Histone acetylation recruits the SWR1 complex toregulate active DNA demethylation in ArabidopsisWen-Feng Niea,b,c,1, Mingguang Leia,b,1, Mingxuan Zhanga,d, Kai Tanga,b, Huan Huanga, Cuijun Zhanga,b, Daisuke Mikia,Pan Liua, Yu Yanga, Xingang Wangb, Heng Zhanga, Zhaobo Langa, Na Liue, Xuechen Xuc, Ramesh Yelagandulaf,Huiming Zhanga, Zhidan Wanga, Xiaoqiang Chaia, Andrea Andreuccig, Jing-Quan Yuc, Frederic Bergerf,Rosa Lozano-Durana, and Jian-Kang Zhua,b,2

aShanghai Center for Plant Stress Biology, Center of Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, 201602 Shanghai, China;bDepartment of Horticulture & Landscape Architecture, Purdue University, West Lafayette, IN 47906; cDepartment of Horticulture, Zhejiang University,310058 Hangzhou, China; dUniversity of Chinese Academy of Sciences, 100049 Beijing, China; eInstitute of Vegetable Science, Zhejiang Academy ofAgricultural Science, 310021 Hangzhou, China; fGregor Mendel Institute, Austrian Academy of Sciences, 1030 Vienna, Austria; and gDepartment of Biology,University of Pisa, 56126 Pisa, Italy

Contributed by Jian-Kang Zhu, June 23, 2019 (sent for review April 25, 2019; reviewed by Aiwu Dong and Lionel Navarro)

Active DNA demethylation is critical for controlling the DNAmethylomes in plants and mammals. However, little is knownabout how DNA demethylases are recruited to target loci, and theinvolvement of chromatin marks in this process. Here, we identify2 components of the SWR1 chromatin-remodeling complex, PIE1and ARP6, as required for ROS1-mediated DNA demethylation, anddiscover 2 SWR1-associated bromodomain-containing proteins,AtMBD9 and nuclear protein X1 (NPX1). AtMBD9 and NPX1 recognizehistone acetylation marks established by increased DNA methylation1 (IDM1), a known regulator of DNA demethylation, redundantlyfacilitating H2A.Z deposition at IDM1 target loci. We show that atsome genomic regions, H2A.Z and DNA methylation marks coexist,and H2A.Z physically interacts with ROS1 to regulate DNA demethyla-tion and antisilencing. Our results unveil a mechanism through whichDNA demethylases can be recruited to specific target loci exhibitingparticular histone marks, providing a conceptual framework tounderstand how chromatin marks regulate DNA demethylation.

gene silencing | histone variant | chromatin remodeling | DNAdemethylation pathway | bromodomain

As a conserved epigenetic mark, DNA methylation at the fifthposition of cytosine plays an important role in plants and

many other eukaryotic organisms (1, 2). A variety of biologicalprocesses, including the control of transposons and other repetitiveDNA elements, generation of epialleles, genomic imprinting, ge-nome interactions, and stress responses, are regulated by DNAmethylation (2–5). In mammals, DNA methylation primarily oc-curs in the symmetric CG context, although non-CG methylationhas been observed in brain tissues and embryonic stem cells (6–8).In plants, DNA methylation occurs in all 3 DNA sequence contexts:CG, CHG, and CHH (where H is C, A, or T) (9, 10). In Arabidopsis,the RNA-directed DNA methylation (RdDM) pathway is re-sponsible for de novo methylation in all sequence contexts (2). CGmethylation is maintained by DNA METHYLTRANSFERASE 1(MET1) (11), while CHG methylation is maintained by the plant-specific DNA methyltransferase CHROMOMETHYLASE 3(CMT3) (12). Asymmetric CHH methylation is maintained byDOMAINREARRANGEDMETHYLTRANSFERASE 2 (DRM2)and CHROMOMETHYLASE 2 (CMT2) (13, 14). DRM2 is directedto specific genomic loci by the RdDM pathway, involving both 24-nucleotide small-interfering RNAs and long noncoding RNAs (4).DECREASE IN DNAMETHYLATION1 (DDM1), a chromatinremodeling ATPase, facilitates CMT2 to catalyze RdDM-independentCHH methylation (14).Genome-wide DNA methylation patterns are tightly controlled

by the opposing actions of DNA methylation and demethylationpathways (2, 4, 15–18). How active DNA demethylation is regu-lated and how DNA demethylases are recruited to their target lociis poorly understood. In plants, active DNA demethylation is me-

diated by 5-methylcytosine DNA glycosylases through a DNA base-excision repair pathway (18–20). There are 4 known 5-methylcytosineDNA glycosylases in Arabidopsis: REPRESSOROF SILENCING1 (ROS1), DEMETER, and DEMETER-LIKE 2 and 3 (DML2and DML3). DME is required for global DNA demethylationand gene imprinting in the endosperm (16, 21). ROS1 isnecessary for active DNA demethylation in vegetative tissuesat thousands of genomic regions and prevents transcriptionalsilencing of endogenous and transgenic loci (17, 20, 22, 23).DML2 and DML3 can excise 5-methylcytosine in vitro andmainly function redundantly with ROS1 in vivo (17). ROS1 istargeted to specific genomic loci through the increased DNAmethylation (IDM) complex, which includes the methyl CpG-binding protein 7 (MBD7), IDM1, IDM2, IDM3, HDP1, andHDP2 proteins (23–29). The IDM complex catalyzes histone

Significance

Transgenes and some endogenous genes are protected fromsilencing by active DNA demethylation. How the demethylaseROS1 targets these specific genomic regions is poorly un-derstood. Here, we show that H2A.Z and components of theconserved SWR1 complex are required for antisilencing. Wefound that ROS1 is recruited to some of its target regions byH2A.Z deposited by the SWR1 complex. Two bromodomain-containing proteins, AtMBD9 and NPX1, are critical for the re-cruitment of SWR1 complex to chromatin through recognitionof histone acetylation marks created by the increased DNAmethylation (IDM) complex, which is known to have a regula-tory role in active DNA demethylation. Thus, we have identi-fied a complete regulatory pathway initiated by the IDMcomplex in active DNA demethylation in Arabidopsis.

Author contributions: W.-F.N. and J.-K.Z. designed research; W.-F.N., M.L., M.Z., C.Z., D.M.,P.L., Y.Y., X.W., Heng Zhang, Z.L., N.L., X.X., Huiming Zhang, Z.W., A.A., and J.-Q.Y.performed research; M.L., M.Z., C.Z., D.M., P.L., Y.Y., X.W., Heng Zhang, Z.L., N.L., X.X.,R.Y., Huiming Zhang, Z.W., and J.-Q.Y. contributed new reagents/analytic tools; W.-F.N.,K.T., H.H., X.C., F.B., R.L.-D., and J.-K.Z. analyzed data; and W.-F.N., F.B., R.L.-D., and J.-K.Z.wrote the paper.

