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Department of Biological Sciences, Sungkyunkwan University, Suwon 16419, Re-public of Korea.*Corresponding author. Email: pjseo1@skku.edu

Lee, Park, Seo, Sci. Signal. 10, eaan0316 (2017) 28 November 2017

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Arabidopsis ATXR2 deposits H3K36me3at the promoters of LBD genes to facilitatecellular dedifferentiationKyounghee Lee, Ok-Sun Park, Pil Joon Seo*

Cellular dedifferentiation, the transition of differentiated somatic cells to pluripotent stem cells, ensures developmen-tal plasticity and contributes to wound healing in plants. Wounding induces cells to form a mass of unorganized plu-ripotent cells called callus at the wound site. Explanted cells can also form callus tissues in vitro. Reversible cellulardifferentiation-dedifferentiation processes in higher eukaryotes are controlledmainly by chromatinmodifications.Wedemonstrate that ARABIDOPSIS TRITHORAX-RELATED2 (ATXR2), a histone lysinemethyltransferase that promotes theaccumulation of histone H3 proteins that are trimethylated on lysine 36 (H3K36me3) during callus formation, pro-motes early stages of cellular dedifferentiation through activation of LATERAL ORGAN BOUNDARIES DOMAIN (LBD)genes. The LBD genes of Arabidopsis thaliana are activated during cellular dedifferentiation to enhance the formationof callus. Leaf explants from Arabidopsis atxr2 mutants exhibited a reduced ability to form callus and a substantialreduction in LBD gene expression. ATXR2 bound to the promoters of LBD genes and was required for the depositionofH3K36me3at thesepromoters. ATXR2was recruited to LBDpromoters by the transcription factorsAUXINRESPONSEFACTOR 7 (ARF7) and ARF19. Leaf explants from arf7-1arf19-2 double mutants were defective in callus formation andshowed reducedH3K36me3 accumulation at LBD promoters. Genetic analysis provided further support that ARF7 andARF19were required for the ability of ATXR2 topromote the expressionof LBDgenes. These observations indicate thatthe ATXR2-ARF-LBD axis is key for the epigenetic regulation of callus formation in Arabidopsis.

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INTRODUCTIONPlant somatic cells have the remarkable capability of dedifferentiating toformunorganizedmasses of pluripotent cells called callus under specificenvironmental conditions, at wound sites, and when explanted intoculture (1). This plasticity of cellular differentiation allows plants tooptimize their growth and development for specific environmentalconditions (2). Callus formation is an early event in cellular dedif-ferentiation, and intricate genetic programs underlie callus formationin Arabidopsis thaliana (3). For example, the hormone auxin and itsdownstream signaling components play key roles in callus forma-tion (4). The transcription factors AUXIN RESPONSE FACTOR 7(ARF7) andARF19 activate several members of the LATERALORGANBOUNDARIES DOMAIN (LBD) gene family to stimulate callus forma-tion (5). The ARF proteins bind directly to the LBD16 and LBD29 pro-moters to stimulate their expression and also indirectly promote LBD17and LBD18 expression by ARF-activated LBD16 and LBD29 (6). Thesefour LBDzinc finger transcription factors facilitate cell cycle progressionby stimulating the expression of E2 PROMOTER BINDING FACTOR a(E2Fa) genes that promote DNA replication and cell division (7).

Chromatin structure is dynamically regulated by covalent chemicalmodifications at nucleosomes, including histone methylation, acetyla-tion, phosphorylation, and ubiquitination and DNA methylation (8).Chromatin context influences the accessibility of transcriptional regu-lators and thereby gene expression (9), facilitating stable but reversiblepatterns of gene expression. Because of the large number of histonemethyltransferases in many plant genomes (10), the histone methyla-tion process has been extensively investigated (11, 12). The effects ofhistonemethylation are variable depending on the position of themod-ified residue and the number of methyl groups added (13). In general,methylation of histone H3 lysine 4 (H3K4) and H3K36 is associated

with transcriptional activation (14), whereas methylation of H3K9,H3K27, andH4K20 is characteristic of repressive epigeneticmarks (15).

SET domain proteins—named after the three founding membersSuVar (3–9), E(z), and Trx from Drosophila melanogaster—have po-tential lysine methyltransferase activity (16) and are grouped into fourmajor classes: (i) enhancer of zeste [E(z)] homologs; (ii) absent, small, orhomeotic disks 1 (Ash1) homologs and related proteins; (iii) trithorax(Trx) homologs and related proteins; and (iv) suppressor of variegation[Su(var)] homologs and relatedproteins (17).H3K4methylation ismainlycatalyzed in yeast (Saccharomyces cerevisiae) by the Set1 class and inD. melanogaster by the Trx class (18–20). In Arabidopsis, 10 proteinshave been identified as putative methyltransferases that not onlydeposit H3K4me3 but also deposit H3K36me3 at a lesser frequency(17, 21–23), based on the structural similarity to SETdomains of Set1 andTrx and the analysis of biochemical activities (21). These 10 proteins areARABIDOPSIS TRITHORAX 1-5 (ATX1-5), ARABIDOPSISTRITHORAX-RELATED 1-4 (ATXR1-4), and ATXR7 (24).

