Transcriptional silencing of 35S driven-transgene is differentially determined depending on promoter methylation heterogeneity at specific cytosines in both plus- and minus-sense strands
- 187 Downloads
De novo DNA methylation triggered by short interfering RNAs is called RNA-directed DNA methylation (RdDM). Transcriptional gene silencing (TGS) through RdDM can be induced using a viral vector. We have previously induced RdDM on the 35S promoter in the green fluorescent protein (GFP)-expressing Nicotiana benthamiana line 16c using the cucumber mosaic virus vector. The GFP fluorescence phenotype segregated into two types, “red” and “orange” in the first self-fertilized (S1) progeny plants by the difference in degree of recovery from TGS on GFP expression. In the second self-fertilized generation (S2 plants), the phenotypes again segregated. Explaining what generates the red and orange types could answer a very important question in epigenetics: How is the robustness of TGS maintained after RdDM induction?
In bisulfite sequencing analyses, we found a significant difference in the overall promoter hypermethylation pattern between the red and orange types in S1 plants but little difference in S2 plants. Therefore, we assumed that methylation at some specific cytosine residues might be important in determining the two phenotypes. To find the factor that discriminates stable, robust TGS from the unstable TGS with incomplete inheritance, we analyzed the direct effect of methylated cytosine residues on TGS. Because it has not yet been demonstrated that DNA methylation at a few specific cytosine residues on known sequence elements can indeed determine TGS robustness, we newly developed a method by which we can directly evaluate the effect of specific methylation on promoter activity. In this assay, we found that the effects of the specific cytosine methylation on TGS differed between the plus- and minus-strands.
We found two distinct phenotypes, the stable and unstable TGS in the progenies of virus-induced TGS plants. Our bisulfite sequencing analyses suggested that methylation at some specific cytosine residues in the 35S promoter played a role in determining whether stable or unstable TGSs are induced. Using the developed method, we inferred that DNA methylation heterogeneity in and between the plus- and minus-strands can differentially determine TGS.
KeywordsCaMV 35S promoter Nicotiana benthamiana RNA-directed DNA methylation VITGS
Apple latent spherical virus
Cucumber mosaic virus
DOMAINS REARRANGED METHYLTRANSFERASE2
double stranded RNA
Green fluorescent protein
NUCLEAR RNA POLYMERASE E1
Post-transcriptional gene silencing
RNA directed DNA methylation
RNA dependent RNA polymerase 2
short interfering RNA
Transcriptional gene silencing
Virus-induced transcriptional gene silencing
DNA methylation is a highly conserved epigenetic hallmark in plant genomes that controls gene expression. Regulation of gene expression by transcriptional gene silencing (TGS) of transgenes and endogenous genes has been extensively studied [1, 2, 3, 4]. In plants, DNA methylation occurs in three sequence contexts: CG, CHG and CHH (where H is A, C or T). The CG methylation is maintained by METHYLTRANSFERASE1 (MET1), whereas CHG and CHH methylation is maintained by plant-specific CHROMOMETHYLASE3 (CMT3) and CHROMOMETHYLASE 2 (CMT2), respectively [1, 2, 3, 4, 5]. In addition, the de novo DNA methyltrasferase, DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) is involved in both maintenance and initiation of DNA methylation .
In plants, DRM2 is required for de novo DNA methylation, which is triggered by short-interfering RNAs (siRNAs) . siRNAs guide DRM2 to the target sequences on the genomic DNA and directs de novo DNA methylation through RNA-directed DNA methylation (RdDM) [7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18], in which two plant-specific RNA polymerases, Pol IV and Pol V play essential roles. The 24-nucleotide (nt) siRNAs are generated and amplified by Pol IV, RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). The 24-nt siRNAs are then loaded onto Argonaute (AGO) for the subsequent DRM2-mediated DNA methylation. AGO4 eventually interacts with the Pol V subunit, NUCLEAR RNA POLYMERASE E1 (NRPE1) and makes a complex with DRM2 to initiate DNA methylation. Recently, RdDM has been classified into two pathways, canonical and non-canonical. In the canonical pathway, a cascade of enzyme reactions (Pol IV-RDR2-DCL3) produces 24-nt siRNAs in concert, whereas several additional small RNAs seem to be involved in RdDM through the non-canonical pathway .
