Pax5–Gfp BAC. Image coutesy of M. Busslinger, Research Institute of Molecular Pathology, Vienna Biocenter, Austria.

The commitment of lymphoid cells to the B cell lineage and maintenance of a B cell phenotype require the transcription factor PAX5, which is expressed by B cells at all stages of differentiation, but is repressed in plasma cells. However, until now little has been known about how the expression of PAX5 is regulated. Meinrad Busslinger and colleagues identify an enhancer region in the Pax5 locus and show that B cell-specific PAX5 activity depends on the stepwise activation of enhancer and promoter elements.

The authors generated transgenic mice expressing large bacterial artificial chromosomes (BACs) containing a green fluorescent protein (Gfp) reporter gene inserted into sequences of Pax5. Mice transgenic for a Gfp reporter BAC comprising −18 kb to intron 6 of Pax5 (see the figure) expressed GFP throughout B cell development but not in plasma cells or non-B cells. When the sequence upstream of the Pax5 promoter was deleted, the GFP expression pattern was retained, showing that any B cell-specific regulatory elements must lie within Pax5. In support of this, deletion of intron 5 resulted in minimal GFP expression by mature B cells.

To localize potential regulatory elements in intron 5 of Pax5, the authors identified four DNase I hypersensitivity sites (HS) in this region (which indicate areas of active transcription). Two of these sites (HS-A and HS-B) were specific to B cells (shown in red). A region of Pax5 from intron 4 to intron 5 containing HS-A and HS-B sites was associated with active chromatin modifications on histone H3 in pro-B cells that coincide with the known histone modification pattern of an active enhancer. A transgene containing the entire Pax5 promoter region fused to a Gfp reporter gene was only expressed by mature B cells when HS-A and HS-B were inserted downstream of the Gfp gene. So, the regulated expression of PAX5 by B cells requires both the promoter region and the newly discovered enhancer region in intron 5.

Seven B cell-specific HS sites were also identified in the Pax5 promoter (shown in red). As the transcriptional regulators E2A (encoded by Tcfe2a ) and EBF1 are thought to act upstream of Pax5 during B cell differentiation, the authors investigated the role of these factors in activating the Pax5 locus. All seven HS sites were absent from the Pax5 promoter in Tcfe2a−/− and Ebf1−/− B cell progenitors, and the chromatin at these sites had an inactive histone modification pattern. As E2A is expressed by Ebf1−/− progenitors (but EBF1 is not expressed by Tcfe2a−/− progenitors), it seems that EBF1 (and not E2A) is required for the formation of HS sites and activation of chromatin in the Pax5 promoter. EBF1 was shown to regulate the Pax5 promoter by binding with high affinity to HS-7.

By contrast, all HS sites in the Pax5 enhancer were still present in Tcfe2a−/− and Ebf1−/− B cell progenitors and had active histone modifications, indicating that the enhancer region is normally active and accessible before EBF1 activity. HS-B of the Pax5 enhancer provided binding sites for PU.1, interferon regulatory factor 4 (IRF4) and IRF8 in pro-B cells and mature B cells, and also binding of nuclear factor-κB in mature B cells.

In summary, the authors propose a model of B cell commitment whereby EBF1 expression at the onset of B cell development regulates chromatin remodelling of the Pax5 promoter, which facilitates interaction of the promoter with the pre-activated enhancer region.