The effects of single and double mutants on selected epigenetically controlled loci. (A) Northern blots of targets previously shown to be upregulated in mom1. Upper panel: AtCyp40 (cyclophilin-40); middle panel: TSIA15 and lower panel: MULE-F19G14. The bottom panel shows 18S RNA as a loading control. (B) Semi-quantitative RT–PCRs of representative endogenous loci. The underlined names indicate the transposon-derived targets. ACT2 is shown as a control.

The levels of DNA methylation and siRNAs in the UBQ3 promoter region that drives LUC expression. (A) Southern blot of genomic DNA digested with methylation-sensitive restriction enzymes and hybridised with a probe covering UBQ3pro. The digestion with the methylation insensitive SmlI enzyme produced a fragment of 1016 bp. S=SmlI, Hp=HpaII, reports on CG/CHG methylation, Ha=HaeIII and Na=NlaIII, reports on CHH methylation. (B) Detection of siRNAs homologous to the transgenic UBQ3pro in momLUC25, moe1 LUC25, moe1 momLUC25 and nrpd1 LUC25. Micro-RNA mir159- and ethidium bromide-stained gels (EtBr) are shown as loading controls.

Characterisation of the moe1-mutant line. (A) Bioluminescence images of LUC25, momLUC25, moe1 LUC25 and moe1 momLUC25 seedlings. All plants are siblings derived from a backcross of moe1 momLUC25 (M3) to LUC25 producing BC1 plants that were self-fertilised and their progeny was genotyped at all three loci (MOE1, MOM1 and LUC25) as described later and depicted in Supplementary Figure 1B. Silencing of LUC25 (upper left) is slightly released in momLUC25 (upper right) but not in moe1 LUC25 plants (lower left). The moe1 momLUC25 plants show strong release of LUC25 silencing (lower right). (B) Semi-quantitative RT–PCR of LUC transcript levels in moe1 momLUC25 M2 individuals as compared with those in LUC25 and momLUC25 controls. ACTIN-2 (ACT2) is shown as a control and no RT lacks RT.

Identification of the moe1 mutation and synergistic effects of the combination of mom1 with mutations in NRPD1 and NRPE1. (A) Semi-quantitative RT–PCR-based transcription analysis of selected genes involved in TGS. ACT2 is shown as a control. (B) PCR on genomic DNA using Pol-IV/V subunit-specific primers as indicated below the image. (C) Genomic map of the 4.4-kb deletion in the moe1 line. (D) Bioluminescence imaging showing the release of silent LUC expression in mutant combinations. In the upper row are LUC25 and momLUC25. The middle and the lower row show nrpe1-2- and nrpd1-derived plants, respectively. Note that neither the nrpd1 nor the nrpe1 single mutant releases LUC silencing. All plants were photographed at the same time.

A genome-wide overview of transcripts upregulated in the different mutants and their epigenetic characteristics. (A) Venn diagram showing the number of upregulated genes. This diagram shows the target overlaps between mom1, nrpe1 and mom1 nrpe1. (B) Genomic overview of the chromosomal localisation of genes activated in the mutants described in this study. Genes upregulated by mom1 are presented as blue squares, nrpe1 in red and genes synergistically upregulated only in the double mutant are shown in green. (C) The number of transposon families transcriptionally upregulated in the mutants analysed in this study. Each superfamily is represented by a different colour, as indicated to the right. (D) siRNA and DNA methylation levels (from wt plants; Zhang et al (2006) and Lister et al (2008)) at genes upregulated by the different mutant combinations (blue=mom1, red=nrpe1, green=mom1 nrpe1 only, grey=random). The 2-kb 5′ and 3′ regions were divided into 100-bp segments and the gene body into 10 even-sized segments covering the whole gene. The data represent percentages of genes having siRNAs and DNA methylation in the corresponding segments. Percentages for randomly selected genes are presented in grey.

The synergistic transcriptional effects of functional loss of MOM1 and factors involved in RdDM. (A) A 10-kb region around APUM9 showing the gene and the ROMANIAT5 transposon that controls its expression. Data were visualised with our online genome browser software, EpiExpress. The expression levels of the different mutants as compared with the wild type are shown in the blue histograms below. Intensities are shown on a log2 scale. LTRs as annotated by Peterson-Burch et al (2004) are represented as red arrows. (B) Qualitative and quantitative confirmation of the tiling array data by northern blotting. Both APUM9 and ROMANIAT5 show strong synergistic transcriptional reactivation in mom1 nrpe1 double mutants (upper and lower panels, respectively). Synergistic effects were also observed in the combination of mom1 with drm2 (right panel). Neither ddm1 nor met1 released ROMANIAT5 transcription to a comparable level (far right). An EtBr-stained gel is shown as loading control. (C) Bisulphite sequencing of the ROMANIAT5 LTR residing in front of APUM9. The bars represent the percentage of cytosines methylated in the corresponding sequence context. (D) siRNA blots detecting 24-nt siRNAs that target the ROMANIAT5 LTRs. An EtBr-stained gel is shown as loading control.