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HSI2/VAL1 PHD-like domain promotes H3K27 trimethylation to repress the expression of seed maturation genes and complex transgenes in Arabidopsis seedlings



The novel mutant allele hsi2-4 was isolated in a genetic screen to identify Arabidopsis mutants with constitutively elevated expression of a glutathione S-transferase F8::luciferase (GSTF8::LUC) reporter gene in Arabidopsis. The hsi2-4 mutant harbors a point mutation that affects the plant homeodomain (PHD)-like domain in HIGH-LEVEL EXPRESSION OF SUGAR-INDUCIBLE GENE2 (HSI2)/VIVIPAROUS1/ABI3-LIKE1 (VAL1). In hsi2-4 seedlings, expression of this LUC transgene and certain endogenous seed-maturation genes is constitutively enhanced. The parental reporter line (WTLUC) that was used for mutagenesis harbors two independent transgene loci, KanR and KanS. Both loci express luciferase whereas only the KanR locus confers resistance to kanamycin.


Here we show that both transgene loci harbor multiple tandem insertions at single sites. Luciferase expression from these sites is regulated by the HSI2 PHD-like domain, which is required for the deposition of repressive histone methylation marks (H3K27me3) at both KanR and KanS loci. Expression of LUC and Neomycin Phosphotransferase II transgenes is associated with dynamic changes in H3K27me3 levels, and the activation marks H3K4me3 and H3K36me3 but does not appear to involve repressive H3K9me2 marks, DNA methylation or histone deacetylation. However, hsi2-2 and hsi2-4 mutants are partially resistant to growth inhibition associated with exposure to the DNA methylation inhibitor 5-aza-2′-deoxycytidine. HSI2 is also required for the repression of a subset of regulatory and structural seed maturation genes in vegetative tissues and H3K27me3 marks associated with most of these genes are also HSI2-dependent.


These data implicate HSI2 PHD-like domain in the regulation of gene expression involving histone modifications and DNA methylation-mediated epigenetic mechanisms.


Transition from seed maturation to seed germination and seedling development involves a complex network of genetic and epigenetic mechanisms that down-regulate the expression of seed maturation genes in seedlings [1] [6]. Seed maturation is under the control of a group of transcriptional activators including LEAFY COTYLEDON1 (LEC1 [7]), LEC1-LIKE (L1L [8]), ABSCISIC ACID INSENSITIVE3 (ABI3 [9]), FUSCA3 (FUS3 [10]) and LEC2 [11], which are collectively called the “LAFL network” [3]. The B3-domain containing transcriptional repressors HIGH-LEVEL EXPRESSION OF SUGAR-INDUCIBLE GENE2 (HSI2) /VP1/ABI3-LIKE1 (VAL1) and its homolog HSI2-LIKE1 (HSL1)/VAL2 act redundantly to repress ectopic activation of embryonic traits during seed germination and seedling development by the “LAFL network” of transcriptional activators [12] [16]. HSI2 was also shown to negatively regulate the expression of β-glucuronidase (GUS) or luciferase (LUC) reporters under the control of seed-maturation specific gene promoters in transgenic Arabidopsis seedlings and vegetative organs [17],[18]. Since many of the genes repressed by HSI2 in vegetative tissues are involved in the maturation phase of seed development, including desiccation tolerance, knock-out hsi2 mutant seedlings show enhanced tolerance to water deficit whereas the overexpression of HSI2 resulted in hypersensitivity to desiccation stress [19]. Recently, it was shown that both fus3 and lec2 loss of function mutants can completely suppress the embryonic phenotype of hsi2/hsl1 double mutant seedlings, while it is partially suppressed in abi3, lec1 and l1l mutants [15]. These results indicate that HSI2 and HSL1 function redundantly to repress the expression of these regulatory genes in seedlings to prevent ectopic expression of embryonic traits during seed germination and vegetative development.

Developmental regulation of gene expression in plants is affected by chromatin mediated epigenetic mechanisms that include DNA methylation, chromatin remodeling, histone variants, and histone modifications [20],[21]. DNA methylation at the 5′position of cytosine plays important roles in transcriptional silencing of transposons, repeat sequences, transgenes and transcribed genes [22]. In addition to DNA methylation, histone modifications also play a vital role in the regulation of both transposons and transcribed genes in plants. Methylation of various lysine residues in the N-terminal tail of histone H3 is a well characterized epigenetic mechanism. In Arabidopsis, mono- (me1), di- (me2) or tri- (me3) methylation of histone H3 occurs mainly at lysine 4 (K4), lysine 9 (K9), lysine 27 (K27) and lysine 36 (K36) [23]. H3K4me3 and H3K36me3 are enriched on actively transcribed genes whereas H3K27me3 marks are associated with developmental repression of transcribed genes. H3K9me2/3 marks, which are associated with DNA methylation and small interfering RNAs (siRNAs), are enriched in heterochromatic regions known to be involved in transcriptional silencing of transposons, repeat sequences and transgenes [23],[24].

HSI2 and HSL1 proteins were predicted to contain a PHD-like domain, a B3-DNA binding domain, a conserved cysteine and tryptophan residue-containing (CW) domain and an ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif [3],[12],[14],[17],[25],[26]. Both CW and PHD protein domains are known to recognize methylated histone marks [23],[27] [30]. Hoppmann et al. [29] showed that the CW domain of HSI2 binds to H3K4me2 and H3K4me3 in vitro and, recently, it was reported that the HSL1 CW domain interacts with the histone deacetylase HDA19 to repress the “LAFL network” genes, including LEC1 and LEC2, by promoting histone deacetylation and the addition of H3K27me3 marks [31]. However, molecular and epigenetic mechanisms underlying the HSI2 PHD-like domain-mediated regulation of gene expression remain to be elucidated.

Previously, we reported a novel HSI2 allele, hsi2-4, in Arabidopsis that harbors a point mutation resulting in an amino acid substitution (C66Y) in the second zinc finger of the HSI2 PHD-like domain. The hsi2-4 mutant seedlings that carry a glutathione S-transferase F8::luciferase (GSTF8::LUC) reporter gene showed constitutively elevated transgene expression [14]. In addition to the LUC transgene, HSI2 PHD-like domain is required for the non-redundant repression of several seed-maturation genes in seedlings. These genes include those that encode both regulatory factors such as FUS3, and AGAMOUS-Like 15 (AGL15) and structural proteins that include cupin family storage protein, oleosins, late-embryogenesis-related proteins and seed storage albumins. Moreover, seed-specific genes that are de-repressed in hsi2-4 mutant seedlings are targets of H3K27me3 marks. Chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) analyses indicated that HSI2 PHD-like domain promotes H3K27me3 marks on transgene GSTF8 promoter and LUC coding sequences to repress transgene expression in parental GSTF8::LUC reporter (WTLUC) seedlings [14]. Both WTLUC and hsi2-4LUC mutant plants harbor two independent transgene loci [14]. One locus, located on chromosome IV, confers kanamycin resistance and luminescence, whereas the second locus, which is on chromosome V, confers only luminescence. Based on kanamycin sensitivity, the chromosome IV and chromosome V loci were named as KanR and KanS, respectively [14].

