Volume 2, Issue 4, Pages 724-737 (July 2009) Histone Acetylation, VERNALIZATION INSENSITIVE 3, FLOWERING LOCUS C, and the Vernalization Response  Bond Donna M. , Dennis Elizabeth S. , Pogson Barry J. , Finnegan E. Jean   Molecular Plant  Volume 2, Issue 4, Pages 724-737 (July 2009) DOI: 10.1093/mp/ssp021 Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 1 VIN3 Expression in Response to Low Temperatures. (A) qPCR measuring the level of VIN3 relative to FDH in seedlings in response to 1, 3, 7, and 28 d of cold exposure (4°C, shaded gray), and in samples placed back into 22°C for 3 or 14 d (white area). All samples are compared to 14-day-old non-vernalized seedlings (0 d). (B) Same as in (A) but expression was measured in response to 1, 7, and 14 d of cold exposure (4°C, shaded gray), then in samples placed back into 22°C for 1 and 7 d (white area), followed by samples re-exposed to cold for 1 and 7 d (shaded gray). Error bars represent S.E.D. For data in (A) and (B), F-test p-values for the terms in the ANOVA analysis suggested significant effects for the days of cold treatment (p < 0.001). Molecular Plant 2009 2, 724-737DOI: (10.1093/mp/ssp021) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 2 Histone Acetylation Increases at the VIN3 Locus in Response to Low Temperatures. (A) Diagram showing the gene structure of VIN3. The solid black line indicates the exons and the thin line indicates the introns. The dashed line represents the untranslated regions. The VIN3.1 (start of transcription) and VIN3.2 (coding region) chromatin regions pulled down by ChIP and amplified by qPCR are marked. (B) ChIP analysis measuring the change in the level of acetylation on histone H3 at region VIN3.1 (solid line) and VIN3.2 (dashed line) relative to SAM in seedlings harvested after 1, 3, 7, and 28 d of cold treatment (4°C, shaded gray), and after the return from cold to 22°C for 3 and 14 d (white area). Test samples are compared to 14-day-old non-vernalized seedlings (0 d). (C) The same as in (B) but measuring the change in the level of acetylation of histone H4 at region VIN3.1 and VIN3.2. Error bars represent S.E.D. For histone H3, F-test p-values for the terms in the ANOVA analysis suggested significant effects for the days of cold treatment (p < 0.001), chromatin site (p < 0.001), and their interaction (p < 0.001). For histone H4, F-test p-values for the terms in the ANOVA analysis suggested significant effects for the days of cold treatment (p < 0.001), but not chromatin site (p  =  0.778) or their interaction (p  =  0.617). Molecular Plant 2009 2, 724-737DOI: (10.1093/mp/ssp021) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 3 No Single HAT Regulates VIN3 Expression. (A) qPCR measuring the level of GCN5, ADA2a, ADA2b, HAG2, HAG3, HAM1, HAM2, HAC1, HAC2, HAC4, HAC5, HAC12, HAF1 relative to FDH in seedlings in response to 28 d (dark gray) of cold exposure (4°C), compared to 14-day-old non-vernalized seedlings (0 d, light gray). HAF2 is not expressed in seedlings (Bertrand et al., 2005). Error bars represent the S.E.D. For HAG3, the F-test p-values for the terms in the ANOVA analysis suggested significant effects for the days of cold treatment (p  =  0.004). (B) qPCR measuring the level of VIN3 relative to FDH in ws-2, gcn5-1, ada2a-1, and ada2b-1 non-vernalized seedlings (0 d) and in response to 1, 7, 14, and 21 d of cold exposure (4°C). The 21-d vernalized samples were set as 100 to consider expression differences in non-vernalized seedlings. Error bars represent S.E.D. For each genotype, F-test p-values for the terms in the ANOVA analysis suggested significant effects for the days of cold treatment (p < 0.001). (C) qPCR measuring the level of VIN3 relative to FDH in Col, hag2-1, ham1-1, ham2-2, hac1-4, hac2-1, hac4-3, hac5-1, hac12-1, haf1-1, haf2-4, Ler, and elo3-1 non-vernalized seedlings (0 d) and in response to 7 and 21 d of cold exposure (4°C). The wild-type Col and Ler 21-d vernalized samples are set as 100, to consider expression differences in non-vernalized seedlings. Error bars represent S.E.D. For each genotype, F-test