Induction of Erythrocyte Protein 4.2 Gene Expression during Differentiation of Murine Erythroleukemia Cells Bahri Karacay* and Long-Sheng Chang * , † , ‡ ,1 * Department of Pediatrics, †Department of Medical Biochemistry, and ‡Department of Pathology, Children’s Hospital and The Ohio State University, Columbus, Ohio 43205-2696 Received November 5, 1998; accepted April 3, 1999 Protein 4.2 (P4.2) is an important component in the erythrocyte membrane skeletal network that regu- lates the stability and flexibility of erythrocytes. Re- cently, we provided the evidence for specific P4.2 ex- pression in erythroid cells during development (L. Zhu et al., 1998, Blood 91, 695–705). Using dimethyl sulfox- ide (DMSO)-induced differentiation of murine eryth- roleukemia (MEL) cells as a model, transcription of the P4.2 gene was found to be induced during ery- throid 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 up- stream of the ATG start codon. The mouse P4.2 pro- moter is TATA-less and contains multiple potential binding sites for erythroid transcription factors GATA-1, NF-E2, EKLF, and tal-1/SCL. Transient trans- fection 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 (nu- cleotide position 88 to 171 relative to the major transcription initiation site designated 1), contain- ing 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 mo- tif rendered the promoter unresponsive to DMSO in- duction. 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 to- gether, these results suggest that the erythroid tran- scription factor GATA-1 plays an important role in the induction of P4.2 gene expression during erythroid cell differentiation. © 1999 Academic Press INTRODUCTION The erythrocyte protein 4.2 (P4.2) 2 is one of the ma- jor components in the membrane skeletal network of red blood cells (RBCs) (for reviews, see Cohen et al., 1993; Yawata, 1994a,b). Although the exact function of P4.2 is not defined, several lines of evidence suggest that it plays an important role in maintaining the stability and flexibility of RBCs. First, individuals whose RBCs are severely deficient in P4.2 experience various degrees of hemolytic anemia (Hayashi et al., 1974; Ideguchi et al., 1990; Nozawa et al., 1974; Rybicki et al., 1988; Bouhassira et al., 1992). Second, genetic analysis of patients’ DNA and RNA reveals the pres- ence of mutations in the P4.2 gene (Bouhassira et al., 1992; Takaoka et al., 1994; Hayette et al., 1995a,b; Iwamoto et al., 1993; Kanzaki et al., 1995a,b; Matsuda et al., 1995). Third, P4.2 has been shown to bind to the cytoplasmic domain of the anion exchanger band 3 and interacts with ankyrin in erythrocytes (Korsgren and Cohen, 1986, 1988; Rybicki et al., 1988, 1995). Fourth, recent biophysical and electron microscopic studies in- dicate that P4.2 may serve as an accessory linking protein to strengthen the interaction between the in- tegral membrane protein, such as band 3, and the cytoskeletal network (Golan et al., 1996; Rybicki et al., 1996; Yawata et al., 1996). Previously, we and others cloned the human and mouse P4.2 cDNAs from reticulocyte cDNA libraries (Korsgren et al., 1990; Sung et al., 1990; Korsgren and Cohen, 1994; Rybicki et al., 1994; Karacay et al., 1995). Amino acid sequences deduced from these P4.2 cDNAs show significant homology with the transglutaminase family of crosslinking enzymes. However, P4.2 lacks the critical cysteine residue required for the enzymatic crosslinking of substrates (Ichinose et al., 1990) and has no transglutaminase activity (Korsgren et al., 1990). Nevertheless, it has been suggested that P4.2 may use the active site to bind other erythrocyte mem- Sequence data from this article have been deposited with the GenBank Data Library under Accession No. AF102234. 1 To whom correspondence should be addressed at the Department of Pediatrics, Children’s Hospital & The Ohio State University, W230, 700 Children’s Drive, Columbus, OH 43205. Telephone: (614) 722-2804. Fax: (614) 722-2716. E-mail: lchang@chi.osu.edu. 2 Abbreviations used: P4.2, protein 4.2 or gene (DNA, RNA) encod- ing P4.2; RBC, red blood cell; MEL, murine erythroleukemia; DMSO, dimethyl sulfoxide; PCR, polymerase chain reaction; EMSA, electro- phoretic mobility shift assay; UTR, untranslated region. Genomics 59, 6 –17 (1999) Article ID geno.1999.5846, available online at http://www.idealibrary.com on 6 0888-7543/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.