The trithorax group (TrxG) proteins, which have the ability to cat-alyze active mark establishment and play roles in the maintenance ofgene transcription, are associated with various physiological processesin Arabidopsis. ATX1 (also known as SDG27), ATX2 (also known asSDG30), and ATXR7 (also known as SDG25) are involved in the acti-vation of FLOWERING LOCUS C (FLC) expression, which suppressesthe floral transition (11). Mutations in theseATX genes suppress in partFRIGIDA-induced flowering delay with decreased FLC expression andreduced H3K4me3 deposition (24–26). Furthermore, ATXR7 furthercontributes to FLC activation by depositing both H3K4me3 andH3K36me3 marks at the locus (24, 27). In addition, ATX1 is involvedin dehydration stress responses (28).ATX1-deficientmutants displayedenlarged stomatal openings, increased water loss, and decreased dehy-dration tolerance with decreased abscisic acid (ABA) accumulation(28). Notably, ATX1 binds to the promoter of the rate-limiting ABAbiosynthetic gene 9-CIS-EPOXYCAROTENOID DIOXYGENASE 3

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(NCED3) and activates its expression with a substantial increase ofH3K4me3 accumulation during dehydration (28). Moreover, ATX1 alsoactivates ABA-independent genes, such as COLD-REGULATED 15A(COR15A),ALCOHOLDEHYDROGENASE 1 (ADH1), andABSCISICACID RESPONSIVE ELEMENTS-BINDING FACTOR 2 (ABF2), inte-grating ABA-dependent and ABA-independent pathways (28). ATXR3is required for global H3K4me3 deposition (29). Consistent withthis, atxr3 mutations affect the expression of many genes and resultin pleiotropic phenotypes including dwarfism, curly leaves, earlyflowering, terminal flowers, and sterility (29, 30). These observationsdemonstrate that diverse aspects of plant development are under thecontrol of TrxG proteins.

Accumulating evidence indicates that epigenetic regulation furtherensures a proper cellular dedifferentiation process by allowing massivereprogramming of gene expression (31–33). Here, we report that, in ad-dition to its global impact on genome-wide H3K36me3 and H3K4me3accumulation, ATXR2 binds to LBD promoters and increasesH3K36me3accumulation at these promoters to stimulate callus induction. ARF7and ARF19 recruit ATXR2 to the LBD promoters and facilitate properhistone modification at these loci. These observations indicate that co-ordination of multiple auxin-related factors underlies callus formationand contributes to the massive transcriptional changes required for arobust transition of cell fate.

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RESULTSCallus formation is reduced in atxr2 mutantsGenome-wide accumulation of H3K4me3 and H3K36me3, epigene-tic marks that are catalyzed by ATXs and ATXRs and characteristicof open chromatin, increases substantially during the leaf-to-callustransition in Arabidopsis (33). Because open chromatin formation isnecessary during callus formation (34), we investigated epigeneticmod-ifications during callus formation. To identify the enzyme(s) regulatingcell fate change through active mark deposition, we analyzed the callusformation capabilities of several atxrmutants using leaf explants oncallus-inducing medium (CIM) (fig. S1).

Among themutants examined, the transferred DNA (T-DNA) inser-tionalmutant atxr2-1 exhibited reduced callus formation (Fig. 1A and fig.S2). Fresh weight measurements revealed that callus formation was sup-pressed by 25% in leaf explants from atxr2-1mutants comparedwith leafexplants from wild-type plants (Fig. 1B). To validate these observations,we obtained the additional T-DNA insertional mutant allele atxr2-3 (fig.S2) and measured callus formation rate (Fig. 1, A and B). The atxr2-3mutant leaf explants also showed reduced callus formation upon callusinduction, similar to atxr2-1 (Fig. 1, A and B). Only atxr2-1was used insubsequent analyses based on these phenotypic similarities.

To provide further support for the role of ATXR2 in callus forma-tion, we generated transgenic plants overexpressing ATXR2 (35S:ATXR2-MYC) and analyzed their callus formation capability (Fig. 1Aand fig. S3). Leaf explants from 35S:ATXR2-MYC transgenic plants ex-hibited more robust callus formation than wild-type leaf explants (Fig.1A and fig. S3). Freshweightmeasurements further supported the rapidprogression of cellular dedifferentiation and proliferation in 35S:ATXR2-MYC transgenic leaf explants (Fig. 1B and fig. S3). Thus,ATXR2 promoted callus formation.

ATXR2 activates LBD gene expressionTo obtain clues about the signaling network involving ATXR2, we ana-lyzed wild-type, atxr2-1, and 35S:ATXR2-MYC calli for the expression

Lee, Park, Seo, Sci. Signal. 10, eaan0316 (2017) 28 November 2017

of genes involved in callus formation, including ARABIDOPSIS RE-SPONSE REGULATOR 1 (ARR1),ARR21, BABY BOOM (BBM), EN-HANCEROF SHOOTREGENERATION 1 (ESR1), ESR2, LBD16, LBD17,LBD18, LBD29, LEAFY COTYLEDON 1 (LEC1), LEC2, WOUND-INDUCEDDEDIFFERENTIATION 1 (WIND1),WIND2, andWIND3.Reverse transcription quantitative real-time polymerase chain reaction(RT-qPCR) analysis showed that accumulation of LBD transcripts wasrepressed in atxr2-1 mutant calli and greatly increased in 35S:ATXR2-MYC calli relative to wild-type calli (Fig. 2A). Reduced LBD expressionwas also observed in atxr2-3 mutant calli (fig. S4). Because LBDs wereexpressed mainly in the callus tissues compared to differentiated leaftissues (fig. S5), it was possible that reduced LBD expression in atxr2-1 mutant calli may be attributable to reduced callus size. However, weharvested the plant materials at 7 DAC (days after explant onto CIM),when callus formation is starting, and thus, no clear difference in callussize was observed between mutants and wild-type plants (fig. S6). Fur-thermore, expression of other genes involved in callus formation wasnot altered in ATXR2-overexpressing (35S:ATXR2-MYC) plants (Fig.2A). In particular, expression of the callus-specific marker WUSCHEL-RELATED HOMEOBOX 5 (WOX5) was also unchanged in atxr2-1 (fig. S7), indicating specific regulation of LBDs by ATXR2.

