The structure of the TNFRSF13C promoter enables differential expression of BAFF-R during B cell ontogeny and terminal differentiation.
The B cell-activating factor of the TNF family receptor (BAFF-R), encoded by the TNFRSF13C gene, is critically important for transitional B cell survival to maturity. Thus, ligation of BAFF-R by BAFF delivers a potent survival signal. Reports implicating the BAFF/BAFF-R signaling axis in the pathogenesis of autoimmune human diseases and B lineage malignancies have largely prompted studies focusing on BAFF expression; however, there is an equally critical need to better understand BAFF-R expression. Initial BAFF-R expression, although characterized in murine B cells, has not yet been reported in human B lymphopoiesis. In this study, we first demonstrate that BAFF-R expression is absent from early precursors and is acquired by bone marrow B cells newly expressing the BCR. We next focused on identifying the specific genomic region that controls BAFF-R expression in mature B cells (i.e., the TNFRSF13C promoter). To accomplish this, we used in silico tools examining interspecies genomic conservation in conjunction with reporter constructs transfected into malignant B and plasma cell lines. DNase protection assays using nuclear extracts from BAFF-R-expressing cells suggested potential regulatory sites, which allowed the generation of EMSA probes that bound NFs specific to BAFF-R-expressing cells. With a more stringent analysis of interspecies homology, these assays identified a site at which a single nucleotide substitution could distinctly impact promoter activity. Finally, chromatin immunoprecipitation assays revealed the in vivo binding of the specific transcription factor c-Rel to the most proximal genomic region, and c-Rel small interfering RNA transfections in BAFF-R-expressing lines demonstrated a coincident knockdown of both c-Rel and BAFF-R mRNA.
Mihalcik
Stephen A
SA
Department of Immunology, Mayo Graduate School, Rochester, MN 55905, USA.
Huddleston
Paul M
PM
Wu
Xiaosheng
X
Jelinek
Diane F
DF
D000818:Animals; D053265:B-Cell Activation Factor Receptor / Q000235:genetics* / Q000378:metabolism; D001402:B-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002454:Cell Differentiation / Q000235:genetics*; D002460:Cell Line; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D002874:Chromosome Mapping; D019295:Computational Biology / Q000379:methods; D024202:Electrophoretic Mobility Shift Assay; D020869:Gene Expression Profiling; D006801:Humans; D015151:Immunoblotting; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D017354:Point Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020569:Proto-Oncogene Proteins c-rel / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D026801:Synteny; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection
D053265:B-Cell Activation Factor Receptor; D020569:Proto-Oncogene Proteins c-rel; D011993:Recombinant Fusion Proteins; C435926:TNFRSF13C protein, human; D014157:Transcription Factors; D008156:Luciferases