Fig 5. Altered Ebf1 chromatin structure results in a blockade of B cell development.

A) Enumeration of B lymphocyte maturation in Stag2 WT and KO mice. Bone marrow was analyzed using flow cytometry for pro- (Cd43⁺) and pre- (Cd43⁻) B-cells in parent gate B220^loIgM⁻ showing B-cell development block in the pro-B to pre-B transition (p<0.003). Immature (B220^loIgM⁺) and recirculating B cells (B220^hiIgM⁺) were analyzed as a percentage of live singlets, which were both markedly reduced in Stag2 KO mice (asterisks indicate statistical significance (student’s t test, **p<0.01, ***p<0.001). B) Methylcellulose colony enriched with IL-7, SCF, and FLT3-L shows reduction in the number of B cell colonies in Stag2 KO bone marrow compared to WT (p<0.003). C) Enumeration of immature B cells (CD34+CD19+) and ratio compared to mature B cells (CD34-CD19+) show reduced immature B cells (p<0.010) and immature:mature ratio in STAG2 mutant MDS patients (p<0.008; n=11) compared to controls (n=15). D-E) Stag2 WT and KO HSPC were infected with lentivirus containing GFP-tagged empty vector, GFP-mycPU.1, or GFP-shPU.1; GFP⁺ cells were plated in either B cell colony methylcellulose or stem cell methylcellulose. Cells were harvested after 7 days and analyzed by flow cytometry for (D) the B marker B220 or (E) stem cell marker cKit. F) Volcano plot for differentially PU.1-occupied loci by chromatin immunoprecipitation sequencing in HSPCs of Stag2 WT and Stag2 KO (data points in red indicate adjusted p<0.1). Loci with decreased in PU.1 occupancy in Stag2 KO to the left (n=246) and loci with increased in PU.1 occupancy in Stag2 KO to the right (n=1). G) HOMER analysis of 246 loci with decreased PU.1 binding shows enrichment for PU.1(ETS) motif (p<10⁹⁸). H) IGV track of the Ebf1 locus for Stag2 WT (n=2) and KO (n=2) shows decreased PU.1 binding at 3 loci (gray boxes).