Trimethylation of histone H3 at lysine 27 (H3K27me3) is a histone marker that is present in inactive gene loci in both plants and animals. Transcription of some of the genes with H3K27me3 should be induced by internal or external cues, yet the dynamic fate of H3K27me3 in these genes during transcriptional regulation is poorly understood in plants. Here we show that H3K27me3 in two cold-responsive genes, COR15A and ATGOLS3, decreases gradually in Arabidopsis during exposure to cold temperatures. We found that removal of H3K27me3 can occur by both histone occupancy-dependent and -independent mechanisms. Upon cold exposure, histone H3 levels decreased in the promoter regions of COR15A and ATGOLS3 but not in their transcribed regions. When we returned cold-exposed plants to normal growth conditions, transcription of COR15A and ATGOLS3 was completely repressed to the initial level before cold exposure in 1 day. In contrast, plants still maintained the cold-triggered decrease in H3K27me3 at COR15A and ATGOLS3, but this decrease did not enhance transcriptional induction of the two genes upon re-exposure to cold. Taken together, these results indicate that gene activation is not inhibited by H3K27me3 itself but rather leads to removal of H3K27me3, and that H3K27me3 can be inherited at a quantitative level, thereby serving as a memory marker for recent transcriptional activity in Arabidopsis.
In eukaryotes, DNA-templated processes such as transcription and DNA repair occur in the context of chromatin, where genomic DNA is wrapped around histone octamers, two sets of H2A, H2B, H3, and H4 (Luger et al., 1997). The core histones can be post-translationally modified, i.e. methylated, acetylated, phosphorylated, or ubiquitinated, at various amino acid residues, particularly those residing in their N-termini (Kouzarides, 2007). Histone modifications play important roles in diverse cellular and developmental processes in plants as well as animals (Bhaumik et al., 2007; Pfluger and Wagner, 2007), and many histone modification sites are conserved between animals and plants (Johnson et al., 2004; Zhang et al., 2007a).
Several lines of evidence indicate that H3K27me3 is important for the regulation of certain developmental genes in Arabidopsis. FUSCA3 and PHERES1, which are expressed during seed development, are repressed with H3K27me3 in the sporophytic tissue of Arabidopsis (Makarevich et al., 2006). Vernalization treatment, a long-term exposure to cold, results in repression of FLOWERING LOCUS C (FLC), a negative regulator for flowering, through the increased level of H3K27me3 (Shindo et al., 2006; Finnegan and Dennis, 2007; Greb et al., 2007). Consistent with this, LHP1 is required for stable maintenance of vernalization-mediated FLC silencing (Mylne et al., 2006; Sung et al., 2006). H3K27me3 also plays a role in repression of other flowering time regulators such as AGL19 and FLOWERING LOCUS T (Schonrock et al., 2006; Jiang et al., 2008). In vegetative tissues, H3K27me3 is required for the repression of AGAMOUS, a floral homeotic regulator (Schubert et al., 2006; Saleh et al., 2007). Depletion of LHP1 also results in ectopic expression of several floral homeotic regulators, including AGAMOUS (Kotake et al., 2003).
In Arabidopsis seedlings, H3K27me3 is found in the transcribed regions of about 4400 genes (Zhang et al., 2007b). Most target genes for H3K27me3 exhibit very low expression levels or tissue-specific expression patterns in Arabidopsis, but some of these genes can change their expression levels in response to environmental and developmental cues (Turck et al., 2007; Zhang et al., 2007b). In particular, stress-responsive genes are rapidly induced during abnormal environmental conditions (Fujita et al., 2006). It is reported that hundreds of stress-responsive genes are targets for H3K27me3 in Arabidopsis (Zhang et al., 2007b). Low temperature evokes chilling or freezing stress, depending on the applied temperature (Thomashow, 1999). Plants exposed to cold temperatures transcriptionally activate a large number of genes; the change in gene expression profile results in alterations of biochemical and physiological pathways, ultimately leading to increased tolerance of plants to hazardous freezing stress (‘cold acclimation’) (Thomashow, 1999; Chinnusamy et al., 2007). Cold acclimation differs from vernalization because vernalization treatment requires several weeks of cold exposure, whereas cold acclimation can be effectively achieved in a couple of days (Sung and Amasino, 2005). Because cold-responsive genes are often strongly induced throughout different tissues upon exposure to cold temperatures, here we used cold-responsive genes to analyze the dynamic fate of H3K27me3, a negative marker for transcriptional activity (Kohler and Villar, 2008), during rapid gene induction in plants. In this study, we investigated the dynamic fate of H3K27me3 at the cold-regulated 15A (COR15A) and galactinol synthase 3 (ATGOLS3) genes during cold acclimation and subsequent return to normal growth temperature in Arabidopsis plants.
