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The chromatin remodeler DDM1 prevents transposon mobility through deposition of histone variant H2A.W

Abstract

Mobile transposable elements (TEs) not only participate in genome evolution but also threaten genome integrity. In healthy cells, TEs that encode all of the components that are necessary for their mobility are specifically silenced, yet the precise mechanism remains unknown. Here, we characterize the mechanism used by a conserved class of chromatin remodelers that prevent TE mobility. In the Arabidopsis chromatin remodeler DECREASE IN DNA METHYLATION 1 (DDM1), we identify two conserved binding domains for the histone variant H2A.W, which marks plant heterochromatin. DDM1 is necessary and sufficient for the deposition of H2A.W onto potentially mobile TEs, yet does not act on TE fragments or host protein-coding genes. DDM1-mediated H2A.W deposition changes the properties of chromatin, resulting in the silencing of TEs and, therefore, prevents their mobility. This distinct mechanism provides insights into the interplay between TEs and their host in the contexts of evolution and disease, and potentiates innovative strategies for targeted gene silencing.

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Fig. 1: DDM1 binds directly to H2A.W through two conserved regions.
Fig. 2: DDM1 is necessary for H2A.W deposition on TEs.
Fig. 3: DDM1 changes the chromatin state of TEs.
Fig. 4: DDM1 binding to H2A.W is required for TE silencing.
Fig. 5: DDM1 deposits H2A.W over TEs to mediate silencing.
Fig. 6: Expression of TEs in ddm1 and other mutants.
Fig. 7: DNA methylation of TEs in ddm1 and F1 Col-0 × ddm1 plants.
Fig. 8: DDM1 silences mobile TEs.

Data availability

The deep-sequencing (ChIP–seq, RNA-seq and EM-seq) data that support the findings of this study have been deposited to the NCBI Gene Expression Omnibus (GEO) under the accession number GSE150436 and the SRA (BioProject ID: PRJNA689609). All other data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

Code availability

Custom code used to process and analyse the deep-sequencing data, as described in the Methods, is available from the corresponding author on request.

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Acknowledgements

F.B. acknowledges support from the staff at the next-generation sequencing and PlantS facilities at the Vienna BioCenter Core Facilities (VBCF), the BioOptics facility and Molecular Biology Services from the Institute for Molecular Pathology (IMP), and the Molecular Biology Services at the GMI. We thank all members of the Berger laboratory for their technical help and H. Wang for sharing material; E. Sasaki, Z. Harvey, M. Borg, A. Marí-Ordóñez, P. Refsing Andersen and J. M. Watson for discussions and reading of the manuscript. This research was supported by the Japan Society for the Promotion of Science (JSPS) Overseas Research Fellowships (to A.O.), the Austrian Science Fund (FWF): M2539-B21 (to A.O.), P26887, P28320, P32054, P30802 and P33380 (to F.B.), and DK1238 (to B.J. and S.A.M.), the Austrian Academy of Sciences (to F.B., Z.J.L., E.A., S.A., R.P. and R.Y.), MEXT/JSPS 15H05963 and 19H00995 (to T.K.) and JST CREST JPMJCR15O1 (to T.K.).

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Contributions

A.O., B.J. and E.A. performed the genome-wide analysis. A.O., B.J. and R.P. performed DNA methylation analysis in ddm1 mutants. A.O., E.A. and S.A. performed RT–qPCR analysis of TEs in F1 DDM1/ddm1 plants and transgenic lines. A.O. and A.L.K. performed biochemical experiments. S.A.M. performed the phylogenic analysis. R.Y. contributed the initial set of ChIP–seq data. Z.J.L. contributed the anti-H1 antibodies and T.K. contributed the design of experiments based on F1 plants. F.B. and A.O. conceived, designed and supervised the project. The manuscript was drafted by F.B., A.O. and B.J. with comments from all of the coauthors.

Corresponding author

Correspondence to Frédéric Berger.

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The authors declare no competing interests.