In addition, we also analyzed the expression kinetics of LBD genesduring callus formation. The LBD genes were rapidly induced upon plac-ing leaves onCIMand showed the greatest expression during the periodof 4 to 7 DAC (Fig. 2B). However, LBD gene induction was reduced inatxr2 mutant calli (Fig. 2B and fig. S4), suggesting that ATXR2 pro-motes LBD expression during the process of callus formation.

On the basis of its similarity to other known histone-modifying en-zymes, ATXR2 is predicted to bind to specific target promoters and

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Fig. 1. Callus formation in leaf explants from atxr2 mutant and ATXR2-overexpressing plants. (A) Callus formation in leaves explanted from young wild-type (Col-0), atxr2-1,atxr2-3, and35S:ATXR2-MYCplants. Scale bar, 5mm.n>30plants ofeach genotype. (B) Fresh weight of calli fromwild-type (Col-0), atxr2-1, atxr2-3, and 35S:ATXR2-MYC plants. Bars indicate the SE of the mean. *P < 0.05 (Student’s t test). n = 30calli of each genotype.

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modify the histones at those loci (24, 25). To determinewhetherATXR2binds to LBD gene promoters, we performed chromatin immuno-precipitation (ChIP) using 35S:ATXR2-MYC transgenic calli. Total pro-tein extracts from control and 35S:ATXR2-MYC transgenic calli wereimmunoprecipitated with an antibody specific for MYC, and theDNA bound to MYC-tagged ATXR2 proteins was analyzed by qPCRassays using primers specific for several regions of LBD promoters (Fig.2C). ATXR2 bound mainly to the LBD16 and LBD29 promoters (Fig.2D). Control ChIP in the absence of theMYC-specific antibody did notenrich LBD16 and LBD29 fragments (fig. S8). To confirm the function-ality of the ATXR2-MYC fusion for LBD activation, we conducted tran-sient expression assays using Arabidopsis protoplasts, which are cellsisolated from leaves and treated to remove the cell wall. The core cis-elements of the LBD promoters were fused in the recombinant reporter

Lee, Park, Seo, Sci. Signal. 10, eaan0316 (2017) 28 November 2017

plasmid, and the construct was expressed with effector constructsexpressing ATXR2 in Arabidopsis protoplasts isolated from wild-typeand atxr2-1 leaves. ATXR2 bound to and transcriptionally activatedtheLBD genes in protoplasts frombothwild-type plants and atxr2-1mu-tants (fig. S9). In addition, the ATXR2-MYC fusion complemented theatxr2-1mutation (fig. S9).

Two other LBD genes, LBD17 and LBD18, were not targeted byATXR2 (Fig. 2D). However, expression of these genes may be influ-enced by LBD16 and LBD29 (6), suggesting that ATXR2 could influ-ence the expression of LBD17 and LBD18 indirectly through its actionon LBD16 and LBD29. These observations account for the reducedexpression of all examined LBDs in atxr2-1 mutant calli. AnotherLBD gene examined as a negative control (LBD1) was not targetedby ATXR2 (fig. S10). These results suggest that ATXR2 specifically

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Fig. 2. Transcriptional activation of LBD genes by ATXR2. (A) Ex-pression profiling of the indicated genes involved in callus formation in35S:ATXR2-MYC and atxr2-1 calli by reverse transcription quantitativereal-time polymerase chain reaction (RT-qPCR) and normalized to eachgene’s expression in wild-type (Col-0) plants. Bars indicate the SE of themean. *P < 0.05 (Student’s t test). n = 3 biological replicates. (B) Kineticsof LBD expression in Col-0 and atxr2-1 leaf explants during callus for-mation at 2, 4, and 7 days after culturing on callus-inducing medium(DAC). Expression of each gene was normalized to the expression ofthat gene at time 2 in Col-0. *P < 0.05 (Student’s t test). n = 3 biologicalreplicates. (C) Promoter analysis of LBD genes. The regions labeled A toMwere identified as putative binding sites for ATXR2. Black lines abovethe labels indicate the regions that were amplified by PCR after chro-

matin immunoprecipitation (ChIP). Black boxes indicate exons. (D) Enrichment of ATXR2 on promoter regions as determined by ChIP-qPCR. Values for qPCR analysis were normal-ized to eIF4a in wild-type empty vector (EV) control plants. Bars indicate the SE of the mean. *P < 0.05 (Student’s t test). n = 3 biological replicates.

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activates a subset of LBD genes by binding directly to the promotersand possibly modifying their chromatin context.

ATXR2 establishes H3K36me3 at LBD promotersSequence analysis places ATXR2 in the Trx family of proteins that pref-erentially and directly conferH3K4me3 accumulation (17). To examinethis possibility, we examined the global accumulation of H3K4me3 inwild-type and atxr2-1 calli. We subjected proteins isolated from calli toimmunoblot analysis with an antibody recognizing H3K4me3. Con-trary to our expectations, H3K4me3 accumulation was only slightlyaltered in atxr2-1 mutant calli (fig. S11). We noted that ATXR2, likeATXR1 and ATXR4, has an interrupted SET domain (17), and thushypothesized that ATXR2 might have biochemical activities that differfrom other SET domain proteins in establishing epigenetic marks. Toexamine this hypothesis, we examined the accumulation of H3K9me3,H3K27me3, and H3K36me3 in wild-type and atxr2-1 calli. Notably,global H3K36me3 accumulation was reduced in atxr2-1 mutant calliat 4 DAC (Fig. 3A), whereas H3K9me3 and H3K27me3 accumulationwas unchanged or increased (fig. S11). The biochemical function ofATXR2 in H3K36me3 deposition was further supported by our obser-vation of increased H3K36me3 accumulation in 35S:ATXR2-MYCtransgenic calli (fig. S12). These results suggest that ATXR2 primarilypromotes global H3K36me3 deposition, and to a lesser degreeH3K4me3, during callus formation.

