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. 2014 Sep 11;10(9):e1004612. doi: 10.1371/journal.pgen.1004612

Figure 2. The effect of the rrp6l1-3 and rrp6l2-3 mutations on expression of FLC sense and antisense transcripts, and on H3K4 methylation.

Figure 2

(A) The diagram of the FLC gene based on analysis of the transcription unit [38] Vertical bars and numbers denote the exons of the FLC sense transcript. The transcription start site of the FLC sense mRNA is indicated by an arrow. Two antisense transcripts, AS I (proximal) and AS II (distal), are depicted below the FLC diagram. Antisense transcripts are alternatively polyadenylated, with a proximal poly(A) site in sense intron 6 and a distal poly(A) site in the sense promoter region. Grey boxes correspond to AS I and II exons, and grey lines correspond to the spliced regions of the antisense RNAs. Horizontal bars (letters a, A to G and 3′-untranslated region, 3′UTR), correspond to the FLC regions used in qRT-PCR and ChIP. Arrowheads correspond to position of primers used for RT-PCR amplification of AS I and II. Blue bars correspond FCA binding region, yellow bar correspond to the FPA binding region. (B) Nascent FLC expression significantly increased in rrp6l1/2 mutants. Expression of FLC was measured by qRT-PCR and is shown relative to FLC expression in Col-0 and Ws wild type plants; (C) Expression of AS I and AS II transcripts in rrp6l1/2 mutants. Expression of AS I and AS II in rrp6l1/2 was compared to their expression in Col-0 wild type plants by qRT-PCR. The antisense transcript levels were normalized by total antisense transcript as described previously [38]. Error bars represent standard deviation (SD). (D) Effect of rrp6l1-3 and rrp6l2-3 mutations on the level of H3K4me3 examined by ChIP using antibodies against H3K4me3 in the various regions of FLC. The level of H3K4me3 in rrp6l1/2 mutants was normalized to the level of H3K4me3 in wild type Col-0 plants. (E) AtRRP6L1 protein physically interacts with the FLC locus. ChIP assays were done using the rrp6l1-3 mutant complemented by a functional AtRRP6L1-TAP transgene. AtRRP6L1-TAP recruitment was normalized to wild type Ws, the background of the rrp6l1-3 mutant. The error bars in ChIP experiments represent the standard error of the mean and correspond to the difference between 2 biological replicates.