays a critical role in hematopoiesis, and therefore we tested whether menin is required for this differentiation process. We generated mouse ES (embryonic stem) cells lacking menin, and induced them to undergo hematopoietic differentiation. We found that the ability to generate hematopoietic colonies was severely affected in cells lacking menin. MLL mediates its role in hematopoiesis by regulating the expression of Hox family members such as Hoxa9 and Hoxc6, and we find decreased expression of these genes in menin-null ES cells. Reduction of menin by injections of antisense oligonucleotides (morpholino derivatives) in embryos allowed us to evaluate the role of menin in zebrafish hematopoiesis; the reduction did not appear to adversely affect blood development in embryos or alter the expression of the critical genes in this process such as Hoxa9 and Gata1. Now that we have isolated a zebrafish menin missense mutant, induced by chemical mutagenesis, efforts are underway to evaluate the role of menin, if any, in zebrafish growth and development. We continue to explore the biological function(s) of menin using other model systems such as Xenopus and Drosophila: transgenic expression and knockout models for MEN1 in Drosophila helped us identify the genetic interactions between menin and Jun/Fos. 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. We have developed high-density BAC arrays and demonstrated their usefulness in evaluating the genomic changes in tumors by the identification of several novel regions amplification and deletion in breast cancer: We intend to use these arrays to study the genomic alterations associated with MEN1 tumors. In addition to the role in tumorigeneis, menin appears to play a critical role in early development as homozygous loss of Men1 alleles in mice results in embryonic lethality (E11.5-E13.5) with defects in the development of several organs including heart. We have established mouse embryo fibroblast (MEF) cell lines lacking menin, and evaluated their characteristic including their expression patterns. Menin-null MEFs display alteration in expression of several genes encoding extracellular matrix proteins. These genes, regulated by TGF-b, are known to be required for early development of heart. We also found that MEF cell lines lacking menin respond poorly to TGF-b. Reexpression of menin in menin-null MEFs, either transiently or stably, resulted in the identification genes regulated by menin. Evaluation of the expression changes associated with menin, and the usefulness of the variety of animal models are likely to reveal the basic biological function(s) of menin.