To assess the importance of ATXR2 in H3K36me3 deposition atLBD loci, we performed ChIP analysis with an antibody specific forH3K36me3 using wild-type, atxr2-1, and 35S:ATXR2-MYC calli. qPCRanalysis showed that H3K36me3 accumulation at the LBD16 andLBD29 promoters was impaired in atxr2-1 mutant calli compared towild-type calli (Fig. 3B) at 2 to 4DAC (Fig. 3C). In contrast, this histonemodification was enhanced in 35S:ATXR2-MYC transgenic calli (Fig.3B). H3K36me3 accumulation at the LBD17 and LBD18 promoterswas unchanged in atxr2-1 and 35S:ATXR2-MYC transgenic calli (Fig.3B), which is consistentwith the binding specificity ofATXR2 (Fig. 2D).In addition, H3K4me3 accumulation at the LBD promoters was un-changed in atxr2-1 mutant calli (fig. S13). These results indicate thatATXR2 promotes H3K36me3 deposition at the LBD loci to ensure ef-ficient callus formation.

ATXR2 interacts with ARFsWe have shown that ATXR2 is recruited to the LBD promoters anddirectly catalyzes histonemodification at cognate regions. One questionthis raises is how ATXR2 recognizes its target promoters. We hypothe-sized thatATXR2may be guided to the LBD promoters by transcriptionfactors that directly bind to nearby regions. ARF7 and ARF19 werestrong candidates, because they bind to the promoters of the LBD16and LBD29 genes (6) and because arf7 arf19 double mutants exhibitabnormal callus formation (5). We therefore decided to test the po-tential physical interactions between ATXR2 and ARF7 or ARF19.

We carried out yeast two-hybrid (Y2H) assays by coexpressingATXR2 fused in-frame to the 3′ end of theGAL4DNAbinding domain(DBD) in yeast cells alongwith theGAL4 activation domain (AD) aloneor fused to ARF7 or ARF19. Cell growth on selective medium showedthatATXR2 interactedwith full-lengthARF19 but not full-lengthARF7(Fig. 4A). We also constructed a series of deletion forms of ATXR2,ARF7, and ARF19 (Fig. 4B). Y2H analysis revealed that the N-terminalregion of ARF19 containing the B3 domain was responsible for the in-teraction with ATXR2 (Fig. 4C). In addition, the middle region ofATXR2, which has the SET domain, associated with both ARF7 and

Lee, Park, Seo, Sci. Signal. 10, eaan0316 (2017) 28 November 2017

ARF19 (Fig. 4D), suggesting that ATXR2 interacts with both theseARFs depending on structural compatibility in yeast cells.

To provide evidence for the interaction of ATXR2 andARFs in vivo,we performed bimolecular fluorescence complementation usingArabidopsis protoplasts. The ATXR2 cDNA sequence was fused in-frame to the 5′ end of a gene sequence encoding the N-terminal halfof yellow fluorescent protein (nYFP), and each ARF gene was fusedin-frame to the 5′ end of a sequence encoding the C-terminal half ofYFP (cYFP). The fusion constructs were then transiently coexpressedin Arabidopsis protoplasts. Yellow fluorescence was visualized exclu-sively in the nucleus in all ATXR2-ARF combinations (Fig. 4E and figs.S14 and S15). Binding of ATXR2 was specific to ARF7 and ARF19, be-cause other ARFs did not associate with ATXR2 (fig. S16). To quantifythe physical interaction betweenATXR2 and these twoARFs, we used a

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Fig. 3. ATXR2 mediates H3K36me3 accumulation at LBD loci during callus for-mation. (A) Global accumulation of H3K36me3 in wild-type (Col-0) and atxr2-1 leafexplants during callus formation. Bands from three independent blots were quantified(right). n =3 biological replicates. (B) H3K36me3 accumulation at the LBD loci indicatedin Fig. 2C. Bars indicate the SE of the mean. *P < 0.05 (Student’s t test). n = 3 biologicalreplicates. (C) Kinetics of H3K36me3 accumulation at the LBD16 and LBD29 promotersduring callus formation in Col-0 and atxr2-1 plants. Bars indicate the SE of themean.*P < 0.05 (Student’s t test). n = 3 biological replicates.

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split Luciferase (Luc) assay. ATXR2 was fused to the amino portionof Luc (nLuc), and ARFs were fused with carboxy portion of Luc(cLuc). Coexpression of ATXR2-nLuc and ARF-cLuc constructs in

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Arabidopsis protoplasts resulted in enhanced Luc activity (Fig. 4F).These results indicate that ATXR2 forms a complex with ARF7 andARF19 in plant cells.

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Fig. 4. ATXR2 interacts with ARFs. (A) Yeast two-hybrid assaysusing ATXR2 fused to the Gal4 DNA binding domain (DBD) andARFs fused to the Gal4 transcriptional activation domain (AD). Full-lengthGAL4wasusedasapositive control. LW,dropoutmediumwith-out leucine (L) or tryptophan (W); LWAH, dropout medium without L,W, adenine (A), or histidine (H). n = 3 biological replicates. (B) Deletionconstructs of ATXR2 and ARFs. Numbers indicate residue positions;black boxes indicate the ARF B3 domain; white boxes indicate the

low-complexity region; gray boxes indicate the coiled-coil region of ARF7; hatched boxes indicate the SET domain of ATXR2. (C) Interactions of ATXR2 with ARF fragments. Barindicate the SE of themean. *P<0.05 (Student’s t test).n=3biological replicates. (D) ARF interactionswithATXR2 fragments. Bars indicate the SE of themean. *P<0.05 (Student’stest).n=3biological replicates. (E) Bimolecular fluorescence complementation (BiFC) assays inArabidopsisprotoplasts transiently expressing the indicated combinations of ATXR2or ARFs YFP (yellow fluorescent protein) fusion constructs. Scale bar, 20 mm. n= 3 biological replicates. DIC, differential interference contrast. (F) Split-luciferase (LUC) assays. Partiafragments of Luciferase (nLuc and cLuc) were fused with ATXR2 or ARFs. Bars indicate the SE of the mean. *P < 0.05 (Student’s t test). n = 3 biological replicates.

