Sequence determinants of DNA binding by the hematopoietic helix-loop- helix transcription factor TAL1: Importance of sequences flanking the E-box core

TAL1 is a helix-loop-helix transcription factor that is essential for hematopoiesis. In vitro DNA binding site selection experiments have previously identified the preferred binding site for TAL1 heterodimers as AACA-GATGGT. TAL1 homodimers do not bind DNA with significant affinity. A subset of other E-box sequences is also bound by TAL1 heterodimers. Here, we present an analysis of TAL1 heterodimer DNA binding specificity, using E- boxes derived from genomic clones, which were isolated by immunoadsorption of K562 erythroleukemia cell chromatin with a TAL1 antibody. We show that TAL1 heterodimer binding to a CAGATG E-box is strongly modulated by nucleotides flanking the E-box. A 10 pair element consisting of the CAGATG E-box and two flanking nucleotides in both the 5' and 3' direction is sufficient for high- affinity binding. Certain mutations of nucleotides in either the 5' (-1 and - 2) or 3' (+1 and +2) direction strongly inhibit binding. The importance of flanking nucleotides also exists in the context of nonpreferred E-boxes recognized by TAL1 heterodimers. Although there are no known target genes for TAL1, the regulatory regions of several genes involved in hematopoiesis contain the preferred E-box CAGATG. However, based on our results, the E- boxes in these potential target genes contain flanking sequences that would be expected to significantly reduce TAL1 heterodimer binding in vitro. Thus, additional stabilizing forces, such as protein-protein interactions between TAL1 heterodimers and accessory factors, may be required to confer high- affinity TAL1 heterodimer binding to such sequences.