Peroxisomal β-oxidation regulates and DNA methylation in Arabidopsis

Lishuan Wanga,1, Chunlei Wangb,1, Xinye Liuc, Jinkui Chenga, Shaofang Lia, Jian-Kang Zhud,e,2, and Zhizhong Gonga,2

aState Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, 100193 Beijing, China; bCollege of Horticulture, Gansu Agricultural University, 730070 Lanzhou, China; cMinistry of Education Key Laboratory of Molecular and Cellular Biology, College of Life Sciences, Hebei Normal University, 050024 Shijiazhuang, China; dShanghai Center for Plant Stress Biology, National Key Laboratory of Plant Molecular Genetics, Center of Excellence in Molecular Plant Sciences, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, 200032 Shanghai, China; and eDepartment of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907

Contributed by Jian-Kang Zhu, April 4, 2019 (sent for review March 12, 2019; reviewed by Bao Liu and Jim Peacock) Epigenetic markers, such as histone acetylation and DNA methylation, DNA methylation is a conserved epigenetic marker important determine organization. In eukaryotic cells, metabolites in genome organization, expression, genomic imprinting, from organelles or the cytosol affect epigenetic modifications. How- paramutation, and X inactivation in organisms (3, ever, the relationships between metabolites and epigenetic modifica- 11–13). DNA methylation patterns are coordinately determined tions are not well understood in plants. We found that peroxisomal by methylation and demethylation reactions in plants and ani- acyl-CoA oxidase 4 (ACX4), an in the fatty acid β-oxidation mals (13, 14). The active removal of 5mC in Arabidopsis is car- pathway, is required for suppressing the silencing of some endoge- ried out by a subfamily of bifunctional DNA glycosylases/ nous loci, as well as Pro35S:NPTII in the ProRD29A:LUC/C24 transgenic represented by REPRESSOR OF SILENCING 1 (ROS1) and line. The acx4 mutation reduces nuclear histone acetylation and in- DEMETER (DME) (15, 16). ROS1 family proteins bind DNA non- creases DNA methylation at the NOS terminator of Pro35S:NPTII and specifically (17) and need other factors to find target genomic regions at some endogenous genomic loci, which are also targeted by the (13). Among these, ROS4/INCREASED DNA METHYLATION 1 demethylation enzyme REPRESSOR OF SILENCING 1 (ROS1). Further- (IDM1) is a plant homeodomain finger-containing histone more, mutations in multifunctional protein 2 (MFP2) and 3-ketoacyl- that catalyzes the acetylation of histone H3 CoA -2 (KAT2/PED1/PKT3), two in the last two steps lysine 18 (H3K18) and lysine 23 (H3K23) to create a favorable of the β-oxidation pathway, lead to similar patterns of DNA hyper- chromatin environment for the recruitment of ROS1 at some methylation as in acx4. Thus, metabolites from fatty acid β-oxidation loci(1,18).ROS4/IDM1,togetherwith other factors, such as ROS5/ in peroxisomes are closely linked to nuclear epigenetic modifications, IDM2, IDM3, methyl-CPG-binding domain 7 (MBD7), Harbinger which may affect diverse cellular processes in plants. Significance β-oxidation | acetyl-CoA | histone acetylation | DNA methylation | gene silencing Small-molecule metabolites from cell organelles or cytosol exert their influence on decision making processes in eukaryotic cells. istone acetylation is important for neutralizing the positive However, the mechanisms underlying the regulation of cellular Hcharges of lysine residues and promoting chromatin relaxation; processes by these metabolites are not well understood. Among it is also required for , DNA replication, histone these small metabolites, acetyl-CoA, a most critical one, is pro- – methylation, and other histone modifications (1 4). are duced in the cytosol and different cell organelles. In plants, acetyl- acetylated by , which transfer acetyl groups CoA produced from fatty acid β-oxidation in peroxisomes plays from acetyl-CoA to histone lysine residues. critical roles in various developmental stages. Here we found that Acetyl-CoA is a central metabolite that can be produced via defects in β-oxidation in peroxisomes affect both histone acety- several metabolic pathways involved in pyruvate, citrate, acetate, lation and DNA methylation in the nucleus. Our work provides β and fatty acid -oxidation metabolism (5). In mammals, acetyl- evidence for retrograde signaling from peroxisomes to regulate CoA in mitochondria is produced from different pathways, in- nuclear epigenetic modifications in higher eukaryotes. cluding the fatty acid β-oxidation (6). In cytosol and nucleus, adenosine triphosphate (ATP)-citrate (ACLY) cleaves Author contributions: L.W., C.W., J.-K.Z., and Z.G. designed research; L.W. and C.W. per- citrate exported from mitochondria to regenerate acetyl-CoA formed research; L.W., C.W., X.L., J.C., and S.L. analyzed data; and L.W., J.-K.Z., and Z.G. that can be used for other biosynthetic processes, such as fatty wrote the paper. acid synthesis and histone acetylation (6). In mouse, conditional Reviewers: B.L., Northeast Normal University; and J.P., Commonwealth Scientific and In- loss of carnitine palmitoyltransferase 1A (CPT1A), which is required dustrial Research Organisation. for the transfer of fatty acid into mitochondria for β-oxidation, The authors declare no conflict of interest. impairs dermal lymphatic formation via histone acetylation in an Published under the PNAS license. ACLY-dependent manner (7). A pyruvate dehydrogenase com- Data deposition: Sequence data referred to in this article have been deposited in the plex can be translocated from mitochondria to nuclei to generate GenBank/EMBL database (accession nos. AT2G36490 for ROS1, AT3G14980 for ROS4/ IDM1, AT5G66750 for DDM1, AT4G11130 for RDR2, AT3G51840 for ACX4, AT3G06860 acetyl-CoA and mediate histone acetylation in mammalian cells for MFP2, AT2G33150 for KAT2, AT1G64230 for UBC28, and AT3G18780 for ACTIN2). in certain conditions (2, 8). In Arabidopsis, the mutations in cytosolic Primary datasets for the whole-genome bisulfite sequences of Col-0, ros1-4, acx4-4, acetyl-CoA carboxylase (ACC1), which converts cytosolic acetyl- acx4-1, mfp2-2, and kat2-3 mutant plants have been deposited in the CoA to malonyl-CoA for elongating the plastid-produced fatty Omnibus (GEO) database (accession no. GSE98214). Histone acetylation ChIP-seq data also have been deposited in the GEO database (accession no. GSE98214). Whole-genome bi- acids, lead to high accumulation of cytosolic acetyl-CoA, spe- sulfite sequencing data of C24 WT, ros1-1, and ros4 plants were obtained from the GEO cifically resulting in increased H3K27 acetylation (H3K27ac) (9). database (accession no. SRP042060). These results underscore the importance of acetyl-CoA in 1L.W. and C.W. contributed equally to this work. histone acetylation in nuclei in both mammals and plants. However, 2To whom correspondence may be addressed. Email: [email protected] or gongzz@cau. in plant cells, plastids, mitochondria, peroxisomes, and cytosol can edu.cn. produce acetyl-CoA (10). Whether impairment of metabolism in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. plant organelles will affect the nuclear epigenetic modifications is 1073/pnas.1904143116/-/DCSupplemental. still being unraveled. Published online May 7, 2019.

