Figure 1. Chromatin accessibility at DSP2.2b-J_H1 rearranged allele.

(a) Schematic of the germline Igh locus in 6312 cells and the DJ_H rearranged Igh locus in 6312-derivative 2B9 cells, which harbor a DSP2.2b-J_H1 junction in one allele and a V_H rearrangement in the other allele (not to scale). The positions of amplicons analyzed by real-time PCR are also shown. Block arrows represent D_H-associated repeat sequences^10,11. (b) ChIP assays were performed using antibodies for modified histones as indicated with chromatin obtained from 6312 (maroon bars) and 2B9 (pink bars) cells. The numbers within the parentheses indicate positions in kb 5′ of the DSP2.2b segment. All samples were assayed in duplicate by real-time PCR and relative abundance (y axis) for each amplicon in the immunoprecipitate was calculated as previously described¹¹. γ-actin promoter and C_γ3 served as controls for active and inactive chromatin, respectively. Data show the average of 2 independent ChIP experiments and error bars indicate standard deviation. (c) DNase I sensitivity analyses of the DSP2.2b-J_H1 allele (pink lines) compared to the germline Igh allele (maroon lines). 2×10⁶ nuclei from 6312 and 2B9 cells were treated with increasing concentrations of DNase I (x axis, 0 to 2 units of DNase I) followed by purification of the genomic DNA. All samples were assayed in duplicate by quantitative real-time PCR and the proportion of intact DNA (y axis) at each DNase I concentration was determined for the indicated amplicon as previously described¹⁸. E_μ corresponds to the known DNase I hypersensitive site in the J_H-C_μ intron, while C_γ3 is DNase I insensitive. Data show the average of 2 independent DNase I sensitivity experiments and error bars indicate standard deviation.