Pax5 is an essential regulator of B cell identity and function. Here, we used transgenesis and deletion mapping to identify a potent enhancer in intron 5 of the Pax5 locus. This enhancer in combination with the promoter region was sufficient to recapitulate the B lymphoid expression of Pax5. The enhancer was silenced by DNA methylation in embryonic stem cells, but became activated in multipotent hematopoietic progenitors. It contained functional binding sites for the transcription factors PU.1, IRF4, IRF8, and NF-kappaB, suggesting that these regulators contribute to sequential enhancer activation in hematopoietic progenitors and during B cell development. In contrast, the promoter region was repressed by Polycomb group proteins in non-B cells and was activated only at the onset of pro-B cell development through induction of chromatin remodeling by the transcription factor EBF1. These experiments demonstrate a stepwise activation of Pax5 in early lymphopoiesis and provide mechanistic insights into the process of B cell commitment.

The B cell regulator Pax5 consists of multiple domains whose function we analyzed in vivo by deletion in Pax5. While B lymphopoiesis was minimally affected in mice with homozygous deletion of the octapeptide or partial homeodomain, both sequences were required for optimal B cell development. Deletion of the C-terminal regulatory domain 1 (CRD1) interfered with B cell development, while elimination of CRD2 modestly affected B-lymphopoiesis. Deletion of CRD1 and CRD2 arrested B cell development at an uncommitted pro-B cell stage. Most Pax5-regulated genes required CRD1 or both CRD1 and CRD2 for their activation or repression as these domains induced or eliminated open chromatin at Pax5-activated or Pax5-repressed genes, respectively. Co-immunoprecipitation experiments demonstrated that the activating function of CRD1 is mediated through interaction with the chromatin-remodeling BAF, H3K4-methylating Set1A-COMPASS, and H4K16-acetylating NSL complexes, while its repressing function depends on recruitment of the Sin3-HDAC and MiDAC complexes. These data provide novel molecular insight into how different Pax5 domains regulate gene expression to promote B cell commitment and development.

The transcription factor Blimp1 is not only an essential regulator of plasma cells, but also a risk factor for the development of autoimmune disease in humans. Here, we demonstrate in the mouse that the Prdm1 (Blimp1) gene was partially activated at the chromatin and transcription level in early B cell development, although mature Prdm1 mRNA did not accumulate due to posttranscriptional regulation. By analyzing a mouse model that facilitated ectopic Blimp1 protein expression throughout B lymphopoiesis, we could demonstrate that Blimp1 impaired B cell development by interfering with the B cell gene expression program, while leading to an increased abundance of plasma cells by promoting premature plasmablast differentiation of immature and mature B cells. With progressing age, these mice developed an autoimmune disease characterized by the presence of autoantibodies and glomerulonephritis. Hence, these data identified ectopic Blimp1 expression as a novel mechanism, through which Blimp1 can act as a risk factor in the development of autoimmune disease.

PAX5 is a tumor suppressor in B-ALL, while the role of PAX5 fusion proteins in B-ALL development is largely unknown. Here, we studied the function of PAX5-ETV6 and PAX5-FOXP1 in mice expressing these proteins from the Pax5 locus. Both proteins arrested B-lymphopoiesis at the pro-B to pre-B-cell transition and, contrary to their proposed dominant-negative role, did not interfere with the expression of most regulated Pax5 target genes. Pax5-Etv6, but not Pax5-Foxp1, cooperated with loss of the Cdkna2a/b tumor suppressors in promoting B-ALL development. Regulated Pax5-Etv6 target genes identified in these B-ALLs encode proteins implicated in pre-B-cell receptor (BCR) signaling and migration/adhesion, which could contribute to the proliferation, survival, and tissue infiltration of leukemic B cells. Together with similar observations made in human PAX5-ETV6+ B-ALLs, these data identified PAX5-ETV6 as a potent oncoprotein that drives B-cell leukemia development.