10576–10585 | PNAS | May 21, 2019 | vol. 116 | no. 21 www.pnas.org/cgi/doi/10.1073/pnas.1904143116 Downloaded by guest on September 27, 2021 transposon-derived protein 1 (HDP1), and HDP2, forms a complex to regulate active DNA demethylation (19–23). MET18 is a com- ponent in the cytosolic iron-sulfur cluster assembly pathway in- volved in the transfer of the Fe-SclustertoROS1,whichis necessary for its function (24). The expression of ROS1 is positively regulated by promoter DNA methylation, which requires a protein complex composed of Su(var)3–9 homologs (SUVHs) and SUVH- interacting DNAJ (SDJ) proteins (25–28). To identify the components required to prevent transgene si- lencing and normal DNA methylation patterns in Arabidopsis,we performed a forward genetic screen for kanamycin (Kan)-sensitive mutants using the ProRD29A:LUC/Pro35S:NPTII transgenic C24 line (18). Several ROS1 alleles, multiple components of the RdDM pathway,ROS4/IDM1,ROS5/IDM2, and MBD7, were identified in this screening (1, 18, 22, 23). Here we identified an antisilencing factor, acyl-CoA oxidase 4 (ACX4), in the fatty acid β-oxidation pathway. In acx4 mutants, overall levels of H3Ac and H4Ac are reduced, and DNA methylation is increased at some genomic loci, resulting in enhanced transcriptional silencing of reporter and some endogenous . The mfp2 and kat2 mutants have similar DNA hypermethylation phenotypes to acx4. Our results uncover a con- nection between fatty acid β-oxidation and epigenetic regulation in plants. Fig. 1. Characterization of the acx4 mutant. (A) The acx4 mutation silenced Results Pro35S:NPTII, as indicated by Kan sensitivity. The acx4-4, ros1-1, and ros4 ACX4 Is a Suppressor of Transcriptional Silencing. To decipher the mutants were grown on MS medium or on MS medium supplemented mechanism that blocks transcriptional gene silencing, we per- with 50 mg/L Kan. (B) The acx4 mutation does not affect the expression of ProRD29A:LUC. The acx4-4, ros1-1, and ros4 mutants grown on MS medium formed a forward genetic screen using an ethyl methanesulfo- PLANT BIOLOGY nate (EMS)-mutagenized population of a C24 transgenic line were treated with 30 μM abscisic acid for 3 h, after which a luciferase assay carrying ProRD29A:LUC and Pro35S:NPTII transgenes, both of was performed using a cold charge-coupled device camera. (C) Relative ex- which are actively expressed (used as the WT) in Arabidopsis (18, pression levels of Pro35S:NPTII in the acx4 mutant and the WT (C24 accession), ros1-1,andros4 seedlings by qPCR. UBC28 served as an in- 22, 23). From this population, a recessive kanamycin (Kan)- acx4 acx4-4 ternal control. (D) Relative expression levels of ProRD29A:LUC in the acx4 sensitive mutant, (hereinafter ), was isolated. Like mutant and WT, ros1-1,andros4 seedlings by qPCR. UBC28 served as an in- ros4, ros5-1,andmbd7 mutants (18, 22, 23), acx4-4 plants exhibited ternal control. (E) Diagram of the region identified as the acx4-4 mutation by silenced NTPII expression (Fig. 1 A and C), but the expression of map-based cloning. (F)ModelstructureoftheAT3G51840 (ACX4) gene. A LUC was unaltered (Fig. 1 B and D), while in ros1-1,both G808-to-A808 mutation occurs in the splice donor site of the third intron of the Pro35S:NPTII and ProRD29A:LUC were silenced (Fig. 1 A–D). AT3G51840 gene in the acx4-4 mutant. Two T-DNA insertion mutants, acx4-1 ACX4 was identified through map-based cloning. We crossed and acx4-3, are shown. The exon and intron are marked by a black box and the acx4-4 mutant in the C24 accession with the WT Columbia- black line, respectively. (G)Theacx4 mutant was complemented by ACX4,as 0 (Col-0) background and used the 2105 F2 plants for mapping. tested on MS medium containing 50 mg/L Kan in three complement lines acx4-4 (Com9, 10, 15). (H)NPTIIproteinlevelsinWT,ros1-1, acx4-4,andthreecom- The mutation was localized on the bottom of chromo- plemented lines, as indicated by immunoblotting with NPTII antibodies. ACTIN some 3 and then narrowed down between bacterial artificial served as a loading control. chromosome clones T18N14 and T25B25 (Fig. 1E). By sequencing candidate genes, a G-to-A mutation was found at position 808 of AT3G51840 (from the first putative ATG), which would change was not obviously affected in the acx4-1 and acx4-3 mutants, as the splicing acceptor site from GT to AT at the end of the third indicated by luciferase and LUC transcript assays (SI Appendix, intron (Fig. 1F). By comparing the cDNAs of AT3G51840 am- Fig. S2 A and B). plified by RT-PCR from WT and acx4-4 lines, we found that the We examined the subcellular localization of ACX4 using cDNA of AT3G51840 from acx4-4 was 16 bp shorter than that transient assays. ACX4 was fused to the C terminus of the red from WT. Furthermore, the acceptor splice site had moved to fluorescent protein mCherry, CD3-990 (a mitochondrial marker) the fourth exon, which would lead to premature termination of was fused to the C terminus of yellow fluorescent protein (YFP) translation and a truncated protein (SI Appendix,Fig.S1A and B). (30), CAT2 (a peroxisomal marker) was fused to the C terminus To further examine whether the mutation in AT3G51840 causes of green fluorescent protein (GFP) (31), and nuclear localization the silencing of the Pro35S:NPTII transgene, WT AT3G51840, signal (NLS) was fused to the N terminus of GFP. Mcherry- including the fragment 2543 bp upstream of the first putative ATG ACX4 was transiently coexpressed with each of the fused codon and 312 bp downstream of the putative stop codon TAA, marker proteins in tobacco (Nicotiana benthamiana) epidermal was cloned and transformed into the acx4-4 mutant. Like WT, cells driven by the cauliflower mosaic virus 35S promoter. three randomly selected independent transgenic lines exhibited ACX4 did not colocalize with CD3-990 and NLS (SI Appendix, Kan resistance, and accumulated the NPTII protein (Fig. 1 G and Fig. S3 A and B) but did colocalize with GFP-CAT2 (SI Appendix, H). Two additional alleles, acx4-1 and acx4-3 (Col-0 accession) Fig. S3C), consistent with immunoelectron microscopy data from (29), were obtained, representing the T-DNA insertion lines a previous study (32). Beta-glucuronidase (GUS) staining of SALK_000879 and SALK_065013, with the T-DNA inserted in transgenic plants expressing ProACX4:GUS revealed ACX4 ex- the third exon and 11th intron of ACX4, respectively (Fig. 1F). We pression throughout the seedling (SI Appendix, Fig. S4). introduced the acx4-1 and acx4-3 mutations into the WT trans- genic plants by crossing. As expected, both mutations resulted in The acx4 Mutation Causes DNA Hypermethylation at the NOS Terminator hypersensitivity to Kan relative to WT with greatly reduced NPTII of Pro35S:NPTII and Some Endogenous Genomic Loci. To test whether transcripts, as determined by quantitative RT-PCR (qPCR) (SI silencing of the Pro35S:NPTII transgene is associated with DNA Appendix,Fig.S2A and B), and the expression of ProRD29A:LUC methylation, we treated the WT, acx4-4, ros1-1,andros4 lines with