COR15A and ATGOLS3 are targets for H3K27me3
We identified the cold-responsive COR15A (Lin and Thomashow, 1992) and ATGOLS3 (Taji et al., 2002) genes from the list of target genes for H3K27me3 (Zhang et al., 2007b). Genome-wide profiling of H3K27me3 in Arabidopsis seedlings indicated that two peaks for the H3K27me3 signature are present in the COR15A and ATGOLS3 loci, respectively (Figure 1a; Zhang et al., 2007b). We performed chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) to observe whether the signature of H3K27me3 could be preserved in whole above-ground tissues of 11-day-old plant seedlings (Figure 1b). We used FUSCA3 as a reference for H3K27me3 because FUSCA3 is expressed in seeds but not in vegetative stages, and it contains extensive domains of H3K27me3 throughout the gene locus (Makarevich et al., 2006; Zhang et al., 2007b). We found a significant level of H3K27me3 in the transcribed regions of COR15A (c1 and c2 regions) and ATGOLS3 (c region) and in the promoter region of ATGOLS3 (p region) (Figure 1b). However, H3K27me3 levels were low in the promoter region of COR15A (p region) and in the upstream intergenic region (u region) of ATGOLS3, which is consistent with the genome-wide profiling of H3K27me3 (Figure 1; Zhang et al., 2007b). We found that H3K27me3 levels were very low in the 5′ transcribed regions of the GAPDH and SAND genes (Figure 1b), genes which are ubiquitously and constitutively expressed (Czechowski et al., 2005). The heterochromatic Ta3 retrotransposon (Johnson et al., 2002) also did not contain a discernible level of H3K27me3 (Figure 1b, Ta3LTR). In order to see if the different H3K27me3 levels in the tested genes result from differences in histone density, we checked histone occupancy by using an antibody against the conserved C-terminus of histone H3. The genomic regions in GAPDH, SAND, and Ta3LTR had histone H3 levels comparable to that in FUS3, indicating that the absence of H3K27me3 in GAPDH, SAND, and Ta3LTR is not a result of lower histone H3 occupancy (Figure 1b). In contrast, relatively low levels of histone H3 were observed in the promoter and intergenic regions of COR15A and ATGOLS3 (COR15Ap, ATGOLS3p, and ATGOLS3u). It thus appeared that the two-fold smaller amount of H3 in ATGOLS3p was partially responsible for the relatively low enrichment of H3K27me3 in the region (Figure 1b).
COR15A and ATGOLS3 differ in their basal expression, but are cold-induced to a similar degree
Because H3K27me3 is thought to be a negative marker for transcription (Kohler and Villar, 2008; Schwartz and Pirrotta, 2008), we first asked if COR15A and ATGOLS3 contain H3K27me3 because these genes are expressed at a lower level compared with GAPDH and SAND. Although ATGOLS3 had the lowest expression among the four genes, the expression of COR15A was comparable to that of SAND (Figure 2a), suggesting that expression level is not the only determinant for H3K27me3 deposition. Alternatively, but not exclusively, it is possible that COR15A is silent in certain cell types where the COR15A locus is a target for H3K27me3. In contrast, we expect that SAND shows low but ubiquitous expression in most cell types because SAND is one of the best marker genes expressed independently of developmental stage and tissue type (Czechowski et al., 2005).
It has been reported that COR15A and ATGOLS3 are rapidly induced by cold exposure (Lin and Thomashow, 1992; Taji et al., 2002). We thus compared the induction kinetics of both genes up to 1 week after cold exposure (Figure 2b). We found that induction of both COR15A and ATGOLS3 was increased by about three orders of magnitude, despite their different basal expression levels (Figure 2a,b). ATGOLS3 was more rapidly induced and reached its maximum expression level in 1 day, whereas 2 days were required for COR15A to reach its maximum expression level (Figures 2b and 4b). After reaching maximum expression, both genes maintained their expression levels quite stably for up to 1 week after cold exposure (Figure 2b).