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ARFs are required for ATXR2 functionConsistent with the physical association of ARFs and ATXR2, the arf7-1arf19-2 double mutant was defective in callus formation (Fig. 5, A andB) and showed a substantial reduction in LBD expression compared towild-type plants (Fig. 5C), similar to the atxr2-1 mutant. ARF7 andARF19 are known to bind directly to the LBD16 and LBD29 promoters(6) and stimulate the expression of these genes as well as other LBDgenes including LBD17 and LBD18 (6). The specific binding of ATXR2to the LBD16 and LBD29 promoters (Fig. 2D) and the interaction be-tween ATXR2 and ARF7 and ARF19 support these observations (Fig.4). The ATXR2-binding sites overlap withARF-binding regions in theLBD16 and LBD29 promoters (Fig. 2D) (6). To verify a role for ARFsin epigenetic activation of LBDs, we analyzed the accumulation ofH3K36me3 at the LBD16 and LBD29 promoters in arf7-1arf19-2mu-tants. Notably, the LBD16 and LBD29 promoters showed reducedH3K36me3 deposition in arf7-1arf19-2 mutant calli (Fig. 5D) com-pared to wild-type calli, whereas H3K4me3 accumulation was notaltered (fig. S17).

To confirm thatATXR2binding toLBD promoterswasmediated byARFs, we generated 35S:ATXR2-MYC/arf7-1arf19-2 plants, in whichprotein accumulation of ATXR2 was similar to 35S:ATXR2-MYC/Col-0, by genetic crosses (fig. S18). ChIP analysis with an antibody spe-cific forMYC revealed that theATXR2proteinwas recruited to theLBDpromoters in a wild-type (Col-0) background, but ATXR2 binding tothe LBD promoters was compromised by introducing the arf7-1 andarf19-2 mutations (Fig. 6A). Accordingly, increased LBD expressionin 35S:ATXR2-MYC transgenic calli was also reduced in the arf7-1arf19-2mutant background (Fig. 6B). Furthermore, the callus forma-tion capability of 35S:ATXR2-MYC was largely dependent on ARFs(Fig. 6, C and D). These results indicate that the ATXR2 and ARF pro-teins are functionally interconnected in promoting LBD-dependent cal-lus formation.

Finally,we examinedwhetherATXR2depends onLBDs for the con-trol of callus formation. 35S:ATXR2-MYC transgenic plants werecrossed with 35S:LBD16-SRDX transgenic plants. Leaf explants of35S:ATXR2-MYC plants exhibited enhanced callus formation (Fig.1A and fig. S19), whereas reduced callus formation was observed in35S:LBD16-SRDX leaf explants (fig. S19) (5). Callus formation of 35S:ATXR2-MYC; 35S:LBD16-SRDX was similar to that of 35S:LBD16-SRDX (fig. S19), indicating that LBDs are epistatic to ATXR2 in the reg-ulation of callus formation (Fig. 6E).

DISCUSSIONReversible transitions in cellular differentiation states require massivegene expression reprogramming (35). Chromatin modification is themost plausible regulatorymechanism that facilitates global gene expres-sion changes, and consistent with this, epigenetic regulation isconsidered as a key molecular scheme underlying cellular reprogram-ming in eukaryotes (36, 37). Several lines of evidence further supportthat chromatin status is closely associated with cell identity. Differen-tiated cells have a closed chromatin status with accumulation of repres-sive marks, whereas embryonic and pluripotent dedifferentiated cellshave a relatively open chromatin status with active mark deposition(38, 39).

Plant dedifferentiation is a multistep process that begins with theformation of partially dedifferentiated founder cells, followed by cal-lus formation and the establishment of pluripotency in callus cells.Widespread epigenetic changes, including modifications of both DNA

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and histones, occur during callus formation. Despite the importanceof chromatin modification during callus formation, few molecularcomponents responsible for this process have been demonstrated inArabidopsis. DNA methylation is likely essential for callus formation(40), and the global cytosinemethylation landscape changes during cal-lus formation (40, 41).ArabidopsisMETHYLTRANSFERASE 1 (MET1)has been implicated in cell fate changes and is an ortholog of mamma-lian DNAMETHYLTRANSFERASE 1 (DNMT1), which maintains CGmethylation globally in the genome (40). MET1-deficient mutantsexhibit defective callus formation with hypomethylation of somegenic loci, including GLUTATHIONE S-TRANSFERASE TAU 10(GSTU10),MITOGEN-ACTIVATED PROTEINKINASE 12 (MAPK12),

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Fig. 5. Functional coordination of ATXR2 with ARF transcription factors. (A) Cal-lus formation in leaf explants from wild-type (Col-0) and arf7-1arf19-2 double-mutantplants. Scale bar, 5 mm. (B) Quantification (fresh weight) of callus formation in leaf ex-plants from Col-0 and arf7-1arf19-2 plants. Bars indicate the SE of the mean. *P < 0.05(Student’s t test). n=30 leaf explants of each genotype. (C) LBD transcript accumulationin Col-0 and arf7-1arf19-2 mutants during callus formation. Expression of each genewas normalized to the expression of that gene at time 1 in Col-0. Bars indicate theSE of the mean. *P < 0.05 (Student’s t test). n = 3 biological replicates. (D) H3K36me3accumulation at LBD loci in Col-0 and arf7-1arf19-2 calli. H3K36me3 abundance wasnormalized to the abundance at time 0 in Col-0. Bars indicate the SE of themean. *P<0.05 (Student’s t test). n = 3 biological replicates.