Reviewers: A.D., Fudan University; and L.N., Institute of Biology of the EcoleNormale Supérieure.

The authors declare no conflict of interest.

Published under the PNAS license.

Data deposition: The data reported in this paper have been deposited in the Gene Ex-pression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no.GSE115170).1W.-F.N. and M.L. contributed equally to this work.2To whom correspondence may be addressed. Email: zhu132@purdue.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1906023116/-/DCSupplemental.

Published online July 30, 2019.

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acetylation at a subset of DNA demethylation target loci.However, how histone acetylation in turn helps recruit DNAdemethylases is unknown, since ROS1 does not physically in-teract with the IDM complex.Histone modifications and histone variants play important

roles in the regulation of gene expression, DNA repair, and avariety of other chromosomal processes (30), but the connectionbetween histone modifications, histone variants, and active DNAdemethylation remains elusive. The histone H2A variant H2A.Zis deposited into chromatin by the SWR1 chromatin-remodelingcomplex, which is conserved in eukaryotes (31–35). H2A.Zspecifically localizes at euchromatin and has a genome-wideanticorrelation with DNA methylation (30, 36, 37).In this study, we performed a forward genetic screen to

identify cellular factors required for ROS1-mediated DNAdemethylation and antisilencing. We isolated 2 SWR1 compo-nents, namely ARP6 and PIE1, and characterized the roles of theSWR1 complex and H2A.Z in regulating DNA demethylation.We discovered that the methyl-DNA-binding protein AtMBD9and the yeast Bromodomain factor 1 (ScBDF1)-related proteinnuclear protein X1 (NPX1) are associated with the plant SWR1complex, and show that AtMBD9 and NPX1 function redundantlyin active DNA demethylation by recognizing specific chromatinmarks generated by IDM1. We have thus uncovered a completeregulatory pathway, initiated by the IDM complex, where the

SWR1 complex, directed by AtMBD9/NPX1, functionally linksthe IDM-deposited histone marks to active DNA demethylationat specific loci in Arabidopsis through the physical recruitment ofROS1 by the SWR1-deposited H2A.Z.

ResultsThe SWR1 Subunits ARP6 and PIE1 Prevent DNA Hypermethylationand Gene Silencing. Active DNA demethylation is critical forpreventing DNA hypermethylation and transcriptional silencingof certain transgenes and endogenous genes (22–25, 38). Toidentify antisilencing mutants in Arabidopsis, we performed aforward genetic screen with transgenic 35S:SUC2 plants, whichexhibit short roots when grown on medium containing sucrosecompared with wild-type Col-0 plants (38) (SI Appendix, Fig.S1A). The 35S:SUC2 plants also express the hygromycinphosphotransferase II (HPTII) and neomycin phosphotransfer-ase II (NPTII) transgenes, which confer resistance to hygromycinand kanamycin, respectively. We isolated 4 mutant alleles withlong roots on sucrose-containing media: arp6-4, arp6-5, pie1-6,and pie1-7 (Fig. 1A and SI Appendix, Fig. S1 B and C). Comparedwith 35S:SUC2 plants, the mutants were sensitive to kanamycinand displayed reduced transgene expression (SI Appendix, Fig.S1 D and E). In addition, AT1G26380 and AT1G62760, 2 en-dogenous loci, which are hypermethylated and silenced in idm1-1and ros1-4 mutant plants (23, 25), also displayed reduced

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Fig. 1. ARP6, PIE1, and H2A.Z prevent transcriptional silencing and DNA hypermethylation. (A) arp6-4, arp6-5, pie1-6, and pie1-7 mutants display long rootson sucrose-containing medium, similar to idm1-9 and ros1-14 (Upper). Seedlings grown on glucose-containing medium show normal root development(Lower). (B) Relative expression of 2 endogenous ROS1-targeted genes, AT1G26380 and AT1G62760, in mutants compared with control 35S:SUC2 plants. (C)Screenshots of DNA cytosine methylation status, H2A.Z deposition, and H3K18Ac at the CaMV35S transgene promoter in 35S:SUC2 control plants or theindicated mutants. At the top is a schematic representation of the 35S:SUC2 transgene, indicating the transcription start site (TSS) and some regulatoryelements (putative transcription factor binding sites and TATA box). The CaMV35S transgene promoter is divided into region A and region B according toLang et al. (25). Scales on the tracks of DNA methylation level are from −1 to +1. Two independent biological replicates of ChIP-seq data are displayed in eachcase. Data for region A are the average of 5 copies (1 in 35S:SUC2, 2 in 2x35S:HPTII, and 2 in 2x35S:NPTII); region B is present in 35S:SUC2 only. (D) The H2A.Ztriple mutants, h2a.z-2 and h2a.z-3, show a long-root phenotype on sucrose-containing medium, similar to pie1-6 mutant plants. (E) Relative expression oftransgenes in the 35S:SUC2 control plants and the indicated mutants. Values are means ± SD of 3 biological replicates relative to transcript levels in 35S:SUC2plants in B and E. **P < 0.01, compared with 35S:SUC2 samples (2-tailed t test).

16642 | www.pnas.org/cgi/doi/10.1073/pnas.1906023116 Nie et al.

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expression in arp6-4, arp6-5, pie1-6, and pie1-7 mutants (Fig. 1B).The reduced expression of AT1G26380 and AT1G62760 in arp6-4and pie1-6 was released by the DNA methylation inhibitor5-Aza-2′-deoxycytidine (5-Aza) treatment (SI Appendix, Fig.S1F). The expression of ARP6-3xFLAG-3xHA or PIE1-3xFLAG-3xHA fusions driven by their native promoters restored expressionof the 35S:SUC2 transgene and the short-root phenotype in arp6-4and pie1-6, respectively (SI Appendix, Fig. S2 A–D). Furthermore,ARP6-3xFLAG-3xHA and PIE1-3xFLAG-3xHA were enrichedat the CaMV35S transgene promoter by chromatin immunopre-cipitation (ChIP) (SI Appendix, Fig. S2 E–G). Thus, ARP6 andPIE1 are associated with chromatin at the transgene promoters,and protect transgenes and endogenous genes from silencing.To determine if transgene silencing in the arp6-4 and pie1-6

mutants is associated with DNA methylation, we examined theDNA methylation levels of the CaMV35S transgene promoter in35S:SUC2, arp6-4 and pie1-6 mutants by bisulfite sequencing.DNA methylation levels increased in arp6-4 and pie1-6 mutantsrelative to 35S:SUC2 plants, similar to what is observed in theantisilencing mutants idm1-9 and ros1-14 (Fig. 1C and SI Ap-pendix, Fig. S3). Furthermore, the DNA methylation inhibitor5-Aza promoted the expression of SUC2, HPTII, and NPTII in35S:SUC2 plants and restored kanamycin resistance and trans-gene expression in arp6 and pie1 mutants (SI Appendix, Fig. S1 Dand E). Taken together, these findings suggest that PIE1 andARP6 prevent transgene silencing by limiting DNA methylation.

