Journal: Nucleic Acids Research

Article Title: Recombination may occur in the absence of transcription in the immunoglobulin heavy chain recombination centre

doi: 10.1093/nar/gkaa108

Figure Lengend Snippet: DNA methylation profiles in the recombination centre. The IgH RC is ∼2.5 kb long and contains the most 3′ D segment, D Q52 , at ∼700 bp from the J H region spread over ∼1.4 kb. The RC is followed by the Eμ enhancer at ∼600 bp from J H4 . CpG methylation of D Q52 , J H1 , CGI and the core Eμ were assayed by bisulphite sequencing. For the ectopic CGI, CpG methylation was determined at the 5′, middle and 3′ parts of the insert. The D Q52 -J H1 region is highlighted in the top scheme. Of the six CpGs analysed in the of D Q52 region, the two 5′ CpGs are located within D Q52 promoter region, the third CpG at the 5′RSS (in red) of D Q52 segment. The fourth and the fifth CpGs are located within the 3′RSS of D Q52 (in red). The sixth is 544 bp away from the first CpG of J H1 segment (the 544 bp sequence contains two CpGs not sequenced and not shown here). Horizontal lines indicate the number and the methylation status of sequenced CpGs and vertical lines, the number of sequenced independent clones. The un-methylated and methylated cytosines are represented by open and filled circles, respectively. The results are summarized in the form of pie charts and the percentage of methylated residues is indicated underneath the indicated elements.

Article Snippet: Purified genomic DNAs from sorted pro-B cells were assayed by sodium bisulphite sequencing by using a bisulphite conversion kit (Diagenode).

Techniques: DNA Methylation Assay, CpG Methylation Assay, Bisulfite Sequencing, Sequencing, Methylation, Clone Assay

Journal: Nucleic Acids Research

Article Title: Recombination may occur in the absence of transcription in the immunoglobulin heavy chain recombination centre

doi: 10.1093/nar/gkaa108

Figure Lengend Snippet: Transcription and DNA methylation are altered on rearranged mutant alleles. ( A ) The top scheme shows a partially rearranged DJ H allele and the resulting DJ H (also called Dμ) spliced transcript as well as the DJ RC. The primers used are shown as red arrows and the transcription start site by a black arrow. Total RNAs from WT and CGI-Eμ pro-B cells were extracted and reverse transcribed. Spliced DJ H transcripts were quantified by qPCR. Actin transcripts were used for normalization. The histograms show the standard deviation ( n ≥ 3). ( B ) DNA methylation profiles of D Q52 J H1 intermediates. CpG methylation was assayed by bisulphite sequencing. Note that the CpG at the 5′RSS (red triangle in the scheme) is heavily methylated and the J H1 CpGs are fully methylated on the CGI-Eμ allele. ( C ) The top scheme shows a fully rearranged gene and the resulting VDJ-Cμ transcript derived from the promoter (P VH ) of the recombined V H gene segment. Red arrows indicate the localization of the primers and the black arrow, the transcription start site. Total RNAs from WT and CGI-Eμ pro-B cells were extracted and reverse transcribed. Spliced VDJ-Cμ transcripts involving distal and proximal V H exons were quantified by qPCR. Actin transcripts were used for normalization. The histograms show the standard deviation (n≥3). Statistical analysis ( t -test). (**) very significant ( P < 0.01), (***) and (****) extremely significant ( P < 0.001 and P < 0.0001 respectively).

Article Snippet: Purified genomic DNAs from sorted pro-B cells were assayed by sodium bisulphite sequencing by using a bisulphite conversion kit (Diagenode).

Techniques: DNA Methylation Assay, Mutagenesis, Reverse Transcription, Standard Deviation, CpG Methylation Assay, Bisulfite Sequencing, Methylation, Derivative Assay