A long-term goal of this research program is to elucidate transcriptional mechanisms involved in the regulation of cellular survival, proliferation, and function in immune responses. Stat transcription factors are critical regulators of these aspects of lymphocyte biology. Stat6 is notable for its tight and unique functional link to signaling by IL-4Ralpha a key constituent of the receptor for the cytokines interleukin (IL)-4 and IL-13. In addition to important roles in immunity, Stat6 is a central regulator of allergic diseases. A requisite role for a C-terminal activation domain has been identified, but far less is known about the molecules and mechanisms by which coregulators integrate Stat6 with the transcriptional apparatus to regulate gene expression. In seeking new molecules which modulate the transcriptional function of Stat6, the complete cDNA encoding a novel protein termed CoASt6 (Collaborator of Stat6) was cloned by yeast 2-hybrid screening for interactions with the full-length Stat6 molecule. CoASt6 is expressed preferentially in lymphoid cells and tissues, and interacts biochemically and functionally with Stat6, including in primary lymphocytes. Although the ability of this protein to enhance Stat6 activation of cis-acting Stat binding sites is more potent than that of any conventional coactivator tested, the CoASt6 sequence is notable for a lack of obvious similarities to conventional coregulator sequence motifs. Our objective is to understand the function of CoASt6. The central hypothesis of the proposal is that CoASt6 is a functionally important cofactor of gene transcription mediated by Stat6. A corollary hypothesis is that CoASt6 can achieve these functions in the intact animal during an immune response, thereby enhancing the function of Stat6. We propose secondarily that the "macro domain" of this protein contributes to its service as a transcriptional collaborator of Stat6, but that maximum activity is achieved by integration of this domain with additional portions of the molecule. To test the hypotheses, three integrated specific aims are proposed: In Aim 1, we will identify functional roles of CoASt6 domains in coregulating Stat6. In Aim 2, we will define how transcriptional enhancement by CoASt6 is integrated with Stat6 and its known cofactors. In Aim 3, we propose to determine functional capacities of CoASt6 in mediating transcriptional and proliferative responses in primary cells and in vivo. These proposed studies are significant at several levels. First, they address a question of general and fundamental significance about the rules which govern how gene expression is regulated and specificity is achieved by Stat transcription factors. Second, they are likely to identify new functional modules involved in transcriptional activation. Taken together, the experiments of these Specific Aims will provide new insights into the regulation of lymphocyte function in immune responses.