p-values for the terms in the ANOVA analysis suggested significant effects for the days of cold treatment (p < 0.001). Molecular Plant 2009 2, 724-737DOI: (10.1093/mp/ssp021) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 4 VIN3 Expression Is Deregulated by HDACi. qPCR measuring the level of VIN3 relative to FDH in seedlings treated with different concentrations of HDACi sodium butyrate (Na But), trichostatin A (TSA), or nicotinamide (nic) for 14 d compared to non-treated control seedlings (No inhibitor). Error bars represent S.E.D. F-test p-values for the terms in the ANOVA analysis suggested significant effects for the different HDACi treatments (p  =  0.002), compared to the no-inhibitor control and each other. Molecular Plant 2009 2, 724-737DOI: (10.1093/mp/ssp021) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 5 The Effects of Nicotinamide on Seedling Growth and VIN3 Expression. (A) Seedlings were germinated on MS agar medium and, after 5 d, were transferred onto new MS agar medium (No inhibitor), or agar medium supplemented with 2.5 or 5 mM nicotinamide (nic) for 14 d. Images were taken at the end of a 14-d nicotinamide treatment. (B) qPCR measuring the level of VIN3 relative to FDH in seedlings after 7, 14, and 21 d (shaded gray) in the presence of 2.5 mM (thick dashed line) or 5 mM nicotinamide (nic, thin dashed line) compared to non-treated control seedlings (No inhibitor, solid line). The 7-d control (No inhibitor) samples were set as 1. After 21 d of treatment, seedlings were transferred to fresh MS agar medium for 7 d to measure the recovery from nicotinamide (white area). Error bars represent S.E.D. F-test p-values for the terms in the ANOVA analysis suggested significant effects for the days of nicotinamide treatment (all time points, p  =  0.002), concentration of nicotinamide (p < 0.001), and their interaction (p  =  0.007). Molecular Plant 2009 2, 724-737DOI: (10.1093/mp/ssp021) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 6 Nicotinamide Induces Histone Acetylation at the VIN3 Locus. (A) ChIP analysis measuring the level of acetylation on histone H3 at region VIN3.1 (light gray) and VIN3.2 (dark gray) relative to SAM in seedlings treated with 2.5 or 5 mM nicotinamide (nic) for 14 d compared to non-treated control seedlings (No inhibitor). (B) The same as in (A) but measuring the level of histone H4 acetylation at region VIN3.1 (light gray) and VIN3.2 (dark gray). Error bars represent S.E.D. For histone H3, F-test p-values for the terms in the ANOVA analysis suggested significant effects for the concentration of nicotinamide (p < 0.001) and chromatin site (p  =  0.029), but not their interaction (p  =  0.425). For histone H4, F-test p-values for the terms in the ANOVA analysis did not suggest any significant effects. Molecular Plant 2009 2, 724-737DOI: (10.1093/mp/ssp021) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 7 SIR2 HDACs Do Not Regulate VIN3 Expression. (A) qPCR measuring the level of SRT1 and SRT2 relative to FDH in seedlings in response to 28 d (dark gray) of cold exposure compared to 14-day-old non-vernalized control seedlings (0 d, light gray). Error bars represent the S.E.D. For SRT2, F-test p-values for the terms in the ANOVA analysis suggested significant effects for the days of cold treatment (p  =  0.044). (B) qPCR measuring the level of SRT1 (light gray) and SRT2 (dark gray) relative to FDH in Col, srt2-1, and individual srt2-1/SRT1synmiRNA T1 transformant lines (line 1–9). The srt2-1 sample is set as 1 for SRT1 and the Col sample is set as 1 for SRT2. These data represent normalized expression in individual T1 seedlings, so no statistical analysis was performed on these results. (C) qPCR measuring the level of VIN3 relative to FDH in Col, srt1-1, srt2-1, and srt2-1/SRT1synmiRNA non-vernalized seedlings (0 d) and in response to 7 and 21 d of cold exposure. The results for the srt2-1/SRT1synmiRNA lines at each time point are an average of four individual T1 transformants. The Col 21-d vernalized sample is set as 100, to consider expression differences in