EXCEED THE SPACE PROVIDED. Transcription of major histocompatibility complex class II (MHCII) genes and thus antigen processing and presentation (APP) to CD4+ T helper cells and their education of MHCII-positive B cells for immunoglobulin class switching and hypermutation is carefully regulated in development and in tissues. Four separate transcription factors are dedicated solely to the transcription of structural MHCII genes, DMA/DMB and the invariant chain (Ii). Of these, the class II transactivator, CIITA plays a key role in turning these genes on and off following the administration of IFN3,. Recently, we discovered that in addition to IFN'y, antigen presenting cells (APC) require another signal that phosphorylates CIITA. Phosphorylation of CIITA leads to its oligomerization, accumulation and increased activity on MHCII promoters. CIITA is also not a DNA-binding protein but is somehow attracted to the MHCII enhanceosome, which assembles on conserved upstream sequences in these promoters. This grant will investigate the precise signals that are required for the full activity of CIITA, what posttranslational modifications occur and how they create super-CIITA complexes that trigger the full APP. First, nucleocytoplasmic shuttling of CIITA will be investigated in great detail. Precise NES and NLS will be mapped and confirmed functionally. Next, phosphorylation, acetylation and ubiquitylation of CIITA will be investigated in great detail. These sites will be mutated appropriately to resemble constitutively phosphorylated and acetylated forms of CIITA. Additionally, they will be changed so that they cannot be modified posttranslationally. Ubiquitin will be fused directly to CIITA. Forced oligomers of CIITA will also be made. All these modified CIITA proteins will be examined for binding the MHCII enhanceosome in vitro and for function in vivo. Moreover, kinases, HATs, HDACs, ubiquitin ligases that participate in these processes will be revealed. Finally, a super-CIITA protein will be created that does not require this second stimulus for full activity. This mutant CIITA protein/s will then program any cell for APP. It will be introduced into cells and into the murine germline, in place of the endogenous CIITA gene (knock-in) and with P 1 or P4 promoters of CIITA for expression in hematopoetic and nonhematopoetic cells, respectively, for induction of full APP following the administration of IFN'y. Collectively, these studies will reveal the normal physiology of CIITA, why it oligomerizes and shuttles, and create new tools to manipulate the immune response in health and disease. PERFORMANCE SITE ========================================Section End===========================================
| Jabrane-Ferrat, N; Campbell, M J; Esserman, L J et al. (2006) Challenge with mammary tumor cells expressing MHC class II and CD80 prevents the development of spontaneously arising tumors in MMTV-neu transgenic mice. Cancer Gene Ther 13:1002-10 |
| Drozina, Gorazd; Kohoutek, Jiri; Nishiya, Tadashi et al. (2006) Sequential modifications in class II transactivator isoform 1 induced by lipopolysaccharide stimulate major histocompatibility complex class II transcription in macrophages. J Biol Chem 281:39963-70 |
| Peterlin, B Matija; Price, David H (2006) Controlling the elongation phase of transcription with P-TEFb. Mol Cell 23:297-305 |
| Kanazawa, Satoshi; Ota, Shusuke; Sekine, Chiyoko et al. (2006) Aberrant MHC class II expression in mouse joints leads to arthritis with extraarticular manifestations similar to rheumatoid arthritis. Proc Natl Acad Sci U S A 103:14465-70 |