H2A.Z Prevents DNA Hypermethylation and Gene Silencing. ARP6and PIE1 are components of the SWR1 complex, which depositsH2A.Z into chromatin (31–35). To investigate the role of H2A.Zin antisilencing, we knocked out the 3 expressed isoforms ofH2A.Z (HTA8, HTA9, and HTA11) in 35S:SUC2 transgenicplants (39–41). We obtained 2 triple mutant lines, h2a.z-2 andh2a.z-3 (SI Appendix, Fig. S4 A and B), which showed develop-mental aberrations (SI Appendix, Fig. S4C). Interestingly, theh2a.z mutants developed long roots on sucrose-containing media(Fig. 1D), and the expression of the SUC2, NPTII, and HPTIItransgenes and of the endogenous genes AT1G26380 and AT1G62760was reduced in these mutants compared with 35S:SUC2 plants(Fig. 1 B and E). Moreover, DNA methylation levels at the 35Spromoter were increased in h2a.z-2 compared with 35S:SUC2plants (Fig. 1C and SI Appendix, Fig. S3), and treatment with5-Aza restored the expression of SUC2 and HPTII transgenes inthe mutants (SI Appendix, Fig. S4D). H2A.Z was enriched at theCaMV35S promoter in 35S:SUC2 plants, and this enrichment wasdecreased in pie1-6, pie1-7, arp6-4, and arp6-5 mutants (Fig. 1Cand SI Appendix, Fig. S5). Thus, H2A.Z is required to protect bothtransgenes and endogenous loci from DNA hypermethylationand silencing.

H2A.Z Promotes ROS1- and IDM1-Dependent DNA Demethylation. Tounderstand the effect of H2A.Z on DNA methylation, we exam-ined the genome-wide DNA methylation profiles in 35S:SUC2plants and in the arp6-4, pie1-6, h2a.z-2, idm1-9, and ros1-14mutants. The overall DNA methylation patterns in genes andtransposons were not altered in arp6-4, pie1-6, or h2a.z-2 mutants,nor in idm1-9 or ros1-14 (SI Appendix, Fig. S6), consistent withobservations that lacking H2A.Z does not substantially perturbgenic DNA methylation (41). However, we identified some spe-cific genomic regions that were hypermethylated in the mutantsrelative to 35S:SUC2 plants (Fig. 2A). These hypermethylatedregions are distributed in genes, transposable elements (TEs), andintergenic regions (SI Appendix, Fig. S7 A and B). The overlap inhyper-differentially methylated regions (DMRs) in different mu-tants was limited, due to the strict statistical criteria used forhyper-DMR calling. There were 135 hypermethylated regionsshared by h2a.z-2, idm1-9, and ros1-14 mutants (Fig. 2B), 140hypermethylated regions shared by h2a.z-2, arp6-4, and pie1-6 (Fig.

2C), and 76 hypermethylated regions shared by ros1-14, idm1-9,h2a.z-2, arp6-4, and pie1-6 (Dataset S2).The effects of ROS1, IDM1, H2A.Z, ARP6, and PIE1 in

preventing DNA hypermethylation are not limited to the 76 ge-nomic regions, since a shared increase in methylation could beobserved in all of the mutants in regions that had not beenclassified as hyper-DMRs (SI Appendix, Fig. S7C). We calculatedthe average DNA methylation levels in all sequence contexts ath2a.z-2–, idm1-9–, and ros1-14–dependent hypermethylated re-gions, and found that DNA methylation levels in all contexts werealso increased at these regions in all of the antisilencing mutants(Fig. 2 D–F and SI Appendix, Fig. S8 A–C). The DNA methylationlevels that were increased at h2a.z-2 hyper-DMRs were also in-creased in the previously published h2a.z triple mutant (41) (SIAppendix, Fig. S8D). At idm1-9–, h2a.z-2–, and ros1-14–dependenthyper-DMRs that correspond to genes or TEs, methylation in-creases also occurred in all sequence contexts (SI Appendix, Fig.S9). These results suggest that H2A.Z deposition by the SWR1complex promotes ROS1- and IDM1-dependent DNA demethylationat some endogenous genomic regions.

H2A.Z Physically Interacts with ROS1. To investigate the mechanismof H2A.Z function in DNA demethylation, we isolated H2A.Z-associated proteins through IP of H2A.Z from 35S:SUC2 plants.In the immunoprecipitate, we identified components of the SWR1complex, including PIE1, ARP6, and SWC6 (Fig. 3A). Inter-estingly, peptides corresponding to ROS1 were also detected asassociated with H2A.Z, but not with H2A.W, another histonevariant in the H2A family (SI Appendix, Fig. S10A). We thenimmunoprecipitated ROS1 from transgenic plants expressing aROS1-3xFLAG-3xHA fusion driven by the ROS1 native promoter,and identified HTA9 and HTA8, 2 H2A.Z isoforms (Fig. 3B and SIAppendix, Fig. S10B). MET18, a protein known to interact withROS1 (42), was also found in these experiments (Fig. 3B). Weconfirmed the interaction between ROS1 and H2A.Z isoforms usinga yeast 2-hybrid (Y2H) assay (minus-3 media) (Fig. 3C) and a lu-ciferase complementation imaging assay in Nicotiana benthamianaleaves (Fig. 3D). Thus, H2A.Z physically interacts with ROS1.

IDM1 Is Required for H2A.Z Deposition at Some Active DNA DemethylationTarget Regions. IDM1 acetylates histone H3 at K14, K18, and K23,facilitating ROS1-dependent DNA demethylation through an asyet unknown mechanism (23). To shed light on the functionalinteraction between IDM1-dependent histone acetylation, H2A.Zdeposition, and ROS1-mediated DNA demethylation, we analyzedthe presence of these epigenetic marks both at the CaMV35Spromoter and genome-wide in the 35S:SUC2 control plants andthe idm1-9, arp6-4, pie1-6, and ros1-14mutants. H2A.Z enrichmentat the CaMV35S promoter was decreased in arp6-4, pie1-6, andidm1-9 mutants, but not in ros1-14 mutants compared with35S:SUC2 plants (Fig. 1C and SI Appendix, Fig. S5A), suggestingthat histone acetylation by IDM1 may be required for H2A.Zdeposition, and that H2A.Z deposition may be upstream of ROS1-mediated DNA demethylation. As expected, the H3K18 acetyla-tion level at the CaMV35S promoter was lower in idm1-9 than inthe 35S:SUC2 control plants (Fig. 1C). H3K18 acetylation levelswere also reduced in arp6-4 (Fig. 1C). In Arabidopsis, H2A.Z ishighly enriched at +1 nucleosomes, while its levels are low atpromoters of genes and TEs (Fig. 4 A and B) (36, 37, 41). Similarpatterns were observed in the deposition of H3K18ac at genes andTEs genome-wide (SI Appendix, Fig. S11A). As observed for theCaMV35S promoter region, H2A.Z enrichment was decreased atboth genes and TEs in the arp6-4 and pie1-6 mutants as well as inidm1-9, but not in ros1-14 (Fig. 4 A and B). The idm1-9 mutantdisplayed significantly decreased H2A.Z and H3K18ac depositionat regions identified as hypermethylated in idm1-9, h2a.z-2, andros1-14 (Fig. 4C and SI Appendix, Fig. S11B). As expected, de-position of H3K18Ac was substantially reduced in the idm1-9

