Fig. 7: E_VH interactome identified.

a Virtual 4C interactions were extracted from KR normalized Hi-C data sets from Rag1^−/− (black lines) and Rag1^−/−E_VH1^−/− (dashed red lines) CD19⁺ pro-B cells. Top: Genomic coordinates (mm10) with the E_VH1 ZOI and Sites (S) I, II, II.5 and III. 4C viewpoints (dashed vertical line) in a 30 kb running window analysis with 10 kb steps from merged biological replicates with E_VH1 interactions (black arcs) shown. Stars (top 15%), circles (top 25%) of locus-wide interactions. New peaks (red arrows) and lost peaks (black arrows) in Rag1^−/−E_VH1^−/− pro-B cells. b Diagram of the Igh locus with genomic distances and FISH probe indicated. Chromatin loops detected in pro-B cells (see Fig. 3a). c–e Source data are provided as a Source Data file. c Representative nuclei from Rag2^−/− and Rag2^−/−E_VH1^−/− CD19⁺ pro-B cells. Short FISH probes, Alexa Fluor 488 (Eμ, green), and Alexa Fluor 647 (E_VH1, blue), Alexa Fluor 555 (E_VH2, red) were hybridized simultaneously to fixed cells in duplicate. Scale bar, 2 μm. d Quantitation of 3D probe configurations in three-color DNA FISH. N = 400 alleles/genotype in two independent experiments. (Upper panel) Nine probe configurations. (Lower panel) The frequency of each probe configuration. P values from two tailed Chi square test. e Median spatial distance was derived for probes engaged in 3-way overlaps shown in panel d inset a. Rag2^−/− (n = 33), Rag2^−/−E_VH1^−/− (n = 54) alleles. Box plots show median (middle line) spatial distances, 25th and 75th percentile (box) and 10th and 90th percentile (whiskers), and outliers (single points) between. P values from two-tailed Wilcoxon signed rank sum test. f Graphically displayed median spatial distances between probes in three-way overlaps from Rag2^−/− (black arrows) and Rag2^−/−E_VH1^−/− (gray arrows) pro-B cells. g Representation of Igh alleles in distinct spatial configurations (a–c, e–g, h, i).