Generation of disease model mice by the alteration of transcription factors regulating immune responses

本研究的目的是通过改变参与免疫反应调节的转录因子NF-AT和STAT的功能来调节体外和体内的免疫反应。在本研究计划开始时，我们分离了人的NF-AT(NF-ATP/c/x)基因，在本研究中我们也分离了相应的小鼠。在核因子-AT家族成员中，我们主要以人和鼠的核因子-ATX为研究对象，对其功能进行分析和改变。首先，我们对人核因子-ATX进行了结构域的定位，并确定了几个不同的功能结构域，包括DNA结合结构域、AP-1相互作用结构域、反式激活结构域、CN调节的抑制结构域(CRI)。对于CRI序列，特别是该结构域的缺失，导致了不依赖于钙信号的核转位。接下来，我们鉴定了小鼠NF-ATX的钙调神经磷酸酶结合域(CNBR)，发现CNBR蛋白片段的过度表达抑制了NF-AT位点的钙调神经磷酸酶结合域作为显性负突变，阻止了MNF-ATX介导的基因激活。STAT6已被证明是Mejor IL-4反应所必需的。我们通过将STAT6与小鼠雌激素受体激素结合域的修饰形式融合，构建了一种有条件的活性形式的STAT6。这种蛋白质激活一种特定的受体结构，以响应雌二醇类似物4-羟基-三苯氧胺。我们还利用JAK2的显性负性突变体成功地调节了GM-CSF诱导的BAF/3细胞的反应，该突变体作用于STAT蛋白的上游。从上述研究中获得的NF-ATX和STAT6的突变形式将有助于免疫反应的调节。我们现在正计划培育表达这些突变体的转基因小鼠，用于分析这些突变体在体内的作用。

The purpose of this study is to regulate the immune responses in vitro and in vivo by the alteration of the function of the transcription factors NF-AT and STAT,which are involved in the regulation of immune responses.We have isolated human NF-AT (NF-ATp/c/x) genes at the beginning of this research program, and during this study we isolated murine counterparts. Among the NF-AT family members, we focused on human and mouse NF-ATx for the analysis and alteration of its function. First, we performed domain mapping of human NF-ATx, and identified several distinct functional domains including the DNA binding domain, AP-1 interaction domain, transactivation domain CN-regulated inhibitory domain (CRI). For CRI sequence, specially deletion of this domain, resulted in nuclear translocation independent of calcium signaling. Next, we identified the calcineurin binding domain (CNBR) of mouse NF-ATx, and found that the over-expression of CNBR protein fragment inhibited NF-AT site calcineurin binding domain acted as a dominant negative mutant that prevents mNF-ATx-mediated gene activation.STAT6 has been shown to be required for mejor IL-4 responses. We constructed a conditionally active form of STAT6 by fusing STAT6 to a modified form of the hormone binding domain of the mouse estrogen receptor. This protein activates a specific receptor construct in response to the bestradiol analog 4-Hydroxy-tamoxifen. We also succeeded in the regulation of GM-CSF-induced response of BAF/3 cells by using a dominant negative mutant of JAK2, which is known to act upstream of STAT proteins.Mutant forms of NF-ATx and STAT6 derived from the above study will be useful fox the regulation of immune responses. We are now planning to generate transgenic mice expressing these mutants for the analysis of the effects of these mutants in vivo.