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Peer review information Nature Cell Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Validation of recombinant DDM1 protein and H2A.X or H2A.W pulldown assay with DDM1 fragments. Related to Fig. 1.

a, SDS-PAGE analysis of purified recombinant DDM1 and DDM1 E334N. Proteins were separated by 10% SDS-PAGE and visualized by Coomassie Brilliant Blue staining. Asterisk indicates the degraded products that could not be removed after purification. (Source data shows one of three representative gels obtained). b, Schematic representation of the ATPase assay. Malachite green solution was used to detect free phosphate liberated from ATP by enzyme-catalyzed hydrolysis. c, Graphical representation of the results of ATPase assays with buffer (green), wild type DDM1 (blue), or DDM1 E334N (red), respectively. DNA concentration is expressed as moles of nucleotides. d, The crystal structure of Drosophila RNA helicase protein VASA49 together with the ATP analog (AMPPNP) (PDB ID: 2DB3). The glutamic acid residue at position 400, which coordinates a catalytic water molecule for stabilizing the interaction with ATP, is indicated. e, Sequence alignment between the Glu400 between Drosophila VASA and Arabidopsis DDM1 centered on GLU400, indicated by the blue arrow. Glu400 in VASA corresponds to Glu334 in Arabidopsis. f, h Schematic representation of the DDM1 fragments used in the pulldown assay. g, i SDS-PAGE analysis of the pulldown assay. His6-tagged DDM1 and its truncations (panel g) or GST-tagged DDM1 fragments (panel i) were incubated with histone dimers containing either H2A.X or H2A.W. After pulldown, samples were analyzed by 15% SDS-PAGE and visualized by Coomassie Brilliant Blue. Data in a, g and i represent three independent experiments. Source data are provided in Source Data Extended Data Fig. 1. Source data

Extended Data Fig. 2 DNA methylation in ddm1. Related to Fig. 2.

Violin plots for the proportion of DNA methylation in CG, CHG, and CHH contexts on protein coding genes (left, n = 27,655), TE fragments not including TEs (middle, n = 25,695), and TEs (right, n = 3,903) in wild type Col-0 (grey) and ddm1 (red).

Extended Data Fig. 3 Genomic distribution of H1, H3K9me2 and H2A.W in ddm1. Related to Figs. 2 and 3.

a, Aggregate profile plots (top) and heatmaps (bottom) of H2A.W, H2A.X, and H1 in Col-0 (grey) and ddm1 (red) over heterochromatic regions, defined by H3K9me2 enrichment. Antibodies against H1 used in this study recognizes both H1.1 and H1.2. b, IGV genome browser snapshot of H1 (black), H2A.W (green), and H3K9me2 (blue) in wild type Col-0 and ddm1. Coloured and grey shading indicate an enriched or depleted signal, respectively. Annotated protein coding genes, TE fragments, and TEs are shown as boxes colored black, pink, or light blue, respectively. c, Aggregate profile plots (top) and heatmaps (bottom) showing distribution of H3K9me2 and H2A.W over TE fragments not including TEs in wild type and ddm1. d, Chromosomal distributions of TEs classified as group 1 (orange; n = 3,257) and group 2 (grey; n = 646) in Fig. 3a for each chromosome. Green boxes represent pericentric heterochromatin. e, f IGV genome browser snapshot of H2A.W (green), H3K9me2 (blue), H3K36me3 (orange), and H3K27me3 (purple) over TEs in wild type Col-0 and ddm1. Coloured and grey shading indicate an enriched or depleted signal, respectively.