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BETA-XYLOSIDASE 1 (BXL1), andWUSCHEL (WUS) (40, 42), as wellas in many nongenic regions (40). Furthermore, chromomethylase 3(cmt3) and domains rearranged methylase (drm) mutants, which havealtered patterns of both CHG and CHH methylation (40), also displayimpaired callus formation (40), underscoring the importance of DNAmethylation in cell fate changes.

Histone modification also plays a key role in callus formation inArabidopsis. Upon callus induction, marks of active chromatin such asH3ac, H3K4me3, and H3K36me3 accumulate globally in the genome(32, 33). In addition to these global effects, local epigenetic modifica-tions at genic regions are also important for robust cellular dedif-ferentiation. Some histone modifiers that act as negative regulators ofgene transcription have been implicated as key drivers of callus forma-tion. For example, transcripts of several genes encoding histone deacet-ylases accumulate during callus formation, and genetic mutations inHISTONE DEACETYLASE 9 (HDA9) or HD-TUINS PROTEIN1 (HDT1) impede callus formation (32). The polycomb repressivecomplex 2 (PRC2) also enhances callus formation by establishing therepressive H3K27me3 mark. PRC2 promotes leaf-to-callus transitionthrough repression of leaf identity genes such as SAWTOOTH1 (SAW1), SAW2, and TEOSINTE BRANCHED1-CYCLOIDEA-PCF10 (TCP10) during callus formation (31).

Here, we report that ATXR2 is a key component that not only pro-motes global deposition ofH3K36me3 but also contributes to epigeneticcontrol of the auxin signaling pathway during callus formation. TheATXR2-ARF-LBD circuitry is a key player in cell fate change. Ectopicexpression of these components results in enhanced callus formation onCIM, whereas the genetic mutants are defective in callus induction.ATXR2 activates LBD expression by catalyzing H3K36me3 depositionat the LBD promoter regions. Notably, ARFs recruit ATXR2 to targetpromoters, and the ARF-ATXR2 complex ensures precise epigeneticmodification at the LBD promoters, adding complexity to callus forma-tion. It remains to be determined whether additional targets of ATXR2,other than the LBDs, play a role in promoting callus formation.Likewise, ATXR2may have additional roles in callus formation becauseit can also catalyze the formation of H3K4me3 marks in addition toH3K36me3 mark. Thus, ATXR2 may make other contributions tothe remarkable capability of cellular dedifferentiation in Arabidopsis.

Note that the roles of TrxG proteins in cellular reprogrammingare well conserved in higher eukaryotes. For example, WD RE-PEAT DOMAIN 5 (WDR5) is the core subunit of the TrxG protein–containing complex in humans, and its expression correlates withthe dedifferentiation state of cells (43). This protein interacts with thepluripotency-promoting transcription factor OCTAMER-BINDINGTRANSCRIPTION FACTOR 4 (OCT4) to promote H3K4me3 accu-mulation and thus stimulate downstream genes, such as POU DO-MAIN, CLASS 5, TRANSCRIPTION FACTOR 1 (POU5F1), NANOG,and SEX DETERMINING REGION Y-BOX 2 (SOX2) (43). Further-more, amouse TrxG protein, Absent, small or homeotic 2-like protein(Ash2l), is also implicated in establishing pluripotency (44). TheAsh2lprotein, which primarily confers the H3K4me3 mark, works togetherwith chromatin remodelers andH3K9 demethylases to globallymain-tain an open chromatin landscape inmouse embryonic stem cells (44).TheATXR2 gene is an example of a TrxG protein possibly performinga similar function in a plant, contributing to the robust callus forma-tion capability of Arabidopsis. On the basis of this initial findingregardingTrxG-dependent cellular dedifferentiation, conservedmecha-nisms underlying cellular dedifferentiation in eukaryotes can be furtherinvestigated.

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Fig. 6. Requirement of ARFs for ATXR2 function. (A) Quantification of ATXR2binding to LBD promoters in arf7-1arf19-2 double-mutant plants by ChIP analysis. En-richment was quantified relative to the amount at each gene promoter in control (EV)plants. Bars indicate the SE of the mean. *P < 0.05 (Student’s t test). n = 3 biologicalreplicates. (B) Expression of LBD16 and LBD29 in calli from arf7-1arf19-2 double-mutantplants overexpressing ATXR2 (35S:ATXR2-MYC/arf7-1arf19-2). Different letters representa significant difference at P < 0.05 [one-way analysis of variance (ANOVA) with Fisher’spost hoc test). n = 3 biological replicates. (C) Callus formation in 35S:ATXR2-MYC/arf7-1arf19-2 leaf explants. n > 30 plants of each genotype. (D) Quantification (freshweight)of callus formation in leaf explants from plants of the indicated genotypes. Differentletters represent a significant difference at P < 0.05 (one-way ANOVAwith Fisher’s posthoc test). Bars indicate the SE of the mean. n = 30 calli of each genotype. (E) Proposedrole of ATXR2 in callus formation. ATXR2 interacts with ARF7 and ARF19 to bind to thepromoters of LBD16 and LBD29. ATXR2 promotes LBD gene expression by catalyzingthe deposition of H3K36me3 at these promoters. ATXR2 indirectly affects the expres-sion of LBD17 and LBD18 through its direct effects on LBD16 and LBD29 expression.These activities facilitate callus formation. CIM, callus-inducing medium.

