DNA methylation status of DNase 1 hypersensitive sites 5′ of DFL16.1.

<p>Three newly identified DNase 1 hypersensitive sites are located at approximately 6–6.5, 4–4.5, and 0.4–1.3 kb 5′ of DFL16.1. Genomic DNA from primary RAG-deficient pro-B cells and DP (CD4⁺ CD8⁺) thymocytes were used in bisulfite mapping experiments to examine CpG methylation. Five amplicons covering regions between 3 kb and 7 kb 5′ of DFL16.1 were modified, cloned, and sequenced. The distribution of CpG dinucleotides within each amplicon are noted in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001475#pbio.1001475.s003" target="_blank">Figure S3</a>. Filled and open circles represent methylated and unmethylated residues, respectively. Pie charts summarize the percentage of methylated alleles at each position.</p

Immunoglobulin heavy chain locus and B cell development.

<p>Schematic of the murine IgH locus showing variable (V_H, blue, n represents approximately 150 V_H gene segments), diversity (D_H, grey, n represents six to nine DSP gene segments), and joining (J_H, orange) gene segment <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001475#pbio.1001475-Chevillard1" target="_blank">[11]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001475#pbio.1001475-Johnston1" target="_blank">[12]</a>. Exons encoding the constant regions of IgM and IgD are indicated as Cμ and Cδ. A promoter 5′ of DQ52, the 3′-most D_H gene segment, is indicated by the yellow oval and the intronic enhancer Eμ by a teal oval. Top line shows the germline (GL) configuration with associated histone modifications in B lineage precursors <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001475#pbio.1001475-Chakraborty1" target="_blank">[23]</a>–<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001475#pbio.1001475-Chowdhury1" target="_blank">[25]</a>. Histone H3 and H4 acetylation are shown in orange and presence of heterochromatic H3K9 methylation by the red line. Vertical red arrows represent the tissue-specific DNase I hypersensitive sites in the germline state. Next two lines show sequential stages of VDJ recombination at the IgH locus. D_H to J_H rearrangement occurs first resulting in a DJ_H junction and, depending on which D_H rearranges, residual upstream unrearranged D_Hs may be present. V_H rearrangement occurs to the DJ_H junction to generate a VDJ_H junction; during this process unrearranged D_Hs are lost from the genome.</p

DNA methylation of IgH alleles in thymocytes.

<p>CD4⁺CD8⁺ thymocytes from C57BL/6 mice were enriched by adsorption to PNA-coated plastic plates. Genomic DNA purified from these cells was used in bisulfite modification analysis. Amplicons corresponding to unrearranged parts of IgH allele (A) and DJ_H junctions (B) were cloned and sequenced. Circles and squares depict CpGs corresponding to D_H and J_H1 cytosines, respectively. Filled circles/squares correspond to methylated cytosines; pie charts summarize the percentage of methylated cytosines at each position except where the number of sequenced alleles falls below 12, indicated by asterisks. Untemplated CpGs incorporated during VDJ recombination were found to be methylated (filled diamonds). Data shown were obtained from two independent preparations of thymocytes, using one mouse per experiment.</p

DNA methylation state of IgH alleles in the absence of the intronic enhancer Eμ.

<p>Pro-B cells were purified from the bone marrow of Eμ-deficient mice <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001475#pbio.1001475-Perlot1" target="_blank">[40]</a>. Genomic DNA was subjected to bisulfite modification followed by PCR amplification of germline gene segments (A) and DJ_H junctions (B). The 220-bp deletion of Eμ is represented as partial blue arcs separated by a gap. Filled and open circles, or squares, indicate methylated and unmethylated cytosines, respectively. Numbers within regions marked as DFL16.1, DSP, and J_H1 denote CpG dinucleotides corresponding to the configuration at the respective unrearranged gene segments. For example, of the five CpGs at unrearranged DFL16.1 only the first two are retained in DFL16.1/J_H1 junctions. Three out of five cytosines used to analyze wild-type alleles remain in this deletion, and their methylation status is shown immediately below the disrupted enhancer. Sequences that substitute for the enhancer also contain CpGs whose methylation status is shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001475#pbio.1001475.s005" target="_blank">Figure S5</a>. (B) Recombined DFL16.1, DSP and DQ52 to J_H1 junctions were amplified, cloned, and sequenced. Untemplated CpGs incorporated during VDJ recombination were found to be methylated (filled diamonds). Data shown are derived from two independent preparations of pro-B cells from Eμ-deleted mice, with four to six mice in each experiment.</p

DNA methylation state of unrearranged and DJ_H recombined alleles in mature B cells.

<p>(A) Mature B cells were purified from spleens of C57BL/6 mice, and the genomic DNA was subjected to bisulfite modification assays. 40% of these cells contain two VDJ_H recombined alleles and the remainder contains one VDJ_H and one DJ_H recombined allele. (B) Amplicons corresponding to unrearranged DFL16.1 gene segment and a region centered 1.3 kb 5′ to DFL16.1 were cloned and sequenced. For comparison, methylation of the same region in pro-B cells derived from RAG2-decificient bone marrow is shown in the bottom panel. Filled and open circles indicate methylated and unmethylated cytosines. Pie charts summarize the percentage of methylated cytosines at each position; data are derived from two independent spleen B cell preparations with two to four mice in each experiment. (C) DJ_H junctions were amplified from bisulfite modified DNA, followed by cloning and sequencing. Circles and squares represent cytosines from D_H and J_H1 gene segment, respectively. Filled and open circles, or squares, indicate methylated and unmethylated cytosines, respectively. Numbers within regions marked as DFL16.1, DSP, and J_H1 denote CpG dinucleotides corresponding to the configuration at the respective unrearranged gene segments. For example, of the five CpGs at unrearranged DFL16.1, only the first two are retained in DFL16.1/J_H1 junctions. Variations in the total number of cytosines are due to imprecise joining during VDJ recombination. Pie charts summarize the percentage of methylated cytosines. The asterisk indicates positions where less than 12 CpGs were observed due to reduced representation caused by junctional diversity.</p

DNA methylation status after the first step of IgH locus recombination.

<p>Pro-B cells were purified from the bone marrow of wild-type C57BL/6 mice. Data shown are derived from two independent preparations of pro- and pre-B cells obtained from six to eight mice in each experiment. This cell population contains a mix of germline and partially rearranged IgH alleles. After bisulfite modification, the genomic DNA was used to amplify unrearranged (A) and DJ_H rearranged junctions containing DFL16.1 and DSP gene segments (B). Cytosines derived from D_H and J_H gene segments are marked as circles and squares, respectively. Filled and open circles, or squares, indicate methylated and unmethylated cytosines, respectively. Numbers within regions marked as DFL16.1, DSP and J_H1 in (B) denote CpG dinucleotides corresponding to the configuration of these residues at the respective unrearranged gene segments. For example, of the five CpGs at unrearranged DFL16.1 only the first two are retained in DFL16.1/J_H1 junctions. The total numbers of CpG dinucleotides are reduced in junctional sequences because some residues are lost during VDJ recombination as described in the text. Additional heterogeneity is due to the imprecise nature of recombination. Pie charts summarize the percentage of methylated cytosines at each position, except where the number of alleles falls below 12 (indicated by asterisks). (C) Methylation state of recombined DJ_H alleles in purified pre-B cells. This population contains a mix of VDJ_H recombined and DJ_H recombined IgH alleles (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001475#pbio-1001475-g005" target="_blank">Figure 5A</a>). Note that the number of circles and squares representing D_H- or J_H-associated CpGs differed in the DJ_H junctions compared to the corresponding unrearranged regions due to junctional variability.</p