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. 2012 Mar 1;8(3):e1002540. doi: 10.1371/journal.pgen.1002540

Figure 6. The Airn CGI plays a major role in Airn transcription and function.

Figure 6

(A) Airn expression by genome-tiling array (left axis) and strand-specific expression analysis by RNA-Seq (right axis) for differentiated S12/+ and S12/CGIΔ-1A cells. Dashed arrows: sharp drop of Airn hybridisation signals in the Airn-specific region (single) and absence after 73 kb (doublet). Below: qPCR assays relative to Airn-TSS with colour code as (B,C). Striped box: overlapping START+RP11 assays. (B) qPCR of total+unspliced Airn in d0/d5/d14 differentiated S12/+ and four S12/CGIΔ clones shows unspliced Airn is reduced by ∼40% at the 5′ end (RP11/154 bp), but when assayed downstream (Airn-middle/53 kb, Airn-end/99 kb) or at positions which include splice variants (START), is reduced by >70% in S12/CGIΔ cells. Shown are mean and standard deviation of three differentiation sets (details as Figure 3A). (C) Airn qPCR in S12/+ and four S12/CGIΔ d14 clones shows that unspliced Airn is reduced by 79–83% at 0.57 kb and ∼85% at 7.3 kb, while spliced Airn reduced by >85%. Shown are mean and standard deviation of three differentiation sets (details as Figure 3A). (D) ChIP for Ser5P/Ser2P RNAPII in S12/+, S12/TDRΔ-1A and S12/CGIΔ-1A d11 cells shows unaffected Airn initiation and elongation (except at Airn-end) in TDRΔ and a sharp RNAPII decrease in the CGIΔ allele. The mean and standard deviation of three technical replicates is shown. Assay Airn-132 controls for background from the overlapping Igf2r transcript, which is 2-fold higher in CGIΔ that fails to repress the paternal Igf2r promoter. Map for qPCR assays as Figure 2. (E) DNA blot analysing methylation of the Igf2r promoter NotI site (see Figure 4A). *methylated fragment in d0 cells originating from feeder-cells. This blot shows that cells carrying a paternal CGIΔ allele contrary to wildtype cells do not gain the methylated 5 kb band on the paternal Igf2r promoter. White lines: indicate the order of samples run on the same gel was changed electronically. (F) qPCR quantifying allelic expression shows absence of Igf2r imprinted expression (Mat∶Pat ratio is close to 1), in four CGIΔ (S12/CGIΔ) cell lines compared to wildtype (S12/+). Three differentiation sets are shown separately due to variability in Mat∶Pat ratios in wildtype controls for each set. Bars represent the mean, error bars the standard deviation of 3 technical replicates (details as Figure 4C).