(a) Structure of the Igh locus indicating the
location of variable (VH), diversity (DH), joining
(JH) and constant (CH) 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 VH gene families, of
which the VHJ558 (black), VH3609 (pink),
VHQ52 (green) and VH7183 (rosa) genes are relevant for
this study. The diagram of the Igh locus is based on the
mm8 genome sequence and the published VH gene assembly19 of the C57BL/6 mouse.
(b-f) Mapping of active chromatin marks in the
DH-Cμ domain (b) and
representative proximal (c) and distal (d,e)
VH 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).