Histone H1 prevents non-CG methylation-mediated small RNA biogenesis in Arabidopsis heterochromatin
- Department of Cell and Developmental Biology, John Innes Centre, United Kingdom
- Institute of Science and Technology, Austria
Figures
Figure 1 with 1 supplement
Histone H1 prohibits RNA-directed DNA methylation of chromomethylase (CMT)-dependent heterochromatic transposons.
(A) The importance of DNA methylation, histone H3 modifications, small RNA (sRNA), H1, and cytosine sequence context to predict CMT transposable element (TE) or DRM TE classes by random forest …
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Figure 1—source data 1
Lists of chromomethylase (CMT)- and DRM-dependent transposons and intermediate transposons in Arabidopsis.
- https://cdn.elifesciences.org/articles/72676/elife-72676-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Chromomethylase (CMT) transposable elements (TEs) gain non-CG DNA methylation and small RNA (sRNA) expression in h1 plants.
(A) Heatmaps of H3K9me, H1, GC (G+C) content, CHG methylation (mCHG), and mCHH at CMT, DRM and intermediate TEs in wt, cmt2 (c2), cmt2cmt3 (c2c3), drm2 (d2), and drm1drm2cmt2cmt3 (ddcc) plants. 3807 …
Figure 2 with 1 supplement
RdDM is preferentially active in linker DNA in h1 plants.
(A, C) Example of a chromomethylase (CMT) transposable element (TE; Chr2: 3,944,600–3,946,400) (A) and DRM TE (Chr2: 6,389,500–6,392,500) (C) with well-positioned nucleosomes (yellow boxes). …
Figure 2—figure supplement 1
RNA-directed DNA methylation (RdDM) is enriched at linker DNA around well-positioned nucleosomes.
Average small RNA (sRNA) expression around well positioned (left) to increasingly poorly positioned nucleosomes at DRM transposable elements (TEs).
Figure 3
Small RNA (sRNA) biogenesis is associated with H3K9me and non-CG methylation.
(A, B) Average H3K9me2 (A) or H3K9me1 (B) (x-axis) and sRNA expression level (y-axis) in wt and h1. Each dot represents the average of 100 transposable elements (TEs) sorted by GC content. (C) …
Figure 4 with 2 supplements
SHH1 is not required for non-CG hypermethylation in h1.
(A) Heatmaps of mCHH and small RNA (sRNA) expression at chromomethylase (CMT) and DRM transposable elements (TEs) in plants with shh1 or clsy mutations. (B) Venn diagram of TEs in indicated …
Figure 4—figure supplement 1
Loss of h1 causes chromomethylase (CMT)2-independent hypermethylation at transposable elements (TEs) with CLSY3/4 small RNA (sRNA) clusters.
Boxplots show mCHH at TEs within indicated CLSY clusters.
Figure 4—figure supplement 2
Heatmaps of wt sRNA expression at chromomethylase (CMT), DRM transposable elements (TEs) and CLSY1/2, CLSY3/4 small RNA (sRNA) clusters in leaves and flowers.
Figure 5 with 1 supplement
Small RNA (sRNA) expression at CLSY3/4 clusters is independent of CG methylation (mCG).
(A) The number of chromomethylase (CMT) transposable elements (TEs; mCHH ≥0.05 in wt) that maintain mCHH in met1 (mCHH ≥0.05 in met1; 2498) or lose mCHH in met1 (mCHH <0.02 in met1; 798). (B, C) …
Figure 5—figure supplement 1
DNA methylation and small RNA (sRNA) expression changes in met1 and h1met1.
(A) H3K9me2 levels around chromomethylase (CMT) transposable elements (TEs) that maintain mCHH (mCHH ≥0.05) in met1 and lose mCHH (mCHH <0.02) in met1. (B) Kernel density distribution of sRNA …
Figure 6 with 1 supplement
Severely reduced H3K9 methylation does not prevent small RNA (sRNA) expansion.
(A-C) Distribution of H3K9 methylation (A, B) and sRNA expression (C) around 5′ ends of chromomethylase (CMT) and DRM transposable elements (TEs) in wt, h1, ddm1, and h1ddm1 plants. (D) Averaged …
Figure 6—figure supplement 1
Small RNA (sRNA) expression and H3K9 methylation changes in h1, ddm1, and h1ddm1.
