by Kyounghee Lee, Ok-Sun Park, and Pil Joon Seo Arabidopsis ATXR2 deposits H3K36me3 at the promoters of LBD genes to facilitate cellular dedifferentiation by Kyounghee Lee, Ok-Sun Park, and Pil Joon Seo Sci. Signal. Volume 10(507):eaan0316 November 28, 2017 ©2017 by American Association for the Advancement of Science

Callus formation in leaf explants from atxr2 mutant and ATXR2-overexpressing plants. 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, and 35S:ATXR2-MYC plants. Scale bar, 5 mm. n > 30 plants of each genotype. (B) Fresh weight of calli from wild-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 = 30 calli of each genotype. Kyounghee Lee et al., Sci. Signal. 2017;10:eaan0316 ©2017 by American Association for the Advancement of Science

Transcriptional activation of LBD genes by ATXR2. Transcriptional activation of LBD genes by ATXR2. (A) Expression profiling of the indicated genes involved in callus formation in 35S:ATXR2-MYC and atxr2-1 calli by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and normalized to each gene’s expression in wild-type (Col-0) plants. Bars indicate the SE of the mean. *P < 0.05 (Student’s t test). n = 3 biological replicates. (B) Kinetics of LBD expression in Col-0 and atxr2-1 leaf explants during callus formation at 2, 4, and 7 days after culturing on callus-inducing medium (DAC). Expression of each gene was normalized to the expression of that gene at time 2 in Col-0. *P < 0.05 (Student’s t test). n = 3 biological replicates. (C) Promoter analysis of LBD genes. The regions labeled A to M were identified as putative binding sites for ATXR2. Black lines above the labels indicate the regions that were amplified by PCR after chromatin immunoprecipitation (ChIP). Black boxes indicate exons. (D) Enrichment of ATXR2 on promoter regions as determined by ChIP-qPCR. Values for qPCR analysis were normalized 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. Kyounghee Lee et al., Sci. Signal. 2017;10:eaan0316 ©2017 by American Association for the Advancement of Science

ATXR2 mediates H3K36me3 accumulation at LBD loci during callus formation. ATXR2 mediates H3K36me3 accumulation at LBD loci during callus formation. (A) Global accumulation of H3K36me3 in wild-type (Col-0) and atxr2-1 leaf explants during callus formation. Bands from three independent blots were quantified (right). n =3 biological replicates. (B) H3K36me3 accumulation at the LBD loci indicated in Fig. 2C. Bars indicate the SE of the mean. *P < 0.05 (Student’s t test). n = 3 biological replicates. (C) Kinetics of H3K36me3 accumulation at the LBD16 and LBD29 promoters during callus formation in Col-0 and atxr2-1 plants. Bars indicate the SE of the mean. *P < 0.05 (Student’s t test). n = 3 biological replicates. Kyounghee Lee et al., Sci. Signal. 2017;10:eaan0316 ©2017 by American Association for the Advancement of Science

ATXR2 interacts with ARFs. ATXR2 interacts with ARFs. (A) Yeast two-hybrid assays using ATXR2 fused to the Gal4 DNA binding domain (DBD) and ARFs fused to the Gal4 transcriptional activation domain (AD). Full-length GAL4 was used as a positive control. LW, dropout medium without leucine (L) or tryptophan (W); LWAH, dropout medium without L, W, adenine (A), or histidine (H). n = 3 biological replicates. (B) Deletion constructs 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. Bars indicate the SE of the mean. *P < 0.05 (Student’s t test). n = 3 biological replicates. (D) ARF interactions with ATXR2 fragments. Bars indicate the SE of the mean. *P < 0.05 (Student’s t test). n = 3 biological replicates. (E) Bimolecular fluorescence complementation (BiFC) assays in Arabidopsis protoplasts transiently expressing the indicated combinations of ATXR2 or ARFs YFP (yellow fluorescent protein) fusion constructs. Scale bar, 20 μm. n = 3 biological replicates. DIC, differential interference contrast. (F) Split-luciferase (LUC) assays. Partial fragments 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. Kyounghee Lee et al., Sci. Signal. 2017;10:eaan0316 ©2017 by American Association for the Advancement of Science

Functional coordination of ATXR2 with ARF transcription factors. Functional coordination of ATXR2 with ARF transcription factors. (A) Callus formation in leaf explants from wild-type (Col-0) and arf7-1arf19-2 double-mutant plants. Scale bar, 5 mm. (B) Quantification (fresh weight) of callus formation in leaf explants 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 accumulation in Col-0 and arf7-1arf19-2 mutants during callus formation. Expression of each gene was normalized to the expression of that gene at time 1 in Col-0. Bars indicate the SE of the mean. *P < 0.05 (Student’s t test). n = 3 biological replicates. (D) H3K36me3 accumulation at LBD loci in Col-0 and arf7-1arf19-2 calli. H3K36me3 abundance was normalized to the abundance at time 0 in Col-0. Bars indicate the SE of the mean. *P < 0.05 (Student’s t test). n = 3 biological replicates. Kyounghee Lee et al., Sci. Signal. 2017;10:eaan0316 ©2017 by American Association for the Advancement of Science

Requirement of ARFs for ATXR2 function. Requirement of ARFs for ATXR2 function. (A) Quantification of ATXR2 binding to LBD promoters in arf7-1arf19-2 double-mutant plants by ChIP analysis. Enrichment 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 biological replicates. (B) Expression of LBD16 and LBD29 in calli from arf7-1arf19-2 double-mutant plants overexpressing ATXR2 (35S:ATXR2-MYC/arf7-1arf19-2). Different letters represent a significant difference at P < 0.05 [one-way analysis of variance (ANOVA) with Fisher’s post 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 (fresh weight) of callus formation in leaf explants from plants of the indicated genotypes. Different letters represent a significant difference at P < 0.05 (one-way ANOVA with Fisher’s post hoc test). Bars indicate the SE of the mean. n = 30 calli of each genotype. (E) Proposed role of ATXR2 in callus formation. ATXR2 interacts with ARF7 and ARF19 to bind to the promoters of LBD16 and LBD29. ATXR2 promotes LBD gene expression by catalyzing the deposition of H3K36me3 at these promoters. ATXR2 indirectly affects the expression of LBD17 and LBD18 through its direct effects on LBD16 and LBD29 expression. These activities facilitate callus formation. CIM, callus-inducing medium. Kyounghee Lee et al., Sci. Signal. 2017;10:eaan0316 ©2017 by American Association for the Advancement of Science