Journal: The EMBO Journal
Article Title: Transcriptional control and exploitation of an immune‐responsive family of plant retrotransposons
Figure Lengend Snippet: H3K27m3 and DNA methylation co‐exist at ATCOPIA93 IGB (integrative genome browser) views showing H3K9m2 levels and H3K27m3 levels in WT and met1 rosette leaves, at ATCOPIA93 EVD and ATR (ChIP‐chip public data, Deleris et al , 2012 ). Orange horizontal bars: protein‐coding genes; horizontal green bars: transposable elements. The LTRs are delineated by pink bars. Vertical blue bars: H3K9m2 signal relative to H3 (two top lanes); vertical purple bars: H3K27m3 signal relative to H3 for each probe. Analysis of H3K27m3 marks at ATCOPIA93 EVD and ATR by ChIP on rosette leaves, followed by qPCR, in wild‐type plants and in clf plants mutated for the H3K27 methyltransferase CURLY LEAF. Data were normalized to the input DNA. ATCOPIA93 CDS is a region in ATCOPIA93 GAG common to EVD and ATR . AT5g17120 is a region in the protein‐coding gene located upstream of EVD . FLC is a region located in the first intron of FLOWERING LOCUS C which shows high levels of H3K27m3 in vegetative tissues and serves as a positive control. TA3 is a transposon and serves as a negative control. Because of technical variability in the ChIP efficiency, one ChIP experiment is presented here and two other independent experiments are presented in Fig EV3 B. ChIPs were performed on a pool of rosette leaves from eight to 10 plants/genotype. Genomic distribution of H3K27m3 marks between EVD and ATR loci by ChIP‐PCR pyrosequencing. Upper panel: Depiction of the pyrosequenced region (in yellow) within the GAG biotinylated qPCR amplicon obtained after H3K27m3 ChIP‐qPCR and purification with streptavidin beads. The position interrogated corresponds to the discriminating SNP between EVD (C/G) and ATR (A/T). Lower panel: The % indicated represents the % of G ( EVD , dark blue bar) or T ( ATR , light blue bar) at that position. The PCR and sequencing primers were designed so that other ATCOPIA93 ‐derived sequences (divergent and presumably nonfunctional) such as AT4G04410 and AT1G43775 cannot be amplified and so that the allelic ratio between the two active ATCOPIA93 copies EVD and ATR only can be evaluated. To verify this, the qPCR GAG product is also amplified from the Input gDNA as a control where a 50–50% ratio is expected. For clarity, an average of two experiments performed on two independent Input and ChIPs samples is shown (error bars represent standard error (SE) of the mean) and individual datasets presented in Fig EV3 C. Methylation status of the DNA captured with H3K27m3 by Sau96I Chop‐qPCR. H3K27m3 ChIP‐DNA from two independent ChIPs was digested with the methylation‐sensitive restriction endonuclease Sau96I which is sensitive to the methylation of the second C at the GGGCCG site in the LTR (as in Fig EV1 ). The values plotted correspond to the ratio between the amount of amplified DNA in the Sau96I digestion and the amount of amplified DNA in the undigested control, as calculated by the formula 2 −(Ct.digestedDNA–Ct.undigestedDNA) and using primers specific for a region of EVD‐ LTR spanning this Sau96I restriction site. Dark and light symbols are used for the first and second experiments, respectively. Results show that the WT ChIP‐DNA had significantly less digestion compared with the ddm1 control; thus, there was more methylation. Source data are available online for this figure.
Article Snippet: Pyrosequencing ATCOPIA93 DNA (ChIP‐DNA, cDNA, or gDNA as a control) was amplified with a biotinylated (forward) primer in the same region where RNA levels were analyzed and containing a SNP between EVD and ATR ; the biotinylated PCR product (40 μl reaction) was pulled down with streptavidin beads (sigma GE17‐5113‐01) and the sense biotinylated strand sequenced with a Pyromark Q24 (Qiagen) on the sequencing mode.
Techniques: DNA Methylation Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control, Negative Control, Polymerase Chain Reaction, Amplification, Purification, Sequencing, Derivative Assay, Methylation