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. 2018 Dec 7;362(6419):1182-1186.
doi: 10.1126/science.aar7854.

A DNA methylation reader complex that enhances gene transcription

C Jake Harris  1 , Marion Scheibe  2 , Somsakul Pop Wongpalee  1 , Wanlu Liu  1 , Evan M Cornett  3 , Robert M Vaughan  3 , Xueqin Li  4 , Wei Chen  4 , Yan Xue  1 , Zhenhui Zhong  1   5 , Linda Yen  1 , William D Barshop  6 , Shima Rayatpisheh  6 , Javier Gallego-Bartolome  1 , Martin Groth  1 , Zonghua Wang  5   7 , James A Wohlschlegel  6 , Jiamu Du  4 , Scott B Rothbart  3 , Falk Butter  8 , Steven E Jacobsen  9   10
Affiliations

A DNA methylation reader complex that enhances gene transcription

C Jake Harris et al. Science. .

Abstract

DNA methylation generally functions as a repressive transcriptional signal, but it is also known to activate gene expression. In either case, the downstream factors remain largely unknown. By using comparative interactomics, we isolated proteins in Arabidopsis thaliana that associate with methylated DNA. Two SU(VAR)3-9 homologs, the transcriptional antisilencing factor SUVH1, and SUVH3, were among the methyl reader candidates. SUVH1 and SUVH3 bound methylated DNA in vitro, were associated with euchromatic methylation in vivo, and formed a complex with two DNAJ domain-containing homologs, DNAJ1 and DNAJ2. Ectopic recruitment of DNAJ1 enhanced gene transcription in plants, yeast, and mammals. Thus, the SUVH proteins bind to methylated DNA and recruit the DNAJ proteins to enhance proximal gene expression, thereby counteracting the repressive effects of transposon insertion near genes.

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Figures

Fig. 1.
Fig. 1.. Comparative interactomics identifies methyl reader proteins.
(A) Heatmap of methyl-binding preferences for proteins identified as significantly enriched in two different underlying DNA sequences per methyl-cytosine (mC) context (mCG, mCHG, mCHH). NA, the protein was not detected. FWA, MEA, SDC, and SUP represent four in vivo methylated loci. Probes are listed in fig. S2. (B) FP binding assays to quantify the interaction of SUVH1 with methylated or unmethylated probes in CG, CHG, and CHH contexts (left) or an amino acid change version, SUVH1Y277A, predicted to abrogate methyl binding (18) (right). Binding affinities are indicated by dissociation constants (Kd) values. Error bars represent SEM of technical replicates. The data are representative of two independent experiments.
Fig. 2.
Fig. 2.. SUVH1 is recruited by RdDM associated mCHH.
(A) SUVH1 enrichment at loci defined by loss of methylation (hypomethylation). Differentially methylated regions (DMRs) in mutant genotypes are indicated. The DRM1 and DRM2 methyltransferases are responsible for mCHH at RdDM target sites, while mCG, mCHG, and heterochromatic mCHH are maintained by MET1, CMT3, and CMT2, respectively (1). *, met1 hypo CG DMRs that overlap with drm1/2 hypo CHH DMRs were removed. (B) SUVH1 enrichment at NRPE1 peaks. (C) SUVH1 enrichment at NRPE1-associated short (<500-bp) vs. long (>5-kb) TEs. (D) Relative importance of genomic features in predicting SUVH1 binding, based on the random forest regressor algorithm. Error bars represent SEM from five random permutations of the training set. (E) Area under receiver–operating characteristic curves (AUC) model accuracy using all features (left) vs. accuracy using mCHH alone (right). (F) Boxplot of SUVH1 enrichment in suvh1, nrpe1, nrpd1, and drm1 drm2 mutant backgrounds at SUVH1 peaks. (G) Scatterplot of SUVH1 over SUVH1Y277A enrichment vs. mCHH methylation percentage at SUVH1 peaks. Line of best fit is shown in blue, with adjusted R2 and P values indicated. Data in the lower panel indicate kernel density for mCHH. Average methylation levels and enrichment are calculated from the 200bp regions surrounding the peak summits.
Fig. 3.
Fig. 3.. SUVH1, SUVH3, DNAJ1, and DNAJ2 interact, colocalize, and are required for the expression of proximal genes.
(A) IP-MS results for tagged lines. Only proteins present in each of the four transgenic [but not wild-type (WT)] pulldowns are presented. NSAF, normalized spectral abundance factor, averaged from two biological replicates. (B) Representative browser track showing ChIP-seq of SUVH1, SUVH3, DNAJ1, and DNAJ2 (normalized reads, FLAG-tagged versions minus WT) (top four lines) and methylation fraction (bottom three lines) at a methylated locus. (C) Pearson’s correlation of genome-wide ChIP-seq profiles at 1kb resolution. H3K23ac from (20) was used as an outgroup control. (D) Scatterplot of FPKM fold change over WT of dnaj1 dnaj2 double vs. suvh1 suvh3 double at genes that were differentially expressed in suvh1 suvh3. Line of best fit is shown in red, with adjusted R2 and P values indicated. (E) Boxplot of expression change for genes proximal to SUVH1 binding sites. n, number of genes. *P < 0.05 (Mann-Whitney test).
Fig. 4.
Fig. 4.. ZF108-DNAJ1 transcriptionally activates mildly expressed proximal loci.
(A) Browser track showing the ZF108-DNAJ1 ChIP-seq profile at FWA. The red arrow indicates the genomic location of the designed ZF108 target binding site. (B) Metaplot of expression change, centred on ZF108-DNAJ1 vs. random peaks. (C) Boxplot of expression changes for genes with promoters proximal to ZF108-DNAJ1 binding sites. n, number of genes. *P < 0.05 (Mann-Whitney test). (D) Observed over expected ratio for overlap of ZF108-DNAJ1 sites with up- or down-regulated ZF108-DNAJ1 gene promoters. (E) Boxplot of expression change for genes that overlap with ZF108-DNAJ1 peaks (upper panel), arranged by ascending WT expression decile (lower panel). Genes that lacked expression in both genotypes were removed. *P < 0.05 (Mann-Whitney test).
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