In this work, we show that HSI2 PHD-like domain represses LUC transgene expression from both KanR and KanS loci by promoting H3K27me3 marks but not DNA methylation and siRNA associated H3K9me2 marks. Expression of Neomycin Phosphotransferase II (NPTII) from the KanR locus is also partially suppressed in an HSI2-dependent mechanism. However, while our data indicate that DNA methylation and histone deacetylation are not involved in the transcriptional repression of transgene loci in WTLUC, the HSI2 PHD-like domain may play a role in the inhibition of seedling growth and development caused by DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-azadC).


Disruption of HSIPHD-like domain affects the expression of both KanRand KanStransgene loci

The GSTF8::LUC reporter construct contains a GSTF8 promoter sequence that controls the transcription of a luciferase expression cassette, along with an NPTII gene under control of the nopaline synthase promoter and terminator sequences, which confers kanamycin resistance in plants (Figure 1A). The parental WTLUC reporter line harbors two independent transgene insertion sites, KanR and KanS. The KanR locus was mapped to chromosome IV, while the KanS locus is located on chromosome V (Table 1) [14]. Active luciferase is expressed by both KanR and KanS loci, conferring a luminescent phenotype; however, only the KanR locus expresses NPTII; thus, plants that harbor only the KanR locus are resistant to kanamycin, while KanS plants are sensitive to this antibiotic.

Figure 1

Genomic structure of GSTF8::LUC transgene and luminescence imaging of WTLUCand hsi2-4 mutant seedlings harboring either KanRor KanStransgene locus or both. A. GSTF8::LUC transgene contain neomycin phosphotransferase (NPTII) coding sequences under the control of nopaline synthase (NOS) promoter and a modified luciferase (LUC+) coding sequences from firefly driven by glutathione S-transferase F8 (GSTF8) promoter conferring kanamycin resistance and luminescence expression respectively in plants. The 3′ ends of both NPTII and LUC+ coding sequences include NOS terminator sequences for transcriptional termination. B. Plants harboring either KanR or KanS transgene locus alone in the wild-type or in hsi2-4 mutant background were obtained by crossing of either WTLUC or hsi2-4LUC into Columbia-0 wild-type and homozygous lines were identified in F2 and F3 generations. Five days old seedlings of various genotypes grown on Murashige and Skoog media plates were imaged using cooled CCD camera after spraying with the substrate luciferin. Pseudocolor image indicates luminescence intensity from lowest (blue) to highest (white).

Table 1 Estimation of LUC copy number in Arabidopsis plants containing either Kan S or Kan R locus or both

To estimate the number of LUC copies at both KanR and KanS loci, real-time quantitative PCR (qPCR) was performed using genomic DNA from WTLUC, KanR and KanS plants. Since both KanR and KanS loci confer luminescence expression, we used PCR primers that are specific to the LUC coding sequences to estimate the copy numbers. The results show that KanS plants contain 2 copies of LUC whereas the KanR locus harbors 5 LUC copies. Independent analysis of WTLUC plants, which contain both KanR and KanS loci, showed seven copies of the LUC transgene (Table 1). Therefore, both KanR and KanS loci are complex and contain multiple copies of the GSTF8::LUC transgene.

Previously, we showed that disruption of the HSI2 PHD-like domain affects the expression of the KanR transgene locus [14] but the effect of this mutation on the KanS locus was not evaluated. Therefore, to further investigate whether the KanS transgene locus is also regulated by the HSI2 PHD-like domain mutation and investigate potential interactions between KanR and KanS transgene loci in WTLUC and hsi2-4LUC mutant plants, these two loci were separated by crossing plants of the WTLUC reporter line and the hsi2-4LUC mutant line into Col-0 wild-type Arabidopsis and subsequent selection for homozygous WTLUC and hsi2-4 lines that carry either the KanR or KanS reporter gene locus.

Comparison of luciferase expression in seedlings homozygous for the isolated KanR and KanS transgene loci in the wild-type background showed that KanR seedlings had higher luminescence signals (Figure 1B) and steady state levels of LUC mRNA (Figure 2) than KanS seedlings. This is in agreement with the relative number of transgene copies at these loci. However, in spite of carrying more luciferase transgene copies than KanR seedlings, WTLUC seedlings, showed significantly lower luminescence signal and LUC transcript levels. On the other hand, analysis of the expression of these transgenes in the hsi2-4 background showed strongly enhanced luciferase expression in all of the lines and the relative levels of both luminescence signal and LUC transcripts corresponded with transgene copy number, with highest levels seen in hsi2-4LUC seedlings and lowest levels in hsi2-4-KanS samples (Figures 1B and 2). This could indicate that, in a wild-type background, the presence of both the KanR and Kans loci may lead to stronger suppression of transgene expression but disruption of the HSI2 PHD-like domain affects the expression of LUC transgenes at both KanR and KanS loci similarly. Thus, the more complete HSI2-mediated repression of the GSTF8::LUC transgenes in WTLUC plants results in stronger relative activation of their expression in the presence of the hsi2-4 mutation.

Figure 2

Transcript levels of endogenous GSTF8 and transgenes in WTLUCand hsi2 mutants carrying either KanRor KanStransgene locus or both. Real-time reverse transcription quantitative PCR was used to determine the relative transcript levels of endogenous GSTF8, LUC and NPTII genes in five day old seedlings of various genotypes. GSTF8 produces two different transcripts with different fragment lengths by alternative start sites namely GSTF8-Long and GSTF8-Short[34]. Expression of GSTF8-Total represents transcripts from both GSTF8-Long and GSTF8-Short versions whereas GSTF8-Long expression level corresponds to GSTF8-Long transcript. EF1α was used for normalization. Data represent means (’sD) of two biological replicates with three technical replicates each. Significant differences in LUC transcript levels between the three luciferase reporter lines in the wild-type background and the respective hsi2-4 mutant background, determined using two-tailed Student’s t-test assuming unequal variances, are indicated by letters (a = p < 0.001 and b = p < 0.0001).

Alternative transcriptional start sites of the endogenous GSTF8 gene result in two different transcripts with different sizes: GSTF8-Long (GSTF8-L) and GSTF8-Short (GSTF8-S) [34]. To determine whether endogenous GSTF8 expression is altered in hsi2 mutant alleles, we performed qRT-PCR using various wild type lines (WTLUC, KanR and KanS) and hsi2 mutant lines (hsi2-2LUC, hsi2-4-KanR, hsi2-4-KanS and hsi2-4LUC). hsi2-2 is a loss-of-function mutant allele that carries a T-DNA insertion in the seventh exon of HSI2 gene (SALK_088606) [12] [14],[17]. To obtain the hsi2-2LUC line, GSTF8::LUC transgenes were introgressed into the hsi2-2 mutant background by genetic crossing. Expression of GSTF8-Total (GSTF8-T) represents both GSTF8-L and GSTF8-S transcripts whereas GSTF8-Long expression represents GSTF8-L transcripts only. As shown in Figure 2, levels of endogenous GSTF8-L and GSTF8-T transcripts were not significantly affected in hsi2-2LUC and hsi2-4LUC plants and NPTII expression was not detected in the KanS reporter line, consistent with the kanamycin sensitivity of these plants. NPTII transcripts were expressed at similar levels in the WTLUC and hsi2-4LUC seedlings but expression of NPTII in KanR seedlings was responsive to the HSI2 PHD-like domain point mutation (Figure 2). Steady-state levels of NPTII transcripts from the KanR locus were about 3-fold higher in hsi2-4 seedlings than in the wild-type background. Taken together, these results indicate that both the GSTF8::LUC and NOS::NPTII transgenes of the T-DNA cassette are partially suppressed by HSI2 in KanR seedlings; however the NPTII genes at the KanS locus may be fully silenced and/or contain loss-of-function mutations. Furthermore, in WTLUC seedlings where the KanS locus is present, expression of both LUC and the NPTII genes at the KanR is more strongly suppressed.