H3K27me3 decreases at COR15A and ATGOLS3 during cold-induced transcription
Because both COR15A and ATGOLS3 are modified by H3K27me3 in their genomic loci (Figure 1) and both show strong transcriptional induction upon cold exposure (Figure 2b) we investigated the fate of H3K27me3 during cold-induced transcription. In all the tested regions (regions c1 and c2 in COR15A, and regions p and c in ATGOLS3), levels of H3K27me3 decreased gradually in proportion to the exposure time to cold (Figure 3a). This differed from the levels of RNA accumulation observed for COR15A and ATGOLS3: after reaching maximum expression levels, the RNA levels did not change substantially (compare Figures 2b and 3a). A decrease of H3K27me3 can be obtained if histone occupancy is reduced during active transcription in the promoter and transcribed region. In order to check for the possibility that the decreased level of H3K27me3 was a result of reduced histone density during strong transcription in these genes, we monitored the level of histone H3 during cold-induced transcription (Figure 3a). In regions c1 and c2 in COR15A and region c in ATGOLS3, a moderate alteration in the level of histone H3 could not explain the gradual decrease in the level of H3K27me3, indicating that the change in H3K27me3 in these regions was largely independent of histone H3 occupancy (Figure 3a). In contrast, we observed a strong decrease in H3 occupancy, particularly at 1 day, in the promoter region of ATGOLS3, indicating that reduced histone occupancy plays an important role in the decrease in H3K27me3 level 1 day after cold exposure (Figure 3a). It is also notable that a decrease of H3K27me3 independent of further reduction in H3 occupancy was seen in the promoter region of ATGOLS3 (ATGOLS3p), particularly between the third and sixth days after cold exposure (Figure 3a). It thus appears that removal of H3K27me3 independent of change in histone occupancy also plays a role during prolonged cold exposure in the ATGOLS3p region.
We next asked if decrease in histone H3 density can also be observed in the promoter region of COR15A (COR15Ap) during cold-induced transcription (Figure 3b). Similar to the case of the ATGOLS3p region (Figure 3a), histone H3 occupancy was reduced 1 day after cold exposure, and a reduced histone occupancy was maintained up to 6 days (Figure 3b). When we monitored histone H3 level in the upstream intergenic region of ATGOLS3 (ATGOLS3u), histone occupancy was also reduced during cold-induced transcription (Figure 3b), suggesting that chromatin remodeling such as histone eviction and nucleosome sliding is not limited to the promoter-proximal region of ATGOLS3 (ATGOLS3p).
Cold-induced transcription of COR15A and ATGOLS3 is repressed upon return to normal growth temperature
Because levels of H3K27me3 in COR15A and ATGOLS3 decreased during cold exposure (Figure 3), we were interested in the fate of H3K27me3 when cold-exposed plants were returned to a normal growth temperature. We first investigated plant growth (Figure 4a) and RNA accumulation (Figure 4b) during cold exposure of plants and subsequent return to normal growth temperature. We exposed 11-day-old plant seedlings to cold (4°C) for 2 days and then returned the plants to normal growth temperature (Figure 4a). We also compared plant growth after cold exposure with control plants grown only at normal growth temperature (Figure 4a). Plant growth was strongly reduced upon cold exposure, and then resumed again after a return to normal growth temperature (Figure 4a). We could roughly estimate that a plant at day 14, having undergone cold exposure for 2 days, was at the stage of a 12–13-day-old plant grown at a normal growth temperature, based on the number of visible leaves and the size of the emerging leaves (Figure 4a). Except for the growth phenotype, we did not find any gross morphological defects in cold-exposed plants (Figure 4a).
