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. 2023 Mar 3;14:1225. doi: 10.1038/s41467-023-36414-2

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — a Schematic of the Igh locus. Eμ is situated between J_H and C_H genes and 3’Eα. The J_H and D_H segments are located between the constant region genes (C_H) and V_H7183 (V_H81X) segments. b Regulatory elements (colored dots) identified in a arrayed along the Hi-C diagonal. Hi-C difference heatmaps at 10 kb resolution from purified CD19⁺ pro-B cells from Rag1^−/− (n = 2) (i) and Rag1^−/−E_VH1^−/− (n = 2) (ii) mice were generated (see Methods). Nested loop domains (dashed vertical lines), stripes (3’->5’, blue triangles; 5’->3’ black triangles), dots (black circles), lost dots (gray circles). Rag1^−/−E_VH1^−/− specific stripes (purple triangles) and dots (purple circles). iii, iv) Schematic of nested loop domains in Hi-C difference heatmaps from Rag1^−/− (iii), and Rag1^−/−E_VH1^−/− (iv) pro-B cells. c The Igh locus includes the interspersed V_H J558/3609 (V_H1-86) families at the 5’ end, the V_H7183 (V_H81X) family at the 3’ end and the intermediate V_H segments (V_H14-2) include the S107 family with nine smaller V_H families. Representative V_H genes are positioned above the locus (chr12, mm10), V_H81X (113578506 − 113578799), V_H14-2 (113994469-113994762), V_H1-2 (114469075-114469371) V_H1-86 (116000028-1160000321). BAC probes, H14, Eμ, RI, RII, RIII are indicated by their colors and are shown with the distances between them. d–f Source data are provided as a Source Data file. d Representative nuclei from fixed Rag2^−/− and Rag2^−/−E_VH1^−/− pro-B cells hybridized with three labeled probe combinations in two biologically independent experiments. Scale bar, 2 μm. Rag2^−/− pro-B alleles for each probe pair: n = 430 (H14-RI), n = 541 (RI-RII), n = 922 (Eμ-RI, RI-RIII, Eμ-RIII). Rag2^−/−E_VH1^−/− pro-B alleles for probe pairs: n = 1213 (H14-RI), n = 400 (RI-RII), n = 566 (Eμ-RI, RI-RIII, Eμ-RIII). e Mean inter-probe distances as a function of genomic distance. Lines indicate connectivity only. The RI anchor probe position, vertical dashed line. f Box plots show the median (middle line), 25th and 75th percentile (box) and 10th and 90th percentile (whiskers) and outliers (single points) of spatial distances between probes and anchor R1. P values from unpaired two-tailed nonparametric Mann–Whitney U test.