Luciferase expression is not affected by DNA methylation or histone deacetylation inhibitors

Complex transgenes with tandem repeats in plants are often subjected to DNA methylation and histone deacetylation mediated transcriptional gene silencing [35]. If the GSTF8::LUC transgene loci in the WTLUC plants are targets of DNA methylation, treatment of these seedlings with an inhibitor of DNA methylation should derepress the luminescence expression similar to that seen in hsi2-4 seedlings. Previous reports showed that treatment with 5 μM/mL of the DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-azadC) was effective in derepressing the transcriptional silencing of auxin-responsive ß-GUS reporter lines [35]. Treatment of Arabidopsis seedlings with 7 μM/mL 5-azadC also caused global changes in gene expression and derepression of silenced transgenes [36],[37]. To investigate whether DNA methylation is involved in repressing GSTF8::LUC transgene expression, WTLUC, hsi2-2LUC and hsi2-4LUC seedlings were grown on media containing 5-azadC at various concentrations. Luminescence expression in WTLUC seedlings was not affected at either 1 or 5 μM/mL 5-azadC concentrations (Figure 3A). Hence, DNA-methylation does not appear to be required for the repression of LUC expression in WTLUC seedlings.

Figure 3

Treatments of WTLUC, hsi2-2LUCand hsi2-4LUCseedlings with DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-azadC) and histone deacteylase inhibitor Trichostatin A (TSA). Seeds were germinated and grown vertically on media plates containing the indicated concentrations of either 5-azadC (A) or TSA (B). Luminescence imaging of 10 days old seedlings was performed using cooled CCD camera after spraying with the substrate luciferin. Pseudocolor images show luminescence intensity from lowest (blue) to highest (white). The experiment was repeated with two technical replicates and representative images are shown.

Histone deacetylation is known to regulate gene expression by transcriptional repression in eukaryotes [38]. Recently, it was reported that treatment of Arabidopsis seedlings with the histone deacetylase inhibitor trichostatin A (TSA) or down-regulation of two histone deacetylase genes, HDA6 and HDA19 by RNA interference resulted in derepression of the embryonic program in germinating seeds and seedlings [39]. Arabidopsis seedlings treated with TSA and histone deacetylase mutants mimic the phenotypes of hsi2-2/hsl1double mutant seedlings [12],[13], indicating that HSI2- and HSL1-mediated repression of the embryonic program could involve histone deacetylation. HSL1 was shown to physically interact with HDA19 via its CW domain and disruption of HSL1 resulted in increased H3K4me3 and decreased H3K27me3 marks on genes that encode transcriptional activators involved in the embryonic program [31]. To test the effects of TSA on the luminescence expression of WTLUC seedlings, WTLUC, hsi2-2LUC and hsi2-4LUC seedlings were grown on media containing 0.1 and 1 μg/mL TSA. Since higher concentrations of TSA resulted in severe growth retardation and developmental delay in all seedlings tested (Figure 3B), only, 0.1 and 1 μg/mL of TSA was used in these assays. Luminescence imaging data showed that treatments of WTLUC seedlings with TSA did not affect their luminescence expression (Figure 3B), indicating that HSI2 PHD-like domain mediated repression of LUC transgene expression in WTLUC seedlings is not dependent on TSA-sensitive histone deacetylation.

hsi2-2LUCand hsi2-4LUCmutant seedlings are partially resistant to DNA methylation inhibitor 5-azadC induced growth inhibition

We noticed that the growth and development of WTLUC seedlings on plates that contained 5 μM/mL 5-azadC was more strongly inhibited than hsi2-2LUC and hsi2-4LUC mutant seedlings (Figure 4A). To further characterize the effects of 5-azadC on hypocotyl and root growth, WTLUC, hsi2-2LUC and hsi2-4LUC seeds were germinated on media containing 0, 1, 5, 10 and 20 μM 5-azadC. After 7 days of incubation on 5-azadC-containing media, all seedlings showed dose-dependent inhibition of growth and development. However, the most severe effects were seen with WTLUC seedlings whereas the growth of hsi2-2LUC and hsi2-4LUC mutant seedlings was less inhibited (Figure 4A). While WTLUC seeds germinated when incubated on media containing 20 μM 5-azadC, subsequent root growth and cotyledon development was almost completely abrogated while both and hsi2-4LUC and hsi2-2LUC mutant seedlings continued to grow and develop, albeit slowly, under these conditions (Figure 4A and B). Comparative measurements of hypocotyl and root growth indicated that hsi2-2LUC and hsi2-4LUC mutant seedlings were about one half as sensitive to 5-azadC-dependent inhibition as WTLUC seedlings at 5, 10 and 20 μM 5-azadC treatments (Figure 4B). These data indicate that, although 5-azadC does not affect the HSI2-dependent suppression of luciferase expression in WTLUC plants, HSI2 does affect sensitivity to 5-azadC-dependent inhibition of seedling development.

Figure 4

Effects of DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-azadC) on the growth and development of WTLUC, hsi2-2LUCand hsi2-4LUCseedlings. Seeds were germinated vertically on media plates containing various concentrations of 5-azadC and pictures were taken 7 days after germination. A. Morphology of seedlings. B. Measurements of hypocotyl and root growths. Hypocotyl and root lengths were measured using ImageJ software. Data represent mean values (’sD) from 10 seedlings. The experiments were repeated with two technical replicates. Letters indicate significant differences between WTLUC and hsi2-2LUC or hsi2-4LUC at each time point (a = p< 0.005, b = p< 0.0001).