We next analyzed the RNA levels of COR15A and ATGOLS3 during cold exposure and after a subsequent return to normal growth temperature (Figure 4b). After a return to normal growth temperature, the mRNA level of COR15A and ATGOLS3 reached the initial level found before cold exposure in 1 day (Figure 4b). It thus appears that a 1-day exposure to normal growth temperature is sufficient for complete repression of cold-induced induction of transcription in COR15A and ATGOLS3. Intriguingly, ATGOLS3 mRNA levels were strongly reduced even at 3 h, while COR15A was only moderately affected at the same time point (Figure 4b). In order to see whether the mRNA level of COR15A at 3 h after a return to normal growth temperature was due to active transcription or post-transcriptional control, we checked the level of non-spliced heterogeneous nuclear RNA (hnRNA), an indicator of promoter activity (Delany, 2001; Offermann et al., 2006). The COR15A hnRNA level was strongly increased within 3 h and was maintained during further exposure to cold (Figure 4b). After a return to normal growth temperature, a reduced but significant level of COR15A hnRNA was detected at 3 h, while complete abolition was achieved in 1 day (Figure 4b). It thus appears that active transcription is partially responsible for the COR15A mRNA level 3 h after a return to normal growth temperature. Complete repression of COR15A and ATGOLS3 in 1 day suggests that active decay of mRNA as well as strong transcriptional repression is necessary. Supporting this, the COR15A mRNA has an estimated half-life of about 8.4 h (Narsai et al., 2007), which is inconsistent with the three-orders of magnitude decrease of the COR15A mRNA in 24 h (Figure 4b).
The cold-induced decrease in H3K27me3 is maintained even after a return to normal growth temperature
Because cold-induced transcription of COR15A and ATGOLS3 was completely repressed by 1 day after a return to normal growth temperature (Figure 4b), we asked if the cold-induced decrease in H3K27me3 level recovers to the initial level before cold exposure, like the mRNA level does, or is maintained irrespective of transcriptional state. Cold exposure for 2 days resulted in a decrease of H3K27me3, and the decrease was maintained in plants 3 days as well as 1 day after a return to normal growth temperature (Figure 5a). This differed from mRNA accumulation, in that COR15A and ATGOLS3 were completely repressed at 1 day and were moderately elevated 3 days after a return to normal growth temperature (Figure 5b). The moderately elevated mRNA level of COR15A and ATGOLS3 at 3 days was likely to be a consequence of different developmental stages due to growth, because basal expression of these genes was slightly higher at later stages (Figure S1). Histone H3 occupancy did not change greatly throughout the experiment, except for the ATGOLS3p region (Figure 5a). Histone H3 level in the ATGOLS3p region appeared to be strictly negatively correlated with ATGOLS3 transcription, suggesting that change in histone occupancy is tightly coupled to transcriptional activity (Figure 5b). We thus conclude that the cold signal resulted in a decrease of H3K27me3 levels in COR15A and ATGOLS3, which could be maintained even after removal of the initial environmental cue.
Because H3K27me3 is thought to be a negative histone marker for transcription (Kohler and Villar, 2008; Schwartz and Pirrotta, 2008), we further examined whether the decrease in the H3K27me3 level induced by previous exposure to cold could positively affect the transcriptional induction of COR15A and ATGOLS3 upon re-exposure to cold. The results showed that there were no substantial differences in the transcriptional induction of COR15A and ATGOLS3 between previously cold-exposed (CNC) and cold-non-exposed (NC) plants (Figure 6). This indicates that the cold-induced decrease of H3K27me3 levels in COR15A and ATGOLS3 does not contribute to the transcriptional induction of the two genes upon re-exposure to cold.
Plants are sessile organisms, but they may undergo changes in physical, chemical, and biological environments, such as temperature, water, nutrition, and pathogens, throughout their life. Abnormal environmental conditions can exert deleterious effects on plant growth and development. In order to tolerate or resist the adverse environment, plants reprogram genome-wide expression profiles in which a large number of stress-responsive genes may play protective roles (Fujita et al., 2006). Therefore, transcriptional regulation plays a key role in every aspect of stress regulation and has been intensively studied (Yamaguchi-Shinozaki and Shinozaki, 2006); however, the histone modification and chromatin remodeling that are probably involved in transcription have not been well addressed in plants.
