Early B-Cell Factor, E2A, and Pax-5 Cooperate To Activate the Early B Cell-Specific mb-1 Promoter

Abstract

Previous studies have suggested that the early-B-cell-specific mb-1(Ig␣) 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.

Pax5 inducesV-to-DJrearrangements and locus contraction of theimmunoglobulin heavy-chaingene

“…More surprisingly, these pro-T cells also expressed the genes Ig␤ (B29), 5, and VpreB (Fig. 4C), which are known to be cooperatively regulated by the transcription factors EBF and E2A (Sigvardsson et al 1997(Sigvardsson et al , 2002O'Riordan and Grosschedl 1999). Consistent with this finding, Pax5 induced expression of the B-cellspecific EBF gene in Ik Pax5/+ pro-T cells to a level that is normally seen in wild-type and Pax5 −/− pro-B cells.…”

Differential Functions for the Transcription Factor E2A in Positive and Negative Gene Regulation in Pre-B Lymphocytes

“…The His and FLAG tags were sequentially used in the affinity purification of E2A bound chromatin. We first evaluated this revised protocol for E2A interaction with the regulatory regions of the 5 surrogate light chain and the mb-1 genes (13,14). Physiological E2A interactions were previously demonstrated at the promoters for both 5 and mb-1 by ChIP-PCR screening (16).…”

Pearson Correlation Analysis of Microarray Data Allows for the Identification of Genetic Targets for Early B-cell Factor

“…The EBF encoding plasmid mediating puromycin resistance was based on the retroviral vector pBabe (30). The mb-1 and 5 promoter constructs have been reported previously (23,31), whereas the VpreB1, CD19, and VpreB3 promoters were cloned by PCR using genomic mouse DNA as template and cloning of the resulting products in the SmaI site of the luciferase reporter plasmid pGL3 basic (Promega). All constructs were verified by sequencing.…”

“…The analysis of control gene expression suggested that this approach allowed for a reasonable approximation of expression patterns (22) giving us a tool to investigate also the coordination of stage-specific gene activation. We have used part of these data to estimate the relative importance of transcription factors in the activation of the mouse mb-1 (Ig␣) promoter by Pearson correlation analysis (23). This promoter contains binding sites for EBF, E47, BSAP, and Ets proteins (23)(24)(25)(26)(27) and when comparing the expression levels of the mb-1 message to the levels of the transcription factors, EBF gave a 98% correlation, whereas BSAP and Ets proteins resulted in lower values (23).…”

“…We have used part of these data to estimate the relative importance of transcription factors in the activation of the mouse mb-1 (Ig␣) promoter by Pearson correlation analysis (23). This promoter contains binding sites for EBF, E47, BSAP, and Ets proteins (23)(24)(25)(26)(27) and when comparing the expression levels of the mb-1 message to the levels of the transcription factors, EBF gave a 98% correlation, whereas BSAP and Ets proteins resulted in lower values (23). This indicated to us that the expression of the mb-1 gene was highly dependent of EBF and that this type of analysis can provide important biological information.…”