Wang et al. PNAS | May 21, 2019 | vol. 116 | no. 21 | 10577 Downloaded by guest on September 27, 2021 the DNA methylation inhibitor 5-Aza-2′-deoxycytidine (5′-Aza). The DNA methylation inhibitor suppressed the Kan sensitivity of the acx4-4, ros1-1,andros4 mutants (SI Appendix,Fig.S5A) and restored the expression of NPTII (SI Appendix,Fig.S5B). Furthermore, the acx4-4 ddm1 double mutant, but not the acx4-4 rdr2 double mutant, released the silencing of NPTII in acx4-4 (SI Appendix, Fig. S5 C and D). Both ddm1 and rdr2 have the same genetic background as acx4-4 (18). Silencing of NPTII in acx4-4 was suppressed by the ddm1, but not the rdr2 mutation (SI Appendix,Fig.S5C and D), suggesting that NPTII silencing is dependent on the DDM1-mediated methylation pathway, but not the RdDM pathway. Published studies indicate that the mutations in the components of RdDM pathway cause silencing of NPTII due to the repression of ROS1 that is positively regulated by DNA methylation in its promoter region (18, 33). Previous studies have indicated that the ros1-1 mutation in- creases DNA methylation in both the RD29A promoter and the 3′-NOPALINE SYNTHASE (NOS) terminator region, but that the ros4-4, ros5-1, and mbd7 mutations increase DNA methyl- ation mainly in the NOS terminator (22, 23). Bisulfite sequencing analyses indicated a slight increase in DNA methylation in the 35S promoter in these mutants (Fig. 2A). Like the ros1-1 and ros4 mutants, acx4-4 showed a clear increase in CG, CHG, and CHH DNA methylation (Fig. 2B)intheNOS terminator. These results suggest that the acx4 mutation leads to DNA hyper- methylation in the NOS terminator and silencing of Pro35S:NPTII. To investigate the effect of the acx4-4 mutation on genomic DNA methylation patterns, whole-genome DNA methylation profiles of acx4-4 (two biological replicates) and the WT seed- lings (22, 23) were compared using next-generation sequencing after bisulfite conversion. Analysis of differentially methylated regions (DMRs) revealed 864 DMRs with increased DNA methylation (hyper-DMRs) in acx4-4, including 405 DMRs with Fig. 2. The acx4 mutation causes hypermethylation of DNA in the NOS hyper-CG methylation, 55 with hyper-CHG methylation, and terminator. (A) Effects of the acx4-4, ros1-1, and ros4 mutations on DNA 404 with hyper-CHH methylation. Heat map analysis of hyper- methylation in the Pro35S:NPTII promoter by bisulfite sequencing. (B) Effects DMRs indicated that the levels of DNA methylation at acx4-4– of the acx4-4, ros1-1, and ros4 mutations on DNA methylation at the NOS specific loci were usually preferentially increased in ros1-1 and region by bisulfite sequencing. (C) Heat map showing the methylation levels ros4 C of the WT, acx4-4, ros1-1, and ros4 plants in hyper-DMR regions of acx4-4 in (Fig. 2 ), We picked several genes to confirm their DNA different sequence contexts. The lighter color indicates a low methylation methylation patterns. Individual bisulfite sequencing revealed level, and the darker color indicates a high methylation level. (D) Expression increased DNA methylation levels of At4G18250, At4G29380, profiles of the hypermethylated genes or genes near DMRs in 7-d-old acx4-4, AT5G52400, AT1G19160,andAT3G51440 in acx4-4 and ros1-1 ros1-1,andacx4-4 ros1-1 seedlings by qPCR. Error bars represent ± SE. n = 3. compared with WT (SI Appendix,Fig.S6A and B). For AT4G29380, only the levels of CHG and CHH DNA methylation were in- creased in acx4-4 and ros1-1, while the level of CG methylation did the Arabidopsis genome with different expression patterns and not change—probably because it was already very high. Analysis of distinct substrate specificities (37). The second and third enzymatic the acx4-4 ros1-1 double mutant revealed no clear additive effects reactions are catalyzed by multifunctional proteins (MFPs), which on DNA methylation for AT1G19160 and AT3G51440 (SI Appendix, exhibit enoyl-CoA hydratase and β-hydroxyacyl-CoA dehydrogenase Fig. S6B). Whole-genome bisulfite sequencing data confirmed that, activities (35, 38). The last step in the pathway is catalyzed by like the ros4 mutant, acx4-4 exhibited increased DNA methylation L-3-ketoacyl-CoA thiolase (KAT), which converts L-3-ketoacyl- only in the NOS terminator region and not in the RD29A pro- CoAs to acetyl-CoAs (35, 39). moter (SI Appendix,Fig.S6C). The DNA methylation of some We next examined whether mutations in the downstream en- endogenous transposons (here we took two transposons ATREP10D zymes of the β-oxidation pathway also lead to silencing of the and HELITRON2 as examples) was increased (SI Appendix,Fig. Pro35S:NPTII transgene. Of the four catalytic activities of MFP2, S6D). These results suggest that ACX4 and ROS1 work in the same two—2-trans enoyl-CoA hydratase for long-chain (C18:0) sub- pathway to regulate DNA methylation of some loci in Arabidopsis. strates and L-3-hydroxyacyl-CoA dehydrogenase for C6:0, C12:0, We next used qRT-PCR to test the expression levels of and C18:0 substrates—are required for β-oxidation (40). MPF2 13 genes (plus 2 kb of sequence on either side) overlapping has one homolog, abnormal inflorescence meristem 1 (AIM1), with hypermethylated DMRs in acx4-4, ros1-1,andacx4-4 ros1-1. whose mutation results in an abnormal inflorescence meristem Transcript levels of these genes were substantially reduced in these phenotype in mature plants (41). The enzyme 3-ketoacyl-CoA mutants, and the acx4-4 ros1-1 double mutant had no additive thiolase-2 (KAT2/PED1/PKT3) catalyzes the last step of the effects over the single mutants (Fig. 2D). These data suggest that, β-oxidation pathway to produce one molecule of acetyl-CoA in like ROS1, ACX4 is critical for preventing the transcriptional si- each repeat cycle; this gene has higher expression than the two lencing of some endogenous genes through DNA methylation. redundant homologs KAT1 and KAT5 (42, 43). kat2 kat5 double mutants recapitulate the aim1 phenotype in inflorescence de- Mutations in the Last Two Enzymes of the β-Oxidation Pathway Cause velopment and fertility (44). Pro35S:NPTII Silencing. The first step in the fatty acid β-oxidation We used the egg-specific CRISPR/Cas9 system (45) to gen- pathway in peroxisomes is catalyzed by ACX enzymes and produces erate the mfp2-cas9-1 and mfp2-cas9-2 mutants in the Pro- trans-2-enoyl-CoAs (34–36). Six ACX genes have been identified in RD29A:LUC/C24 transgenic line (WT) (SI Appendix, Fig. S7 A

10578 | www.pnas.org/cgi/doi/10.1073/pnas.1904143116 Wang et al. Downloaded by guest on September 27, 2021 and D). Meanwhile, we obtained two T-DNA insertion alleles, showed that AT4G28910 was expressed at lower levels in acx4-4, Salk_098016 (mfp2-2) (40) and GK_787F01 (mfp2-10), and one mfp2-cas9-1,andmfp2-cas9-2 mutants compared with the WT T-DNA insertion allele, Salk_024922 (kat2-3), in which MFP2 (Fig. 3G). Taken together, our results suggest that the β-oxidation and KAT2 expression were eliminated (SI Appendix, Fig. S7 A– pathway in peroxisomes regulates gene silencing and DNA C). We introduced these mutations (in Col-0 accession) into methylation in Arabidopsis. ProRD29A:LUC/C24 transgenic line by crossing. Consistent with NPTII expression levels (Fig. 3 A and C), the mfp2-cas9-1 and MFP2 and KAT2 Target Some Common Loci as ACX4 for DNA mfp2-cas9-2 mutants were more Kan-sensitive than the WT and Methylation. To determine whether MFP2 and KAT2 also func- less sensitive than acx4-4, but did not exhibit altered LUC ex- tion in modulating DNA methylation at the whole genome level, pression (Fig. 3 B and D). Similarly, the mfp2-2, mfp2-10, and we performed bisulfite sequencing analyses and identified 1,153 acx4-1 mfp2-2 kat2-3 kat2-3 mutations caused more Kan sensitivity than the WT, but DMRs in , 1,135 DMRs in ,and686DMRsin the ProRD29A:LUC transgene was not affected (SI Appendix, compared with the WT (all in the Col-0 background), respectively. A Among these, 1,069 were hypermethylated and 84 were hypo- Fig. S8 ). Bisulfite sequencing analysis showed increased DNA acx4-1 methylation at the NOS region of the Pro35S:NPTII gene in mfp2- methylated in , 1,045 were hypermethylated and 90 were cas9-1 mfp2-cas9-2 hypomethylated in mfp2-2, and 623 were hypermethylated and and mutants compared with the WT, although the kat2-3 SI Appendix DNA methylation at these regions was a little lower compared 63 were hypomethylated in ( ,Fig.S9). More than 60% of these hyper-DMRs were located in genic regions in with that in acx4-4 (Fig. 3E). The lesser effect of mfp2 mutation acx4-1, mfp2-2,andkat2-3, whereas only 40% were located in on DNA methylation compared with acx4 suggests a redundancy genic regions in ros1-4 (Col-0) (Fig. 4A and SI Appendix,Fig.S9). of the MFP2 homologs in the β-oxidation pathway. Transcrip- Furthermore, 60.5% of 1,069 hyper-DMRs in acx4-1, 60.7% of tionally silent information (TSI) is regulated by the DDM1 1,045 hyper-DMRs in mfp2-2, and 76.1% of 623 hyper-DMRs in pathway, but not by the RdDM pathway (46, 47). qPCR dem- kat2-3 overlapped with those in ros1-4 (Fig. 4B). Approximately onstrated that the transcript levels of TSI were reduced in mfp2- mfp2-2 2 mfp2-10 kat2-3 50% of the 1,069 hyper-DMRs in and 60% of the 623 hyper- , , and mutants compared with Col-0 plants; a DMRs in kat2-3 were hypermethylated in acx4-1 (Fig. 4B). similar reduction was observed in acx4-4 compared with the WT mfp2-2 kat2-3 ros1-4 SI Appendix B Profiling the methylation levels of , , and in ( , Fig. S8 ). We found increased CG DNA methyl- the loci that were hypermethylated in acx4-1 revealed increased AT4G28910 mfp2-cas9 ation at the promoter in two independent DNA methylation levels at these loci compared with Col-0 (Fig. mutant alleles, and in the acx4-4 mutant (Fig. 3F). qPCR analysis 4C). Boxplots indicate that at those hyper-DMRs that appear PLANT BIOLOGY specific for acx4-1, the average DNA methylation levels in the CG context but not the CHG or CHH contexts were higher in the mfp2-2, kat2-3,andros1-4 mutants than in Col-0 plants (Fig. 4D), suggesting that fatty acid β-oxidation preferentially affects genomic regions in the CG context. These data indicate that mutations in ACX4, MFP2,andKAT2 affect the DNA methylation levels of some common genomic regions (SI Appendix,Fig.S10A–E).