Here, we show that H3K27me3, the conserved histone marker for Polycomb group proteins in both plants and animals (Kohler and Villar, 2008), is removed in two cold-responsive genes, COR15A and ATGOLS3, during cold-induced transcription. In the transcribed regions of COR15A and ATGOLS3, H3K27me3 gradually decreases during cold-induced transcription without notable depletion of the H3 histone. In contrast, in the promoter region of ATGOLS3, a decrease of H3K27me3 is coupled with a loss of histone H3, particularly at earlier time points during cold-induced transcription. At the same time, removal of H3K27me3 independent of removal of H3 is also observed in the promoter region of ATGOLS3. Based on our results, we propose three, possibly non-exclusive, mechanisms that contribute to the removal of H3K27me3 in the promoter and transcribed regions of actively transcribed genes. First, chromatin remodeling resulting in reduced histone occupancy can be used to eliminate H3K27me3 in transcriptionally activated promoter regions (Figure S2). In general, a higher occupancy of histones in promoter regions negatively influences transcription (Workman and Kingston, 1998). Consistent with this, lower nucleosome density has often been found in the promoter regions of constitutively expressed eukaryotic genes (Barrera and Ren, 2006). In contrast, certain inducible genes contain nucleosomes in the promoter region, which should be remodeled or removed for transcriptional induction (Williams and Tyler, 2007). One of the best examples is the effect of phosphate deficiency on the PHO5 promoter in yeast, where four positioned nucleosomes are disassembled by the histone H3/H4 chaperone Asf1 (Adkins et al., 2004). Evolutionarily conserved ATP-dependent chromatin remodelers such as SWI/SNF can also change histone occupancy through histone eviction, nucleosome sliding, and other nucleosome restructuring in eukaryotic promoter regions (Kwon and Wagner, 2007; Clapier and Cairns, 2009). In pea plants, illumination results in increased accessibility of nuclease to the promoter and enhancer regions of the plastocyanin gene, suggesting that nucleosome disassembly may occur in these regions (Chua et al., 2001). Under drought conditions, histone density decreases in the promoter regions of RD20 and RAP2.4 but not in those of RD29A and RD29B in Arabidopsis (Kim et al., 2008). Here, we investigated H3 occupancy to show that chromatin remodeling resulting in reduced histone occupancy may occur in the promoter regions of ATGOLS3 and COR15A during cold-induced transcription. Second, H3K27me3 can be eliminated by a jumonji-class enzyme(s) specific for H3K27 that can be targeted to the promoter and transcribed regions (Figure S2). Two jumonji-class enzymes, UTX and JMJD3, have recently been identified as histone demethylases for both dimethylated and trimethylated H3K27 (Agger et al., 2007; De Santa et al., 2007; Hong et al., 2007; Lan et al., 2007). In Drosophila, these enzymes can be targeted to promoters and coding regions by specific transcriptional activators and the general transcription machinery (Smith et al., 2008). In humans, UTX can be found in association with Set1-like H3K4 methyltransferase complexes that are targeted to the 5′ ends of certain actively transcribed genes (Cho et al., 2007; Issaeva et al., 2007; Lee et al., 2007). Among the 21 predicted jmjC domain-containing proteins in Arabidopsis, one or more might be an enzyme(s) responsible for the demethylation of H3K27me3 (Lu et al., 2008). Third, H3K27me3 can be removed via histone replacement in the promoter and transcribed regions (Figure S2). It is known that the histone variant H3.3 replaces the canonical histone H3.1 in actively transcribed genes (Mito et al., 2005). In Arabidopsis, the histone variant H3.2 (H3.3 in animals) does not appear to contain a detectable level of H3K27me3 (Johnson et al., 2004). Therefore, we expect that the histone variant H3.2 replaces trimethylated H3.1 at Lys27 during active transcription in plants (Ahmad and Henikoff, 2002; Schwartz and Ahmad, 2005).
