C-Myb and GATA-3 Cooperatively Regulate Th2 Cytokine Gene Expression, with the Most Profound Effect On IL-13.

Abstract 3669

Poster Board III-605

The c-myb proto-oncogene encodes a transcription factor, c-Myb, which regulates genes important for early myeloid, B, and T lymphoid cell development. Our group has recently shown that c-Myb binds a conserved site in the GATA-3 promoter, upregulating its expression and forming a complex that plays an important role in human Th2 cell development. We have also observed that silencing c-Myb in primary human peripheral blood CD4+ cells under Th2 cell promoting conditions decreases the expression of the Th2 cytokines, IL-4, IL-5 and IL-13. We wondered if this was a direct effect of c-Myb alone, or was also due to an effect of the c-Myb/GATA-3 complex? The human IL-4, IL-5 and IL-13 genes are located within a 150kb locus that is called the “Th2 cytokine gene” on Chromosome 5. The Th2 cytokine locus has numerous DNase I hypersensitive sites (DHSs) which act as regulatory elements for Th2 cytokine gene expression. Among these DHSs, the conserved GATA-3 response element (CGRE), the conserved noncoding sequence 1 (CNS-1), the intronic enhancer (IE) and the hypersensitive site V (HSV) are all known to be activated by GATA-3 directly. Interestingly, sequence analysis identified c-Myb binding sites in close proximity to GATA binding sites within all 4 DHSs. To determine if c-Myb, and GATA-3, occupied these putative binding sites, we performed ChIP assays and found that anti-c-Myb antibody, for example, enriched the CGRE locus 30-fold compared to control IgG. Similar experiments revealed that both c-Myb and GATA-3 bound to all 4 of the elements in human naïve CD4+ T-cells under Th2 promoting conditions. To determine the role of c-Myb and GATA-3 in Th2 cytokine gene expression, we performed reporter assays in 293T, Jurkat and human CD4+ T-cells using IL-4, IL-13 and IL-5 promoter constructs with, or without, the 4 DHSs. c-Myb weakly (2-2.5-fold), but significantly, activated both the IL-4 and IL-5 promoters in 293T cells in the absence of DHSs. In contrast, GATA-3 did not activate the IL-4 and IL-5 promoters without the enhancer elements, though it weakly activated the IL-4 promoter through CNS-1. GATA-3 activated the minimal IL-13 promoter 5-fold, and the addition of c-Myb increased promoter activation 10-fold. In marked contrast to these results, an IL-13 promoter construct containing the CGRE enhancer was activated 65-fold by GATA-3 alone, and 160-fold by the addition of c-Myb. The CGRE locus has only one canonical Myb binding site, but Myb's strong enhancer effect on IL-13 expression was completely lost when the CGRE element's Myb binding site was mutated. Mutating the GATA binding sites decreased IL-13 promoter activity by ∼50%. These results suggest that c-Myb and GATA-3 regulate IL-13 expression directly, and in a cooperative manner. These results lead us to hypothesize that the binding of a c-Myb and GATA-3 complex to the CGRE element changes the chromatin structure of the IL-13 locus which acts as a switch to promote IL-13 expression. Though the most dramatic effects of c-Myb were on IL-13 expression, because c-Myb binds to the other three enhancers, and activates both the IL-4 and IL-5 promoters as well, we hypothesize that c-Myb directly contributes to all Th2 cytokine gene expressions, including IL-13. Finally, a ChIP assay showed that MLL also bound to the locus of CGRE. Since we have already shown that c-Myb can bind to MLL through Menin those results suggest, in aggregate, the possibility of histone modification in Th2 cytokine locus by MLL, GATA-3 and c-Myb. This possibility is under active investigation in our laboratory.