The acx4 Mutant Has Reduced Histone Acetylation. ACX4 catalyzes the conversion of fatty acyl-CoAs to trans-2-enoyl CoA. This is thought to be the predominant way by which the rate of acetyl-CoA flux is controlled through β-oxidation in peroxisomes. We asked whether the level of acetyl-CoA is reduced in acx4 mutants. High- performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) was used to measure the accumulation of acetyl- CoA and its related metabolites butyryl-CoA and hexanoyl-CoA, the main substrates for ACX4. Both butyryl-CoA and hexanoyl- CoA accumulated to much higher levels, but the level of acetyl- CoA was significantly decreased in acx4-4 compared with the WT (Fig. 5A). We next conducted immunoblotting assays to assess whether defective β-oxidation in the acx4-4 mutant would change the level of histone acetylation in vivo. Compared with WT, levels of all three acetylated (Ac) core histones (H2B, H3, and H4) were decreased in the acx4-4 mutant (Fig. 5 B and C). Immunos- Fig. 3. MFP2 prevents transcriptional gene silencing of Pro35S:NPTII.(A) taining nuclei using H3Ac or H4Ac antibodies showed that levels The mfp2 CRISPR/Cas9 mutants are more sensitive to Kan than the WT, but more of these acetylated histones were significantly reduced in acx4-4 resistant to Kan than acx4-4. Seeds were planted on MS medium supplemented acx4-4 D with 150 mg/L Kan and cultured for 2 wk before being photographed. (B)The compared with the WT or complemented (Fig. 5 and mfp2 CRISPR/Cas9 mutants do not affect ProRD29A:LUC.(C)Themfp2 CRISPR/ E). Furthermore, chromatin immunoprecipitation (ChIP)-PCR Cas9 mutants and acx4-4 reduced Pro35S:NPTII expression levels compared with assays with antibodies for the acetylated histones H2B, H3, and the WT, as determined by qPCR analyses. UBC28 was used as an internal H4 revealed that levels of H3Ac and H4Ac, but not of H2BAc, control. (D) qPCR analysis of the expression levels of ProRD29A:LUC in the were substantially reduced at both the 35S promoter and NOS indicated genotypes. UBC28 served as an internal control. (E) Compared with the regions (SI Appendix, Fig. S11). These results indicate that his- WT, increased methylation levels were observed in the NOS region of mfp2 tone acetylation is reduced in the acx4-4 mutant and suggest that CRISPR/Cas9 and acx4-4 mutants, according to bisulfite sequencing analysis. (F) peroxisome-derived acetyl-CoA is important for histone acetylation. Bisulfite sequencing data showing methylation levels of the AT4G28910 pro- moter in the WT and indicated mutant plants. (G) qPCR analysis of expression levels of the AT4G28910 gene in the WT and different mutant plants. UBC28 Decreased Histone H3K18Ac Is Associated with DNA Hypermethylation acx4 served as an internal control. Data are means ± SE (n = 3) with three replicate in in Mutant. Recent studies have demonstrated that the DNA one experiment. Three independent experiments were done with similar results. demethylation complex recognizes acetylated H3K18 and H3K23 *P < 0.05. at some target loci for DNA demethylation, suggesting that histone

Wang et al. PNAS | May 21, 2019 | vol. 116 | no. 21 | 10579 Downloaded by guest on September 27, 2021 Fig. 4. MFP2 and KAT2 target some common loci as ACX4 for DNA methylation. (A) Comparison of the genomic regions of hyper-DMRs in acx4-1, mfp2-2, kat2-3, and ros1-4 mutants. All mutants are from the Col-0 accession. TE, transposable element. (B) Num- bers of overlapping hyper-DMRs in acx4-1, mfp2-2, kat2-3, and ros1-4 mutants. (C) Heat map comparing the methylation levels in acx4-1 hyper-DMRs with the same regions in mfp2-2, kat2-3, and ros1-4 mu- tants. Columns represent the indicated genotypes; rows represent differentially methylated loci. Hyper- DMRs in acx4-1 relative to the WT were used to compare DNA methylation levels with the same re- gions in mfp2-2, kat2-3, and ros1-4. Light yellow in- dicates a low methylation level, and black indicates a high methylation level. (D) Boxplots representing methylation levels of Col-0, acx4-1, mfp2-2, kat2-3, and ros1-4 mutants in those regions that are hyper- DMRs in acx4-1 in different sequence contexts. Two- tailed Student’s t test, *P < 0.05.

acetylation is required for active DNA demethylation (1, 20–23). We next plotted ChIP-seq reads on acx4-4 hyper-DMRs in WT To better evaluate whether ACX4-dependent generation of acetyl- and acx4-4 and found that the enrichment levels of H3K18Ac CoA selectively affects acetylation of histone H3 at lysine residues, around hyper-DMRs, but not other histone acetylation makers, we analyzed the levels of histone H3 acetylation at K9, K14, K18, were clearly decreased in acx4-4 relative to WT (Fig. 6D). In K23, and K27 in acx4-4 and WT by immunoblotting. The levels of agreement with this finding, boxplots show much lower H3K9Ac, H3K18Ac, and H3K23Ac were moderately reduced, but H3K18Ac levels of nearby hyper-DMRs in acx4-4 compared E ros1-1 those of H3K14Ac and H3K27Ac were unchanged (Fig. 6 A and B with WT (Fig. 6 ). Several hyper-DMRs shared by and acx4-4 and SI Appendix,Fig.S12). To determine which histone acetylation were selected for further investigation by ChIP-qPCR acx4 (qPCR) assays, which revealed lower H3K18Ac levels in the acx4-4 marks are associated with DNA hypermethylation in mutant, A B we conducted ChIP followed by sequencing (ChIP-seq) in WT and mutant compared with WT (Fig. 7 and ). As negative controls, no changes were found at ACTIN2, AT1G10950,andAT1G01260. acx4-4 using antibodies against H3K9Ac, H3K14Ac, H3K18Ac, These results suggest that reduced histone H3K18 acetylation in and H3K23Ac. For H3K14Ac, H3K18Ac, and H3K23Ac ChIP-seq acx4-4 is correlated with DNA hypermethylation. experiments, two biological replicates were performed. Pearson’s SI Appendix correlation between two replicates are shown in ,Fig.S13.In Overexpression of ATP-Citrate Lyase A and B Together Can Rescue the our analysis, 15,421 H3K9Ac, 8,460 H3K14Ac, 9,040 H3K18Ac, Kanamycin Sensitivity of acx4-4. Acetyl-CoA is impermeable to and 14,884 H3K23Ac peaks were identified in the WT. Consistent membranes. The foregoing data suggest that mutations of genes with the immunoblotting results, we found that ChIP-seq reads on in the β-oxidation pathway reduce the acetyl-CoA level in per- H3K9Ac, H3K18Ac, and H3K23Ac peaks, but not those on oxisomes, which may in turn decrease the acetyl-CoA level in H3K14Ac peaks, were decreased in acx4-4 relative to WT cytosol and affect histone acetylation and DNA methylation or (Fig. 6C). affect other metabolisms, such as the reduced production of

Fig. 5. The acx4 mutation reduces histone acetylation. (A) Relative levels of acetyl-CoA, butyryl-CoA, and hexanoyl-CoA determined by HPLC-MS/MS in 10-d-old seedlings. SD ± SE (n = 3) of three independent biological repeats is shown. Two-tailed Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001. (B)Overall levels of H2BAc, H3Ac, and H4Ac as determined by immunoblotting. Total proteins extracted from the WT and acx4-4 seedlings were used for immunoblot- ting with anti-H2BAc, -H3Ac, and -H4Ac antibodies. H3 was detected as a loading control. (C) Quantified mean data normalized to H3 showing decreased lev- els of acetylated H2B, H3, and H4 in acx4-4 compared with the WT. Error bars represent ± SE from three independent experiments (n = 3). (D and E)Levelsof acetylated H3 (D)andH4(E)inWT,acx4-4,andthe complemented acx4 line 9, as indicated by immuno- fluorescence under confocal microscopy. Acetylated- H3/H4 colocalized with the nuclear stain DAPI (blue). Representative images (Left) and quantified mean data (Right) are shown. Values are means ± SE, n = 40. **P < 0.01.

10580 | www.pnas.org/cgi/doi/10.1073/pnas.1904143116 Wang et al. Downloaded by guest on September 27, 2021 Fig. 6. The hyper-DMRs in acx4-4 are associated with reduced H3K18Ac. (A)Theacx4-4 mutant reduces histone acetylation. Histone proteins were extracted and used for immunoblotting with different anti- bodies. Histone H3 served as a loading control. (B) Quantified mean data normalized to H3 showing the relative levels of different H3 acetylation modi- fications in acx4-4 compared with the WT. Error bars represent ± SE from three independent experiments. n = 3. (C) Comparison of ChIP-seq reads with different histone acetylation antibodies between acx4-4 and the WT. −2kband+2 kb represent 2 kb upstream and 2 kb downstream of the peak middle point site, re- spectively. (D) Correlation analyses of DMRs with dif- ferent histone acetylation modifications between acx4-4 and the WT. Only the enrichment levels of H3K18Ac are found to be decreased in acx4-4 relative to the WT. −2kband+2 kb represent 2 kb upstream and 2 kb downstream of middle point site of the hyper-DMR region, respectively. (E) Boxplots showing that the H3K18Ac levels of nearby hyper-DMRs are significantly lower in acx4-4 than in WT. 50 bp, 100 bp, 150 bp, and 200 bp represent the distances upstream

and downstream of the middle point site of the PLANT BIOLOGY hyper-DMR region. Two-tailed Student’s t test, **P < 0.01.

