Of Mice and Men: Using Mutations to Characterize Disease

在以前的研究中，我们发现Mitf编码一个bHLH-Zip转录因子，它是黑素细胞、破骨细胞和肥大细胞功能所必需的。随后，人类MITF被克隆，并显示在常见的人类色素沉着和听力障碍Waardenburg综合征2型（WS 2）中发生突变。因此，小鼠Mitf的克隆导致了一个新的重要的人类疾病基因的鉴定。我们在与Mitf密切相关的三个基因（Tfe 3，Tfeb和Tfec）中进行了生殖系突变，这些基因的蛋白质产物与MITF相互作用。我们还产生了携带这四种突变的不同组合的小鼠。我们的研究表明，Tfeb是胎盘血管化所必需的，而Tfe 3在破骨细胞中起作用。令人惊讶的是，我们没有发现这四个基因之间有任何遗传相互作用。基于MYC-MAX-MAD范例，这是出乎意料的。Tfeb和Tfe 3都是人类疾病基因，我们的研究可能有助于揭示这些蛋白质在人类疾病中的功能。我们还发现，Mitf的表达是复杂的，至少有13种不同的亚型。其他研究表明，MITF在许多信号转导途径中发挥作用，并经历几种翻译后修饰。为了进一步评估这些修饰在体内的需要，我们使用BAC重组工程在小鼠Mitf基因内产生突变，阻断这些修饰，然后分析它们在携带BAC和Mitf无效等位基因的动物中的作用。我们也在继续一个敏感的，F1占主导地位的屏幕抑制和增强MITF。在初步研究中，我们已经确定了一个非常有趣的基因内抑制，我们现在的特点。瘙痒是人类自身免疫疾病的少数单基因小鼠模型之一。以前，我们表明，痒编码一种新的HECT结构域的E3连接酶。在最近的合作研究中，我们发现瘙痒功能的丧失导致体外T细胞向Th 2分化的偏向。在最近的移植研究中，我们也发现瘙痒小鼠骨髓中的细胞可以将疾病传给致死辐射的野生型宿主。我们现在正在确定一种特定类型的淋巴细胞是否足以治疗瘙痒病，以及瘙痒小鼠是否存在巨噬细胞功能缺陷，这些缺陷可能使它们倾向于Th 2偏好。痒是果蝇deltex抑制因子Su（dx）的小鼠直系同源物，Su（dx）是Notch信号传导的负调节因子。在最近的研究中，我们发现携带激活的Notch 1转基因的小鼠患有与瘙痒小鼠相似的自身免疫性疾病。我们还发现，携带激活的Notch 1转基因的瘙痒小鼠具有比单独的瘙痒或转基因小鼠更严重且发生更早的自身免疫性疾病。这些发现非常有趣，因为它们表明瘙痒和Notch 1可能在相同的自身免疫性疾病途径中起作用。该通路的进一步表征可能对治疗人类自身免疫性疾病具有重要意义。最后，我们还确定ITCH和密切相关的E3连接酶WWP 1和WWP 2之间的功能重叠，通过检查携带这三个基因突变组合的小鼠的表型。半显性Crc突变的纯合子小鼠在发育过程中死于严重的神经管缺陷，与Lp小鼠中观察到的相似。Crc和Lp在神经管闭合水平上遗传性地相互作用，表明它们在相同的信号通路中起作用。Lp编码果蝇旺/stbm基因（Vangl 2）的同源物，其是Wnt信号传导途径的潜在成员，而Crc编码Scrb 1，其是果蝇scribble的直系同源物，是含有PDZ结构域的基因。在苍蝇，涂鸦是需要正确定位的顶端结构域蛋白质和上皮极性的产生。

In previous studies we showed that Mitf encodes a bHLH-Zip transcription factor that is required for melanocyte, osteoclast, and mast cell function. Subsequently, human MITF was cloned and shown to be mutated in a common human pigmentation and hearing disorder, Waardenburg syndrome type 2 (WS2). The cloning of mouse Mitf thus led to the identification of a new important human disease gene. We made germ line mutations in three genes that are closely related to Mitf and whose protein products interact with MITF (Tfe3, Tfeb, and Tfec). We also generated mice that carried different combinations of these four mutations. Our studies showed that Tfeb is required for placental vascularization, while Tfe3 functions in the osteoclasts. Surprisingly, we failed to show any genetic interaction between these four genes. This was unexpected based on the MYC-MAX-MAD paradigm. Both Tfeb and Tfe3 are human disease genes and our studies may help to show how these proteins function in human disease. We also showed that Mitf expression is complex; there are at least 13 different isoforms. Others have shown that MITF functions in a number of signal transduction pathways and undergoes several posttranslational modifications. To further evaluate the requirement for these modifications in vivo, we are using BAC recombineering to create mutations within the mouse Mitf gene that block these modifications and then analyze their effects in animals carrying the BAC and a null allele of Mitf. We are also continuing a sensitized, F1 dominant screen for suppressors and enhancers of MITF. In preliminary studies, we have identified a very interesting intragenic suppressor that we are now characterizing. itch represents one of the few single gene mouse models of human autoimmune disease. Previously, we showed that itch encodes a novel HECT-domain-containing E3 ligase. In recent collaborative studies, we showed that loss of itch function leads to a bias towards Th2 differentiation of T cells in vitro. In recent transplantation studies we also showed that cells residing in the bone marrow of itch mice can confer the disease to lethally irradiated wild type hosts. We are now determining whether a particular class of lymphocytes is sufficient for itch disease and whether itch mice have defects in macrophage function that might predispose them to the Th2 bias. itch is the mouse ortholog of Drosophila Suppressor of deltex, Su(dx), a negative regulator of Notch signaling. In recent studies, we found that mice carrying an activated Notch1 transgene have an autoimmune disease that is similar to that seen in itch mice. We also showed that itch mice carrying the activated Notch1 transgene have an autoimmune disease that is more severe and occurs much earlier than itch or transgenic mice alone. These findings are very interesting because they suggest that both itch and Notch1 may function in the same autoimmune disease pathway. Further characterization of this pathway may have important implications for treating human autoimmune diseases. Finally, we are also determining the functional overlap between ITCH and the closely related E3 ligases WWP1 and WWP2 by examining the phenotype of mice carrying combinations of mutations in these three genes. Mice homozygous for the semidominant Crc mutation die during development from severe neural tube defects similar to those observed in Lp mice. Crc and Lp genetically interact at the level of neural tube closure, suggesting that they function in the same signaling pathway. Lp encodes a homolog of the Drosophila vang/stbm gene (Vangl2), a potential member of the Wnt signaling pathway, while Crc encodes Scrb1, a PDZ domain-containing gene that is the ortholog of Drosophila scribble. In flies, scribble is required for the correct localization of apical domain proteins and for the generation of epithelial polarity.