Stepping up PAX5 Expression

ReseaRch highlights

B CELL DEvELOPmENt Stepping up PAX5 expression

The commitment of lymphoid cells to To localize potential regulatory factors in activating the Pax5 locus. the B cell lineage and maintenance of a elements in intron 5 of Pax5, the All seven HS sites were absent from B cell phenotype require the transcrip- authors identified four DNase I the Pax5 promoter in Tcfe2a–/– and tion factor PAX5, which is expressed hypersensitivity sites (HS) in this Ebf1–/– B cell progenitors, and by B cells at all stages of differentia- region (which indicate areas of the chromatin at these sites had tion, but is repressed in plasma cells. active transcription). Two of these an in active histone modification However, until now little has been sites (HS-A and HS-B) were specific pattern. As E2A is expressed by known about how the expression of to B cells (shown in red). A region Ebf1–/– progenitors (but EBF1 is not PAX5 is regulated. Meinrad Busslinger of Pax5 from intron 4 to intron 5 expressed by Tcfe2a–/– progenitors), and colleagues identify an enhancer containing HS-A and HS-B sites it seems that EBF1 (and not E2A) region in the Pax5 locus and show that was associated with active chrom- is required for the formation of HS B cell-specific PAX5 activity depends atin modifications on histone H3 sites and activation of chromatin in on the stepwise activation of enhancer in pro-B cells that coincide with the the Pax5 promoter. EBF1 was shown and promoter elements. known histone modification pattern to regulate the Pax5 promoter by The authors generated transgenic of an active enhancer. A transgene binding with high affinity to HS-7. mice expressing large bacterial artifi- containing the entire Pax5 promoter By contrast, all HS sites in the cial chromosomes (BACs) containing region fused to a Gfp reporter gene Pax5 enhancer were still present in a green fluorescent protein (Gfp) was only expressed by mature B cells Tcfe2a–/– and Ebf1–/– B cell progenitors reporter gene inserted into sequences when HS-A and HS-B were inserted and had active histone modifications, of Pax5. Mice transgenic for a Gfp downstream of the Gfp gene. So, the indicating that the enhancer region is reporter BAC comprising –18 kb regulated expression of PAX5 by normally active and accessible before to intron 6 of Pax5 (see the figure) B cells requires both the promoter EBF1 activity. HS-B of the Pax5 expressed GFP throughout B cell region and the newly discovered enhancer provided binding sites for development but not in plasma cells enhancer region in intron 5. PU.1, interferon regulatory factor 4 or non-B cells. When the sequence Seven B cell-specific HS sites (IRF4) and IRF8 in pro-B cells and upstream of the Pax5 promoter was were also identified in the Pax5 mature B cells, and also binding of deleted, the GFP expression pattern promoter (shown in red). As the nuclear factor-κB in mature B cells. was retained, showing that any B cell- transcriptional regulators E2A In summary, the authors propose specific regulatory elements must lie (encoded by Tcfe2a) and EBF1 are a model of B cell commitment within Pax5. In support of this, dele- thought to act upstream of Pax5 whereby EBF1 expression at the onset tion of intron 5 resulted in minimal during B cell differentiation, the of B cell development regulates chro- GFP expression by mature B cells. authors investigated the role of these matin remodelling of the Pax5 promoter, which facilitates interaction of +1 Intron 5 –18 kb +72 kb the promoter with the pre-activated 1A 1B 2 Gfp 3 4 5 6 enhancer region. Kirsty Minton

ORIGINAL RESEARCH PAPER Decker, T. et al. Pax5–Gfp BAC. Image Stepwise activation of enhancer and promoter coutesy of M. Busslinger, regions of the B cell commmitment gene Pax5 in Research Institute of Molecular Pathology, early lymphopoiesis. Immunity 2 Apr 2009 HS 12354 678 HS ABCD Vienna Biocenter, Austria. (doi:10.1016/j.immuni.2009.01.012) Promoter Enhancer