non-vernalized seedlings. For the T-DNA insertion lines, F-test p-values for the terms in the ANOVA analysis suggested significant effects for the days of cold treatment (p < 0.001). For the SRT1synmiRNA, the standard deviations of the mean expression of the combined individual lines in the different conditions are given. Molecular Plant 2009 2, 724-737DOI: (10.1093/mp/ssp021) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 8 FLC Expression Is Repressed in Response to Nicotinamide and srt2-1/SRT1synmiRNA Lines, which Correlates with an Early-Flowering Phenotype. (A) qPCR measuring the level of FLC relative to FDH in seedlings after 7, 14, and 21 d (shaded gray) in the presence 2.5 mM (thick dashed line) or 5 mM nicotinamide (nic, thin dashed line) compared to non-treated control seedlings (No inhibitor, solid line). The 7-d no-inhibitor control samples were set as 1. After 21 d of treatment, seedlings were transferred to fresh MS agar medium for 7 d to measure the recovery from nicotinamide (white area). Error bars represent S.E.D. F-test p-values for the terms in the ANOVA analysis suggested significant effects for the days of nicotinamide treatment (p < 0.001), concentration of nicotinamide (p < 0.001), and their interaction (p  =  0.007). (B) Flowering time (total leaf number) of C24 seedlings after treatment with 2.5 and 5 mM nicotinamide (nic) for 14 d, then transferred onto fresh MS agar medium without nic, compared to non-treated control seedlings (No inhibitor). Error bars represent standard deviation. (C) qPCR measuring the level of FLC relative to FDH in Col:FRI (light gray) and vin3-4 (dark gray) seedlings after treatment with 2.5 or 5 mM nicotinamide (nic) compared to non-treated control seedlings (No inhibitor). vin3-4 contains a T-DNA in the second intron of VIN3 that interrupts the VIN3 transcript (Supplemental Figure 3). Error bars represent S.E.D. F-test p-values for the terms in the ANOVA analysis suggested significant effects of nicotinamide treatment (p < 0.001), but not genotype (p  =  0.484) or their interaction (p  =  0.384). (D) qPCR measuring the level of FLC relative to FDH in Col (light gray), srt2-1 (dark gray), and individual srt2-1/SRT1synmiRNA T1 transformant lines (line 1–9; black). For Col and srt2-1, error bars represent S.E.D. F-test p-values for the terms in the ANOVA analysis suggested significant effects of the genotype (p  =  0.013). The data for the srt2-1/SRT1synmiRNA lines represent normalized expression in the nine individual T1 seedlings, so no statistical analysis was performed on these results. The average expression in these lines is presented (Ave.), and the standard deviation of the mean expression of the combined individual lines is given. Molecular Plant 2009 2, 724-737DOI: (10.1093/mp/ssp021) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 9 There Is No Change in the Expression of Genes from the Autonomous Pathway or Members of the PRC2 Complex that Regulates FLC Expression in Nicotinamide-Treated or srt2-1 /SRT1synmiRNA Plants. (A) qPCR measuring the expression of FCA, FLD, FLK, FPA, FVE, LD, REF6 relative to FDH in Col:FRI seedlings after treatment with 2.5 mM (dark gray) or 5 mM nicotinamide (black) compared to non-treated control seedlings (No inhibitor; light gray). Error bars represent the S.E.D. (B) qPCR measuring the expression of FCA, FLD, FLK, FPA, FVE, LD, REF6 relative to FDH in Col (light gray) and two individual srt2-1/SRT1synmiRNA T1 transformant lines (line #8, dark gray and line #9, black). These data represent normalized expression in the two individual T1 seedlings, so no statistical analysis was performed on these results. (C) Same as (A) but measuring the expression of CLF, SWN, FIE, VRN2, VIL1, and LHP1. Error bars represent the S.E.D. (D) Same as (B) but measuring the expression of CLF, SWN, FIE, VRN2, VIL1, and LHP1. These data represent normalized expression in the two individual T1 seedlings, so no statistical analysis was performed. Molecular Plant 2009 2, 724-737DOI: (10.1093/mp/ssp021) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions