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应答调节的转录因子NF-AT和STAT的功能来调节体外和体内的免疫应答。在本研究项目开始时，我们已经分离了人类NF-AT （NF-ATp/c/x）基因，在研究过程中，我们分离了小鼠的相应基因。在NF-AT家族成员中，我们重点分析了人和小鼠NF-ATx的功能变化。首先，我们进行了人类NF-ATx的结构域定位，并确定了几个不同的功能域，包括DNA结合域，AP-1相互作用域，cn调节抑制域（CRI）。对于CRI序列，特别是该结构域的缺失，导致核易位不依赖于钙信号。接下来，我们鉴定了小鼠NF-ATx的calcineurin结合域（CNBR），发现CNBR蛋白片段的过表达抑制NF-AT位点calcineurin结合域作为显性负突变体，阻止了mnf - atx介导的基因激活。STAT6已被证明是主要IL-4应答所必需的。我们通过将STAT6融合到小鼠雌激素受体的激素结合域的修饰形式，构建了STAT6的有条件活性形式。这种蛋白激活了一种特定的受体结构，以响应雌二醇类似物4-羟基他莫昔芬。我们还利用JAK2显性负突变体成功地调控了gm - csf诱导的BAF/3细胞的应答，JAK2已知作用于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应答的调节。我们现在正计划产生表达这些突变体的转基因小鼠，以分析这些突变体在体内的影响。

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.