LUC and NPTIItransgene expression is associated with changes in histone methylation marks

To examine the histone methylation properties along the transgene cassette and the role of HSI2 PHD-like domain in regulating those marks and transgene expression, ChIP-qPCR analyses were performed using 5 day old seedlings of various genotypes. Antibodies specific to H3K4me3, H3K9me2, H3K27me3 and H3K36me3 marks were used, along with PCR primers that specifically amplify sequences from the endogenous (native) and transgene GSTF8 promoters and LUC and NPTII coding regions. For the specific amplifications of E1 and E2 PCR fragments only from the endogenous GSTF8 promoter sequence during ChIP-qPCR, at least one PCR primer that binds outside of the −495 bp endogenous GSTF8 region that is not part of the GSTF8::LUC transgene cassette was used. Also, to make sure the PCR products of T1 and T2 fragments are only amplified from the GSTF8 transgene promoter sequence, at least one primer that binds outside of the −495 bp region in the GSTF8::LUC transgene cassette was used ([14], Figure 5A). PCR amplification specificities of E1, E2, T1 and T2 fragments were confirmed using Col-0 wild-type and WTLUC. Among the histone methylation marks, H3K4me3 and H3K36me3 are associated with actively transcribed genes, while H3K9me2 is a repressive mark commonly enriched on transposable elements and repetitive sequences [24]. H3K27me3 is a repressive mark associated with transcribed genes that are under tissue-specific or developmental regulation [40] [42]. Preimmune immunoglobulin G (IgG) was used as a negative control for non-specific binding and all genomic DNA fragments tested show very low background levels of enrichment when chromatin samples were immunoprecipitated with IgG (Figure 5B). FUS3 was used as a positive control for H3K27me3, while actin2/7 (ACT2/7) was used as a negative control for H3K27me3 and as a positive control for H3K4me3 and H3K36me3. TA2 was used as a positive control for H3K9me2 and as a negative control for H3K4me3 and H3K36me3. In agreement with our previous report [14], chromatin from the transgene GSTF8 promoter region, and both 5′and 3′ ends of the LUC coding sequences was highly enriched in H3K27me3 marks in WTLUC seedlings (Figure 5B) while endogenous GSTF8 promoter sequences showed consistently low levels of H3K27me3 (Figure 5B). Chromatin from the 5′region of the NPTII coding sequence was also highly H3K27me3 enriched in WTLUC seedlings (Figure 5B). A substantial decrease in H3K27me3 levels was detected on chromatin from the transgene GSTF8 promoter sequences and LUC and NPTII coding sequences in hsi2-4LUC seedlings that carry a point mutation in HSI2 PHD-like domain (Figure 5B). Though the transgene sequences tested showed considerable H3K9 dimethylation, unlike H3K27me3, no significant differences in H3K9me2 enrichment were seen between chromatin from WTLUC and hsi2-4LUC seedlings at any of the sites tested (Figure 5B). Therefore, among the histone methylation marks associated with transcriptional suppression, only H3K27me3 was dependent on the HSI2 PHD-like domain.

Figure 5

Chromatin immunoprecipitation (ChIP) and quantitative PCR (qPCR) analyses of H3K4me3, H3K9me2, H3K27me3 and H3K36me3 levels on endogenous GSTF8 promoter and transgene chromatin in WTLUCand hsi2-4LUCmutant. A. Genomic structures of endogenous GSTF8 gene and GSTF8::LUC transgene showing the locations of amplified regions by ChIP-qPCR. B. qPCR analyses of chromatin samples from 5-day old seedlings of WTLUC and hsi2-4LUC that were immunoprecipitated using either specific antibodies recognizing indicated histone methylation marks or IgG (non-specific binding control). Data is expressed as percentage of immunoprecipitated DNA relative to input DNA. ACT2/7 (H3K4me3 and H3K36me3), FUS3 (H3K27me3) and TA2 (H3K9me2) serve as positive controls whereas TA2 (H3K4me3 and H3K36me3) and ACT2/7 (H3K9me2 and H3K27me3) were used as negative controls. Data represent means (’sD) from two biological replicates with three qPCR replicates each. Significant differences in enrichment between WTLUC and hsi2-4LUC for each genomic region tested were determined using two-tailed Student’s t-test assuming unequal variances and P values are indicated by letters (a = p < 0.05, b = p < 0.005, c = p < 0.0005, d = p < 0.0001).

Histone methylation marks H3K4me3 and H3K36me3, which are associated with chromatin from actively transcribed genes, were enriched at all of the transgene sequences assayed in hsi2-4LUC seedlings, relative to WTLUC (Figure 5B). These marks were particularly abundant at the proximal transgene GSTF8 promoter and 5′LUC coding sequences but significant enrichment was also seen at the endogenous GSTF8 promoter and NPTII coding sequence.

To examine whether the hsi2-4-dependent changes in histone methylation marks are associated with both the KanR and KanS transgene loci, ChIP-qPCR analyses were performed on various regions of the endogenous GSTF8 gene and the GSTF8::LUC transgene in KanR, KanS, hsi2-4-KanR and hsi2-4-KanS seedlings (Figure 6). As in chromatin from WTLUC seedlings, higher levels of H3K27me3 marks at transgene GSTF8 promoter sequences and at LUC and NPTII coding sequences were detected in wild-type seedlings carrying either the KanR or KanS transgene locus than in corresponding hsi2-4-KanR or hsi2-4-KanS mutant seedlings. Thus, the significant decrease in H3K27me3 levels at the GSTF8::LUC transgene associated with homozygosity for the hsi2-4 allele was seen at both insertion sites. While chromatin from transgene sequences generally had higher levels of H3K9me2 marks than the endogenous GSTF8 gene, no significant change was seen between these genotypes.

Figure 6

ChIP-qPCR analyses of H3K4me3, H3K9me2, H3K27me3 and H3K36me3 enrichments on endogenous GSTF8 promoter and GSTF8::LUC transgene regions in WTLUCand hsi2-4 carrying either KanSor KanRlocus. Chromatin samples were prepared using five day old seedlings from various genotypes. ChIP-qPCR data represents mean values (’sD) of three PCR reactions obtained from each of two independent immunoprecipitations. Significant differences between wild-type lines and corresponding mutant lines (KanS versus hsi2-4-KanS, KanR versus hsi2-4-KanR) were determined using two-tailed Student’s t-test assuming unequal variances and P values are indicated by letters (a = p < 0.05, b = p < 0.005, c = p < 0.0005, d = p < 0.0001).

Enrichment of H3K4me3 and H3K36me3 was seen in chromatin at both KanR and KanS loci in hsi2-4 seedlings (Figure 6). This enrichment was most pronounced at LUC coding sequences rather than in promoter regions and significant enrichment was also seen in chromatin of the NPTII gene at the KanR locus. Therefore, disruption of HSI2 PHD-like domain resulted in increased activation marks on 5′and 3′ end of LUC coding sequences in both KanR and KanS backgrounds. However, increased H3K36me3 marks on the NPTII coding sequences were observed only in hsi2-4-KanR seedlings (Figure 6).

