Extended Data Fig. 9: Generation and analysis of conditional Eed mutant mice.

a, Generation of a floxed (fl) Eed allele. The Eed^fl-Neo allele was generated by replacing exon 6 of the Eed gene with the following sequences in the 5′ to 3′ direction: (i) a loxP-flanked Eed cDNA fragment (from exon 6 to the 5′ part of the 3′UTR) linked to six copies of the SV40 polyadenylation (pA) region, (ii) a DNA fragment containing the 3′ splice site and 5′ sequences of exon 6 fused in-frame to the coding sequence of gfp followed by an SV40 pA sequence and (iii) a frt-flanked DNA fragment containing the mouse phosphoglycerate kinase 1 (Pgk1) promoter linked to the neomycin (Neo) resistance gene. Brackets indicate the two homology regions used for ES cell recombination. The frt (blue) and loxP (red) sites (arrowheads) of the Eed^fl-Neo allele were used to generate the Eed^fl and Eed^∆ alleles by sequential Flpe- and Cre-mediated deletion in vivo. The six SV40 pA sequences downstream of the last Eed exon prevented RNA splicing to the gfp exon before Cre-mediated deletion, as demonstrated by flow cytometry (e, right). A genotyping gel image of Eed^+/+, Eed^fl/+, and Eed^fl/fl mice is shown to the right. b, Selective loss of H3K27me3 at the Wapl promoter in Wapl^∆P1/∆P1 pro-B cells (light blue) compared to Wapl^+/+ pro-B cells (black). The H3K27me3 signal is precipitously lost 220 bp upstream of the P1 site. One of two ChIP-seq experiments is shown. c, Loss of Ezh2 and Pax5 binding at the Wapl promoter in mature B cells after two days of activation. d, Co-precipitation of Myc-tagged Ezh2 or IRF4 by streptavidin (SA) pulldown of biotinylated Pax5-Bio from nuclear extracts prepared from HEK-293T cells that were transiently transfected with expression vectors encoding Pax5-Bio-IRES-BirA, Myc-Ezh2, Eed and Suz12 or Pax5-Bio-IRES-BirA and Myc-IRF4. The input (1/100) and protein precipitates were analysed by immunoblotting with antibodies detecting Myc and Pax5. The band indicated by an asterisk may correspond to endogenous Myc. One of four experiments is shown with marker proteins (kilodaltons). e, Flow cytometric analysis of bone marrow from Rag1^cre/+Eed^fl/fl (blue) and control (black) mice. Rag1^cre/+Eed^fl/+, Rag1^cre/+, Eed^fl/+ and Eed^+/+ mice were used as control. The relative frequencies of the indicated cell types (left), which were determined in six experiments, are shown as mean values with s.e.m. and were analysed by multiple t-tests (unpaired and two-tailed with Holm–Sidak correction). GFP expression (right) is shown for Rag1^cre/+Eed^fl/fl pro-B cells in contrast to control Eed^fl/+ pro-B cells. f, Scatter plot of gene expression differences between Eed-deficient and control pro-B cells. Eed-activated (blue) and Eed-repressed (red) genes were defined by an expression difference of >3-fold, an adjusted P value of <0.05 and a TPM value of >5 in Eed-deficient or control pro-B cells, respectively (Supplementary Table 2). The expression data are based on 5 (Rag1^cre/+Eed^fl/fl) and 4 (control; 3 × Rag1^cre/+Eed^fl/+, 1 × Eed^fl/fl) RNA-seq experiments. g, Functional classification and quantification of the proteins encoded by Eed-activated and Eed-repressed genes (Supplementary Table 2). The bar size indicates the percentage of activated or repressed genes in each functional class relative to the total activated or repressed genes, respectively. Numbers in the bars refer to the genes in each functional class. h, Expression of selected genes in Eed-deficient (grey) and Eed-expressing (black) pro-B cells. The expression of the indicated genes, which are not differentially expressed according to the definition in f, is shown as mean TPM value with s.e.m. i, j, VDJ-seq analysis of Rag1^cre/+Eed^fl/fl and control pro-B cells. i, The mean percentages of uniquely identified DJ_H and VDJ_H sequences were determined based on 4 (Rag1^cre/+Eed^fl/fl) and 2 (control; 1 × Eed^fl/+, 1 × Eed^+/+) experiments. j, The V_H gene usage is shown as mean percentage of all VDJ_H rearrangement events with s.e.m. n, number of experiments. Each dot corresponds to one mouse (e, h, i) or one gene (f).

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