Extended Data Fig. 5. Cohesin stabilization on chromatin by Pax5-dependent Wapl repression in pro-B cells.

a, Wapl mRNA expression in developing and mature B and T cells of wild-type mice. The indicated B and T cell types were sorted from the bone marrow (pro-B, pre-B cells), thymus (DN, DP, CD4⁺, CD8⁺ T cells) or spleen (mature B, CD4⁺ and CD8⁺ T cells). The Wapl mRNA expression of the different cell types was determined by RT-qPCR analysis relative to Tbp expression and is shown relative to that of pro-B cells (set to 1). The expression data are shown as mean values with SEM, based on four RT-qPCR experiments per cell type. b, Wapl protein expression in ex vivo sorted Rag2 ^–/– pro-B cells from the bone marrow and wild-type mature B cells from the spleen and lymph nodes, as determined by immunoblotting of 3-fold serially diluted whole-cell extracts with antibodies detecting Wapl or TBP. One of two experiments is shown with marker proteins (kilodaltons). c, B cell development in the bone marrow of Smc3-Gfp transgenic (green) and wild-type (black) mice, as determined by flow cytometry. The relative frequencies of the indicated cell types are shown as mean values with SEM (left). GFP expression in selected cell types is shown (right). d, Interaction of the Smc3-GFP protein with other cohesin subunits in short-term cultured Smc3-Gfp pro-B cells. Endogenous Smc1, Scc1 (Rad21), Wapl, Pds5a and Pds5b proteins were co-precipitated with Smc3-GFP from whole-cell extracts of Smc3-Gfp or wild-type pro-B cells with an anti-GFP antibody. The input (1/10) and protein precipitate were analysed by immunoblotting with antibodies detecting the indicated cohesin proteins. Smc3ac, acetylated Smc3. One experiment was performed. e, Images of one of 16 or 24 iFRAP experiments performed with Smc3-Gfp pro-B or mature B cells, respectively. The fluorescence intensities of SiR-Hoechst (pink) and GFP (green) are false-coloured. Scale bar, 10 μm. f, Identification of open chromatin regions at the Wapl locus by ATAC-seq of the indicated progenitor and B cell types. MPPs, ALPs, pro-B and mature B cells were sorted from wild-type bone marrow or lymph nodes (mature B). Activated B cells and plasmablasts were generated by in vitro LPS stimulation of mature B cells. hCD2^– (Pax5^–) and hCD2⁺ (Pax5⁺) BLPs were sorted from bone marrow of Pax5 ^ihCd2/ihCd2 mice, and Pax5 ^Δ/Δ progenitors from Vav-Cre Pax5 ^fl/fl Rag2 ^–/– mice. n, number of mice. Each dot corresponds to one mouse (a,c). The different cell types are defined in Methods.