Volume 23, Issue 14, Pages 1324-1329 (July 2013) VAL- and AtBMI1-Mediated H2Aub Initiate the Switch from Embryonic to Postgerminative Growth in Arabidopsis  Chao Yang, Fabian Bratzel, Nora Hohmann, Marcus Koch, Franziska Turck, Myriam Calonje  Current Biology  Volume 23, Issue 14, Pages 1324-1329 (July 2013) DOI: 10.1016/j.cub.2013.05.050 Copyright © 2013 Elsevier Ltd Terms and Conditions

Current Biology 2013 23, 1324-1329DOI: (10.1016/j.cub.2013.05.050) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 1 Phenotype and Gene Expression Pattern Comparison of Different Mutants (A–C) Wild-type (WT), val1/2, and atbmi1a/b strong mutants at 10 days after germination (DAG), respectively. Scale bars represent 2 mm. (D) RT-PCR analysis of the expression levels of ABI3, FUS3, LEC1, LEC2, STM, WUS, BBM, WOX5, and AG in 10 DAG WT, atbmi1a/b strong and intermediate mutants, val1/2 strong and weak mutants (left panel), and atbmi1a/b/c mutants (right panel). ACTIN2 was used as internal control. emf2-1 seedlings at 10 DAG were used as a positive control for AG expression. (E) Expression levels of AtBMI1A, AtBMI1B, and AtBMI1C in the aerial parts and roots of 10 DAG WT seedlings. (F and G) atbmi1a/b/c mutants at 10 DAG. Scale bars represent 2 mm. (H) Pull-down assay of VAL1-3XFLAG, using GST or different GST-tagged proteins as indicated (upper panel), and coIP of VAL1-3XFLAG and AtBMI1C-HA using anti-FLAG M2 antibody (bottom panel). Pull-down and coIP fractions were analyzed by immunoblotting using anti-FLAG and anti-HA antibodies, respectively. (I and J) emf2-1 and emf1-2 mutants at 7 DAG, respectively. The presence of sessile leaves in emf2-1 mutants is indicated (arrows). (K and L) atbmi1a/b/emf2-1 and atbmi1a/b/emf1-2 mutants at 7 DAG, respectively. Pickle-like roots (solid arrows) and secondary roots (dashed arrows) are indicated. (M and N) val1/2/emf2-1 and val1/2 mutants at 20 DAG, respectively. (O and P) clf28/swn7 mutants at 7 DAG. Sessile leaves (short arrows), pickle-like root (solid arrow), and secondary root (dashed arrow) are indicated. Scale bars represent 2 mm. (Q) Expression analysis of ABI3, FUS3, and LEC1 in the aerial parts and roots of WT seedlings and atbmi1a/b, atbmi1a/b/c, emf1-2, emf2-1, clf28/swn7, atbmi1a/b/emf2-1, and atbmi1a/b/emf1-2 mutants at 7 DAG. ACTIN2 was used as internal control. See also Figure S1. Current Biology 2013 23, 1324-1329DOI: (10.1016/j.cub.2013.05.050) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 2 Flow Cytometry Analysis of WT, clf28/swn7, and atbmi1a/b/c Plants after Germination (A) Flow cytometry experiments with the indicated different plants at 7 and 30 DAG. (B) Percentage of nuclei with 2C, 4C, 8C, and 16C of DNA content in the different plants at 7 and 30 DAG. Error bars represent SD between replicates. Current Biology 2013 23, 1324-1329DOI: (10.1016/j.cub.2013.05.050) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 3 ChIP Analysis of H3K27me3 Levels in WT and atbmi1a/b, atbmi1a/b/c, val1/2, clf28/swn7, emf2-1, and emf1-2 Mutants (A) Structure of the genes and location of the region amplified by quantitative chIP-PCR. Boxes and lines represent exons and introns, respectively. (B) Levels of H3K27me3 at the transcriptional start site (TSS) of the different genes in WT and mutants at 10 DAG. The results show the recovery of immunoprecipitated material using antibody (IP) and no antibody (mock control) as percentage of input. ACTIN7 was used as an active gene control. (C) Levels of H3K27me3 at different regions of FUS3, STM, and AG in WT and atbmi1a/b/c mutants. (D) Levels of H3K27me3 at the TSS of the different genes in WT and atbmi1a/b intermediate and strong mutants’ aerial parts at 10 DAG. Significant differences compared to WT levels in (C) and (D) are shown by asterisks (one-tailed Student’s t test, ∗p ≤ 0.05). Error bars represent SD between replicates. Current Biology 2013 23, 1324-1329DOI: (10.1016/j.cub.2013.05.050) Copyright © 2013 Elsevier Ltd Terms and Conditions

Figure 4 ChIP Analysis of H2Aub Levels in WT and atbmi1a/b, atbmi1a/b/c, val1/2, clf28/swn7, and emf1-2 Mutants (A) WT and atbmi1a/b histone-enriched extract (left panel) and nuclear extract (middle panel) blots probed with anti-hH2Aub antibody, and WT histone extract (right panel) blot probed with anti-ubiquitin antibody, showing the presence of different bands with the predicted MW of several H2Aub isoforms. Similar amounts of samples were loaded as indicated by the loading control. MW markers (kDa) H2Bub and H2Aub bands are indicated. (B) Levels of H2Aub at the TSS of the genes in WT and mutants at 10 DAG. The results show the recovery of immunoprecipitated material using antibody (IP) and no antibody (mock control) as percentage of input. ACTIN7 was used as an active gene control. Error bars in (B) and (C) represent SD between replicates. (C) Levels of H2Aub at different regions of FUS3, STM, and AG in WT seedlings. (D) Global levels of H2Aub in WT, atbmi1a/b, and clf28/swn7 at 10 DAG. Left panel shows immunoblots probed with anti-ubiquitin antibodies, and right panel shows immunoblot probed with anti-hH2Aub antibodies. Similar amounts of histones were loaded as indicated by the loading control. MW markers (kDa), H2Bub, and H2Aub are indicated. The different WT, atbmi1a/b, and clf28/swn7 samples represent independent biological replicates. See also Figure S2. Current Biology 2013 23, 1324-1329DOI: (10.1016/j.cub.2013.05.050) Copyright © 2013 Elsevier Ltd Terms and Conditions