Our study also reveals that the decreased level of H3K27me3 following cold exposure can be maintained for up to 3 days after transcriptional attenuation. This suggests that the quantitative level of H3K27me3 can be inherited to some degree through cell division. However, the decrease in H3K27me3 alone does not appear to be sufficient for transcriptional reactivation of COR15A and ATGOLS3. One possibility is that transcriptional activation machinery is only present during cold exposure and is thus not available for re-induction of COR15A and ATGOLS3 during cold attenuation. Another possibility is that the H3K27me3 present in COR15A and ATGOLS3 cannot repress these genes directly. It has been reported that H3K27me3 is not sufficient for gene repression in Arabidopsis (Schubert et al., 2006). Actually, we show that the cold-induced decrease in the H3K27me3 level does not affect the transcriptional induction of COR15A and ATGOLS3 upon re-exposure to cold temperatures. Moreover, the promoter activity of COR15A is quite stable during cold exposure, whereas the H3K27me3 level in COR15A decreases gradually depending on the length of time for which the plants are exposed to the cold. In Arabidopsis, LHP1 binds to H3K27me3 in vitro (Zhang et al., 2007c; Exner et al., 2009) and is required for silencing of genes with H3K27me3 (Mylne et al., 2006; Sung et al., 2006; Exner et al., 2009). A recent genome-wide study of LHP1 showed that COR15A and ATGOLS3 are not targets for LHP1 binding (Zhang et al., 2007c). Rather, a subset of genes, in which H3K27me3 usually extends over a 1-kb region, serves as the target for LHP1 binding (Zhang et al., 2007c). Thus, it is likely that the extent of H3K27me3 present in COR15A and ATGOLS3 is not enough for LHP1 binding, which may be essential for silencing. In FLOWERING LOCUS C, which can be repressed by vernalization treatment, the silenced state requires a threshold level of H3K27me3 and differs from the silencing phase, where there is accumulation of inactive histone modifications such as H3K27me3 (Shindo et al., 2006). In addition, the quantitative level of H3K27me3 correlates with the natural variation in the vernalization requirement (Shindo et al., 2006). We therefore speculate that H3K27me3 levels in COR15A and ATGOLS3 represent an intermediate stage (silencing phase) along the way to the silenced state, and that transcriptional activation of these genes upon short-term cold exposure leads to a decrease in H3K27me3, thereby lowering the probability that these genes will be silenced. Similarly, we observed that the PR1, PR2, and PR5 genes, which are typical marker genes induced following pathogen stress, carry H3K27me3 but are not targets for LHP1 (Zhang et al., 2007b,c). We thus envisage that previous exposure of plants to certain environmental stresses may negatively affect the level of H3K27me3 and lower the chance of stress-responsive genes being silenced. This is likely to be beneficial for plants because they are sessile and may be exposed repeatedly to the same environmental conditions. An irreversible silencing of stress-responsive genes might hinder the rapid response of plants to adverse environmental conditions.
Arabidopsis thaliana Col-0 seeds were surface-sterilized, imbibed for 3 days at 4°C, and then grown at 22°C on solid 0.5× MS salts with a 16-h light/8-h dark cycle in a growth room. For cold treatment, 11-day-old plant seedlings were transferred at 11 a.m. to a 4°C cold chamber equipped with a continuous dim light (∼200 lux). Photographs of Arabidopsis were taken with an Olympus SZX7 microscope equipped with a DP70 camera (http://www.olympus-global.com).
RNA isolation and reverse transcription (RT)
Total RNA was extracted from whole above-ground plant tissues with an RNeasy blue kit (iNtRON Biotechnology, http://eshop.intronbio.com/). One microgram of total RNA was used in 20 μl of a cDNA synthesis reaction with ImProm-II reverse transcriptase (Promega, http://www.promega.com/) according to the manufacturer’s protocol. The resulting 20-μl reaction mixture was diluted to 240 μl, and 8 μl was used for quantitative real-time PCR analysis.
Chromatin immunoprecipitation was performed essentially as described previously (Gendrel et al., 2002) with the following modifications. Nuclear extract was obtained from whole above-ground plant tissues. Chromatin was sonicated with a BIORUPTOR (Cosmo Bio, http://www.cosmobio.co.jp/) for 3 min with a 50% duty cycle and high power output, such that we obtained 200–600 bp of fragmented chromatin DNA. Chromatin was precipitated with antibodies against the H3 C-terminus (ab1791; Abcam, http://www.abcam.com/) and H3 (trimethyl K27) (07-449; Millipore, http://www.millipore.com/). After reverse cross-linking and proteinase K treatment, input (5%) and immunoprecipitated DNA were purified with a PCR purification kit (Solgent, http://www.solgent.com/). The resulting DNA was subjected to quantitative real-time PCR analysis.