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mutant at the 76 shared hyper-DMRs (Fig. 4D). H2A.Z deposition atthe idm1-9–dependent hypermethylated regions was higher inros1-14 than in idm1-9 (Fig. 4 C, Left). It has been previouslyshown that the idm1-dependent hypermethylated regions are asubset of ros1-dependent hypermethylated regions (23). Takentogether, these results suggest that H2A.Z functions downstreamof IDM1 and upstream of ROS1 at idm1-9 hyper-DMRs to pre-vent DNA hypermethylation and silencing. However, at some ofthe hyper-DMRs, particularly IDM1-independent genomic re-gions, the H2A.Z levels appeared lower also in ros1mutant plants(Fig. 4C). The expression of ARP6 or PIE1 was not reduced inros1-4 mutant plants (SI Appendix, Fig. S11C). These results in-dicate that ROS1-mediated DNA demethylation may somehowfeedback-regulate the deposition of H2A.Z. The decrease in H3K18acetylation levels in arp6-4 at the hyper-DMRs (SI Appendix, Fig.S11B) indicates a possible mutual reinforcement between SWR1-dependent H2A.Z deposition and IDM1-dependent histone acety-lation. The above data led us to propose that the histone acetylationmarks created by IDM1 may promote the deposition of H2A.Z atcertain genomic regions, and that H2A.Zmay help recruit ROS1 foractive DNA demethylation at these loci. We found that 3xFLAG-3xHA-tagged ROS1 enrichment was reduced at several tested lociin h2a.z compared with 35S:SUC2 plants (SI Appendix, Fig. S12),supporting the notion that H2A.Z is required for ROS1 recruit-ment to specific DNA demethylation target loci.

H2A.Z and DNA Methylation Marks Coexist at the Shared Hyper-DMRs.In the 76 hyper-DMRs shared by ros1-14, idm1-9, arp6-4, pie1-6,

and h2a.z-2, DNA methylation levels were substantially increasedin all 3 sequence contexts (CG, CHG, and CHH) compared withthe 35S:SUC2 control, while H2A.Z deposition was markedlydecreased (Fig. 5 A and B). A strong genome-wide anticorrelationbetween DNA methylation and H2A.Z deposition has been pre-viously described (37). Interestingly, as shown in Fig. 5 A and B,the 76 shared hyper-DMRs are an exception to this rule, simul-taneously displaying moderate levels of DNA methylation andH2A.Z deposition in the 35S:SUC2 control plants. With the aimof determining if the coexistence of DNA methylation and H2A.Zmarks is a distinctive feature of this subset of genomic regions, weperformed a simulation analysis in which we replaced each of the76 hyper-DMRs with another genomic region of the same lengthand with the same level of methylation (Fig. 5C), and examined thelevel of H2A.Z deposition at this group of surrogate loci in 35:SUC2plants. Notably, H2A.Z deposition was much lower at the simula-tion regions selected by their methylation level (Fig. 5D). In anothersimulation analysis, we replaced each of the 76 hyper-DMRs withanother genomic region of the same length and with the same levelof H2A.Z deposition (Fig. 5D), and found that the DNA methyl-ation level at the simulation regions was close to zero and there wasno increase in any of the antisilencing mutants (Fig. 5E). Therefore,DNA methylation and H2A.Z deposition were anticorrelated at thesimulation regions. These results support that the coexistence ofDNA methylation and H2A.Z marks is a distinct feature of theshared hyper-DMRs, consistent with our model that the IDMcomplex is recruited to genomic regions with DNA methylation (23,24), and that the histone acetylation marks generated by the IDM

GenotypeHyperDMR

overlapwith ros1-14

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overlapwith arp6-4

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Hypo DMR

ros1-14 2746 46idm1-9 782 438 (56.0%) 75arp6-4 952 524 (55.0%) 329 (34.6%) 104pie1-6 792 516 (65.2%) 292 (36.9%) 358 (45.2%) 64h2a.z-2 559 417(74.6%) 167 (29.9%) 226 (42.7%) 224 (40.1%) 59

atmbd9 npx1 259 178 (68.7%) 82 (32.0%) 125 (48.3%) 106 (40.9%) 105 (40.5%) 21

A

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110 32 312 135 303 282

2025 140

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Fig. 2. ARP6, PIE1 and H2A.Z function in active DNAdemethylation. (A) Number of DMRs identified inarp6-4, pie1-6, h2a.z-2, idm1-9, and ros1-14, andoverlap of hyper-DMRs between them. The P valuesof tests at these overlaps are <1 − 10−4. Further in-formation about mapping, coverage, depth, andconversion rate can be found in Dataset S1. (B)Overlap of hyper-DMRs among h2a.z-2, idm1-9, andros1-14. (C) Overlap of hyper-DMRs among h2a.z-2,arp6-4, and pie1-6. (D–F) Heatmap representation ofthe methylation level at h2a.z-2 hyper-DMRs (D),idm1-9 hyper-DMRs (E), and ros1-14 hyper-DMRs (F)in 35S:SUC2, arp6-4, pie1-6, h2a.z-2, idm1-9, andros1-14 mutants. Results from 6 (35S:SUC2 controllines) or 2 (mutants) independent biological repli-cates are shown.

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complex are important for the SWR1 complex to deposit H2A.Z atthese regions.

The Bromodomain Proteins AtMBD9 and NPX1 Are Associated withthe SWR1 Complex and Function in Active Demethylation andAntisilencing. Although our results suggest that IDM1 promotesH2A.Z deposition at the hypermethylated regions in antisilencingmutants (Fig. 4C), we did not detect a direct interaction betweenIDM1—or its acetylation products—and the SWR1 complexsubunits ARP6 or PIE1. To further characterize the connectionbetween IDM1 and the SWR1 complex, we immunoprecipitatedPIE1 from plants expressing a PIE1-3xFLAG-3xHA fusion drivenby the PIE1 native promoter and subjected it to LC-MS/MS, withthe aim of identifying potential unidentified components that maymediate an interaction with IDM1. Using this approach, we iso-

lated a number of proteins putatively associated with the Arabi-dopsis SWR1 complex (SI Appendix, Fig. S13A). While most ofthese proteins are homologs of the yeast SWR1 complex compo-nents (43), several had not been previously reported in Arabidopsis,such as NPX1 and AtMBD9. NPX1, a bromodomain-containingprotein, is conserved in plants and has sequence similarity toScBDF1 (SI Appendix, Fig. S13 B and C). AtMBD9 is a member ofthe MBD family and also contains a bromodomain, which canrecognize acetylated lysines in histones (44, 45). Probing a histonepeptide array with the N-terminal part of NPX1, which contains thebromodomain (amino acids 1 to 509), revealed that the NPX1bromodomain binds histone H3 peptides acetylated at K14 andK18, as well as other histone acetylation marks, but not H3 peptideswith the repressive H3K9me2 mark (SI Appendix, Fig. S14 A–C).Mutating a conserved residue in the acetylation binding pocket of

