cis-acting sequences required for inducible interleukin-2 enhancer function bind a novel Ets-related protein, Elf-1.
The recent definition of a consensus DNA binding sequence for the Ets family of transcription factors has allowed the identification of potential Ets binding sites in the promoters and enhancers of many inducible T-cell genes. In the studies described in this report, we have identified two potential Ets binding sites, EBS1 and EBS2, which are conserved in both the human and murine interleukin-2 enhancers. Within the human enhancer, these two sites are located within the previously defined DNase I footprints, NFAT-1 and NFIL-2B, respectively. Electrophoretic mobility shift and methylation interference analyses demonstrated that EBS1 and EBS2 are essential for the formation of the NFAT-1 and NFIL-2B nuclear protein complexes. Furthermore, in vitro mutagenesis experiments demonstrated that inducible interleukin-2 enhancer function requires the presence of either EBS1 or EBS2. Two well-characterized Ets family members, Ets-1 and Ets-2, are reciprocally expressed during T-cell activation. Surprisingly, however, neither of these proteins bound in vitro to EBS1 or EBS2. We therefore screened a T-cell cDNA library under low-stringency conditions with a probe from the DNA binding domain of Ets-1 and isolated a novel Ets family member, Elf-1. Elf-1 contains a DNA binding domain that is nearly identical to that of E74, the ecdysone-inducible Drosophila transcription factor required for metamorphosis (hence the name Elf-1, for E74-like factor 1). Elf-1 bound specifically to both EBS1 and EBS2 in electrophoretic mobility shift assays. It also bound to the purine-rich CD3R element from the human immunodeficiency virus type 2 long terminal repeat, which is required for inducible virus expression in response to signalling through the T-cell receptor. Taken together, these results demonstrate that multiple Ets family members with apparently distinct DNA binding specificities regulate differential gene expression in resting and activated T cells.
Thompson
C B
CB
Howard Hughes Medical Institute, University of Michigan Medical Center, Ann Arbor 48109.
Wang
C Y
CY
Ho
I C
IC
Bohjanen
P R
PR
Petryniak
B
B
June
C H
CH
Miesfeldt
S
S
Zhang
L
L
Nabel
G J
GJ
Karpinski
B
B
D000328:Adult; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004742:Enhancer Elements, Genetic; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics*; D008745:Methylation; D008969:Molecular Sequence Data; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050783:Proto-Oncogene Proteins c-ets; D013601:T-Lymphocytes / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; C494041:ETS1 protein, human; D007376:Interleukin-2; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D014157:Transcription Factors; D004247:DNA