Essential role of the Pax5 C-terminal domain in controlling B cell commitment and development

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. ChIP-Seq: Peak calling and read signal comparison between Pax5(+/+), Pax5(DCRD1/DCRD1), Pax5(DCRD1,2/DCRD1,2) and Pax5(Prd/floxed) conditions (Pax5 antibody); SB(+/+), Pax5(DCRD1/-) conditions (Brg1 antibody). ATAC-Seq: Peak calling and read signal comparison between Pax5(+/+), Pax5(DCRD1/DCRD1), Pax5(DCRD1,2/DCRD1,2) and KO (vav-cre Pax5(fl/fl)) conditions. Two replicates each. RNA-Seq: Comparative gene expression profiling analysis of RNA-seq data for wild type Pro-B cells (Pax5(+/+) and various central (Pax5(DOP/DOP), Pax5(DHD/DHD), Pax5(DOP,HD/DOP,HD)) ,C-terminal (Pax5(DCRD1/-),Pax5(DCRD2/-),Pax5(DCRD1,2/-),Pax5(DCRD1/DCRD1),Pax5(DCRD2/DCRD2),Pax5(DCRD1,2/DCRD1,2) mutants and KO (vav-cre Pax5(fl/fl)). Furthermore comparisons between vav-cre Pax5(floxed/+), vav-cre Pax5(Prd/floxed) and vav-cre Pax5(floxed/floxed). At least two replicates per condition. Various other tracks are reported only for expression analysis (TPMs) (LMPP-,ALP-,BLP-,Pre-B-,Immature B-,DN1-,DN2a-,DN2b-,DN3a-,DN3b-cells).