some phytohormones, as some products of β-oxidation may go to sequencing revealed that the DNA methylation of endogenous different metabolite pathways to produce MeJA, SA, or auxin target gene AT4G18250 was recovered to WT level in both lines (48, 49). Under different hormone treatments, such as NAA, SA, #6 and #12 in F2 Kan-resistant seedlings (Fig. 8F). These results or MeJA, the Kan sensitivity of acx4-4 mutant was not changed indicate that increasing acetyl-CoA content rescued the acx4-4 Kan (SI Appendix, Fig. S14A), indicating that Pro35S:NPTII silencing sensitivity. is not regulated by these phytohormones. We further added some possible precursors for acetyl-CoA Discussion biosynthesis, such as succinate, acetate, oxoglutarate, and citric Acetyl-CoA is a central metabolite produced from fatty acids, acid, to the growth medium to see whether these chemicals could glucose, and amino acids. It is a donor of single acetyl groups for rescue the Kan-sensitive phenotype of acx4-4, but found that they histone acetylation and is crucial for cell growth and pro- could not. We then searched for a strategy to increase cytosolic liferation (39, 51, 52). Because it is impermeable to membranes, acetyl-CoA in vivo. In the cytosol, ATP-citrate lyase (ACL) is the acetyl-CoA can be produced in different organelles, such as sole key enzyme for generating acetyl-CoA (50). Unlike in animals, peroxisomes, mitochondria, and plastids (5). In these organelles, in which ACL has a monomeric structure, plant ACL consists of acetyl-CoA must be condensed to other molecules before being two distinct subunits, ACLA (45 kDa) and ACLB (65 kDa), and the exported to the cytosol. However, whether organellar acetyl- heterooctomer holoenzyme is expected to have an A(4)B(4) con- CoA can affect nuclear DNA methylation in pants is unknown. figuration (50). We speculated that if ACL is overexpressed in In this study, we provide genetic and molecular evidence showing acx4-4 mutant, more acetyl-CoA will be produced, which may that a deficiency of short-acid β-oxidation can reduce histone compromise the Kan-sensitive phenotype of acx4-4.Weexpressed acetylation and increase DNA methylation at a subset of geno- ACLA1-GFP (AT1G10670) and ACLB2- (AT5G49460) mic loci in Arabidopsis, suggesting a close connection between under the control of 35S promoter in acx4-4. We obtained trans- organelle metabolites and nuclear epigenetic modifications. genic lines and tested their Kan sensitivity, but did not find any DNA methylation and histone modifications are primary difference between acx4-4 and acx4-4 expressing ACLA1-GFP epigenetic markers for the regulation of chromatin stability and or ACLB-MYC (SI Appendix,Fig.S14B and C), suggesting transcription (12, 53). In our genetic screen, we identified the that overexpressing one subunit of ACL does not affect the ROS4/IDM1, ROS5/IDM2, and MBD7 genes for their roles in silenced Pro35S:NPTII. DNA demethylation in the ProRD29A:LUC/C24 transgenic line We then crossed different transgenic lines of ACLA1-GFP (18, 22, 23). The current favored model suggests that MBD7 binds with those of ACLB2-MYC and obtained F1 seeds. Protein to genomic regions with high CG methylation density and physi- immunostaining using GFP and MYC antibodies and qPCR in- cally associates with ROS4/IDM1 to create acetylated H3K18 and dicated that the ACLA1-GFP/ACLB2-MYC F1 #12 and #6 lines H3K23 marks, which in turn facilitate recruitment of the DNA showed high levels of both ACLA1-GFP and ACLB2-MYC pro- demethylation enzyme ROS1 (1, 18). These studies suggest that tein and high transcript levels of the two genes (Fig. 8 A and B). histone acetylation is required for DNA demethylation enzymes to target The acetyl-CoA content in ACLA1-GFP/ACLB2-MYC F1 #12 genomic loci to prevent DNA hypermethylation and gene silencing. was three times that in the WT (Fig. 8C). Both lines #12 and #6 Using the same ProRD29A:LUC screening system, we identi- had the Kan-sensitive phenotype of acx4-4 rescued and expressed fied ACX4 and found that compared with WT, acx4 mu- higher levels of NPTII than acx4-4 (Fig. 8 D and E). Bisulfite tants have greatly reduced histone acetylation levels. In acx4

Wang et al. PNAS | May 21, 2019 | vol. 116 | no. 21 | 10581 Downloaded by guest on September 27, 2021 Fig. 7. Comparison of hyper-DMRs and reduced H3K18Ac at some loci in acx4, ros1-1, and WT. (A) DMRs and H3K18Ac levels in different loci among acx4-4 and WT. The DNA methylation levels in ros1-1 were included. (B) ChIP-qPCR confirmation of H3K18Ac levels in different DMRs between acx4-4 and WT. Values are means ± SE of three replicates in one experiment. Three independent experiments were done with similar results. Two-tailed Student’s t test, *P < 0.05, **P < 0.01, ***P < 0.001, n.s., no significant difference.

hyper-DMRs, the levels of H3K18Ac were reduced relative to mitochondrial acetyl-CoA synthetases (ACSS2 and ACSS1) are re- the WT (Fig. 6 D and E), suggesting a close connection between quired for acetate-induced histone acetylation under hypoxia in DNA hypermethylation and histone acetylation at these loci. A cells (56). Acetyl-CoA produced by the β-oxidation pathway comparison of DNA hypermethylated loci showed that in acx4, in peroxisomes may have a similar role in modulating nuclear histone hyper-DMRs partially overlap with those identified in the ros1 acetylation in plants, which in turn would affect DNA methylation. mutant (Fig. 4B), supporting the notion that ACX4 functions in We found that the levels of all three acetylated core histones ROS1-mediated DNA demethylation. The presence of other (H2B, H3, and H4) were down-regulated in acx4 compared with hyper-DMRs in the acx4 mutant that did not increase in the ros1 the WT (Fig. 5 B and C), demonstrating that ACX4 is important mutant (Figs. 2C and 4C)suggeststhatintheseloci,DNA for histone acetylation. However, along with affecting DNA demethylation is controlled by ROS1 paralogs, or histone acetylation methylation, histone acetylation usually has a generally positive directly affects DNA methylation, as seen in had6 mutants (54). effect on gene expression. The globally reduced histone acetylation The expression levels of hypermethylated genes, or genes located in acx4 mutants would also affect the expression of genes not near hyper-DMRs, were reduced in the acx4-4 and ros1-1 mu- related to DNA methylation, which merits future exploration. Since tants, and the acx4-4 ros1-1 double mutant had no additive effect acetyl-CoA can be produced in various metabolic pathways in plant on gene expression relative to the single mutants (Fig. 2D), cells (5) and other organisms (39), further studies are needed to suggesting the importance of ACX4 in preventing transcriptional demonstrate whether these other pathways are also involved in gene silencing by DNA demethylation. histone acetylation and DNA demethylation. We found that acx4-1, mfp2-2, and kat2-3 mutants have similar In plants, the β-oxidation of storage fatty acids is required to hyper-DMRs, suggesting that β-oxidation in peroxisomes is crucial establish seedlings during germination. This is coupled with the for modulating DNA methylation in nuclei. In plants and microbes, glyoxylate cycle to convert acetyl-CoA into succinate. Succinate β-oxidation occurs in peroxisomes, while in mammalian cells, it can be used for amino acid biosynthesis or can be converted into occurs mainly in mitochondria. In mammalian cells, the addition of sucrose by gluconeogenesis. Sucrose can then be catabolized into butyrate, a short-chain fatty acid that can be oxidized in mitochondria, pyruvate by glucose oxidation and converted to acetyl-CoA by induces ACL-mediated histone acetylation (55). Both cytosolic and the pyruvate dehydrogenase complex in mitochondria. Acetyl-CoA

10582 | www.pnas.org/cgi/doi/10.1073/pnas.1904143116 Wang et al. Downloaded by guest on September 27, 2021 cannot be reflected so quickly after addition of sucrose or other compounds in the medium. Another possibility is that active DNA demethylation is preferentially active during early development, such that once high levels of DNA methylation are established during seed development, they cannot be decreased by later addi- tion of the precursors for acetyl-CoA to the seedlings. We found that when acx4-4 expressedbothACLA1-GFPandACLB2-MYC, more acetyl-CoA was produced, and the Kan-sensitive phenotype and DNA hypermethylation were rescued, suggesting that acetyl- CoA plays a critical role in mediating DNA demethylation and antisilencing. The status of cellular metabolism is closely connected to epi- genetic modifications (59, 60). Nutrition is well known to affect development and diseases through metabolism that modulates epigenetic modifications in mammals (61). Further elucidation of the connections between peroxisomal metabolism and chro- matin regulation will enhance our understanding of epigenetic regulation and development. Methods Plant Growth Conditions and Mutant Screening. Seeds sterilized with 0.5% NaClO were sown onto MS medium plates containing 2% (wt/vol) sucrose and 0.8% (wt/vol) agar. After 3–4 d at 4 °C, the plates were transferred to a growth chamber at 22 °C under long-day conditions (23-h light/1-h dark). Seven-day-old seedlings were transferred to soil and cultivated in a green- house at 20 °C under long-day conditions (16-h light/8-h dark). The WT C24, ros1-1, ros4, rdr2, ddm1, acx4-4, mfp2-cas9-1, and mfp2-cas9- 2 mutants (C24 background) mentioned in this study all carried the RD29A-

