ACD15, ACD21, and SLN regulate the accumulation and mobility of MBD6 to silence genes and transposable elements

DNA methylation mediates silencing of transposable elements and genes in part via recruitment of the Arabidopsis MBD5/6 complex, which contains the methyl-CpG binding domain (MBD) proteins MBD5 and MBD6, and the J-domain containing protein SILENZIO (SLN). Here, we characterize two additional complex members: α-crystalline domain (ACD) containing proteins ACD15 and ACD21. We show that they are necessary for gene silencing, bridge SLN to the complex, and promote higher-order multimerization of MBD5/6 complexes within heterochromatin. These complexes are also highly dynamic, with the mobility of MBD5/6 complexes regulated by the activity of SLN. Using a dCas9 system, we demonstrate that tethering the ACDs to an ectopic site outside of heterochromatin can drive a massive accumulation of MBD5/6 complexes into large nuclear bodies. These results demonstrate that ACD15 and ACD21 are critical components of the gene-silencing MBD5/6 complex and act to drive the formation of higher-order, dynamic assemblies at CG methylation (meCG) sites.

The PDF file includes: Figs. S1 to S6 Legend for table S1 Tables S2 and S3 Other Supplementary Material for this manuscript includes the following: Table S1 Fig. S1.ChIP-seq and RNA-seq analysis of ACD15 and ACD21.A) Venn diagram of ChIP-seq peaks showing large overlap between samples.The peak sets indicated with circles (putative MBD5/6 unique, ACD15 unique, or MBD5/6/ACD15 unique peaks) were selected and visualized with heatmaps (right).We noted that at each peaks set groups, enrichment of most proteins was observed, thus suggesting that these regions are bound by all components of the MBD5/6 complex, despite not reaching our stringent significance threshold to be called as peaks.The heatmap shows log2(fold-change) over no-FLAG control.B) Scheme of ACD15 and ACD21 genes showing the location of the guide RNAs used for CRISPR/Cas9 mediated mutant generation.The table below shows the mutations obtained in each line.C) Bar plots showing the number of differentially expressed TEs (DE-TEs) or differentially expressed genes (DEGs) in the indicated genotypes.D) Upset plots showing the intersection of the upregulated genes or TEs found for each genotype.The largest intersection group constitutes loci upregulated in all six mutant lines.S3. qPCR primer sequences.List of qPCR primers, forward (Fw) and reverse (Rev), along with DNA sequences (5'-3').

Fig
Fig. S2.Organization of the MBD5/6 complex structure.(A-I) Correlation between MBD6-RFP signal and either ACD15-YFP, ACD21-CFP, or SLN-CFP signal in the indicated mutant backgrounds (underlined).Images represent individual zstack slices of roots from plants co-expressing MBD6 with either ACD15, ACD21, and SLN.Scatter plots indicate signal intensity for each fluorescent protein at each pixel of the image shown.Correlation coefficient: Pearson.Scale bars = 20 µM.(J-K) AlphaFold Multimer predicted structure of MBD5/6 complex with two copies each of MBD6, ACDC15, ACD21, and SLN along with confidence score map of the predicted complex.(L) Cartoon representation of the core dimeric MBD5/6 complex based on the AlphaFold Multimer prediction.The figure was created with Biorender.com.

Fig. S3 .
Fig. S3.ACD15, ACD21, and SLN protein levels across plant lines.(A) ACD15-YFP nuclear intensity in wild-type vs sln mutant plants.Comparisons were made using two-tailed t tests (NS: P>=0.05,N=52 and 50 respectively).(B) Western blot analysis of ACD15-YFP protein levels along with ponceau stain as a loading control.(C) ACD21-CFP nuclear intensity in wild type vs sln mutant plants.Comparisons were made using two-tailed t tests (NS: P>=0.05,N=50 per genotype).(D-E) Western blot analysis of ACD21-CFP protein levels along with ponceau stains as loading controls.(F) Western blot analysis of SLN-CFP protein levels along with ponceau staining as a loading control.

Table S1 . (Separate File)
Table showing merged IP-MS experiments.The numbers reported correspond to MS/MS counts.Col0 samples are no-FLAG negative controls.Each independent experiment is annotated with a shaded color.

Table S2 . Gene identifiers for MBD5/6 complex.
All members of the MBD5/6 complex are listed along with gene identifiers associated with each gene for online A. thaliana database (https://www.arabidopsis.org/).