Protein Accession Uniquepeptides

Rep1 Rep2

Coverage(%)

Rep1 Rep2

ROS1 AT2G36490 94 67 62.38 54.34HTA9 AT1G52740 1 1 6.72 5.22HTA8 AT2G38810 2 1 19.12 6.62

MET18 AT5G48120 1 1 1.50 1.50

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Rep1 Rep2HTA9 AT1G52740 3 2 36.57 16.34PIE1 AT3G12810 24 16 18.25 14.60ARP6 AT3G33520 3 0 9.26 0ROS1 AT2G36490 4 3 5.81 2.08SWC6 AT5G37055 2 2 12.87 12.87

H2A.Z Purification (anti-HTA9)

ROS1 Purification (anti-Flag)

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Fig. 3. H2A.Z interacts with ROS1. (A) Selectedproteins detected by LC-MS/MS following IP using ananti-HTA9 antibody, which immunopurifies H2A.Zfrom Col-0 plants. Proteins included in this table havebeen previously reported (PIE1 and ARP6) or con-firmed as interactors of H2A.Z in Y2H or split lucifer-ase assays in this work (ROS1). (B) Unique identifiedpeptides detected by LC-MS/MS following IP usinganti-FLAG antibody in ros1-14/pROS1:ROS1-3xFLAG-3xHA transgenic plants. Proteins included in this tablehave been confirmed as interactors of ROS1 by in-dependent methods or those previously reported (42).Results obtained in 2 independent biological repli-cates are shown (Rep1-Rep2) in A and B. (C) ROS1 andH2A.Z interact in a Y2H assay. BD, GAL4 binding do-main; AD, GAL4 activation domain. (D) ROS1 andH2A.Z interact in a split luciferase complementationassay. The indicated proteins were transiently expressedfused to the N-terminal or the C-terminal part of theluciferase protein (nLuc or cLuc, respectively) in N.benthamiana leaves.

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35S:SUC2

Fig. 4. H2A.Z is differentially deposited athyper-DMRs in antisilencing mutants. (A and B)Average enrichment of H2A.Z in genes (A) or TEs(B) delimited by a TSS and a transcription termi-nation site (TTS). Information about the rawpairs, concordantly mapped pairs and uniquelymapped pairs can be found in Dataset S3. (C)Metaplot and heatmap representations of theH2A.Z ChIP signal at the hyper-DMRs of idm1-9,h2a.z-2, and ros1-14 in 35S:SUC2 control plantsand idm1-9, ros1-14, arp6-4, and pie1-6 mutants.(D) Metaplot and heatmap representations ofthe H3K18Ac ChIP signals at the hyper-DMRsshared by idm1-9, ros1-14, arp6-4, pie1-6, andh2a.z-2 mutants.

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the bromodomain (V185A) abolished its binding to acetylated his-tone H3 peptides (SI Appendix, Fig. S14 C and D).To investigate whether NPX1 and AtMBD9 are involved in DNA

demethylation, we generated mutant alleles in the 35S:SUC2 back-ground (SI Appendix, Fig. S15A). Both the npx1-4 and atmbd9-4mutants showed weak root phenotypes on sucrose-containingmedia, compared with 35S:SUC2 plants (Fig. 6A). The expres-sion of SUC2, NPTII, and HPTII transgenes was also reduced innpx1-4 and atmbd9-4, but the reduction was less pronounced than inidm1-9 or ros1-14 (Fig. 6B). The atmbd9 npx1 double-mutant,however, developed long roots on sucrose-containing media,which were longer than those of npx1-4 or atmbd9-4 single-mutantplants and comparable to those of idm1-9 or ros1-14 (Fig. 6A), in-dicating that functional redundancy exists between AtMBD9 andNPX1. The expression of SUC2, HPTII, and NPTII was reduced inatmbd9 npx1 double-mutant plants, similar to idm1-9 and ros1-14mutants (Fig. 6B). The expression of the IDM1- and ROS1-targetedendogenous genes AT1G26380 and AT1G62760 was also reduced inatmbd9 npx1 compared with 35S:SUC2 plants, as in arp6-4, pie1-6,h2a.z-2, idm1-9, and ros1-14 (Fig. 1B). The expression of NPX1-3xFLAG-3xHA and AtMBD9-3xFLAG fusions driven by their na-tive promoters restored the short-root phenotype in atmbd9 npx1,while mutation of the conserved amino acid in the bromodomain ofNPX1 abolished this restoration (SI Appendix, Fig. S15 B and C).

DNA methylation levels increased at the CaMV35S transgene pro-moter in atmbd9 npx1 plants relative to 35S:SUC2 plants, similar tothe antisilencing mutants arp6-4, pie1-6, h2a.z-2, idm1-9, and ros1-14(Fig. 1C and SI Appendix, Fig. S3). Furthermore, 5-Aza restoredkanamycin resistance in the atmbd9 npx1 double-mutant (SI Ap-pendix, Fig. S15D). DNA methylation levels in all sequence con-texts at h2a.z-2 and idm1-9 hyper-DMRs were increased also inatmbd9 npx1 (SI Appendix, Fig. S15E). Thus, NPX1 and AtMBD9have a redundant role in antagonizing transcriptional silencing,and are necessary for protecting specific genomic regionsfrom hypermethylation.The bromodomain of AtMBD9 binds H3K14Ac, H3K18Ac,

and H3K23Ac peptides in a pull-down assay, and mutating aconserved residue (D1209A) within the bromodomain abolishedthis binding (Fig. 6C). IP-MS experiments using tagged AtMBD9confirmed that AtMBD9 is associated with PIE1 and SWR1components (SI Appendix, Fig. S16). In Y2H assays and luciferasecomplementation imaging in N. benthamiana leaves, we foundthat both NPX1 and AtMBD9 interact with PIE1 (SI Appendix,Fig. S17). These results indicate that AtMBD9 and NPX1 asso-ciate with the plant SWR1 complex and may function together asreaders of histone acetylation marks.Several examples of the shared hyper-DMRs in atmbd9 npx1