Fig. 8. Overexpression of ACLA1 and ACLB2 together can rescue the Kan- LUC and Pro35S:NPTII transgenes (16, 18). The T-DNA lines ros1-4, SALK_000879 PLANT BIOLOGY sensitive phenotype of acx4-4.(A) Protein levels detected with different (acx4-1), SALK_065013 (acx4-3), SALK_098016 (mfp2-2), GK_787F01 (mfp2-10), antibodies in different F1 seedlings of acx4-4 expressing both ACLA1-GFP and SALK_024922 (kat2-3) were obtained from the Arabidopsis Stock Center. and ACLB2-MYC. The F1 seedlings were obtained by crossing the different The acx4-4 mutant was identified from an EMS-mutagenized WT pop- lines of acx4-4 expressing ACLA1-GFP with those expressing ACLB2-MYC. ulation as described previously (18). In brief, since WT seedlings can grow on ACTIN served as a loading control. (B) Relative expression levels of ACLA1 MS medium containing 50 mg/L Kan, mutants that did not grow well on this and ACLB2 in two F1 lines as determined by qPCR. (C) Relative levels of medium were selected and transferred to MS medium for recovery. Their Acetyl-CoA in F1 seedlings. Values are means ± SE (n = 3) of three in- Kan-sensitive phenotype was confirmed in the next generation. Putative mu- dependent biological repeats is shown. Two-tailed Student’s t test, **P < tants were crossed with a Columbia accession (gl1), and Kan-sensitive plants 0.01. (D) Comparison of Kan-resistant phenotypes among WT, acx4-4, and from the F2 seedlings were assayed by PCR (using primers Pro35S:NPTII-Fand two F1 lines on MS containing 25 mg/L Kan. (E) Relative transcriptional levels Pro35S:NPTII-R). Mutants (approximately 2,000) containing the T-DNA inser- of NPTII in WT, acx4-4, and two F1 lines. (F) Bisulfite sequencing analyses of tion were selected for mapping. DNA methylation for endogenous gene AT4G18250 in WT, acx4-4, and two For the complementation experiment, the genomic sequence of ACX4 − + acx4-4 lines expressing both ACLA1 and ACLB2 that were isolated from from 2,543 to 3,546 bp was cloned into the SacI and SmaI (XmaI) sites of F2 seedlings with Kan resistance. pCAMBIA1300; this sequence contains the 2,543-bp promoter, the coding region, and the 190-bp 3′ region. Using Agrobacterium tumefaciens GV3101, this construct was transformed into the acx4-4 mutant. Transgenic plants can enter the tricarboxylic acid cycle in mitochondria to produce were selected and analyzed for sensitivity to Kan. citrate, which can be exported to the cytosol and converted to acetyl-CoA by ACL (10). However, in later developmental stages, Individual Loci DNA Methylation Analysis. Genomic DNA was extracted using β the Qiagen DNeasy Plant Mini Kit. DNA methylation at individual loci was neither -oxidation nor the glyoxylate cycle is considered essential, analyzed using bisulfite sequencing. because photosynthesis can provide the carbohydrates and precur- For bisulfite sequencing, 500 ng of genomic DNA was treated with the EZ sors required for protein and nucleic acid biosynthesis. In our study, Methylation-Gold Kit (Zemo Research), following the manufacturer’s pro- we used Murashige and Skoog (MS) medium supplemented with tocol. Approximately 75 ng of bisulfite-treated DNA was used for PCR with 2% sucrose (sufficient for heterotrophic seedling growth) to isolate the specific primers listed in SI Appendix,TableS1. PCR products were cloned the acx4 mutant. Contrary to previous studies indicating that ex- into the pMD18-T vector (Takara Bio), and at least 15 independent clones from ogenous application of sucrose can largely rescue the growth defects each sample were sequenced for each region. of various mutants in the β-oxidation pathways (29, 32, 57), our study indicates that Kan-sensitive acx4 phenotypes could not be Real-Time qPCR. Total RNA was extracted from 7-d-old seedlings using TRIzol reagent (Invitrogen), and contaminating DNA was removed using RNase-free rescued by sucrose or by any other possible precursors for acetyl- DNase I (Takara Bio). Approximately 4 μg of mRNA was used for first-strand CoA biosynthesis, such as succinate, acetate, oxoglutarate, and citric cDNA synthesis using Moloney Murine Leukemia Virus Reverse Transcriptase acid. Peroxisomal β-oxidation not only functions in fatty acid ca- (Promega) in a 20-μLreactionvolume,and5μL of a 1:20 dilution of the cDNA tabolism, but also is required for the metabolism of hormones and reaction mixture was used as a template in a 20-μL PCR with SYBR Green amino acids (58). Similarly, exogenous application of hormones, Master Mix (Takara Bio) on a Step One Plus machine (Applied Biosystems) with including jasmonate, salicylate, and NAA, could not rescue the three technical replicates. UBC28 served as an internal control. RNA transcript phenotype of Kan sensitivity in acx4 (SI Appendix,Fig.S14A). These levels were determined by qPCR in a 20-μL reaction mixture, with TUB8 as an results suggest that exogenous addition of these components cannot internal control. The primers used for PCR are listed in SI Appendix,TableS1. bypass the β-oxidation defect in the acx4 mutant to rescue the si- Histochemical GUS Staining. The ACX4 promoter fragment (from −2,620 to lencing phenotype. We speculate that the metabolites provided by − β 1 bp) and the GUS-coding fragment were cloned into the pCAMBIA1391 -oxidation in cells might be used more efficiently than exogenous vector. The recombinant plasmid was introduced into A. tumefaciens strain sources for biosynthesis of the acetyl-CoA donor for histone acet- GV3101 and then transformed into C24 WT plants. Histochemical GUS staining ylation, or that active DNA demethylation is a slow process that was conducted on the T2 transgenic plants as described previously (62).

Wang et al. PNAS | May 21, 2019 | vol. 116 | no. 21 | 10583 Downloaded by guest on September 27, 2021 Histone Extraction and Immunoblotting. Ten-day-old seedlings were ground to cloned into the pCAMBIA1300 vector to generate NLS-GFP. The peroxisomal a fine powder in liquid nitrogen and suspended in nuclear isolation buffer marker GFP-CAT2 was obtained from a previous study (31). The transient expres-