H3K27me3 levels are significantly decreased on a subset of seed-maturation genes in hsi2LUCmutant seedlings

Some members of the LAFL clade of regulatory genes that control the expression of seed maturation genes [3] are misregulated in hsi2 mutant seedlings [14]. LEC1 and ABI3 are ectopically expressed in hsi2-2 but not in hsi2-4 seedlings, while FUS3 is upregulated in both hsi2-2 and hsi2-4 lines [14]. These results suggested to us that the HSI2-dependent negative regulation of LEC1 and ABI3 in seedlings does not require the PHD-like domain, while suppression of FUS3 could be dependent on the PHD-like domain of HSI2. To determine if correlations exist between these expression patterns and histone modifications, ChIP-qPCR analysis of these genes was carried out on chromatin samples from WTLUC, hsi2-2LUC and hsi2-4LUC seedlings using antiH3K27me3 (Figure 7). Consistent with our hypothesis, significant reductions in H3K27me3 chromatin marks were detected in association with ABI3 and LEC1 genomic sequences only in chromatin from hsi2-2LUC but not hsi2-4LUC mutant seedlings and genes such as LEC2 and L1L, which are not misregulated in either hsi2 mutant allele alone, also showed no change in H3K27me3 marks in these mutant backgrounds. However, H3K27me3 marks associated with FUS3 sequences were not altered in either mutant background. Thus, the effects of HSI2 on FUS3 expression do not appear to depend on alterations in H3K27me3. On the other hand, the enrichment of H3K27me3 marks detected on both 5′and 3′ coding sequences of AGL15 (At5g13790) in WTLUC seedlings was significantly decreased in both hsi2-2LUC and hsi2-4LUC backgrounds, which is consistent with the increased expression of AGL15 in these mutants [14]. Therefore, the HSI2 PHD-like domain does appear to be required for both the repressed expression and H3K27 hypermethylation of the AGL15 locus.

Figure 7

ChIP-qPCR analyses of H3K27me3 levels on the master transcriptional regulators of seed-maturation program and seed maturation related target genes of HSI2 PHD-like domain in WTLUCand hsi2LUCmutant seedlings. Chromatin samples were prepared using five day old WTLUC, hsi2-2LUC and hsi2-4LUC seedlings. Immunoprecipitations were performed using either IgG (non-specific binding control) or anti-H3K27me3 antibody. Data represent means (’sD) obtained from three qPCR reactions each from three independent immunoprecipitations from three different biological replicates. Significant differences in H3K27me3 enrichments between WTLUC and hsi2LUC mutants (WTLUC versus hsi2-2LUC, WTLUC versus hsi2-4LUC) are indicated by letters (a = p < 0.05, b = p < 0.005, c = p < 0.0005, d = p < 0.0001). ABSCISIC ACID-INSENSITIVE3 (ABI3, At3g24650), FUSCA3 (FUS3, At3g26790), LEAFY COTYLEDON1 (LEC1, At1g21970), LEAFY COTYLEDON2 (LEC2, At1g28300), LEC1-LIKE (L1L, At5g47670), AGAMOUS-Like 15 (AGL15, At5g13790), At2g41260 (Late-embryogenesis-abundant protein), At3g22640 (cupin family seed storage protein), At1g04660 (glycine-rich protein), At2g29370 (NAD(P)-binding Rossmann-fold superfamily protein), At5g45830 (DOG1, DELAY OF GERMINATION1), At2g34700 (putative proline-rich glycoprotein).

As previously reported, a number of seed maturation-related structural genes are derepressed in hsi2-4 mutant seedlings [14]. To investigate whether the increased expression of these genes in hsi2 mutant seedlings correlates with changes in associated H3K27me3 marks, ChIP-qPCR analyses were performed on chromatin from WTLUC, hsi2-2LUC and hsi2-4LUC mutant seedlings using primers specific to At2g41260 (Late-embryogenesis-abundant (LEA) protein), At3g22640 (cupin family seed storage protein), At1g04660 (glycine-rich protein), At2g29370 (NAD(P)-binding Rossmann-fold superfamily protein), At5g45830 (Delay of Germination1, DOG1) and At2g34700 (proline-rich glycoprotein). These seed maturation genes were previously reported to be targets of H3K27 hypermethylation [41] and our results confirmed that H3K27me3 marks were enriched, relative to IgG, on the 5′coding sequences of these genes (Figure 7). In comparison to WTLUC, significant decreases in H3K27me3 enrichments were detected in both hsi2-2LUC and hsi2-4LUC mutant seedlings on chromatin associated with 5′genomic sequences of DOG1 and At2g34700, and at both 5′and 3′ sequences of At3g22640 (Figure 7). On the other hand, H3K27me3 levels on At1g04660 and At2g29370 were significantly decreased only in the hsi2-2LUC background and not in chromatin from hsi2-4LUC seedlings (Figure 7). These data indicate that, as with the regulatory genes described above, derepression of seed maturation-specific gene expression in hsi2 mutant seedlings often corresponds with decreased accumulation of H3K27me3 marks that may or may not depend on the presence of an intact HSI2 PHD-like domain. However, as with FUS3, the LEA-like protein gene At2g41260, which is expressed at elevated levels in both hsi2-2 and hsi2-4 mutant plants [14], is strongly enriched for H3K27me3 but these marks are not significantly reduced in either hsi2 mutant.


Despite detailed genetic and functional characterization, the molecular mechanisms that underlie HSI2- and HSL1-mediated repression of seed maturation program in seedlings are still not fully understood [12] [16]. HSI2 contains a PHD-like domain [3],[12],[14],[26] and PHD domains can act as “readers” of the histone methylation status of target genes to regulate their expression [30]. Through characterization of a novel mutant allele, hsi2-4, which affects the expression of GSTF8::LUC transgenes and certain seed maturation genes [14], we provide evidence that the HSI2 PHD-like domain is involved in regulating the expression of some genes by altering histone modifications.

Quantitative PCR data indicates that both KanR and KanS transgene loci in the WTLUC reporter gene line contain multiple copies of the GSTF8::LUC transgene. The KanR locus is more complex than KanS, harboring five copies of the transgene, while the KanS locus includes two (Table 1). Transgene loci in plants that harbor multiple and complex transgene repeats at a single locus were frequently targeted by DNA methylation-associated H3K9me2 marks and also histone deacetylation mediated transcriptional gene silencing [35],[43] [45]. However, treatment of WTLUC seedlings with either the DNA methylation inhibitor 5-azadC or the histone deacetylase inhibitor TSA failed to derepress the LUC expression in WTLUC seedlings (Figure 3A and B). Also, ChIP-qPCR analyses showed no differences in DNA methylation-associated H3K9me2 histone methylation marks on transgene sequences between WTLUC and hsi2-4 mutants that harbor either individual KanR and KanS loci or both (Figures 5B and 6). Based on these data, it appears that DNA methylation and histone deacetylation mechanisms are not involved in HSI2 PHD-like domain-mediated repression of transgene expression in WTLUC seedlings.

Although 5-azadC did not affect LUC expression in WTLUC seedlings (Figure 3A), hsi2 mutant seedlings treated with various concentrations of 5-azadC maintained root and hypocotyl growth better than WTLUC seedlings under the same conditions (Figure 4A and B). These results could indicate that HSI2 is somehow involved in the inhibition of seedling growth and development caused by 5-azadC and, since this effect was observed with both the PHD-like domain mutant allele hsi2-4 and the hsi2-2 T-DNA knock-out allele, it appears that the PHD-like domain may be required for this 5-azadC-dependent inhibition of growth. LEC1, an HSI2 and HSL1 target gene and member of the “LAFL network”, was shown to be regulated by DNA methylation [46],[47] and the embryonic phenotypes of gain-of-function lec1 mutants were enhanced by treatment with a DNA methylation inhibitor 5-azacytidine [46]. Hence, the partial rescue of seedling growth in hsi2 mutants in the presence of 5-azadC could be an indirect effect of changes in the DNA methylation status of HSI2-targeted regulatory genes, including LEC1.