as well as in the ros1-14, idm1-9, arp6-4, pie1-6, and h2a.z-2

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Fig. 5. Correlation beteween DNA demethylationand H2A.Z enrichement at hyper-DMRs shared byidm1-9, ros1-14, arp6-4, pie1-6, and h2a.z-2 mutants.(A) DNA methylation levels at the hyper-DMRsshared by idm1-9, ros1-14, arp6-4, pie1-6, andh2a.z-2 mutants in 35S:SUC2 and indicated mutants.(B) H2A.Z level at the hyper-DMRs in A in 35S:SUC2and indicated mutants. (C) DNA methylation levels atsimulated regions with same length and DNAmethylation in 35S:SUC2 control plants (correspond-ing to the hyper-DMRs in A) in 35S:SUC2 and in-dicated mutants. (D) H2A.Z level at the shared hyper-DMRs in A, simulations with same H2A.Z and simu-lations with same DNA methylation in 35S:SUC2control plants. Compared with the simulated regionswith the same DNA methylation, H2A.Z was largelyenriched at the common hyper-DMRs shared byidm1-9, ros1-14, arp6-4, pie1-6, and h2a.z-2 mutantsin 35S:SUC2 control plants. (E) DNA methylationlevels at simulated regions with same length andH2A.Z enrichment in 35S:SUC2 control plants (corre-sponding to the hyper-DMRs in A) in 35S:SUC2 andindicated mutants.

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mutants are shown using integrative genomics viewer screenshots,which also display the ChIP-seq results for H2A.Z deposition,H3K18ac enrichment, and nucleosome density in these genomicregions targeted for active DNA demethylation (Fig. 6D and SIAppendix, Fig. S18). ChIP-qPCR assays confirmed that H2A.Zdeposition at several tested DNA hypermethylated loci was re-duced in atmbd9 npx1, as well as in idm1-9 (SI Appendix, Fig. S19).

DiscussionIn this study, we showed that ARP6 and PIE1 are 2 cellular factorsthat inhibit DNA hypermethylation at specific genomic regionsand prevent transcriptional gene silencing. In Arabidopsis, muta-tions in the SWR1 subunits PIE1 or ARP6 are known to causesimilar developmental phenotypes as those seen in h2a.z mutants,including early flowering, reduction in plant size, curly leaves,shortened siliques, and reduced fertility (39, 41, 46–49), consistentwith the role of the SWR1 complex in depositing H2A.Z. Ourresults suggest that the SWR1 complex, in addition to regulatingplant development, also functions in active DNA demethylation.H2A.Z interacts with ROS1, and h2a.z mutant plants show DNAhypermethylation and gene-silencing phenotypes. Thus, the his-tone variant H2A.Z is important for active DNA demethylation atspecific genomic regions.Our results show that the Arabidopsis SWR1 complex associates

with 2 bromodomain-containing proteins, NPX1 and AtMBD9,which recognize acetylated histone marks deposited by the IDM

complex. The presence of double bromodomains in the yeastBDF1 highlights the significance of histone acetylation in H2A.Zdeposition. In addition, the recruitment of the SWR1 complex tochromatin in yeast is regulated by the sequence-specific tran-scription factor Swc2/YL-1 (43). The MBD domain of AtMBD9may be important for targeting the SWR1 complex to certainmethylated genomic regions with histone acetylation marks todeposit H2A.Z. Chromatin modifiers are often associated withMBD proteins. In mammals, MBD2 is part of the histone deacetyla-tion complex MeCP1 (50). In Arabidopsis, the chromatin remodelingprotein DDM1 binds in vitro and colocalizes in vivo with AtMBD5, -6,and -7 (51). Our study on AtMBD9 and NPX1 provides insight intothe mechanism of recruitment of the SWR1 complex, throughrecognition of both euchromatic (histone acetylation) and het-erochromatic (DNA methylation) marks.Previous work showed that the histone acetyltransferase

IDM1 exists in a protein complex that specifically recognizescertain methylated genomic regions through the MBD domainsof its subunits MBD7 and IDM1, and through the DNA bindingdomain ofHDP2 (24). Our results here suggest that the bromodomain-containing proteins NPX1 and AtMBD9 recognize acetylatedhistone marks established by IDM1 and help attract the SWR1complex to chromatin for H2A.Z deposition at the 35S:SUC2transgene and specific endogenous genomic loci. In these partic-ular regions, we propose that H2A.Z interacts with ROS1 andmediates its recruitment for active DNA demethylation to prevent

0 0.75

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Fig. 6. AtMBD9 and NPX1 antagonize transcriptional silencing redundantly by recruiting the SWR1 complex to chromatin through the recognition ofacetylated histone marks. (A) Root phenotype of npx1-4, atmbd9-4, and atmbd9 npx1 mutants on sucrose-containing medium (Upper) or glucose-containingmedium (Lower). (B) Relative expression of transgenes in the 35S:SUC2 control plants and the indicated mutants. Values are means ± SD of 3 biologicalreplicates. *P < 0.05, **P < 0.01, compared with atmbd9 npx1 plants. NS, not significantly different compared with atmbd npx1 plants (2-tailed t test). (C)Biotinylated peptide pull-down assays showing in vitro binding of the recombinant AtMBD9 bromodomain to acetylated histone H3 peptides. (D) Examples ofDNA methylation status, H2A.Z deposition, H3K18Ac signal, and nucleosome density at several selected shared hyper-DMRs in antisilencing mutants. Scales onthe tracks of DNA methylation level are from −1 to +1. The highlighted region (in the square) indicates regions with differential DNA methylation, H2A.Zsignal and H3K18Ac signal. For DNA methylation, 2 biological replicates in each mutant were performed and 1 representative result is shown.

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hypermethylation and the spread of DNA methylation (Fig. 7).These specific genomic regions are characterized by a coexistenceof DNAmethylation and H2A.Z marks, and thus are an exceptionto the general rule of anticorrelation between H2A.Z depositionand DNA methylation (37). IDM1-dependent acetylation and SWR1-directed H2A.Z deposition could possibly act through a mutualreinforcement mechanism during active DNA demethylationsince the H3K18 acetylation mark at the 35S:SUC2 transgene andat some endogenous ROS1 targets were reduced in the arp6-5 andatmbd9 npx1 mutants (Fig. 1C and SI Appendix, Fig. S11B).Since our model only applies to a subset of genomic regions

demethylated by ROS1, additional mechanisms for directingROS1 for locus-specific demethylation must exist. H2A.Z hasbroad genomic distribution and a wide range of functions (36, 37,39–41, 52–54), many of which are clearly unrelated to activeDNA demethylation, since the antisilencing/DNA demethylationmutants do not show any hypermethylation at the +1 nucleo-somes, where H2A.Z is highly enriched (Fig. 4A and SI Appen-dix, Fig. S20), and incorporation or removal of H2A.Z can becarried out by specific histone chaperones in yeasts and meta-zoans (55, 56). ROS1 has domains that are important in therecognition of substrate DNA (57); this and perhaps other as yetunknown factors may function together with H2A.Z in recruitingROS1 to the aforementioned particular genomic regions thatbreak the rule of anticorrelation between DNA methylation andH2A.Z deposition.Despite the growing body of knowledge concerning the nature

and genome-wide landscape of diverse epigenetic modifications,the interplay and hierarchy between the various epigenetic

modifications are not well understood. In particular, little isknown about how active DNA demethylation may be regulatedby chromatin marks. The results presented here offer an exampleof how chromatin marks can determine the DNA methylationstatus through the sequential actions of chromatin modifiers,shedding light on the coordinated control of gene expression,and providing a conceptual framework for the interplay betweenchromatin modifications and active DNA demethylation.