(250 mM sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2,15mM sion assay in tobacco and image acquisition were also described previously (68). Pipes pH 6.8, and 0.8% Triton X-100). The mixture was centrifuged, and the pellet was resuspended in 0.4 M H2SO4 and incubated for at least 1 h on ice. Whole-Genome Bisulfite Sequencing and Data Analysis. For plants in the The preparation was centrifuged again, followed by the addition of a 20-fold C24 background, genomic DNA was isolated from 7-d-old seedlings using the volume of acetone to precipitate the histone protein. The preparation was Qiagen DNeasy Plant Mini Kit and sent to BGI (Shenzhen, China) for bisulfite kept at −20 °C overnight, after which the proteins were dissolved in 4 M urea treatment, library preparation, and sequencing using the Illumina HiSeq and then used for protein blot analysis with the following antibodies: anti- 2000 sequencing system. Raw reads obtained from sequencing were trimmed H2BAc (ab1759; Abcam), anti-H3 (17–10046; EMD Millipore), anti-H3Ac (7–615; using SolexaQA software (69). Clean reads were mapped to the Arabidopsis EMD Millipore), anti–Pan-H3Ac (ab47915; Abcam), anti-H3K9ac (07–352; EMD reference sequence (TAIR 10) using Bismark, allowing two mismatches. DMR Millipore), anti-H3K14ac (07–353; EMD Millipore), anti-H3K18ac (ab1191; identification was performed as described previously (23). Bins applied in Abcam), anti-H3K23ac (07-355; EMD Millipore), and anti-H4Ac (17–630; EMD this study were 100 bp from the reference genome. DMR identification used Millipore). Anti-H3 immunoblot was used as a loading control. the standard of an absolute difference in methylation levels in the CG, CHG, and CHH contexts of at least 0.4, 0.2, and 0.1, respectively, and the Benjamini– Immunofluorescence Assay. An immunofluorescence assay was performed Hochberg-corrected FDR was <0.01 (Fisher’s exact test). using 10-d-old seedlings. Nuclei were fixed in 4% paraformaldehyde and For plants in the Col-0 background, genomic DNA was extracted from 14-d- blocked with 3% BSA in PBS. The primary antibody H3Ac (7–615; EMD Mil- old seedlings using the DNeasy Plant Mini Kit and sent to BGI for bisulfite lipore) was diluted at 1:100, and H4Ac (17–630; EMD Millipore) was diluted treatment, library preparation, and sequencing using the Illumina HiSeq at 1:200, incubated overnight at 4 °C, and finally incubated with rabbit Alexa 4000 system. For library preparation, 5 μg of genomic DNA was sonicated Fluor 594 (A11012m; Invitrogen)-conjugated secondary antibodies for 2 h at into 100- to 300-bp fragments, which were end-repaired and ligated with 37 °C. Chromatin was counterstained with DAPI in Prolong Gold (Invitrogen). adenine at their 3′ ends. After ligation with Illumina DNA adaptors, unme- Images were acquired with a Leica TCS SP8 STED 3X confocal microscope at thylated cytosine residues were converted to uracils using the Qiagen Epi- 100× magnification. Tect Bisulfite Kit. For data analysis, clean reads were mapped to the TAIR 10 genome using ChIP Assays. ChIP assays were performed as described previously (63) using 10-d- Bismark, allowing up to two mismatches. DMR identification was performed as old seedlings and the following antibodies: anti-H2BAc (ab1759; Abcam), anti- described previously (70). In brief, the DNA methylation level in every 200-bp H3Ac (17–615; EMD Millipore), anti-H4Ac (17–630; EMD Millipore), anti-H3K9ac window at 50-bp intervals was compared between WT and mutant seedlings (07–352; EMD Millipore), anti-H3K14ac (07–353; EMD Millipore), anti-H3K18ac using Fisher’s exact test with P ≤ 0.05. FDRs were estimated using the Benjamini– (ab1191; Abcam), anti-H3K27ac (07-360; EMD Millipore), and anti-H3K23ac (07- Hochberg correction of calculated Fisher’s P values. Windows with seven or 355; EMD Millipore). ChIP products were eluted into 50 μL of Tris-EDTA buffer more differentially methylated cytosines (defined as C with P < 0.01 in Fisher’s anddilutedtoaratioof1:5,andthena2-μL aliquot of this dilution was used for exact test) and a >1.5-fold change in DNA methylation levels were retained and each qPCR reaction. The primers used for qPCR are listed in SI Appendix, Table S1. combined if the gap size was no more than 100 bp to generate DMRs. Finally, DMR length was adjusted to start at the first mC and end at the last mC. ChIP-Seq and Data Analysis. The ChIP DNA concentration was determined using a fluorescence-based quantification method (Qubit 3.0 system; Life Metabolite Measurement. Ten-day-old Arabidopsis seedlings grown on plates Technologies). High-throughput sequencing libraries were prepared using containing MS medium were used to measure acetyl-CoA, butyryl-CoA, and the NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina (E6240; New hexanoyl-CoA levels. Sample preparation and procedures for measuring England BioLabs) according to the manufacturer’s instructions. Sequencing metabolites were described previously (71). The experiments were per- was performed on an Illumina HiSEq 2500 instrument with single-end 50-bp formed with three biological replicates. reads. The reads were aligned to the TAIR10 Arabidopsis reference genome using Bowtie (64) with default parameters. Reads that perfectly and uniquely Data Availability. Sequence data referred to in this article can be found in the mapped to the genome were retained for further analysis. For each ChIP-seq, GenBank/EMBL databases under the following accession numbers: AT2G36490 for mapped reads were pooled from both replicates. Wiggle (WIG) format was ROS1, AT3G14980 for ROS4/IDM1, AT5G66750 for DDM1, AT4G11130 for generated using MACS program (65) and visualized using IGV (66). Acetylation- RDR2, AT3G51840 for ACX4, AT3G06860 for MFP2, AT2G33150 for KAT2, enriched peaks were identified using SICER (67) with a window size setting AT1G64230 for UBC28, and AT3G18780 for ACTIN2. Primary datasets for the of 200 bp, gap size of 200 bp, false discovery rate (FDR) ≤0.05, and fold whole-genome bisulfite sequences of Col-0, ros1-4, acx4-4, acx4-1, mfp2-2, change ≥1.5. The genome-wide occupancies, boxplots, and Pearson corre- and kat2-3 mutant plants have been deposited in the Gene Expression lations were performed with R packages. Omnibus (GEO) database (accession no. GSE98214), as have histone acetylation ChIP-seq data (accession no. GSE98214). Whole-genome bisulfite sequencing Localization of the mCherry-ACX4 Fusion Protein. The red fluorescent protein data of C24 WT, ros1-1,andros4 plants were obtained from the GEO database mCherry was fused to the N terminus of ACX4 under the control of the 35S (accession no. SRP042060) (23). promoter, and this construct was cloned into a modified pCAMBIA1300 vector. The vector carrying YFP-CD3-990 was generated in a previous study (30). NLS ACKNOWLEDGMENTS. This study was supported by the Natural Science was fused to the N terminus of GFP under the control of the 35S promoter and Foundation of China (Grant 31330041).

1. Qian W, et al. (2012) A histone acetyltransferase regulates active DNA demethylation 11. He XJ, Chen T, Zhu JK (2011) Regulation and function of DNA methylation in plants in Arabidopsis. Science 336:1445–1448. and animals. Cell Res 21:442–465. 2. Sutendra G, et al. (2014) A nuclear pyruvate dehydrogenase complex is important for 12. Law JA, Jacobsen SE (2010) Establishing, maintaining and modifying DNA methyl- the generation of acetyl-CoA and histone acetylation. Cell 158:84–97. ation patterns in plants and animals. Nat Rev Genet 11:204–220. 3. Tariq M, Paszkowski J (2004) DNA and histone methylation in plants. Trends Genet 20: 13. Zhang H, Lang Z, Zhu JK (2018) Dynamics and function of DNA methylation in plants. 244–251, and erratum (2005) 21:36. Nat Rev Mol Cell Biol 19:489–506. 4. Vogelauer M, Rubbi L, Lucas I, Brewer BJ, Grunstein M (2002) Histone acetylation 14. Wu SC, Zhang Y (2010) Active DNA demethylation: Many roads lead to Rome. Nat Rev regulates the time of replication origin firing. Mol Cell 10:1223–1233. Mol Cell Biol 11:607–620. 5. Shen Y, Wei W, Zhou DX (2015) Histone acetylation enzymes coordinate metabolism 15. Gehring M, et al. (2006) DEMETER DNA glycosylase establishes MEDEA polycomb and gene expression. Trends Plant Sci 20:614–621. gene self-imprinting by allele-specific demethylation. Cell 124:495–506. 6. Sivanand S, Viney I, Wellen KE (2018) Spatiotemporal control of acetyl-CoA metab- 16. Gong Z, et al. (2002) ROS1, a repressor of transcriptional gene silencing in Arabi- olism in chromatin regulation. Trends Biochem Sci 43:61–74. dopsis, encodes a DNA glycosylase/lyase. Cell 111:803–814. 7. Wong BW, et al. (2017) The role of fatty acid β-oxidation in lymphangiogenesis. 17. Parrilla-Doblas JT, Ponferrada-Marín MI, Roldán-Arjona T, Ariza RR (2013) Early steps Nature 542:49–54. of active DNA demethylation initiated by ROS1 glycosylase require three putative 8. Nagaraj R, et al. (2017) Nuclear localization of mitochondrial TCA cycle enzymes as a helix-invading residues. Nucleic Acids Res 41:8654–8664. critical step in mammalian zygotic genome activation. Cell 168:210–223.e11. 18. Li X, et al. (2012) Antisilencing role of the RNA-directed DNA methylation pathway 9. Chen C, et al. (2017) Cytosolic acetyl-CoA promotes histone acetylation predominantly and a histone acetyltransferase in Arabidopsis. Proc Natl Acad Sci USA 109:11425– at H3K27 in Arabidopsis. Nat Plants 3:814–824. 11430. 10. Fatland BL, et al. (2002) Molecular characterization of a heteromeric ATP-citrate lyase 19. Duan CG, et al. (2017) A pair of transposon-derived proteins function in a histone that generates cytosolic acetyl-coenzyme A in Arabidopsis. Plant Physiol 130:740–756. acetyltransferase complex for active DNA demethylation. Cell Res 27:226–240.

