Fig. 2: Contact-dependent γH2Ax profiles in a tractable cell model.

a Southern blotting analysis for DSBs targeted to the Eβ enhancer in Tcrb. Bands corresponding to uncut (top band) or cut (bottom band) loci are indicated. Genomic DNA was harvested 4 h after Lig4^−/− or Rag2^−/− v-abl cells were nucleofected with an RNP targeting Eβ. See Supplementary Fig. 2A for replicates. b UCSC genome browser tracks showing γH2Ax CR-seq performed 2 h after Lig4^−/− or Rag2^−/− v-abl cells were nucleofected with RNP-Eβ (n = 2, representative of independent replicates). Tcrb gene segments, neighboring genes (red arrows) and the DSB location (lightning bolt) are shown at the top. TAD locations are indicated on bottom. c UCSC genome browser tracks showing the Tcrb loci in pro- (63–12 cell line) or pre-lymphocyte cell lines (p5424). For each panel, the locations of gene segments, regulatory elements, and RNP target (lightning bolt and dashed line) are shown at the top. Tracks represent values for γH2Ax CR-seq (red, RPKM, n = 3, representative of independent replicates), V.4C (gray, interaction count, n = 2 merged independent replicates). In panels with interactome data, V.4C is extracted from the DSB site (dark blue line). c, e include subtraction plots (labeled Δ) for γH2Ax and V.4C data, showing differences in (mean of independent replicates) signal from pro- versus pre-lymphocytes. d Box and whisker plot showing γH2Ax in 25 kb bins after pre- (p5424: gray) or pro- (63–12: gray) lymphocyte cell lines were nucleofected with an RNP-Eβ. Data points show three biologically independent replicates. Means, quartiles, and outlier limits (1.5 × interquartile range) are indicated by the median line, box and whiskers, respectively. Relative locations are indicated above the graph. *p < 0.05, two-sided Paired Student’s T test. Enrichment and statistics for all bins across the Tcrb locus is shown in Supplementary Fig. 2C. e UCSC genome browser tracks showing the Myc locus, as in (c). Green tracks represent H3K27ac CR-seq (green, RPKM, n = 1). Cell type-specific H3K27ac+ regions near the Myc Super-Enhancer (SE) are indicated by colored dots, showing positions for pre- (black) or pro- (gray) lymphocyte enhancers.