Data presented here clearly indicate that the HSI2 PHD-like domain is involved in suppressing the expression of GSTF8::LUC transgenes in both KanR and KanS transgene loci (Figure 2). Furthermore, the levels of LUC expression seen in hsi2-4 plants that carry these reporter complexes correlate with transgene copy number, with relatively low levels of LUC expression seen in hsi2-4, KanS plants that contain two GSTF8::LUC copies and correspondingly higher levels expression in hsi2-4, KanR or hsi2-4LUC lines with five and seven total reporter gene copies, respectively. The direct correlation between derepressed LUC expression and the number of reporter gene copies means that the luminescence of mutant plants with compromised HSI2-dependent repression will be amplified in a high copy number reporter gene line, making these mutants far more apparent in a luminescence-based mutant screen. On the other hand, LUC expression in wild-type plants that carry the KanR locus alone showed higher levels of LUC transcripts than did WTLUC seedlings (Figure 2). These results appear to indicate that the presence of both KanR and KanS loci in the same genome may lead to stronger transcriptional suppression of the GSTF8::LUC reporter genes than when the KanR locus is present alone. No NPTII transcripts were detected in either KanS or hsi2-4-KanS seedlings (Figure 2), which is consistent with the kanamycin sensitivity of these plants. However, hsi2-4-KanR seedlings showed 3-fold higher expression of NPTII transcripts relative to wild-type KanR seedlings but this was not seen in corresponding hsi2-4LUC and WTLUC seedlings (Figure 2). Thus, as with the LUC reporter gene, the co-existence of KanR and KanS loci is associated with stronger transcriptional repression of NPTII gene expression. To better understand the function of the HSI2 PHD-like domain, interactions between activation-associated and repressive histone methylation marks at the KanR and KanS transgene loci were evaluated by ChIP-qPCR assays. Transgene GSTF8 promoter sequences, along with LUC and NPTII coding sequences were highly enriched in H3K27me3 marks in WTLUC seedlings (Figures 5B and 6) and significantly lower amounts of H3K27me3 were observed on these transgene sequences in the hsi2-4 mutant background (Figure 5B). Similar histone modification patterns were observed in seedlings harboring individual KanR or KanS loci (Figure 6). Thus, the PHD-like domain of HSI2, which is required to repress the expression of these transgene complexes, is also necessary for the appearance of H3K27me3 marks on these loci. In contrast, H3K4me3 and H3K36me3 histone methylation marks, which are associated with active gene expression and have been shown to inhibit H3K27me3 marks on transcribed genes in both animals and plants [48] [50], were enriched on these transgene sequences in hsi2-4 seedlings, relative to those with the wild-type HSI2 allele. Thus, the decrease in H3K27me3 marks on transgene sequences in both KanR and KanS loci in the hsi2-4 mutant background is associated with both increased expression (Figure 2) and increased accumulation of H3K4me3 and H3K36me3 marks (Figures 5B and 6). Developmental repression of transcribed genes is often associated with H3K27me3 marks [41],[42] but emerging evidence also suggests that H3K27me3 may act as an alternative to DNA methylation-associated H3K9me2 in transposable elements and repetitive sequence silencing [40],[51] [54]. Turck et al. [40] showed that the chromodomain-containing H3K27me3 “reader” protein LHP1 (LIKE HETEROCHROMATIN PROTEIN 1) is enriched on tandemly duplicated genes, such as the nine closely linked chitinase/glucosylase-18 genes (At4g19720-At4g19820) on chromosome 4 of Arabidopsis, but not on segmentally duplicated genes. Expressed genes that flank tandemly duplicated gene loci are also not associated with LHP1. Both KanR and KanS loci contain multiple GSTF8::LUC transgenes at individual loci (Table 1) and expression of genes that flank the KanR transgene locus does not differ between WTLUC and hsi2-4LUC seedlings [14]. However, similarities in HSI2-dependent H3K27me3 accumulation at the KanR and KanS loci and the corresponding relative changes in LUC expression in hsi2-4 plants lead us to speculate that transcriptional repression is mediated by the GSTF8::LUC transgene itself and is not dependent on tandem T-DNA insertions. The presence of multiple transgene copies results in high levels of expression that accentuates the apparent repressive effect of HSI2 when its activity is compromised by mutation. However, since LUC expression in WTLUC seedlings is lower than in KanR seedlings, the presence of these two unlinked loci appears to have synergistic effects on reporter gene silencing.

In contrast to the GSTF8::LUC reporter genes, expression of native GSTF8 transcripts derived from either the “long” or “short” transcriptional start sites show no significant increase in the hsi2-4LUC mutant background (Figure 2). The most parsimonious explanation for the discrepancy between native GSTF8 expression and the expression of the GSTF8::LUC reporter gene is that the isolated GSTF8 promoter sequence used in the GSTF8::LUC gene construct, which corresponds with the short promoter as defined by Thatcher et al. [34], could contain cis-acting suppressor elements that are masked in the context of the native gene. Support for this explanation can be seen in Figure 4B of Veerappan et al. [14]. In this experiment, LUC expression (measured as luminescence) was assayed in WT Col-0 and mutant hsi2-4 Arabidopsis plants newly transformed with either short-GSTF8::LUC or long-GSTF8::LUC gene constructs (Col-S, Col-L and hsi2-4-S and hsi2-4-L, respectively). No significant differences in luminescence were apparent between Col-L and hsi2-4-L plants but LUC expression driven by the short GSTF8 promoter in hsi2-4-S plants was substantially elevated relative to that detected in Col-S plants.

Polycomb group (PcG) proteins are evolutionarily conserved multi-protein complexes required for developmental repression of gene expression by chromatin based mechanisms. PcG proteins in plants comprise of two major complexes: Polycomb Repressive Complex 1 (PRC1) and PRC2 [4] [6],[55]. Arabidopsis PRC1 proteins BMI1 and RING1 were shown to have histone H2A mono ubiquitination (H2Aub) activity in vitro[16],[56],[57], whereas PRC2 complex proteins catalyze the deposition of H3K27me3 marks to promote developmental repression in animals and plants [58] [60].

