Molecular Genetics--Mitf-Tfe Family of bHLH-Zip

小鼠小眼炎转录因子(MITF)突变影响四种细胞类型的发育：黑素细胞、肥大细胞、破骨细胞和眼睛的色素上皮细胞。这些突变是表型多样的，可以按等位基因序列排列。在人类中，MITF突变会导致2A型Waardenburg综合征(WS2A)和Tietz综合征，这两种常染色体显性疾病会导致耳聋和色素减退。因此，MITF小鼠是研究人类疾病的一个重要的模型系统。在过去的一年里，我们描述了小鼠MITF基因的完整外显子/内含子结构，并证明它与人类基因相似。我们还表明，小鼠基因的转录比之前认为的更复杂，能够产生至少13种不同的异构体。这些异构体中的一些缺失了蛋白质的重要功能结构域，提示它们可能在MITF功能和信号转导中发挥抑制作用。此外，我们还确定了6个小眼症等位基因的分子基础。其中两个突变是首次描述的(MITF(mi-enu198)和MITF(mi-x39))，其他突变(MITF(mi-ws)、MITF(mi-bws)、MITF(mi-ew)和MITF(mi-di))已被描述，但突变的分子基础尚未确定。根据我们获得的基因组和转录数据进行分析，很明显，这些突变是由于RNA加工或转录缺陷造成的。有趣的是，其中三个突变(MITF(mi-x39)、MITF(mi-bws)和MITF(mi-ws))产生的蛋白质缺少体外研究中确定的蛋白质的重要功能域，进一步证实了这些域在整个动物中的生物学作用。 在MITF小鼠中观察到的破骨细胞缺陷只在携带强显性MITF等位基因的纯合子动物中可见。MITF蛋白是一种碱性-螺旋-环-螺旋-亮氨酸拉链(BHLHZip)转录因子，与相关的TFE3、TFEB和TFEC蛋白形成异二聚体。对MYC/Max/mad bHLHZip蛋白的研究表明，不同家族成员的功能效应主要是由异源二聚体介导的。最近，我们发现在体内不同的bHLHZip亚家族之间，异二聚体相互作用的重要性有很大的不同。虽然MITF或TFE3基因的丢失单独不会导致骨化症，但它们的联合丢失会导致严重的骨化症。不能检测到对其他细胞类型的额外影响，TFEB和TFec都不影响表型，这表明这种合作关系是破骨细胞中MITF和TFE3所特有的。此外，我们的结果表明，某些形式的人类骨化症可能是由于多个基因位点的突变所致，所产生的表型既与剂量有关，也与等位基因有关。

Mouse microphthalmia transcription factor (Mitf) mutations affect the development of four cell types: melanocytes, mast cells, osteoclasts, and pigmented epithelial cells of the eye. The mutations are phenotypically diverse and can be arranged in an allelic series. In humans, MITF mutations cause Waardenburg syndrome type 2A (WS2A) and Tietz syndrome, autosomal dominant disorders resulting in deafness and hypopigmentation. Mitf mice thus represent an important model system for the study of human disease. During the past year, we described the complete exon/intron structure of the mouse Mitf gene and showed it to be similar to the human gene. We also showed that the mouse gene is more transcriptionally complex than previously thought and is capable of generating at least 13 different isoforms. Some of these isoforms are missing important functional domains of the protein, suggesting that they might play an inhibitory role in Mitf function and signal transduction. In addition, we determined the molecular basis for six microphthalmia alleles. Two of the mutations were described for the first time (Mitf(mi-enu198) and Mitf(mi-x39)), while the others (Mitf(mi-ws), Mitf(mi-bws), Mitf(mi-ew), and Mitf(mi-di)) have been described but the molecular basis for the mutation not determined. When analyzed in terms of the genomic and transcriptional data we obtained, it is apparent that these mutations result from RNA processing or transcriptional defects. Interestingly, three of the mutations (Mitf(mi-x39), Mitf(mi-bws), and Mitf(mi-ws)) produce proteins that are missing important functional domains of the protein identified in in vitro studies, further confirming a biological role for these domains in the whole animal. The osteoclast defects observed in Mitf mice are only seen in homozygous animals carrying strong dominant Mitf alleles. The MITF protein, a basic-helix-loop-helix-leucine zipper (bHLHZip) transcription factor, forms heterodimers with the related TFE3, TFEB, and TFEC proteins. Studies of the MYC/MAX/MAD bHLHZip proteins have suggested that the functional effects of the different family members are chiefly mediated by heterodimers. Very recently, we have shown that the importance of heterodimeric interactions varies dramatically between different bHLHZip subfamilies in vivo. While loss of either the Mitf or Tfe3 genes alone does not lead to osteopetrosis, their combined loss results in severe osteopetrosis. No additional effects on other cell types can be detected and neither Tfeb nor Tfec contribute to the phenotype, suggesting that this partnership is specific to Mitf and Tfe3 in osteoclasts. Furthermore, our results suggest that some forms of human osteopetrosis may be due to mutations at multiple loci where the resulting phenotype is both dose and allele dependent.