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的功能来调节体内外的免疫应答。在NF-AT家族成员中，我们重点研究了人和小鼠的NF-ATx，以分析其功能和改变。首先，我们对人NF-ATx进行了结构域定位，并鉴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、转录激活结构域、CN调节抑制结构域（CRI）。对于CRI序列，特别是该结构域的缺失，导致不依赖于钙信号的核转位。随后，我们鉴定了小鼠NF-ATx的钙调神经磷酸酶结合域（CNBR），发现CNBR蛋白片段的过表达抑制了NF-AT位点的钙调神经磷酸酶结合域作为一种显性负性突变体，阻止了mNF-ATx介导的基因激活。我们通过将STAT 6融合到小鼠雌激素受体的激素结合结构域的修饰形式，构建了STAT 6的条件活性形式。这种蛋白质激活一个特定的受体结构，以响应贝伐他汀类似物4-羟基-他莫昔芬。我们还成功地利用JAK 2的显性失活突变体调节了GM-CSF诱导的BAF/3细胞的免疫应答，该突变体作用于STAT蛋白的上游，由此获得的NF-ATx和STAT 6的突变体将有助于调节免疫应答。我们现在正计划产生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.