Experimental ProceduresPlant Materials, Mutant Screening, and Map-Based Cloning. 35S:SUC2 plants inthis study refer to transgenic plants previously reported; the EMS-mutagenizedlibrary was generated and screened for mutants based on the long-rootphenotype (38, 58). To map the mutant genes, mutants were crossed towild-type plants of the Landsberg ecotype. Seedlings were grown verticallyon 1/2 Murashige and Skoog (MS) plates with 1% sucrose with 20 μg/LHygromycin and 1% agar and showed segregation of long and short rootplants. We screened for mutants with a long-root phenotype among 7-d-oldseedlings. Genetic mapping and gene cloning was performed as describedpreviously (38). The T-DNA mutants arp6-1 (SAIL_599_G03), pie1-5 (SALK_096434),npx1-1 (SAIL_123_A10), npx1-2 (WiscDsLox422C12), atmbd9-2 (SALK_121881), andatmbd9-3 (SALK_039302C) were ordered from the Arabidopsis Biological ResourceCenter (https://www.arabidopsis.org) and genotyped by PCR. Plants were grown inlong-day (16-h light/8-h dark) or short-day (8-h light/16-h dark) conditions at 22 °C.

DNA Methylation Inhibitor 5-Aza Treatment. For tests of antibiotic resistance,plants were grown on glucose-containing 1/2 MS medium with 50 mg/Lkanamycin. For 5-Aza treatments, both 50 mg/L kanamycin and 20 μM 5-Azawere added to 1% glucose-containing 1/2 MS medium. An equal volume ofDMSO was added to the medium as control. The seedlings were photo-graphed and harvested for RNA extraction after 14 d.

Acetylation

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Fig. 7. Working model for the role of SWR1 and H2A.Z in active DNA demethylation. The methylated genomic target regions of active DNA demethylationare recognized by the MBD-containing proteins in the IDM complex (i). The histone H3K14Ac, H3K18Ac and H3K23Ac marks generated by IDM1 are rec-ognized by the bromodomain proteins AtMBD9 and NPX1 in the SWR1 complex (ii). The SWR1 complex deposits H2A.Z to the targeted methylated regions.H2A.Z may also reinforce histone H3 acetylation (iii). H2A.Z interacts with and recruits ROS1 to initiate active DNA demethylation (iv). The model explains thebehavior of particular genomic regions where moderate levels of DNA methylation and H2A.Z deposition coexist.

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Mutant Plant Complementation. For the complementation of mutants, ARP6,PIE1, or NPX1 genomic DNA with 2-kb upstream region (as the native pro-moter region) was amplified from genomic DNA of Col-0 with primers listedin SI Appendix, Table S1 and cloned into the pENTR/D-TOPO vector (Invi-trogen). After sequencing, through LR recombination reaction, the genomicDNA was subcloned into the pEarly305 vector (with a 3xFLAG-3xHA tag at theC terminus) using LR clonase II (Invitrogen). AtMBD9 genomic DNA with 2-kbupstream region (as the native promoter region) was amplified from genomicDNA of Col-0 with primers listed in SI Appendix, Table S1. The full-length ofgenomic AtMBD9 DNA was divided into 2 fragments and the 2 fragmentswere digested with KpnI and SalI, and SalI and SbfI, respectively. The 2 frag-ments were cloned to pCambia 1305 vector (with a 3xFLAG tag at the Cterminus) by T4 DNA ligase (New England Biolabs). Unsegregated T3 plantsgrowing on 1% sucrose-containing medium and that were identified as ho-mozygous complementation lines were then used for the additional experi-ments. The ros1-14/proROS1:ROS1-3xFLAG-3xHA transgenic plants used for IPand ChIP assays were generated in a previous publication (59). The ros1-14h2a.z/proROS1:ROS1-3xFLAG-3xHA transgenic plants were generated bycrossing ros1-14/proROS1:ROS1-3xFLAG-3xHA with h2a.z mutant plants. Theh2a.z mutant was described previously (41).

Generation of h2a.z Mutants in the 35S:SUC2 Transgene Background. To gen-erate the h2a.z mutant alleles using the CRISPR-Cas9 system, 3 20-bp sgRNAoligos targeting HTA8, HTA9, or HTA11, were inserted into the BbsI sites of18T-AtU6-chim, 18T-AtU3b-chim, and 18T-At7SL-chim, respectively. To cas-cade the 3 single-guide RNA (sgRNAs) together, the pAtU6-sgRNA fragmentwas digested with HindIII and XhoI, the pAtU3b-sgRNA fragment with XhoIand XbaI and the pAt7SL-sgRNA fragment with XbaI and XmaI from theirhost vectors. These 3 fragments were inserted into the HindIII and XmaI sitesof the psgRNA-Cas9-At to obtain the p3×sgRNA-Cas9-At and then p3×sgRNA-Cas9-At was subcloned into the pCAMBIA1300 binary vector (60). The con-struct was transformed into 35S:SUC2 plants using Agrobacterium tumefa-ciens GV3101 by the standard floral-dip method (61). Seeds from T4 plantscontain homozygous h2a.z mutations and were genotyped by PCR or se-quencing (JIELI BIOTEC).

Real-Time qPCR. Total RNA was extracted from 0.1 g of 14-d-old Arabidopsisseedlings with the RNeasy plant kit (Qiagen). Two micrograms of mRNA wasconverted to cDNA with M-MuLV Reverse Transcriptase (New EnglandBiolabs) and the cDNAs were used as templates for real-time PCR with iQSYBR green supermix (Bio-Rad).

Whole-Genome Bisulfite Sequencing and Data Analysis. Fourteen-day-oldseedlings grown on 1/2 MS medium with 1% glucose and 0.8% agar wereused for extraction of genomic DNA, according to the manufacturer’s in-structions (Qiagen). Six replicates were performed in 35S:SUC2 plants. Tworeplicates were performed for h2a.z-2, idm1-9, ros1-14, arp6-4, pie1-6, andatmbd9npx1. Bisulfite conversion, library construction, and deep sequencingwere performed by the Genomics Core Facility at the Shanghai Center forPlant Stress Biology, China.

For data analysis, data were trimmed using trimmomatic (62) with pa-rameters “LEADING:20 TRAILING:20 SLIDINGWINDOW:4:15 MINLEN:50.”Clean reads were mapped to the Arabidopsis thaliana TAIR 10 genome usingBSMAP with parameters “-m 0 -x 1000 -w 2”, and the python script of BSMAPwas used to remove potential PCR duplicates and extract methylation ratios (63).

