Early B-cell factor, E2A, and Pax-5 cooperate to activate the early B cell-specific mb-1 promoter.
Previous studies have suggested that the early-B-cell-specific mb-1(Igalpha) promoter is regulated by EBF and Pax-5. Here, we used in vivo footprinting assays to detect occupation of binding sites in endogenous mb-1 promoters at various stages of B-cell differentiation. In addition to EBF and Pax-5 binding sites, we detected occupancy of a consensus binding site for E2A proteins (E box) in pre-B cells. EBF and E box sites are crucial for promoter function in transfected pre-B cells, and EBF and E2A proteins synergistically activated the promoter in transfected HeLa cells. Other data suggest that EBF and E box sites are less important for promoter function at later stages of differentiation, whereas binding sites for Pax-5 (and its Ets ternary complex partners) are required for promoter function in all mb-1-expressing cells. Using DNA microarrays, we found that expression of endogenous mb-1 transcripts correlates most closely with EBF expression and negatively with Id1, an inhibitor of E2A protein function, further linking regulation of the mb-1 gene with EBF and E2A. Together, our studies demonstrate the complexity of factors regulating tissue-specific transcription and support the concept that EBF, E2A, and Pax-5 cooperate to activate target genes in early B-cell development.
Sigvardsson
Mikael
M
Department for Stem Cell Biology, Lund, Sweden.
Clark
Dawn R
DR
Fitzsimmons
Daniel
D
Doyle
Michelle
M
Akerblad
Peter
P
Breslin
Thomas
T
Bilke
Sven
S
Li
Ronggui
R
Yeamans
Carmen
C
Zhang
Gongyi
G
Hagman
James
J
D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D001402:B-Lymphocytes / Q000502:physiology*; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D001665:Binding Sites; D051925:CD79 Antigens; D002460:Cell Line; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D051757:PAX5 Transcription Factor; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011947:Receptors, Antigen, B-Cell / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D015703:Antigens, CD; D051792:Basic Helix-Loop-Helix Proteins; D051925:CD79 Antigens; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009687:Nuclear Proteins; D051757:PAX5 Transcription Factor; D011947:Receptors, Antigen, B-Cell; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C497881:CD79A protein, human; C497882:Cd79a protein, mouse; C081976:EBF1 protein, human; C489921:Ebf1 protein, mouse; C497281:PAX5 protein, human; C497282:Pax5 protein, mouse; C407909:TCF3 protein, human