The GSTF8 promoter sequence used in the GSTF8::LUC reporter contains an octopine synthase (OCS) sequence element at −460 (Additional file 1) that is known to be required for transcriptional activation in response to a variety of biotic and abiotic stress signals [61]. This OCS element is flanked by OCS element binding factor 5 (OBF5)and OCS element binding proteins 1 (OBP1) elements that were shown to bind proteins of the DNA binding with One Finger (DOF) family of transcription factors and are reported to act either as positive or negative regulatory factors in various plant genes [62]. A putative myeloblastosis2 (MYB2) binding element is also located at −311 but its potential function is unknown. Sequence elements with a potential role in PRC2-based silencing can also be identified in the GSTF8 promoter. By analyzing the co-distribution of the ubiquitous Arabidopsis PRC2 protein FERTILIZATION INDEPENDENT ENDOSPERM (FIE) and H3K27me3 marks, Deng et al. [63] identified four sequence motifs that could act as PRC2 binding sites. Two of these motifs are found in the GSTF8 promoter, a putative GAGA box located between −123 and −131 relative to the transcription start site and a TTC repeat element located in the 5′untranslated region between +36 and +56. GAGA elements were found to be specifically associated with FIE and H3K27me3 enriched sites and these sequences are also found in polycomb response elements (PREs) of Drosophila [63]. However, while GAA (reverse complement of TTC) sequence motifs were found to be associated with genomic regions that bind both FIE and H3K27me3, they were also enriched in random promoter sequences and were, therefore, not considered to be specific PRC2 binding sites [63].

The role of the GAGA element in the transcriptional regulation of the PRC2-repressed LEC2 gene was confirmed by Berger et al. [64]. However, mutational analysis showed that, in this context, the GAGA element acted as a required cis-activating element, which was associated with a distinct cis-repressing element, termed repressive LEC2 element (RLE) that apparently consists of two component sequences. Comparison of the LEC2 RLE sequence with the GSTF8 promoter identified a duplicated element identical to the 5′component of the LEC2 RLE. As in the LEC2 gene, the putative GSTF8 RLE-like sequence is located immediately downstream of the GAGA element (Additional file 1). Whether this putative GAGA-RLE motif plays a role in the transcriptional regulation of the GSTF8 promoter in a transgene context is not known.

ChIP-qPCR analyses were carried out to investigate whether LAFL network genes and other seed maturation genes that are up-regulated in hsi2 mutant seedlings are also associated with HSI2-dependent changes in H3K27me3 marks. With the exception of L1L, all the tested seed maturation genes are enriched with H3K27me3 marks in WTLUC seedlings (Figures 7) in agreement with previous reports [41],[58]. Significant reductions in H3K27me3 levels, relative to WTLUC seedlings, were observed at some gene loci in both hsi2-2LUC and hsi2-4LUC seedlings, while other genes showed reductions only in hsi2-2LUC seedlings and a few showed no changes in either hsi2 mutant background. In general, these HSI2-dependent differences in H3K27me3 marks correlate well with HSI2-dependent changes in gene expression. For example, among the regulatory genes tested, ABI3 and LEC1, which are expressed at elevated levels in hsi2-2 seedlings but not in hsi2-4 seedlings [14], showed correspondingly decreased levels of H3K32me3 enrichment in chromatin from hsi2-2LUC plants but not from hsi2-4LUC plants (Figure 7). On the other hand, H3K27me3 marks on AGL15 were strongly decreased in both hsi2-2LUC and hsi2-4LUC mutant plants (Figure 7) and expression of this gene is also upregulated in both hsi2 mutant backgrounds. These results can be interpreted to indicate that HSI2-dependent transcriptional repression and H3K27 hypermethylation of AGL15 is mediated by the PHD-like domain while that of ABI3 and LEC1 may be mediated by other HSI2 domains, such as the CW domain, which was shown to interact with HDA19 to promote histone deacetylation and H3K27me3 marks to repress seed maturation genes [31].

Similar patterns can be seen in the structural (non-regulatory) seed maturation gene sample. The putative glycine-rich protein gene (At1g04660) is more strongly expressed in hsi2-2LUC plants than in hsi2-4LUC plants [14] and H3K27me3 marks at this site are correspondingly reduced in hsi2-2LUC but not hsi2-4LUC mutants. On the other hand, genes for a cupin-like protein (At3g22640), DOG1 (At5g45830) and a proline-rich glycoprotein (At2g34700) are similarly up-regulated in hsi2-2LUC and hsi2-4LUC plants and decreased H3K27me3 marks are also apparent in both mutant lines.

Two of the genes in our sample group do not show correlations between HSI2-dependent transcriptional repression and H3K27me3 marks. These genes, FUS3 and the LEA protein gene At2g41260, are expressed at elevated levels in both hsi2-2LUC and hsi2-4LUC backgrounds and accumulate substantial H3K27me3 in WTLUC plants but these marks are not diminished in either hsi2 mutant. Therefore, derepression of these genes in hsi2 mutant plants does not appear to require the depletion of H3K27me3. It seems likely that, in addition to the PRC2-mediated accumulation of H3K27me3 marks, other mechanisms are involved in the repression of these genes.

AGL15 is a member of the MIKC subfamily of MADS domain transcription factors that is preferentially expressed in developing embryos. Ectopic overexpression of AGL15 results in enhanced somatic embryogenesis [65],[66]. AGL15 acts upstream of LAFL network genes and several LAFL genes are direct regulatory targets of AGL15 [67],[68]. DOG1 is a seed-specific gene and plays a critical role in promoting seed dormancy by integrating environmental signals [69],[70]. Recently, AGL15 and DOG1 were shown to be targets of H3K27me3 marks, which could be mediated by PRC1 proteins [58],[71]. Our data shows that HSI2 regulates the expression of AGL15 and DOG1 in seedlings by promoting H3K27me3 marks possibly via PRC1-PRC2 complex which requires HSI2 PHD-like domain.

HSI2 was shown to directly interact with PRC1 complex proteins AtBMI1A/B/C and is required for the deposition of H2Aubi and H3K27me3 marks on “LAFL network” seed-maturation genes including LEC1, FUS3 and ABI3[16]. Disruption of PRC2 complex genes in Arabidopsis led to decreased H3K27me3 levels, activation of “LAFL network” transcription factor genes and ectopic expression of embryonic traits during seed germination and vegetative development [58],[72],[73]. Thus, it is possible that HSI2 interacts with PRC1 proteins like AtBMI1 to recruit PRC2 proteins such as the histone methyltransferase CURLY LEAF (CLF), to deposit H3K27me3 marks on the GSTF8::LUC transgene loci. AtRING1a was also shown to physically interact with the PRC2 core component CLF [74] and several reports have demonstrated the involvement of PHD-PRC2 and PHD-PRC1-PRC2 complexes in deposition of H3K27me3 marks to promote transcriptional repression of gene expression in plants [16],[71],[75]. Similarly, HSI2 could be part of a repression complex that involves the HSI2 PHD-like domain, PRC1 and PRC2 complex proteins to promote high levels of H3K27me3 marks on native seed maturation genes and GSTF8::LUC transgene loci to repress their expression during the seed to seedling developmental phase transition.


HSI2 contains a putative PHD domain, which could act as a “reader” of histone methylation marks. In this work, we show that HSI2 PHD-like domain regulates both LUC and NPTII transgenes from two independent transgene loci. Transcriptional repression of both of these transgene loci by HSI2 PHD-like domain is associated with repressive histone methylation marks H3K27me3 but not siRNA and DNA methylation associated H3K9me2 marks. In addition to the transgenes, HSI2 is also required for the repression of a subset of seed maturation genes in seedlings by promoting H3K27me3 marks in a PHD-like domain dependent and independent manner.