Identification of DMRs. Identification of DMRs was performed as describedpreviously (25) with some modifications. We had 3 batches of BS-Seq of35S:SUC2 wild-type (each batch has 2 replicates). We combined 2 replicatesof each sample. Only cytosines with a depth of at least 4 in the libraries wereretained for further analysis. Differentially methylated cytosines (DMC) wereidentified if the P value from the 2-tailed Fisher’s Exact test was not higherthan 0.05. We used a 200-bp sliding window with sliding step of 50 bp. DMCnumbers were counted in each window. A region was selected as an anchorregion if it had at least 2 DMCs. The actual boundary of each anchor regionwas then adjusted as the locations of first and last DMCs in the region. If thedistance between 2 anchor regions was ≤100 bp, they were combined into alarger region. Only regions containing at least 10 DMCs were selected ascandidate DMR. Each mutant was compared with each of the 3 35S:SUC2wild-types and only the intersection of the 3 candidate DMRs set arereported as final DMRs. Further information about the identified hyper-DMRs can be found in Dataset S2.

ChIP Assay and Data Analysis. The ChIP assay was performed as describedpreviously (36) with few modifications. Five grams of seedlings were cross-

linked with 1% formaldehyde in PBS buffer and ground in liquid nitrogen.Sonicated chromatin was incubated overnight with anti-H2A.Z (36), anti-H3K18ac (ab1191, Abcam), or anti-FLAG (F1804, Sigma). Immunoprecipi-tated DNA was treated with RNase A (Qiagen) and purified with theQIAquick purification kit (Qiagen). For individual locus detection, ChIP productswere diluted with 80 μL of TE buffer, and inputs were diluted with TE bufferat the ratio of 1:100; 2 μL were used for each qPCR and 3 technical replicateswere performed. Primers are listed in SI Appendix, Table S1.

For ChIP-seq, the DNA was sequenced at the Genomics Core Facility of theShanghai Center for Plant Stress Biology, Shanghai, China. The quality of thesequencing data were first checked with FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The first 8 bp of each read and reads after86 bp (H2AZ ChIP)/90 bp (H3K18Ac) were trimmed. Clean paired-end readswere mapped to the TAIR10 A. thaliana genome using Bowtie2 (64) withparameters “–very-sensitive–no-unal–no-mixed–no-discordant -k 2” andonly uniquely aligned reads were kept. Duplicated reads were removedusing the SAMtools “rmdup” command (65).

The normalization method for H2A.Z ChIP-Seq was performed as pre-viously described (66). Data were normalized so that the H2A.Z signal insideof background regions (defined as one-quarter of TEs with lowest ChIPsignal) were the same. Briefly, TEs that do not overlap with genes were usedas input for feature counts to count the ChIP fragment number in 35S:SUC2wild-type. Then the TEs were sorted by value (=ChIP_fragment_number/TE_length) and the lowest 25% TEs were chosen as background regions. Thefragments were pooled together in the background and the backgroundsignal in each genotype was calculated as signal = [(ChIP_background_rep1/ChIP_total_rep1+ChIP_background_rep2/ChIP_total_rep2)/2]/[Input_background/Input_total]. The normalization factors were calculated as norm_factor_mut =signal_mut/ signal_WT.

Histone Peptide Array and Pull-Down Assays. The coding sequence of N-terminal NPX1 (1 to 509 aa), including the bromodomain, was amplifiedand cloned into the pET28a vector under the direction of the One StepCloning Kit (Vazyme Biotech). The bromodomain mutation site was in-troduced by site-direct mutagenesis. The constructs were transformed intoEscherichia coli BL21 for fusion protein expression. The His-tag fusion pro-teins were purified with Ni-NTA His Bind Resin (Novagen).

The coding sequence of the bromodomain of AtMBD9was amplified usingthe primers in SI Appendix, Table S1 and cloned to pGEX-4T-1 vector. GST-tag fusion proteins were purified as indicated previously (38). The bromo-domain mutation site was introduced by site-directed mutagenesis.

MODified Histone Peptide Array (Active Motif) was used to screen thespecific histonemarks for NXP1 binding. For pull-down assays, the biotinylatedhistone H3K9me2, H3K14Ac, H3K18Ac, or H3K23Ac peptides (EpiCypher) wereincubated with 5 μg of His-Tag fusion or GST-Tag fusion proteins. The outputsof the incubation were detected by Western blot.

Y2H Assay. In brief, the cDNA sequences were cloned into pGADT7-AD orpGBKT7-BD vectors and the pair of genes to be tested for interaction werecotransformed into the yeast strain AH109. Yeast cells expressed bindingdomain-ROS1 and activation domain-HTA2, -6, -8, -9, or -11. Y2H assays wereperformed as described previously (67).

IP and LC-MS/MS Analysis. For IP, about 10 g of floral tissue for each epitope-tagged transgenic line were used. Dynabeads (10003D, Invitrogen) conju-gated with FLAG antibody (F1804, Sigma), anti-H2A.W (36), and anti-H2A.Z(36), respectively, were applied for IP. Affinity purification was performed asdescribed in a previous publication (68); the protein samples were subjectedto LC-MS/MS analysis as detailed in a previous publication (25). For the IPsamples of ROS1 or PIE1, to increase the number of detected peptides andthe subsequent protein sequence coverage, the bound-bead proteins werefirst eluted by acid elution with 0.1 M glycine HCl at pH 3.5, and then thebound-bead proteins subsequently were digested on-bead by the additionof 100 μl of 100 mM NH4HCO3 containing 1 μg of Lys-C (Promega) overnightat 37 °C (69). All peptides were purified using StageTips before LC MS/MSanalysis. We combined the 2 LC MS/MS results from products of glycineelution and the products of on-beads digestion together as the final IP resultof PIE1 or ROS1.

Split Luciferase Complementation Assays. Split luciferase complementationassays were performed using A. tumefaciens GV3101 carrying different con-structs in 4-wk-old tobacco leaves. The coding sequences of the indicatedproteins were cloned into pCAMBIA-cLUC and pCAMBIA-nLUC vectors. Lucif-erase activity was detected at 48 h postinfiltration. The detailed procedure is asdescribed previously (25).

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Data Access. The MNase-Seq data are downloaded from a previously publishedpaper (70); the Sequence Read Archive accession number is SRP120232. All high-throughput sequencing data generated in this studywere submitted to theNationalCenter for Biotechnology Information’s Gene Expression Omnibus (GSE115170).

ACKNOWLEDGMENTS. We thank Dr. Daniel Zilberman for kindly providingthe T-DNA insertion h2a.z mutant seeds. This work was supported by theChinese Academy of Sciences and the Gregor Mendel Institute (F.B.). W.-F.N.was partly supported by China Scholarship Council.

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