Extended Data Fig. 10: Changes of chromosomal architecture and gene expression in Wapl+/+ and Wapl∆P1,2/∆P1,2 pro-B cells.
From: Wapl repression by Pax5 promotes V gene recombination by Igh loop extrusion
a, iFRAP analysis of splenic mature B cells from Wapl∆P1,2/+ (blue) and Wapl+/+ (pink) mice carrying the Smc3-gfp transgene. The difference in fluorescence intensity between bleached and unbleached regions is plotted against time. The mean values are indicated by lines and the s.d. by shading. n, number of cells analysed in three experiments per genotype. b, Frequency distribution of intrachromosomal contacts as a function of the genomic distance using logarithmically increased genomic distance bins, as determined by Homer analysis of Hi-C data generated with short-term-cultured pro-B cells (grey) and ex vivo-sorted splenic mature B cells (pink) from wild-type mice. c, Hi-C contact matrices of a zoomed-in region on chromosome 12 (mm9: 72,500,000–78,500,000; upper row) and 16 (21,500,000–28,500,000; lower row), displayed at a 10-kb bin resolution for Pax5∆/∆ progenitors, Wapl+/+ and Wapl∆P1,2/∆P1,2 pro-B cells. Black dots indicate loop anchors identified with Juicebox, and the intensity of each pixel represents the normalized number of contacts between a pair of loci30. The maximum intensity is indicated in the lower left of each panel. d, Density distribution of the loop length in Pax5∆/∆ progenitors, Wapl+/+ and Wapl∆P1,2/∆P1,2 pro-B cells, as determined with HiCCUPS of Juicer. The median loop length (in kb) is shown for each genotype. e, Scatter plot of gene expression differences, based on two RNA-seq experiments per genotype. Genes upregulated (red) or downregulated (blue) in Wapl∆P1,2/∆P1,2 pro-B cell compared to Wapl+/+ pro-B cells were defined by an expression difference of >2-fold, an adjusted P value of <0.05 and a TPM value of >5 in at least one of the two pro-B cell types (Supplementary Table 3). f, Functional classification and quantification of the proteins encoded by upregulated (red) and downregulated (blue) genes in Wapl∆P1,2/∆P1,2 pro-B cells relative to Wapl+/+ pro-B cells (Supplementary Table 3). See Extended Data Fig. 9g for detailed explanation. g, Expression of selected genes in Wapl∆P1,2/∆P1,2 (grey) and Wapl+/+ (black) pro-B cells. The expression of the indicated genes, which are not differentially expressed according to the definition in e, is shown as mean TPM value. h, Schematic depicting a ‘stable’ loop formed by loop extrusion across the entire Igh locus in pro-B cells. The forward CBEs (in the VH gene cluster and IGCR region) and the reverse CBEs (in the IGCR and 3′CBE regions) are indicated by red and blue arrows, respectively. The RAG-bound recombination centre, which is located at the DJH-rearranged gene segment29, is indicated in grey. The convergent orientation of the 12-RSS (recognition signal sequence with a 12-bp spacer, black arrowhead) of the DH segment and the 23-RSS (with a 23-bp spacer, green arrowhead) of the VH genes is essential for mediating RAG-cleavage and deletional joining16. The loop-extruding cohesin ring (orange) is arrested at convergent CBEs. In a ‘stable’ loop, the correct alignment of the RSS elements of a VH gene and the DJH-rearranged gene segment probably occurs by local diffusion. Arrowheads symbolize the RSS element consisting of the heptamer, nonamer and intervening spacer. i, Correct alignment of the RSS elements of a VH gene and the DJH-rearranged gene segment may be mediated by the ongoing process of loop extrusion. Orange and black arrows indicate the direction of movement of cohesin and DNA, respectively. j, Misalignment of the RSS elements of an inverted VH gene and the DJH-rearranged gene segment during loop extrusion, which prevents RAG-mediated cleavage.