Extended Data Fig. 4 RT-qPCR analyses of TEs in complementation lines expressing DDM1 mutant forms. Related to Fig. 4.

a, RT-PCR analyses of transgenic lines. cDNA (+RT) and DNaseI-treated RNA (-RT) were prepared from each plant and used as template for RT-PCR analyses. ACT2 was used as a control for RT-PCR. PCR products were separated by agarose gel and visualized by SYBR Green I staining. b, Bar plots showing the results of RT-qPCR as fold change of enrichment over ACT2 at TEs analyzed in Extended Data Fig. 6c,d from wild type Col-0 (grey), ddm1 (red), ddm1 expressing DDM1-3xFLAG (purple), ddm1 expressing DDM1 ∆N-3xFLAG (green), ddm1 expressing DDM1 ∆C-3xFLAG (orange), or ddm1 expressing DDM1 ∆NC-3xFLAG (brown), respectively. Presented data are means of n = 3 independent biological replicates (each biological replicate has 2 technical repeats); error bars represent SD within biological replicates. Source data are provided in Source Data Extended Data Fig. 4. Source data

Extended Data Fig. 5 Genomic distribution of H2A.W in complementation lines. Related to Fig. 4.

a, b, d, e IGV genome browser snapshot of the distribution for H2A.W and H3K9me2 over TEs (panels a and b), protein coding gene (panel d) and TE fragment (panel e) in wild type, ddm1, ddm1 expressing DDM1 FL, DDM1 ∆N, DDM1 ∆C, and DDM1 ∆NC. c, Aggregate profile plots (top) and heatmaps (bottom) showing distribution of H2A.W over TEs in wild type, ddm1, ddm1 expressing DDM1 FL, DDM1 ∆N, DDM1 ∆C, and DDM1 ∆NC.

Extended Data Fig. 6 ChIP-qPCR analyses of H3K9me2 or H2A.W over TEs and Expression of TEs in F1 DDM1/ddm1. Related to Fig. 5.

a, b, Bar plots showing the results of ChIP-qPCR as fold change of H3K9me2 (panel a) or H2A.W (panel b) enrichment over H3 at TEs analyzed in Extended Data Fig. 6c,d from wild type Col-0 (grey), F1 DDM1/ddm1 (blue), or ddm1 (red), respectively. Presented data are means of n = 3 independent biological replicates (each biological replicate has 3 technical repeats); error bars represent SD within biological replicates. The numbers below each TE show the expression level of TEs (log2FoldChange(F1/ddm1)) compared to ddm1 mutant plants. c-e, Bar plots showing relative expression levels of TEs classified as inactivated (blue) or neutral (pink) measured by RT-qPCR from wild type Col-0 (grey), F1 DDM1/ddm1 (blue), or ddm1 (red), respectively. The expression levels of each TE are presented as a fold change over ACT2. Presented data are means of n = 3 independent biological replicates (each biological replicate has 2 technical repeats); error bars represent SD within biological replicates. The dashed lines represent 50% of the ddm1 fold change over ACT2. Source Data are provided in Source Data Extended Data Fig. 6. Source data

Extended Data Fig. 7 Transcriptome of TEs in ddm1 and other mutants. Related to Fig. 6.

Heatmap of the z-transformed expression levels of differentially expressed TEs (n = 1,878) in mutants from wild type Col-0, suvh4;5;6, met1, and ddm1.

Extended Data Fig. 8 DNA methylation of TEs in ddm1 and F1 cross Col-0 x ddm1. Related to Fig. 7.

Heatmaps showing weighted DNA methylation levels for TEs sorted in categories ‘Inactivated’, ‘neutral’ and ‘other’ in wild type Col-0, ddm1, artificial F1 cross [(Col-0 + ddm1)/2] and F1 cross Col-0 X ddm1 for a CG, b CHG, and c CHH contexts.

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Supplementary Tables

Supplementary Table 1: Sequence list for the DDM1 orthologues across species, which were used for alignment in this study (Fig. 1a and Supplementary Information). Supplementary Table 2: Sequences for primers used in the current study

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Source Data Extended Data Fig. 1

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Original gels.

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Osakabe, A., Jamge, B., Axelsson, E. et al. The chromatin remodeler DDM1 prevents transposon mobility through deposition of histone variant H2A.W. Nat Cell Biol 23, 391–400 (2021). https://doi.org/10.1038/s41556-021-00658-1

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