Plant materials, growth conditions and chemical treatments

Arabidopsis thaliana Columbia-0 (Col-0; CS60000) wild-type was obtained from Arabidopsis Biological Resources Center. WTLUC and hsi2-4LUC, which contain both KanR and KanS transgene loci in Col-0 background were described before [14]. The other genotypes that were used in this study including KanR, KanS, hsi2-4-KanR and hsi2-4-KanS were obtained by crossing the WTLUC and hsi2-4LUC into the Col-0 wild-type. The hsi2-2LUC line harbors GSTF8::LUC transgenes in the HSI2 T-DNA knock-out allele hsi2-2 (SALK_088606) background. For all the experiments described here, plants were grown under continuous illumination at 24°C on 0.3% Phytagel plates containing 0.5X Murashige and Skoog (MS) salt, 0.5 g/L MES (2-(N-morpholino) ethanesulfonic acid, 1X Gamborg vitamin mix and 1 % sucrose (pH adjusted to 5.7). 5-Aza-2′-deoxycytidine (5-azadC, A3656; Sigma) and Trichostatin A (T8552; Sigma) stocks were prepared using dimethyl sulfoxide and methanol respectively, and added directly to the MS media plates. Hypocotyl and root length measurements were made using Image J software ( Digital photos of seedlings grown on 5-azadC plates were taken along with a ruler of known length. Hypocotyl length was measured from the tip of the apical meristem to the junction between hypocotyl and root, while root length was measured from the hypocotyl/root junction to the tip of the primary root.

Luminescence imaging, genetic crosses and genotyping

Luminescence imaging was performed using Andor iKON-M DU934N-BV CCD camera (Andor Technology). After spraying with 1 mM D-luciferin potassium salt (Gold Biotechnology) containing 0.01% Triton X-100 solution, seedlings were kept in the dark for 5 minutes and imaging was performed with a 5 minute exposure. Andor SOLIS (I) imaging software (Andor Technology) was used for the acquisition of luminescence images and processing. To separate KanR and KanS loci, WTLUC and hsi2-4LUC mutant were crossed into Col-0 wild-type plants. Successful crosses were identified based on luciferase imaging in the F1 generation and plants were allowed to self-pollinate. Progeny lines homozygous for the KanS locus were identified based on kanamycin sensitivity whereas plants homozygous for the KanR locus were identified by PCR genotyping using T-DNA and genomic primers. To genotype hsi2-4 mutation, a previously described CAPS marker [14] was used.

Preparation of total RNA, cDNA synthesis and real-time reverse transcription quantitative PCR

Total RNA extraction and real-time reverse transcription quantitative-PCR (RT-qPCR) analysis was performed as described in Veerappan et al. [14]. Primers used in RT-qPCR are listed in Additional file 2.

Estimation of LUCtransgene copy numbers by real-time quantitative PCR

To determine the copy numbers of LUC transgenes in WTLUC, KanR and KanS lines, real-time quantitative PCR (qPCR) was performed as described before [32],[33]. All qPCR reactions were performed using AB StepOnePlus Real-Time PCR System (Applied Biosystems) in 10 μl volume containing different amounts of DNA, 0.2 μM of each primers and 5 μl iTaqTM SYBR green supermix (Bio-Rad). Several sets of primers were tested for optimal performance. Temperature cycling conditions were 95°C for 10 minutes, 40 cycles for 15 seconds at 95°C and 1 minute at 60°C. Each DNA sample was tested in triplicates with three different DNA concentrations. Calibration curves were also performed in triplicates with five different DNA concentrations. Ct values were calculated using StepOne Software v2.1 (Applied Biosysytems). Concentrations of DNA samples were measured using Nanodrop 2000 (Thermoscientific) and the exact copy numbers of the template genome in the reactions were calculated using the following website:, applying the formula: number of copies = (amount * 6.022×1023)/(length * 1×109 * 650). The calibration curves for LUC were created using the plasmid DNA pBI121-GSTF8::LUC as a template. At5g47480, a single copy gene from Arabidopsis, was used as an internal control for normalization of the data. PCR primers used in the estimation of transgene copy numbers can be found in Additional file 2.

Chromatin immunoprecipitation and quantitative PCR analyses

Chromatin immunoprecipitation (ChIP) and quantitative PCR (qPCR) analyses were performed as described by Veerappan et al. [14]. Percentage of immunoprecipitated DNA relative to the total chromatin input was calculated for various samples using qPCR. Antibodies used for ChIP: normal rabbit IgG (Millipore, 12–370), anti-H3K4me3 (Millipore, 07–473), anti-H3K9me2 (Abcam, ab1220), anti-H3K27me3 (Millipore, 07–449) and anti-H3K36me3 (Abcam, ab9050). Primers used for ChIP PCR analyses are listed in Additional file 2.

Authors’ contributions

VV designed and performed genetic crosses, luminescence imaging, qRT-PCR, DNA methylation and histone deacetylase inhibitor experiments, chromatin immunopreciptation and qPCR analyses. NC and AR designed and performed the estimation of LUC copy numbers in various GSTF8::LUC transgene reporter lines. VV and RDA coordinated all the experiments, wrote and edit the manuscript. All authors read and approved the final manuscript.

Additional files



High-level expression of sugar inducible gene2






Plant homeodomain






:LUC: Glutathione S-transferase F8::luciferase


Neomycin phosphotransferase II


Leafy cotyledon1


Leafy cotyledon2




Abscisic acid insensitive3








Cysteine and tryptophan residue-containing


Ethylene-responsive element binding factor-associated amphiphilic repression


Small interfering RNAs


Chromatin immunoprecipitation


Quantitative PCR


Wild-type luciferase


Kanamycin resistant


Kanamycin sensitive








Trichostatin A




Immunoglobulin G


Trimethylation of histone H3 at lysine 27


Trimethylation of histone H3 at lysine 4


Trimethylation of histone H3 at lysine 36


Dimethylation of histone H3 at lysine 9


Late embryogenesis abundant


Delay of germination1


Like heterochromatin protein1


Polycomb group


Polycomb repressive complex 2


Polycomb repressive complex 1


H2A mono ubiquitination


Octopine synthase


OCS element binding factor 5


OCS element binding proteins 1


DNA binding with one finger


Fertilization independent endosperm




Polycomb response elements


Repressive LEC2 element


Curly leaf


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The authors would like to thank Drs. Mohamed Fokar, Miyoung Kang and Million Tadege, for critically reading and providing helpful comments on the manuscript. We also thank Ms. Katie Pranger for her careful editing. This work was supported by the Oklahoma Agricultural Experiment Station, a grant from the Samuel Roberts Noble Foundation and an endowment from the Walter Sitlington Foundation to RDA.

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Correspondence to Randy D Allen.

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Additional file 1: Analysis of GSTF8 promoter sequence for the identification of putative cis -elements.(PDF 138 KB)

Additional file 2: List of PCR primers used in this study.(PDF 66 KB)

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  • HSI2
  • VAL1
  • AGL15
  • DOG1
  • Transgene silencing
  • Seed-maturation
  • DNA methylation
  • Histone methylation
  • H3K27me3
  • 5-aza-2′-deoxycytidine