Defining In Vivo Transcription Factor Complexes of the Murine CD21 and CD23 Genes

Abstract

The expression of the CD21 and CD23 genes is coincident with differentiation from transition 1 B cells (T1) to transition 2 B cells (T2). To define constituents controlling CD21 and CD23 expression, we conducted chromatin immunoprecipitation analyses for candidate transcription factors. We found constitutive binding of Oct-1, NFAT species, YY1, NF-κB-p52, Pax5, E2A, and RBP-Jκ to CD21 sequences and NF-κB-p52, Pax5, NFAT species, E2A, and RBP-Jκ to CD23 promoter sequences. Splenic T and B cell subsets displayed constitutive binding of YY1, NF-κB-p52, Pax5, and Oct-1 proteins to CD21 sequences in B cells but no specific binding of NFATc3 or Pax5 in T cells. Similarly, CD23 sequences demonstrated constitutive binding of NF-κB-p52 in splenic T and B cells but only Pax5 in B cells. Of the various NFAT species, only a subset were found forming constitutive DNA/protein complexes with the CD21, CD23, and IL-2 gene sequences. Maturing B cells in the marrow possess stable Pax5 complexes on CD19, CD21, and CD23 gene promoters in the nuclei of such cells, even though only CD19 is expressed. The similarity of genetic controlling elements between the CD21 and CD23 genes does not suggest a mechanism for alternative regulation of these genes; however, separation of splenic B cell subsets into T1, T2, marginal zone (MZ), and mature follicular B cells, followed by quantitative RT-PCR, demonstrated the lack of appreciable CD23 transcripts in CD21+ MZ cells. We propose an alternative derivation of MZ cells as maturing directly from T1 cells, leaving CD23 transcriptionally inactive in that lineage of cells.

Canonical NF-κB signaling is uniquely required for the long-term persistence of functional mature B cells

“…1C). CD23, used to discriminate T1 and T2 cells (15), has been reported to be an NF-κB target gene (16). We therefore confirmed the identity of the mutant transitional populations using an independent marker, CD93, which is expressed at lower levels on T2 cells (3,9) (Fig.…”

Defining a transcriptional fingerprint of murine splenic B-cell development

“…19,59 One such protein, Pax5, has been implicated in controlling Cr2 and Fcer2a expression, 23 and we have documented that Pax5 binds to the genes in vivo using mature splenic B cells. 19 Interestingly, Pax5 is also bound to these two genes as well as CD19 in immature marrow B cells that express the CD19 protein but not the CR1/CR2 or CD23 proteins, suggesting that the Cr2 and Fcer2a genes may be transcriptionally repressed in such cells.…”

“…Semiquantitative RT-PCR for C2ta using P 32 -dCTP was performed as described before 45 and PhosphorImager quantification was done (STORM820 PhosphorImager and Image Quant analysis; Molecular Diagnostics, Inc., Sunyvale, CA, USA) as detailed earlier. 19 …”

“…17,18 The Cr2 and Fcer2a genes share a number of transcriptional control elements including those for Pax5, nuclear factor (NF)-kB, nuclear factor of activated T cells family members, E box protein E2A and the Notch signaling family member retinol binding protein-Jk. [19][20][21][22] Mature B cells engineered to lack Pax5 (through a CD19-credependent deletion) show decreased expression of the CR1/CR2 proteins and CD23. 23 Earlier we have demonstrated that Pax5 binds equally to the Cr2 and Fcer2a genes in expressing mature splenic B cells as well as marrow B cells, which do not express Cr2 and Fcer2a.…”

“…23 Earlier we have demonstrated that Pax5 binds equally to the Cr2 and Fcer2a genes in expressing mature splenic B cells as well as marrow B cells, which do not express Cr2 and Fcer2a. 19 This finding suggests that these two genes either lack a key activating transcription factor to allow their expression at the T1/T2 stages or are blocked from transcription in marrow B cells by a transcriptional silencing protein whose function is lost during B-cell differentiation.…”