An NFAT-dependent enhancer is necessary for anti-IgM-mediated induction of murine CD5 expression in primary splenic B cells.
CD5 is a 67-kDa membrane glycoprotein the expression of which in murine splenic B cells is induced by surface IgM cross-linking. To analyze this induction, we transiently transfected primary splenic B cells with luciferase reporter constructs driven by various wild-type and mutated CD5 5'-flanking sequences. The transfected cells were subsequently cultured in medium with or without F(ab')2 anti-IgM (anti-IgM), and luciferase expression was assayed. Using this approach, we identified a 122-bp enhancer element necessary for anti-IgM-mediated induction of the CD5 promoter. Electrophoretic mobility shift assays indicated that four inducible and four constitutive complexes form on the enhancer fragment in nuclear extracts of primary B cells. Supershift assays revealed that two of the inducible complexes contained NFATc. Point mutations that abolished NFAT binding severely impaired enhancer function. Thus, CD5 is a target of NFAT in B cells. A third inducible complex required an intact H4TF-1 site. One of several constitutive complexes required an intact Ebox site while a second required an intact putative ets binding site. Mutation of the H4TF-1, Ebox, and Ets sites, in the presence of wild-type NFAT sites, significantly reduced the activity of the enhancer. Therefore, the induction of B cell CD5 expression requires NFAT binding and binding to at least one of three additional sites in the CD5 enhancer.
Berland
R
R
Department of Pathology, Program in Immunology, Tufts University School of Medicine, Boston, MA 02111, USA. Rberland@opal.tufts.edu
Wortis
H H
HH
D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000502:physiology*; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018956:CD5 Antigens / Q000096:biosynthesis* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D004742:Enhancer Elements, Genetic / Q000276:immunology*; D005786:Gene Expression Regulation / Q000276:immunology; D006657:Histones / Q000235:genetics; D007075:Immunoglobulin M / Q000276:immunology; D008213:Lymphocyte Activation / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011401:Promoter Regions, Genetic / Q000276:immunology; D011518:Proto-Oncogene Proteins / Q000235:genetics; D050783:Proto-Oncogene Proteins c-ets; D013154:Spleen / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism / Q000502:physiology
D000888:Antibodies, Anti-Idiotypic; D018956:CD5 Antigens; D004268:DNA-Binding Proteins; C059062:H4TF-1 protein, human; D006657:Histones; D007075:Immunoglobulin M; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D014157:Transcription Factors; C044203:anti-IgM