Figure 5. Mapping of active histone modifications along the Igh locus in Rag2^−/− pro-B cells.

(a) Structure of the Igh locus indicating the location of variable (V_H), diversity (D_H), joining (J_H) and constant (C_H) gene segments together with the intronic (Eμ) enhancer, the 5’ DNase I hypersensitive sites (red) and 3’ regulatory region (3’RR; red). Different colors indicate the members of distinct V_H gene families, of which the V_HJ558 (black), V_H3609 (pink), V_HQ52 (green) and V_H7183 (rosa) genes are relevant for this study. The diagram of the Igh locus is based on the mm8 genome sequence and the published V_H gene assembly¹⁹ of the C57BL/6 mouse. (b-f) Mapping of active chromatin marks in the D_H-C_μ domain (b) and representative proximal (c) and distal (d,e) V_H gene regions of the Igh locus as well as at the 5’ end of the Ikzf1 (Ikaros) gene (f) in Rag2^−/− pro-B cells. Antibodies recognizing acetylated lysine 9 (H3K9ac), dimethylated lysine 4 (H3K4me2) or trimethylated lysine 4 (H3K4me3) on histone H3 were used for ChIP-chip analysis³¹ of Rag2^−/− bone marrow pro-B cells, which were expanded in vitro for 5 days in the presence of IL-7 and OP9 stromal cells. The ChIP-precipitated DNA was amplified with the T7-based linear amplification protocol³¹ prior to fluorescent labeling and co-hybridization with the input DNA probe onto a high-density 50-mer oligonucleotide array containing the mouse Igh locus at 100-bp resolution (produced by NimbleGen Systems). The logarithmic ratio (log₂) of the hybridization intensities between antibody-precipitated and input DNA (bound/input) is shown as a bar for each oligonucleotide on the microarray. The results shown are representative of two ChIP-chip experiments, which were performed with independently prepared DNA probes from Rag2^−/− pro-B cells. A scale bar is shown in kb. Supplementary Fig. 15 shows the result of a similar ChIP-chip analysis, which was performed with DNA probes amplified with the WGA method³². The mm8 sequence coordinates for the displayed regions of the Igh locus on chromosome 12 are 116,520,000-113,586,000 (a), 113,938,000-113,852,500 (b), 114,234,000-114,086,000 (c), 115,441,500-115,087,000 (d) and 115,859,000-115,780,000 (e).