Induction of Erythrocyte Protein 4.2 Gene Expression during Differentiation of Murine Erythroleukemia Cells

Abstract

Protein 4.2 (P4.2) is an important component in the erythrocyte membrane skeletal network that regulates the stability and flexibility of erythrocytes. Recently, we provided the evidence for specific P4.2 expression in erythroid cells during development (L. Zhu et al., 1998, Blood 91, 695–705). Using dimethyl sulfoxide (DMSO)-induced differentiation of murine erythroleukemia (MEL) cells as a model, transcription of the P4.2 gene was found to be induced during erythroid differentiation. To examine the mechanism for this induction, we isolated the mouse P4.2 genomic DNA containing the 5′ flanking sequence and defined the location of the P4.2 promoter. Transcription of the mouse P4.2 gene initiates at multiple sites, with the major initiation site mapped at 174 nucleotides upstream of the ATG start codon. The mouse P4.2 promoter is TATA-less and contains multiple potential binding sites for erythroid transcription factors GATA-1, NF-E2, EKLF, and tal-1/SCL. Transient transfection experiments demonstrated that a 1.7-kb mouse P4.2 promoter fused with the luciferase coding regions was induced in DMSO-treated MEL cells. Deletion analysis showed that a 259-bp P4.2 promoter DNA (nucleotide position −88 to +171 relative to the major transcription initiation site designated +1), containing a GATA-binding site at position −29 to −24, could still respond to the induction in differentiated MEL cells. Importantly, mutations in the −29/−24 GATA motif rendered the promoter unresponsive to DMSO induction. Electrophoretic mobility shift assay revealed that GATA-1 could bind to the −29/−24 GATA motif and this was confirmed by the observation that the nuclear protein bound to the motif was supershifted by an anti-GATA-1 monoclonal antibody. Taken together, these results suggest that the erythroid transcription factor GATA-1 plays an important role in the induction of P4.2 gene expression during erythroid cell differentiation.