Research Project
7192388
[unreadable] DESCRIPTION (provided by applicant): Normal B cell function requires the appropriate expression of multiple lineage and developmental-stage specific glycoconjugates. Further, the glycan portion of at least one such glycoconjugate requires a 2,6-linked sialic acid residue synthesized by the ST6Gal I sialyltransferase, as evidenced by the profoundly compromised B cell function seen in ST6Gal I KO mice. The ST6Gal I gene has at least eight separate promoters giving rise to tissue and developmental-stage specific transcripts differing only in their 5' untranslated regions (UTRs).We have shown that ST6Gal I activity in B cells is modulated, at least in part, by changes in isotype transcription and/or mRNA stability in response to signaling through various B cell receptors. More specifically, we have shown that the abundance of the 5'UTR sequence X1, arising from use of the ST6Gal I P2a promoter, is increased in response to interleukin 4 receptor (IL-4R) signaling and is at least partially dependent on STAT6, while the 5'UTR sequence Q-O, arising from use of the P3 promoter is increased by IL-2R signaling and decreased by signaling through the B cell receptor (BCR). A survey of appropriate immune signaling molecules for their ability to affect mRNA abundance levels will be conducted using well defined populations of purified immune cells and cell lines. Signals exhibiting differential quantitative effects on ST6Gal I gene expression will be selected for further investigation. Multiple experimental approaches for assessing mRNA stability and translational efficiency will be examined for indicator so regulation beyond that of transcriptional control and a novel hypothesis for the role of the divergent 5'- untranslated regions of the ST6Gal I mRNA will be evaluated. Extracellular signals exhibiting quantitative control of ST6Gal I mRNA expression will be dissected and the molecular systems responsive to such signals identified. Differential promoter usage will be quantitatively assessed for each immune relevant ST6Gal I promoter and the specific sequence elements dictating tissue and development-specific transcription will be identified. The involvement of specific transcription factors will be demonstrated using gene deletion and sequence mutants. An in depth understanding of the controls regulating this important enzyme may eventually lead to new modalities for the treatment of immune based disease. Previous studies with gene knockout mice have shown that the ST6Gal I sialyltransferase gene is required for the normal immune function of mature B cells. This gene has multiple promoters giving rise to multiple mRNAs differing only in their 5'- untranslated regions and promoter usage is strictly regulated by tissue and developmental stage-specific controls. The experiments of this proposal are designed (1) to identify the immune specific developmental control mechanisms that regulate ST6Gal I mRNA expression in B cells and to explicate their modes of action, (2) to identify functional roles for the specific 5'-untranslated regions of these varied mRNAs, and (3) to identify the stimuli responsive elements in the B cell-specific promoters and in the 'general' promoter for this gene. An in depth understanding of the controls regulating this important enzyme may eventually lead to new modalities for the treatment immune based disease. [unreadable] [unreadable] [unreadable]
