The structure of the TNFRSF13C promoter enables differential expression of BAFF-R during B cell ontogeny and terminal differentiation

Abstract

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.

FIGURE 1. BAFF-R surface expression on bone marrow B cell populations

A, Gating strategy identifying BM B lineage cell subpopulations. B lineage cells were first identified with CD19 expression. Mature recirculating B cells were identified through light chain (LC) expression and the absence of CD10, while immature transitional B cells were identified as LC⁺ and CD10⁺. Earlier B lineage cells were LC⁻ and CD10⁺. These early B cells were segregated to a pro-B population characterized by surface CD34, and a pre-B population identified by its lack of CD34. B, The B cell populations were co-stained for the above markers as well as for BAFF-R surface expression or an isotype-matched control antibody. The upper row depicts the BAFF-R surface staining (empty histogram) compared to the isotype control (filled histogram) for that population. The lower row shows a contour plot of the BAFF-R staining in these same populations with gating around the BAFF-R⁺ population. The data are representative of four independently stained bone marrow samples.

FIGURE 2. Organization and conservation of the genomic TNFRSF13C locus in context

A, Representation of the human genomic TNFRSF13C locus at 22q13.2, above the murine locus located at 15qE1, to scale. The diagram represents genes as boxes along the chromosome, represented as a line, in appropriate orientation designated by the arrows. B, VISTA Browser graph showing the percent homology of horse (upper) and mouse (lower) genomes to the human genome, which is acting as the baseline. The values of the histograms are calculated based on sequence lengths of 100 bp. Sequence lengths >100 bp with >70% sequence conservation are indicated by shading. The color of the shading corresponds to the coding, non-coding, or untranslated identity of the homologous sequence, labeled above.

FIGURE 3. Luciferase reporter activity in human B lineage cell lines

A, Surface expression of BAFF-R (empty histogram) on Loukes and RAMOS B cell lines and on ALMC-1 and KAS-6/1 plasma cell lines versus isotype-matched staining (filled histogram). B, Luciferase reporter activity in the cell lines 48 h after transfection with the pGL3-Basic reporters in which the 5′ regions adjacent to the TNFRSF13C gene have been inserted. The upstream regions begin at approximately 2.5, 2.0, 1.5, 1.0, and 0.5 kb upstream of the gene’s transcriptional start site (TSS) and each end 40 bp 3′ of the TSS, 6 bp upstream of the coding sequence. The activity is measured as the ratio of specific promoter activity to empty vector normalized to Renilla luciferase activity in the same cells. Asterisks indicate statistically significant differences between the activity of the promoter insert and the empty pGL3-Basic reporter, p < 0.05. C, Luciferase reporter activity in the RAMOS and Loukes cell lines 48 h after transfection with the pGL3-Basic reporter with the complete −0.5 kb TNFRSF13C promoter inserted, inserts with specific regions of the −0.5 kb promoter deleted, or no insert. Activity was measured as in B. Asterisks represent statistically significant differences in the reporter vectors when compared with the complete −0.5 kb reporter, p < 0.05.

FIGURE 4. DNase protection assay

Using probes generated from the pGL3-Basic constructs created for the reporter assays in conjunction with various primary cell nuclear B cell extracts (tonsillar, peripheral blood, and splenic B cells, in repetitions of the assay), footprinting regions were identified as those which faded more quickly than surrounding bands as the DNase and protein concentration were adjusted. These footprints are noted by the solid black arrows to the right of the lanes. The far left band is a GA tracker, showing the locations of guanine and adenine bases within the sequence. The concentration of DNase during treatment is noted above, while protein concentration is noted with the wedges of increasing concentration at 0, .013, .065, and .13 mg/mL nuclear extract. A and B, Excerpts from representative exposures of two different probes following DNase digestion, probes spanning regions from −676 to −193 and −243 to +40 relative to the TSS, respectively.

FIGURE 5. EMSAs in the region of greatest promoter activity

A, VISTA Browser graphs showing percent homology of the horse (upper) and mouse (lower) genomes to the human genome, which is acting as the baseline. The region shown to have the greatest promoter activity based on the luciferase reporters, from +40 to −88 bp (relative to the TNFRSF13C TSS) is shown. The values of the histograms are calculated based on sequences of 100 bp. Sequence lengths ≥5 bp with 100% sequence conservation, i.e. at least 5 consecutive bases that exactly match the human genome, are indicated by shading. As in Fig. 2B, the color of the shading corresponds to the coding, non-coding, or untranslated identity of the homologous sequence, labeled above. Beneath the histograms, regions identified by the DNase protection assays are identified in the black bars, while EMSA probes encompassing these sites and covering the entire region are shown in gray. B, EMSAs demonstrating the differential shifts of the probes encompassing the region using nuclear extracts generated from the B (Loukes, RAMOS) and plasma (ALMC-1, KAS-6/1) cell lines. Arrowheads indicate shifts present in the B cell line but not the plasma cell line extracts.

FIGURE 6. Mutant luciferase reporter activity in human B lineage cell lines

Luciferase reporter activity in the RAMOS and Loukes cell lines 48 h after transfection with the pGL3-Basic reporter with the complete −0.5 kb TNFRSF13C promoter (“Complete Reporter”), the region of highest activity deleted (Δ( −88/+40)), point mutations at specific conserved sites (−75C>G, +17C>G), or no insert. Activity was measured as in Fig. 3B. Asterisks represent statistically significant differences in the reporter vectors when compared with the complete −0.5 kb reporter, p < 0.05.

FIGURE 7. c-Rel binds TNFRSF13C genomic DNA endogenously and affects BAFF-R expression in B cell lines

A, Western blot of the nuclear extracts of the plasma and B cell lines as well as normal primary peripheral blood (PB) B cells. Extracts were blotted for c-Rel and for Histone H1 as a nuclear control. B, Chromatin immunoprecipitation (ChIP) of the region upstream of the BAFF-R gene with c-Rel and Pax5 antibodies prepared from RAMOS cells. The enrichment values represent amounts relative to the amount of chromatin in the non-specific polyclonal Ab IP. C, Following c-Rel knockdown in Loukes and RAMOS cells with c-Rel-specific siRNA or GC-content-matched non-specific control siRNA, c-Rel and BAFF-R mRNA were measured with qRT-PCR. Amounts were calculated according to the ΔΔC_T method compared to 18S rRNA and normalized to expression in the cells treated with the GC-content control non-specific siRNA. Asterisks indicate a significant change in gene expression in the test c-Rel siRNA compared to control non-specific siRNA, p < 0.05, in three replicates.