Cloning and functional characterization of early B-cell factor, a regulator of lymphocyte-specific gene expression.
Early B-cell factor (EBF) was identified previously as a tissue-specific and differentiation stage-specific DNA-binding protein that participates in the regulation of the pre-B and B lymphocyte-specific mb-1 gene. Partial amino acid sequences obtained from purified EBF were used to isolate cDNA clones, which by multiple criteria encode EBF. The recombinant polypeptide formed sequence-specific complexes with the EBF-binding site in the mb-1 promoter. The cDNA hybridized to multiple transcripts in pre-B and B-cell lines, but transcripts were not detected at significant levels in plasmacytoma, T-cell, and nonlymphoid cell lines. Expression of recombinant EBF in transfected nonlymphoid cells strongly activated transcription from reporter plasmids containing functional EBF-binding sites. Analysis of DNA binding by deletion mutants of EBF identified an amino-terminal cysteine-rich DNA-binding domain lacking obvious sequence similarity to known transcription factors. DNA-binding assays with cotranslated wild-type and truncated forms of EBF indicated that the protein interacts with its site as a homodimer. Deletions delineated a carboxy-terminal dimerization region containing two repeats of 15 amino acids that show similarity with the dimerization domains of basic-helix-loop-helix proteins. Together, these data suggest that EBF represents a novel regulator of B lymphocyte-specific gene expression.
Howard Hughes Medical Institute, San Francisco, California.
J
J
Hagman
C
C
Belanger
A
A
Travis
C W
CW
Turck
R
R
Grosschedl
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD; D001402:B-Lymphocytes / Q000378:metabolism*; D001483:Base Sequence; D015152:Blotting, Northern; D051925:CD79 Antigens; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation / Q000502:physiology*; D005803:Genes, Immunoglobulin; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis; D011947:Receptors, Antigen, B-Cell / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D015703:Antigens, CD; D051925:CD79 Antigens; C497881:CD79A protein, human; C497882:Cd79a protein, mouse; D004268:DNA-Binding Proteins; C081976:EBF1 protein, human; C489921:Ebf1 protein, mouse; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D011947:Receptors, Antigen, B-Cell; D011994:Recombinant Proteins; D015534:Trans-Activators; D004247:DNA