Boxplots of H3K9 methylation levels (A) and sRNA expression (B) at chromomethylase (CMT) transposable elements (TEs) in wt, h1, ddm1, and h1ddm1 plants.
Figure 7 with 1 supplement
Non-CG DNA methylation and H3K9 methylation are decoupled in h1c2c3.
(A) Number of chromomethylase (CMT) transposable elements (TEs) with H3K9 methylation (average H3K9me1 [K9me1] or H3K9me2 [K9me2] >0.5) in cmt2cmt3 (c2c3) or h1cmt2cmt3 (h1c2c3) plants. (B) Example …
Figure 7—figure supplement 1
Analysis of chromatin features at chromomethylase (CMT) transposable elements (TEs).
(A) Boxplots of DNA methylation and H3K9 methylation levels at CMT TEs in wt, cmt2cmt3 (c2c3), and h1cmt2cmt3 (h1c2c3) plants. (B) Cytosine context density (total GC content, CG, CCG, CWG, CWA, and …
Figure 8 with 1 supplement
Small RNA (sRNA) expression specifically correlates with non-CG methylation.
(A, C) Boxplots of H3K9me2, CHG methylation (mCHG), and sRNA expression changes in h1 vs. wt (A) and h1c2c3 vs. c2c3 (C). (B, D) Correlation among H3K9 methylation, DNA methylation, and sRNA …
Figure 8—figure supplement 1
H3K9 methylation, DNA methylation, and small RNA (sRNA) expression in h1 and h1c2c3 plants.
(A, C) Boxplots of H3K9me1, mCG, and mCHH change in h1 vs. wt (A) and h1c2c3 vs. c2c3 (C). (B, D) Boxplots of H3K9 methylation, DNA methylation, and sRNA expression levels at chromomethylase (CMT) …
Figure 9
CLSY1/2-dependent small RNA (sRNA) expression is associated with non-CG methylation.
(A) Boxplots of H3K9me1, H3K9me2, mCHH, and sRNA expression levels at CLSY1/2-dependent sRNA clusters in h1c2c3 plants. sRNA clusters were classified by H3K9 methylation level (H3K9me1 >0.5, H3K9me2 …
Figure 10
Histone H1 prevents non-CG methylation-mediated small RNA (sRNA) biogenesis in Arabidopsis heterochromatin.
(A) In wt plants, H1 binds to GC-rich chromomethylase (CMT) transposable elements (TEs) to restrict access of RNA polymerase IV (Pol IV). Pol IV binds to DRM TEs and produces sRNA. (B) In h1 plants, …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | anti-H3K9me1 (Rabbit polyclonal) | Millipore | 07-450RRID:AB_310625 | 1:200 |
| Antibody | anti-H3K9me2 (Mouse monoclonal) | Abcam | ab1220RRID:AB_449854 | 1:200 |
| Commercial assay or kit | Library construction (Native ChIP) | Tecan | 3460-24 | |
| Commercial assay or kit | Bisulfite conversion | QIAGEN | 59,104 | |
| Commercial assay or kit | Library construction (bisulfite sequencing) | New England Biolabs | E7645 and E7335S | |
| Commercial assay or kit | Library construction (small RNA) | Illumina | RS-200-0012 and RS-200-0024 | |
| Software, algorithm | cutadapt | doi:10.14806/ej.17.1.200 | RRID:SCR_011841 | |
| Software, algorithm | bowtie | doi:https://doi.org.10.1186/gb-2009-10-3-r25 | RRID:SCR_005476 | |
| Software, algorithm | deepTools2 | doi:10.1093/nar/gkw257 | ||
| Software, algorithm | dzlabtools | doi:10.1126/science.1172417 | https://zilbermanlab.net/tools/ | |
| Software, algorithm | RandomForestExplainer | doi:10.1198/jasa.2009.tm08622 | ||
| Software, algorithm | IGV | doi:10.1038/nbt.1754 | RRID:SCR_011793 | |
| Software, algorithm | Gene Cluster 3.0 | doi:10.1093/bioinformatics/bth078 | ||
| Software, algorithm | corrplot | doi:10.1198/000313002533doi:10.1080/00031305.1996.10474371 | ||
| Software, algorithm | Treeview | doi:10.1093/bioinformatics/bth078 | RRID:SCR_016916 |