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MATERIALS AND METHODSPlant materials and growth conditionsA. thaliana (Columbia-0 ecotype) was used for all experiments unlessotherwise specified.Arabidopsis seeds were surface sterilized and sownon 0.7% agar plates containing half-strengthened Murashige andSkoog media. Plants were grown under long-day conditions (16-hourlight/8-hour dark cycles) with white fluorescent light (120 mmolphotons m−2 s−1) at 22° to 23°C. The arf7-1arf19-2 mutant was de-scribed previously (6). The atxr2-1 (SAIL-600-E07) and atxr2-3(SALK-095652)mutants were isolated from a T-DNA insertionalmu-tant pool deposited in the Arabidopsis Biological Resource Center(ABRC; https://abrc.osu.edu/).

To produce transgenic plants overexpressing the ATXR2 gene,we subcloned a full-length cDNA into the modified binary pBA002vector under the control of the CaMV 35S promoter. Agrobacteriumtumefaciens–mediated Arabidopsis transformation was thenperformed.

For callus induction, leaf explants of third leaf from 2-week-oldplants were placed on CIM [B5 medium supplemented with 2,4-dichlorophenoxyacetic acid (0.5 mg/ml) and kinetin (0.05 mg/ml)],followed by incubation at 22°C in the dark for additional 2weeks. Thirtycalli of each genotype were collected to measure fresh weight. Threeindependent measurements were averaged. Statistically significant dif-ferences between wild-type and transgenic or mutant calli are de-termined by Student’s t test.

RT-qPCR analysisTotal RNA was extracted using TRI reagent (Takara Bio) accordingto the manufacturer’s recommendations. RT was performed usingMoloney Murine Leukemia Virus reverse transcriptase (Dr. Protein)with oligo(dT20) to synthesize first-strand cDNA from 2 mg of totalRNA. The cDNAs were diluted to 100 ml with Tris-EDTA (TE) buffer,and 1 ml of diluted cDNA was used for PCR amplification.

RT-qPCR reactions were performed in 96-well blocks using theStepOnePlus Real-Time PCR System (Applied Biosystems). The PCRprimers used are listed in table S1. The values for each set of primerswere normalized relative to the EUKARYOTIC TRANSLATION INI-TIATION FACTOR 4A1 (eIF4A) gene (At3g13920). All RT-qPCR re-actions were performed in biological triplicates using total RNAsamples extracted from three independent replicate samples. The com-parative DDCt method was used to evaluate the relative quantities ofeach amplified product in the samples. The threshold cycle (Ct) wasautomatically determined for each reaction with the analysis softwareset using default parameters. Specificity of the RT-qPCR reactions wasdetermined by melt curve analysis of the amplified products.

Chromatin immunoprecipitationPutative ATXR2 binding sites were predicted on the basis of the Web-based promoter analysis. Promoter regions containingmultiple putativecis-elements were chosen as candidate binding sites. The epitope-taggedtransgenic plant samples were cross-linked with 1% formaldehyde,ground to powder in liquid nitrogen, and then sonicated. The sonicatedchromatin complexes were precipitated with salmon sperm DNA/protein A agarose beads (Millipore) and antibodies recognizingMYC, H3K4me3, H3K27me3, and H3K36me3 (Millipore). Precipi-tated DNA was purified using phenol/chloroform/isoamyl alcoholand sodium acetate (pH 5.2). The abundance of specific precipitatedDNA fragments was quantified by qPCR using the primer pairs listedin table S2. Values were normalized according to input DNA abun-

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dance. Values for control plants were set to 1 after normalizationagainst eIF4a for qPCR analysis.

Yeast two-hybrid assaysY2H assays were performed using the BD Matchmaker system(Clontech). Full-length or truncated cDNAs of ARF7 and ARF19were cloned into the pGADT7 vector for GAL4 AD fusion. Full-length or truncated cDNAs of ATXR2 were cloned into the pGBKT7vector for GAL4 DBD fusion. Full-sized GAL4 transcription factorwas expressed as a positive control (Clontech). The yeast strainAH109 harboring the LacZ and His reporter genes was used. Theexpression constructs were cotransformed into yeast AH109 cells,and transformed cells were selected by growth on SD/-Leu/-Trp me-dium and SD/-Leu/-Trp/-His/-Ade. Interactions between proteins wereanalyzed by measuring b-galactosidase activity using o-nitrophenyl-b-D-galactopyranoside as substrate.

Preparation of Arabidopsis protoplastsLeaves from 4-week-old plants were cut into 0.5-mm pieces using afresh razor blade. Twenty leaves were digested in 15 ml of enzyme so-lution [0.8% cellulase (Yakult), 0.2% macerozyme (Yakult), 0.4 Mmannitol, 10 mM CaCl2, 20 mM KCl, 0.1% bovine serum albumin,and 20 mM MES (pH 5.7)], vacuumed for 20 min, and incubated inthe dark for 5 hours at 22° to 23°C. Protoplasts were then passedthrough 40-mm stainless mesh and collected after a gentle wash withW5 media (154 mMNaCl, 125 mM CaCl2, 5 mM KCl, 2 mMMES,5 mM glucose adjusted to pH 5.7 with KOH).

Bimolecular fluorescence complementation assaysThe ATXR2 gene was fused in-frame to the 5′ end of a gene sequenceencoding the C-terminal half of enhanced YFP (EYFP) in the pSATN-cEYFP-C1 vector (E3082). The ARF cDNA sequences were fused in-frame to the 5′ end of a gene sequence encoding the N-terminal halfof EYFP in the pSATN-nEYFP-C1 vector (E3081). Using the poly-ethylene glycol (PEG) method of transformation, the expressionconstructs were transfected into Arabidopsis protoplasts that had beengenerated by standard methods (45). Expression of the fusion con-structs was monitored by fluorescence microscopy using the ZeissLSM510 confocal microscope (Carl Zeiss).

