TAL1 Regulates Expression of the Band 3 Gene in Erythroid Progenitors through Both Upstream Regulatory and Proximal Promoter Elements.

The TAL1 (also known as SCL and TCL-5) protein is a class II basic helix-loop-helix (bHLH) transcription factor that plays an important role during embryonic and adult hematopoiesis. We previously established that the Protein 4.2 (P4.2) gene is a physiologic target of TAL1 in erythroid cells and showed that tandem E box-GATA elements in its proximal promoter mediated TAL1-directed gene activation. Through database searches, we identified two E box-GATA consensus sequences in the proximal promoter of another erythroid membrane skeleton gene, that encoding the Band 3 protein. Identical to what was observed for P4.2, overexpression of wild-type TAL1 slightly increased while enforced expression of a DNA binding-defective TAL1 mutant severely reduced endogenous Band 3 gene expression in murine erythroleukemia (MEL) cells induced to differentiate with dimethyl sulfoxide (DMSO). Overexpression of Ldb1 significantly inhibited and a LIM protein interaction-defective Ldb1 mutant virtually ablated Band 3 mRNA accumulation in DMSO-induced MEL cells. Quantitative chromatin immunoprecipitation (ChIP) analysis with Tal1 antibody confirmed that Tal1 was associated with the proximal promoter of the Band 3 gene in MEL cells, while analysis of approximately 28 kb of genomic sequence spanning the Band 3 gene, including 5 kb 5′ and 3′ of the gene, revealed another consensus E box-GATA element in intron 16; however, no significant TAL1 binding was detected in this region by ChIP analysis. To identify potential non-conventional TAL1 binding site(s), scanning ChIP analysis was applied to the entire 28 kb Band 3 genomic region, and a region ~2.5 kb upstream of the major transcriptional start site was found to bind significantly more TAL1 protein than the proximal promoter. Fine mapping of TAL1 binding to this region by ChIP analysis using more highly sheared DNA and smaller sized amplicons narrowed TAL1 binding to a region of ~100 bp, which we designated as the Band 3 upstream regulatory region (B3URE). Luciferase reporter assays in transiently transfected MEL cells with vectors containing genomic sequence revealed the presence of an orientation- and position- independent repressor in this upstream region, with a 107 bp fragment retaining nearly all the repressing activity of a larger, 700 bp fragment. In contrast, coexpression of TAL1, E47, GATA-1, LMO2, and Ldb1 in COS cells led to transactivation of a reporter gene linked to the promoter-proximal E box-GATA element. These data suggest that TAL1 acts as a repressor on the upstream B3URE in undifferentiated cells and then, as differentiation proceeds, as an activator on the E box-GATA elements in the proximal promoter.