MBD5 and MBD6 couple DNA methylation to gene silencing through the J-domain protein SILENZIO

Abstract

DNA methylation is associated with transcriptional repression of eukaryotic genes and transposons, but the downstream mechanism of gene silencing is largely unknown. Here we describe two Arabidopsis methyl-CpG binding domain proteins, MBD5 and MBD6, that are recruited to chromatin by recognition of CG methylation, and redundantly repress a subset of genes and transposons without affecting DNA methylation levels. These methyl-readers recruit a J-domain protein, SILENZIO, that acts as a transcriptional repressor in loss-of-function and gain-of-function experiments. J-domain proteins often serve as co-chaperones with HSP70s. Indeed, we found that SILENZIO's conserved J-domain motif was required for its interaction with HSP70s and for its silencing function. These results uncover an unprecedented role of a molecular chaperone J-domain protein in gene silencing downstream of DNA methylation.

Figure 1:. MBD5 and MBD6 are CG specific methyl-readers in vitro and in vivo.

A) Binding curves of MBD6 with DNA oligos methylated (m) or unmethylated (u) in the indicated contexts, measured by fluorescence polarization (N=3, standard error of the mean [SEM]). B) Diagram of DNA curtain assay and representative image of YOYO-1 stained methylated (mCG) and unmethylated (uCG) DNA (green) bound by Cy3-labeled MBD6 (magenta). (−) chrome diffusion barriers. Scale bar - 5 μm. C) Distribution of MBD6 binding events along mCG DNA overlayed with the distribution of mCG density (green line). Error bars: 95% confidence intervals (CI) by bootstrap. D) Correlation scatterplot of MBD6 binding to methylated curtains and mCG density (1 kb bins). R: Pearson. E) Genome-wide correlation between DAP-seq and mCG density (400 bp bins). Trend lines calculated by locally weighted polynomial regression (loess curves). F) Homology models of MBD5 and MBD6. The two arginine residues of the 5mC–Arg–G triads (R1 and R2) are shown in the sequence alignment. G) Example ChIP-seq peaks at regions of dense CG methylation. H) Loess curves of ChIP-seq enrichment and methylation density (400 bp bins overlapping Pol V ChIP-seq peaks). E,H) Shaded area: 95% CI.

Figure 2:. MBD5 and MBD6 redundantly repress a subset of genes and transposons downstream of DNA methylation.

A) Boxplot of polyA RNA-seq for different mutants. Shown are the transcripts (genes and transposons) upregulated in mbd5 mbd6. B) Scatterplot comparing polyA RNA-seq with GRO-seq data at mbd5 mbd6 T-DNA differential transcripts. R and p-value: Spearman. Shaded area: 95% CI. C) Heatmap of mbd5 mbd6 T-DNA differential transcripts, showing polyA RNA-seq and BS-seq data (average methylation ratio at 400 bp windows around the TSS). D) RT-qPCR analysis of FWA expression normalized to IPP2. Dots: individual plants. Error bars: SEM. E) Genome browser tracks at FWA. The GRO-seq enrichment at the FWA promoter likely corresponds to Pol V transcription. F) Number of promoter methylated genes and TEs, upregulated in different mutants.

Figure 3:. SLN represses transcription downstream of MBD5 and MBD6.

A) IP-MS spectral counts of FLAG-tagged MBD5 and MBD6. All proteins displayed were not detected in the no-FLAG negative control (see Table S1). B) RNA-seq data at FWA. C) Scatterplot of the union of mbd5 mbd6 CRISPR and sln differential transcripts. R and p-value: Spearman. Blue line: linear model fit. Shaded area: 95% CI. D) Heatmap of ChIP-seq data (log2 fold change over no-FLAG control). E) Example methylated site bound by MBD5, MBD6, and SLN, in the indicated backgrounds. F) Cartoon showing SLN’s ectopic recruitment to unmethylated FWA via fusion to ZF108. G) RT-qPCR analysis of FWA expression and McrBC-qPCR analysis of FWA promoter methylation in T1 lines expressing low or high levels of ZF108-SLN (western blot in Figure S15A). Dots: individual plants. P-value: t-test. RT-qPCR data (normalized to IPP2) is relative to fwa epiallele plants. H) Flowering time (number of leaves produced before flowering) of segregating T2 populations from three transgenic lines expressing high levels of ZF108-SLN, comparing transgene positive to null segregant (negative) plants.

Figure 4:. SLN silencing function requires the conserved HPD tripeptide.

A) RT-qPCR analysis of FWA expression (normalized to IPP2) in T1 lines expressing SLN or SLN^H94Q in the sln mutant background. p-values: t-test. Error bars: SEM. Dots: individual plants. B) IP-MS spectral counts of wild-type and H94Q mutant SLN-FLAG (representative of two independent experiments, see Table S1). C) ChIP-seq of FLAG-tagged SLN and SLN^H94Q (log2 fold change over the no-FLAG control).