Defining the Functional Network of Epigenetic Regulators in Arabidopsis thaliana  Luo Chongyuan , Durgin Brittany G. , Watanabe Naohide , Lam Eric   Molecular Plant  Volume 2, Issue 4, Pages 661-674 (July 2009) DOI: 10.1093/mp/ssp017 Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 1 Differential Expressions of Luciferase and NPTII Transcripts in Chromatin Charting Lines. (A) Luciferase imaging of Chromatin Charting lines. Bright-field (BF) and bioluminescence (BL) images were separately acquired from an MS agar plate containing wild-type Arabidopsis, CCP4.20, CCP4.80, CCP4.211, CCT 383, CCT 396, and CCT 431 plants, which were vertically grown for 12 d before imaging. Merged image of BF and BL is also shown. The color scale of luminescence, from the lowest of 4.8 × 106 photons s−1 (dark blue) to the highest of 9.0 × 107 photons s−1 (light red), is shown in the BL image. (B) Northern blot analysis of NPTII transcripts in CC lines. Ten micrograms of total RNA isolated from shoots of 14-day-old plants were blotted and hybridized with 32P-labeled NPTII probe. A methylene blue-stained membrane was shown as a loading control. (C) RT–PCR analysis of Luc and Actin2 transcripts. RNA samples used in (B) were subjected to this analysis. Relative expression levels of Luc and Actin2 (Act2) transcripts were estimated by agarose gel electrophoresis followed by ethidium bromide staining. RT–PCR for Luc was carried out with three different cycles (28, 30, 32) under specific conditions. An Act2 cDNA fragment was amplified under specific conditions and used as a positive control. Inverted images of ethidium bromide-stained gels were shown. (D) Integration sites of transgene constructs in lines CCP4.211, CCT431, CCT396, and CCT383. The x-axis represents the first 100 kbp of chromosome 2. Positions of red bars indicate the integration site of each CC line and the lengths of red bars represent RLA (y-axis) in shoots from 12–14-day-old plants. Horizontal bars indicate regions of rDNA (green), Ty3/Gypsy Retrotransposons (blue) and expressed genes (gray). Molecular Plant 2009 2, 661-674DOI: (10.1093/mp/ssp017) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 2 Effects of RNAi-Mediated Gene Silencing of Eight Chromatin Modifiers on Luciferase Expression in Shoot Tissues of CC Lines. (A) Schematic overview of screening of T1 plants transformed with dsRNA constructs in CC lines. (B) Detection of relative Luc activity in shoots of 2-week-old T1 seedlings. The relative Luc activity (RLA) was measured in vivo for T1 seedlings of four CC lines transformed with RNAi constructs that could suppress LHP1, MOM1, CMT3, DRD1, DRM2, SUVH2, CLF, or HD1. Each value represents the mean and standard error of six representative T1 seedlings for each RNAi construct. RLA of non-transformants of CCP4.211, CCT383, CCT396, and CCT431 lines was shown as control. More than three-fold higher (line marked with 3X) or less than three-fold lower (line marked with 0.3X) RLA in comparison with untransformed CC plants were considered as significant changes in RLA for each. Molecular Plant 2009 2, 661-674DOI: (10.1093/mp/ssp017) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 3 Effects of RNAi-Mediated Gene Silencing of Eight Chromatin Modifiers on Luciferase Expression in Root Tissues of CC Lines. Detection of relative Luc activity in roots of 2-week-old T1 seedlings. The relative Luc activity (RLA) was measured in vivo for roots of T1 seedlings from four CC lines transformed with RNAi constructs that could suppress LHP1, MOM1, CMT3, DRD1, DRM2, SUVH2, CLF, or HD1. Each value represents the mean and standard error of six representative T1 seedlings for each RNAi construct. RLA of non-transformants of lines CCP4.211, CCT383, CCT396, and CCT431 was shown as control. More than three-fold higher (line marked with 3X) or less than three-fold lower (line marked with 0.3X) RLA in comparison with untransformed CC plants were considered as significant changes in RLA for each. Molecular Plant 2009 2, 661-674DOI: (10.1093/mp/ssp017) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 4 Effects of RNAi-Mediated Silencing of Eight Epigenetic Regulators on Expression of Endogenous Loci. Expression of endogenous genes AGAMOUS (AG), FLOWER LOCUS C (FLC), AT2G15790 (CyP40), centromeric satellite repeats (180 bps) and transposable elements Arabidopsis Mutator Like Element 1 (AtMu1), SINE retroelement AtSN1 and Athila LTR were analyzed by RT–PCR in line CCP4.211 and two representative T1 plants in the CCP4.211 background for each construct. Rosette leaves of 30-day-old CCP4.211 and T1 plants were used for RNA isolation. An Act2 cDNA fragment was amplified to serve as control. Inverted images of ethidium bromide-stained gels are shown. Molecular Plant 2009 2, 661-674DOI: (10.1093/mp/ssp017) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 5 Hierarchical Clustering of Chromatin Modifiers Using Molecular Phenotypes Resulting from RNAi-Mediated Silencing of Epigenetic Regulators. Heat maps representing qualitative changes of RLA caused by RNAi-mediated silencing of epigenetic regulators (listed to the right of the heat maps) in four CC lines and two tissue types (listed on the top of heat maps). In (A), more than three-fold higher or less than three-fold lower than the RLA of untransformed CC lines is considered significant up-regulation (red in heat maps) or down-regulation (green in heat maps). In (B), more than two-fold higher or less than two-fold lower than RLA of untransformed CC lines is considered significant changes. Changes in expression of endogenous loci were estimated by RT–PCR results shown in Figure 4 and are also represented in the heat maps. Black blocks indicate insignificant changes of RLA and expression levels of endogenous loci, while missing data are shown as gray blocks. The gene tree at the left of the heat maps represents the similarity between effects (change of RLA and endogenous loci expression) of RNAi-mediated silencing of different epigenetic regulators. Longer internode distance indicates less similarity while shorter internode distance indicates higher similarity. Molecular Plant 2009 2, 661-674DOI: (10.1093/mp/ssp017) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions