Of Mice and Men: Using Mutations to Characterize Disease

在以前的研究中，我们发现MITF编码一种bHLH-Zip转录因子，这是黑素细胞、破骨细胞和肥大细胞功能所必需的。随后，人类MITF被克隆并被证明在一种常见的人类色素沉着和听力障碍--Waardenburg综合征2型(WS2)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。我们对与MITF密切相关且其蛋白产物与MITF相互作用的三个基因(TFE3、TFEB和Tfec)进行了胚系突变。我们还培育了携带这四种突变不同组合的小鼠。我们的研究表明，TFEB是胎盘血管形成所必需的，而TFE3在破骨细胞中起作用。令人惊讶的是，我们没有显示出这四个基因之间的任何遗传交互作用。根据MYC-MAX-MAD范式，这是意想不到的。TFEB和TFE3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如何在人类疾病中发挥作用。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在许多信号转导通路中发挥作用，并经历了几次翻译后修饰。为了进一步评估体内对这些修改的要求，我们正在使用BAC重组工程在小鼠MITF基因内创建阻止这些修改的突变，然后分析它们在携带BAC和MITF零等位基因的动物中的影响。我们还在继续对MITF的抑制子和增强子进行敏化的F1显性筛选。在初步研究中，我们已经确定了一种非常有趣的基因内抑制因子，我们现在正在对其进行表征。瘙痒是为数不多的人类自身免疫性疾病的单基因小鼠模型之一。在此之前，我们已经证明了Ich编码了一种新的含有Hect结构域的E3连接酶。在最近的合作研究中，我们发现在体外，瘙痒功能的丧失会导致T细胞偏向Th2分化。在最近的移植研究中，我们还表明，驻留在瘙痒小鼠骨髓中的细胞可以将疾病传递给受到致死性照射的野生型宿主。我们现在正在确定特定类别的淋巴细胞是否足以治疗瘙痒疾病，以及瘙痒小鼠是否存在巨噬细胞功能缺陷，这可能使它们容易患Th2偏向。ITCH是果蝇Deltex抑制因子SU(DX)的同源小鼠，SU(DX)是Notch信号的负调控因子。在最近的研究中，我们发现携带激活的Notch1转基因的小鼠患有一种与瘙痒小鼠类似的自身免疫性疾病。我们还表明，携带激活的Notch1转基因基因的瘙痒小鼠患上了一种更严重的自身免疫性疾病，比单纯瘙痒或转基因小鼠发生得更早。这些发现非常有趣，因为它们表明瘙痒和Notch1可能在相同的自身免疫性疾病途径中发挥作用。这一途径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，我们还通过检测携带这三个基因组合突变的小鼠的表型，确定了瘙痒和密切相关的E3连接酶WWP1和WWP2之间的功能重叠。携带半显性CRC突变纯合子的小鼠在发育过程中死于严重的神经管缺陷，与LP小鼠中观察到的情况类似。CRC和LP在神经管闭合的基因水平上相互作用，表明它们在相同的信号通路中发挥作用。Lp编码果蝇vang/stbm基因(Vangl2)的同源基因，Vangl2是Wnt信号通路的潜在成员，而CRC编码含有PDZ结构域的基因Scrb1，Scrb1是果蝇Scribble的同源基因。在果蝇中，涂鸦是正确定位顶端结构域蛋白和产生上皮极性所必需的。

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.