10584 | www.pnas.org/cgi/doi/10.1073/pnas.1904143116 Wang et al. Downloaded by guest on September 27, 2021 20. Lang Z, et al. (2015) The methyl-CpG-binding protein MBD7 facilitates active DNA 45. Wang ZP, et al. (2015) Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently demethylation to limit DNA hyper-methylation and transcriptional gene silencing. generates homozygous mutants for multiple target genes in Arabidopsis in a single Mol Cell 57:971–983. generation. Genome Biol 16:144. 21. Qian W, et al. (2014) Regulation of active DNA demethylation by an α-crystallin do- 46. Zhang J, et al. (2016) The second subunit of DNA polymerase delta is required for main protein in Arabidopsis. Mol Cell 55:361–371. genomic stability and epigenetic regulation. Plant Physiol 171:1192–1208. 22. Wang C, et al. (2015) Methyl-CpG-binding domain protein MBD7 is required for active 47. Steimer A, et al. (2000) Endogenous targets of transcriptional gene silencing in – DNA demethylation in Arabidopsis. Plant Physiol 167:905 914. Arabidopsis. Plant Cell 12:1165–1178. 23. Zhao Y, et al. (2014) REPRESSOR OF SILENCING5 encodes a member of the small heat 48. Wasternack C, Song S (2017) Jasmonates: Biosynthesis, metabolism, and signaling by shock and is required for DNA demethylation in Arabidopsis. Plant Cell proteins activating and repressing transcription. J Exp Bot 68:1303–1321. – 26:2660 2675. 49. Bussell JD, Reichelt M, Wiszniewski AAG, Gershenzon J, Smith SM (2014) Peroxisomal 24. Duan CG, et al. (2015) MET18 connects the cytosolic iron-sulfur cluster assembly ATP-binding cassette transporter COMATOSE and the multifunctional protein ab- pathway to active DNA demethylation in Arabidopsis. PLoS Genet 11:e1005559. normal INFLORESCENCE MERISTEM are required for the production of benzoylated 25. Gong Z (2019) A SUVH-DNAJ/SDJ protein complex activates the expression of metabolites in Arabidopsis seeds. Plant Physiol 164:48–54. promoter-methylated genes in Arabidopsis. J Integr Plant Biol 61:90–92. 50. Fatland BL, Nikolau BJ, Wurtele ES (2005) Reverse genetic characterization of cytosolic 26. Harris CJ, et al. (2018) A DNA methylation reader complex that enhances gene acetyl-CoA generation by ATP-citrate lyase in Arabidopsis. Plant Cell 17:182–203. transcription. Science 362:1182–1186. 51. Cai L, Sutter BM, Li B, Tu BP (2011) Acetyl-CoA induces cell growth and proliferation 27. Xiao X, et al. (2019) A group of SUVH methyl-DNA binding proteins regulate ex- by promoting the acetylation of histones at growth genes. Mol Cell 42:426–437. pression of the DNA demethylase ROS1 in Arabidopsis. J Integr Plant Biol 61:110–119. 52. Öst A, Pospisilik JA (2015) Epigenetic modulation of metabolic decisions. Curr Opin 28. Zhao QQ, Lin RN, Li L, Chen S, He XJ (2019) A methylated-DNA-binding complex re- – quired for plant development mediates transcriptional activation of promoter Cell Biol 33:88 94. methylated genes. J Integr Plant Biol 61:120–139. 53. Jacob Y, et al. (2010) Regulation of heterochromatic DNA replication by histone – 29. Adham AR, Zolman BK, Millius A, Bartel B (2005) Mutations in Arabidopsis acyl-CoA H3 lysine 27 methyltransferases. Nature 466:987 991. oxidase genes reveal distinct and overlapping roles in beta-oxidation. Plant J 41:859– 54. Earley KW, et al. (2010) Mechanisms of HDA6-mediated rRNA gene silencing: Suppression of 874. intergenic Pol II transcription and differential effects on maintenance versus siRNA-directed 30. Nelson BK, Cai X, Nebenfuhr A (2007) A multicolored set of in vivo organelle markers cytosine methylation. Genes Dev 24:1119–1132. for co-localization studies in Arabidopsis and other plants. Plant J 51:1126–1136. 55. Donohoe DR, et al. (2012) The Warburg effect dictates the mechanism of butyrate- 31. Li J, et al. (2015) A chaperone function of NO CATALASE ACTIVITY1 is required to mediated histone acetylation and cell proliferation. Mol Cell 48:612–626. maintain catalase activity and for multiple stress responses in Arabidopsis. Plant Cell 56. Gao X, et al. (2016) Acetate functions as an epigenetic metabolite to promote lipid 27:908–925. synthesis under hypoxia. Nat Commun 7:11960. 32. Hayashi H, et al. (1999) A novel acyl-CoA oxidase that can oxidize short-chain acyl-CoA 57. Eastmond PJ, Graham IA (2000) The multifunctional protein AtMFP2 is co-ordinately in plant peroxisomes. J Biol Chem 274:12715–12721. expressed with other genes of fatty acid beta-oxidation during seed germination in 33. Lei M, et al. (2015) Regulatory link between DNA methylation and active demethy- Arabidopsis thaliana (L.) Heynh. Biochem Soc Trans 28:95–99. lation in Arabidopsis. Proc Natl Acad Sci USA 112:3553–3557. 58. Reumann S, Ma C, Lemke S, Babujee L (2004) AraPerox: A database of putative 34. Kindl H (1993) Fatty acid degradation in plant peroxisomes: Function and biosynthesis Arabidopsis proteins from plant peroxisomes. Plant Physiol 136:2587–2608. – of the enzymes involved. Biochimie 75:225 230. 59. Keating ST, El-Osta A (2015) Epigenetics and metabolism. Circ Res 116:715–736. PLANT BIOLOGY 35. Pan R, Liu J, Hu J (December 21, 2018) Peroxisomes in plant reproduction and seed- 60. Meng J, et al. (2018) METHIONINE ADENOSYLTRANSFERASE4 mediates DNA and related development. J Integr Plant Biol10.1111/jipb.12765. histone methylation. Plant Physiol 177:652–670. 36. Shockey JM, Fulda MS, Browse JA (2002) Arabidopsis contains nine long-chain acyl- 61. Carrer A, Wellen KE (2015) Metabolism and epigenetics: A link cancer cells exploit. coenzyme a synthetase genes that participate in fatty acid and glycerolipid metab- Curr Opin Biotechnol 34:23–29. – olism. Plant Physiol 129:1710 1722. 62. Xia R, et al. (2006) ROR1/RPA2A, a putative replication protein A2, functions in epi- 37. Graham IA, Eastmond PJ (2002) Pathways of straight and branched chain fatty acid genetic gene silencing and in regulation of meristem development in Arabidopsis. catabolism in higher plants. Prog Lipid Res 41:156–181. Plant Cell 18:85–103. 38. Preisig-Müller R, Gühnemann-Schäfer K, Kindl H (1994) Domains of the tetrafunc- 63. Saleh A, Alvarez-Venegas R, Avramova Z (2008) An efficient chromatin immunopre- tional protein acting in glyoxysomal fatty acid beta-oxidation: Demonstration of cipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants. epimerase and activities on a peptide lacking hydratase activity. J Biol Nat Protoc 3:1018–1025. Chem 269:20475–20481. 64. Langmead B, Trapnell C, Pop M, Salzberg SL (2009) Ultrafast and memory-efficient 39. Pietrocola F, Galluzzi L, Bravo-San Pedro JM, Madeo F, Kroemer G (2015) Acetyl co- alignment of short DNA sequences to the . Genome Biol 10:R25. enzyme A: A central metabolite and second messenger. Cell Metab 21:805–821. 65. Zhang Y, et al. (2008) Model-based analysis of ChIP-seq (MACS). Genome Biol 9:R137. 40. Rylott EL, et al. (2006) The Arabidopsis thaliana multifunctional protein gene (MFP2) 66. Robinson JT, et al. (2011) Integrative genomics viewer. Nat Biotechnol 29:24–26. of peroxisomal beta-oxidation is essential for seedling establishment. Plant J 45:930– 67. Zang C, et al. (2009) A clustering approach for identification of enriched domains 941. – 41. Richmond TA, Bleecker AB (1999) A defect in beta-oxidation causes abnormal in- from histone modification ChIP-seq data. Bioinformatics 25:1952 1958. florescence development in Arabidopsis. Plant Cell 11:1911–1924. 68. Liu Q, et al. (2010) DNA replication factor C1 mediates genomic stability and tran- – 42. Castillo MC, León J (2008) Expression of the beta-oxidation gene 3-ketoacyl-CoA scriptional gene silencing in Arabidopsis. Plant Cell 22:2336 2352. thiolase 2 (KAT2) is required for the timely onset of natural and dark-induced leaf 69. Cox MP, Peterson DA, Biggs PJ (2010) SolexaQA: At-a-glance quality assessment of senescence in Arabidopsis. J Exp Bot 59:2171–2179. Illumina second-generation sequencing data. BMC Bioinformatics 11:485. 43. Germain V, et al. (2001) Requirement for 3-ketoacyl-CoA thiolase-2 in peroxisome 70. Zhang H, et al. (2013) DTF1 is a core component of RNA-directed DNA methylation development, fatty acid beta-oxidation and breakdown of triacylglycerol in lipid and may assist in the recruitment of Pol IV. Proc Natl Acad Sci USA 110:8290–8295. bodies of Arabidopsis seedlings. Plant J 28:1–12. 71. Purves RW, Ambrose SJ, Clark SM, Stout JM, Page JE (2015) Separation of isomeric 44. Wiszniewski AAG, Bussell JD, Long RL, Smith SM (2014) Knockout of the two evolu- short-chain acyl-CoAs in plant matrices using ultra-performance liquid chromatography tionarily conserved peroxisomal 3-ketoacyl-CoA thiolases in Arabidopsis recapitulates coupled with tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life the abnormal inflorescence meristem 1 phenotype. J Exp Bot 65:6723–6733. Sci 980:1–7.

Wang et al. PNAS | May 21, 2019 | vol. 116 | no. 21 | 10585 Downloaded by guest on September 27, 2021