Purification of SEF1 proteins binding to transcriptional enhancer elements active in T lymphocytes.
Binding sites for SL3-3 enhancer factor 1 (SEF1) are important for the transcriptional activity in T lymphocytes and the tumorigenicity of SL3-3 murine leukemia virus. SEF1 is also implicated in the activity of many other leukemia, lymphoma, and sarcoma virus enhancers, and enhancers of genes for T cell receptor-CD3 subunits. We have purified several proteins binding to SEF1 sites from bovine thymus using a five-step purification procedure. The proteins migrated as 19 distinct bands representing molecular masses from 23 kDa to about 200 kDa in SDS-polyacrylamide gel electrophoresis. Ten DNA binding proteins, with molecular masses between 23 and 67 kDa, could be isolated after separation by SDS-polyacrylamide gel electrophoresis. The DNA binding specificities of these proteins were similar and corresponded to that of the SEF1 binding activity in nuclear extracts. Each of these isolated SEF1 proteins also bound to the essential delta-E3 element of the human T cell receptor delta enhancer. Antibodies against one SEF1 protein only reacted with the protein used for immunization, which indicates a limited homology between at least some SEF1 proteins. We also present data suggesting that SEF1 proteins exist in multiple forms with differences in their DNA binding specificity, and that high affinity DNA binding of the SEF1 proteins requires protein phosphorylation.
Thornell
A
A
Department of Applied Cell and Molecular Biology, University of Umeå, Sweden.
Holm
M
M
Grundström
T
T
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017252:CD3 Complex / Q000378:metabolism; D002417:Cattle; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D010766:Phosphorylation; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D013601:T-Lymphocytes / Q000378:metabolism*; D013950:Thymus Gland / Q000378:metabolism; D014157:Transcription Factors / Q000302:isolation & purification* / Q000378:metabolism; D014158:Transcription, Genetic
D017252:CD3 Complex; D004268:DNA-Binding Proteins; D009363:Neoplasm Proteins; D011948:Receptors, Antigen, T-Cell; D014157:Transcription Factors; D004247:DNA