The PCR primers were designed with the primer3 program (http://frodo.wi.mit.edu/primer3/input.htm) with default parameters (oligo size, 20 nucleotides (nt) and Tm, 60°C), with an expected 0.2-kb product. p region qPCR primer set for COR15A ChIP: 5′-TGTTGGCCGACATACATTTG-3′ and 5′-TTTCAGGCCACGTGTAATCA-3′; c1 region qPCR primer set for COR15A ChIP: 5′-TGGCTTCTTCTTTCCACAGC-3′ and 5′-ATGTTGCCGTCACCTTTAGC-3′; c2 region qPCR primer set for COR15A ChIP and RT: 5′-CAGATGGTGAGAAAGCGAAAG-3′ and 5′-GACCCTACTTTGTGGCATCC-3′; u region qPCR primer set for ATGOLS3 ChIP: 5′-GGCTTTTGGCCAGTTAACAA-3′ and 5′-CCAAAGCTAATTTGCTGCTC-3′; p region qPCR primer set for ATGOLS3 ChIP: 5′-CCCTCAGCTTCTTCTTCGTG-3′ and 5′-AAGATGCCCGAAATGATGAC-3′; c region qPCR primer set for ATGOLS3 ChIP and RT: 5′-TCTTCCTGACGGCAACTTCT-3′ and 5′ GGGAGGGACAACTTTGAGTG-3′; FUSCA3 ChIP-qPCR primer set: 5′-GTGGCAAGTGTTGATCATGG-3′ and 5′-AGTTGGCACGTGGGAAATAG-3′; GAPDH (GAPC-2) ChIP-qPCR primer set: 5′-TGTTGATCTGCGATTCTTCG-3′ and 5′-CAACCAAACGACCGATTCTT-3′; SAND ChIP-qPCR primer set: 5′-ATTTCTGCAATGGCGACTTC-3′ and 5′-GTTAGCCGATGCAACCTCAT-3′; Ta3LTR ChIP-qPCR primer set: 5′-CTATCTGGCCCCAGACGTAG-3′ and 5′-CGAGTAAGCCTCGCTCTGAT-3′; COR15A hnRNA RT-PCR primer set: 5′-TGGCTTCTTCTTTCCACAGC-3′ and 5′-AAACATTGCCTGCTAAGATCC-3′; GAPDH RT-qPCR primer set: 5′-CTCTTCGGTGAGAAGCCAGT-3′ and 5′-CAGCAGCCTTGTCTTTGTCA-3′; SAND RT-qPCR primer set: 5′-CAGGCAAACCGATATATTCCA-3′ and 5′-TAGCTGAAAATCCAGCAAGC-3′. The PP2A RT-qPCR primers are as published previously (Czechowski et al., 2005).
Real-time PCR (qPCR)
Real-time PCR analysis was performed in iQ SYBR Green Supermix with an iCycler iQ5 cycler (Bio-Rad, http://www.bio-rad.com/) according to the manufacturer’s protocol. The PCR amplification was performed up to 45 cycles with 95°C, 57°C, and 72°C for 20 sec at each step. Primer specificity for PCR amplification was tested by agarose gel electrophoresis. After PCR amplification, melting curve analysis was performed to verify amplification of a single PCR product. Serial dilutions of input DNA and cDNA mixtures were used for standard curve generation and primer efficiency (E) for ChIP and RT products, respectively. The ChIP enrichment for H3K27 trimethylation and H3 was quantified by comparing the threshold cycle (CT) of the ChIP sample with that of the INPUT (5% of input) and by then normalizing the level with the FUS3 level: Es(CT of INPUT−CT of sample ChIP)/Ef(CT of INPUT−CT of FUS3 ChIP), where Es and Ef refer to the primer efficiencies (>1.8) for sample and FUS3 genomic sites, respectively. The relative mRNA levels were quantified with respect to that of PP2A (At1g13320), which is a superior reference gene that is expressed independently of developmental stages and abiotic stresses in Arabidopsis (Czechowski et al., 2005).
This work was supported in part by the biogreen 21 program (20070301034005), Rural Development Administration, Republic of Korea and by the Korea Research Foundation Grant (KRF-2008-313-C00843) funded by the Korean Government.