Immunoblot analysisHarvested plant materials were ground in liquid nitrogen, and totalcellular extracts were suspended in SDS–polyacrylamide gel electropho-resis (PAGE) sample loading buffer. The protein samples were thenanalyzed by SDS-PAGE (10% gels) and blotted onto Hybond-P+mem-branes (Amersham Pharmacia). Epitope-tagged proteins were immu-nologically detected using antibodies specific for H3K4me3, H3K9me3,H3K27me3, or H3K36me3 (Millipore).

Transient gene expression assaysFor transient expression assays using Arabidopsis protoplasts, reporterand effector plasmids were constructed. The reporter plasmid containsaminimal 35S promoter sequence and theGUS gene. The core elementson the LBD promoters were inserted into the reporter plasmid. Toconstruct effector plasmids, cDNAs were inserted into the effectorvector containing the CaMV 35S promoter. Recombinant reporterand effector plasmids were cotransformed into Arabidopsis protoplastsby PEG-mediated transformation. The GUS activities were measuredby a fluorometric method. A CaMV 35S promoter–Luc construct was

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also cotransformed as an internal control. The Luc assay was performedusing the Luciferase Assay System kit (Promega).

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SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/10/507/eaan0316/DC1Fig. S1. Callus formation in leaf explants from atxr1 and atxr4 mutants.Fig. S2. ATXR2 expression in atxr2 mutants.Fig. S3. ATXR2 expression in 35S:ATXR2-MYC transgenic plants.Fig. S4. Transcript accumulation of LBDs in atxr2-3 mutant calli.Fig. S5. Spatial expression of LBD16 in leaf explants and calli.Fig. S6. Phenotype of leaf explant–derived callus at 7 DAC.Fig. S7. Transcript accumulation of WOX5 in atxr2-1 calli.Fig. S8. ChIP assays using antibody-free resin.Fig. S9. Transient expression assays.Fig. S10. Binding of ATXR2 to the LBD1 promoter.Fig. S11. Accumulation of H3K4me3, H3K9me3, and H3K27me3 in atxr2-1 mutants duringcallus induction.Fig. S12. Accumulation of H3K4me3 and H3K36me3 in 35S:ATXR2-MYC calli.Fig. S13. H3K4me3 accumulation at the LBD promoters in atxr2-1.Fig. S14. Interactions of ATXR2 with ARF7 and ARF19.Fig. S15. Interactions of ATXR2 with deletion constructs of ARF7 and ARF19.Fig. S16. Interactions of ATXR2 with other ARFs.Fig. S17. Accumulation of H3K4me3 at LBD promoters in arf7-1arf19-2 mutant calli.Fig. S18. Protein accumulation of ATXR2 in 35S:ATXR2-MYC/Col-0 and 35S:ATXR2-MYC/arf7-1arf19-2.Fig. S19. Callus formation of leaf explants from 35S:ATXR2-MYC x 35S:LBD16-SRDX plants.Table S1. Primers used for PCR.Table S2. Primers used for ChIP assays.

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Acknowledgments: We thank the Nottingham Arabidopsis Stock Centre (NASC) and theArabidopsis Biological Resource Center (ABRC) for Arabidopsis mutant seeds used in thiswork and M. S. Choi for critical reading of the manuscript. Funding: This work wassupported by the Basic Research Laboratory (2017R1A4A1015620) and Basic Science

Lee, Park, Seo, Sci. Signal. 10, eaan0316 (2017) 28 November 2017

Research (NRF-2016R1D1A1B03931139) programs provided by the National ResearchFoundation of Korea and by the Next-Generation BioGreen 21 Program (PJ01119204) providedby the Rural Development Administration. This paper was also supported by SEOK CHUNResearch Fund, Sungkyunkwan University, 2016. Author contributions: P.J.S. conceived anddesigned the experiments. P.J.S. wrote the paper with the help of K.L. K.L. and O.-S.P.conducted experiments and contributed to the study design. K.L. analyzed the data.Competing interests: The authors declare that they have no competing interests.

Submitted 22 February 2017Accepted 10 October 2017Published 28 November 201710.1126/scisignal.aan0316

Citation: K. Lee, O.-S. Park, P. J. Seo, Arabidopsis ATXR2 deposits H3K36me3 at the promotersof LBD genes to facilitate cellular dedifferentiation. Sci. Signal. 10, eaan0316 (2017).

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dedifferentiation genes to facilitate cellularLBD ATXR2 deposits H3K36me3 at the promoters of Arabidopsis

Kyounghee Lee, Ok-Sun Park and Pil Joon Seo

DOI: 10.1126/scisignal.aan0316 (507), eaan0316.10Sci. Signal. 

remarkable developmental plasticity of plant cells.controlling cell potency and differentiation in both plants and animals, and these findings contribute to understanding therecruited to the promoters by the transcription factors ARF7 and ARF19. Epigenetic regulation is a key mechanism

promoters, stimulated the accumulation of lysine-methylated histones at these promoters, and wasLBDlocalized to genes, which encode transcription factors that promote cell cycle progression. ATXR2LBDstimulating the expression of

byArabidopsis thalianamethyltransferase ATXR2 promoted cellular dedifferentiation during callus formation in . found that the histone lysineet alwound sites but can also be induced by specific laboratory culture conditions. Lee

Some plant cells can dedifferentiate to form a mass of pluripotent cells called callus. This not only occurs atEpigenetic control of dedifferentiation

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