Assuming that RdDM is triggered by siRNAs from a promoter sequence, there are two options to induce specific DNA methylation artificially: transgene-induced or virus-induced. The first involves the creation of transgenic plants where double-stranded RNAs (dsRNAs) against the target promoter sequence are generated, for example, by an inverted repeat construct [20, 21, 22], while the second involves the virus-induced transcriptional gene silencing (VITGS), which is quite convenient because we do not have to produce transgenic plants . However, there is a large difference between the two methods in that transgene-induced siRNAs are generated even in the progeny plants but virus-driven siRNAs are not detectably carried into the next generation without viral infection.
A substantial number of viruses has been developed for VITGS, e.g., apple latent spherical virus (ALSV) [24, 25, 26], tobacco rattle virus [27, 28], barley stripe mosaic virus  and potato virus X [30, 31]. We have previously developed the cucumber mosaic virus (CMV)-based vector, CMV-A1 for both PTGS and TGS [32, 33, 34, 35, 36, 37]. Using CMV-A1, we could successfully induce VITGS against some endogenous genes as well as a transgene [33, 38]. When we inoculated the green fluorescent protein (GFP)-expressing transgenic Nicotiana benthamiana (16c) with the CMV-A1 constructs containing various sizes of the 35S promoter sequences, we found that the GFP expression levels were downregulated due to RdDM and that the sizes of the virus-integrated sequences were important for DNA methylation .
The CaMV 35S promoter is such a strong promoter that it has been widely used for gene expression in plants. Several sequence domains affecting promoter activity have already been identified. The activation sequence factors 1 and 2 (as-1 and as-2), which are located in subdomains A1 and B1, respectively, have been found as a transcription factor-binding site [40, 41, 42, 43, 44, 45]. Subdomains B2 and B4 have been also reported to be important for the promoter activity . However, it has not yet been demonstrated that specific DNA methylation on those sequence elements can control the promoter activity.
In our observation of the VITGS against the 35S promoter in 16c plants, we noticed that the virus-free S1 progeny plants comprised two phenotypes when exposed to UV light: red coloration (RED) resulting from autofluorescence of chlorophylls due to loss of GFP fluorescence by TGS and orange coloration (ORN) from the combined chlorophyll autofluorescence and GFP fluorescence. Most of the S2 progeny plants from the S1 ORN plants were ORN plants, but occasionally RED plants appeared. Virus vector-derived siRNAs corresponding to the 35S promoter sequence would not be generated in the progeny plants because progenies are no longer infected by the recombinant virus. We raise the following questions regarding unknown mechanisms for maintenance and initiation of the DNA methylation: What discriminates the RED and ORN plants? How are stable and unstable TGS controlled? Our preliminary analyses of the overall DNA methylation status of the 35S promoter in the two phenotypes indicated that there was not much difference between the RED and ORN plants, suggesting that methylation at specific cytosine residues on the target sequence may be important for inheritance of TGS. To answer the above questions, we here analyzed the DNA methylation in our VITGS-induced 16c plants in detail.
Differential induction of VITGS depending on the sizes of target sequences
Analysis of methylation of 35S promoter in second-generation self-fertilized progenies from VITGS plants
Among S2 progenies, we found that the leaf veins (Vein) fluoresced brighter yellow than the other parts (leaf without vein, LwV) (Fig. 4a and b). We separated Vein and LwV and compared the GFP expression levels in those tissues by qRT-PCR (Fig. 4c). As we expected from the GFP fluorescence intensity, GFP was expressed in S2:208 ORN at a level of about 1/3 of that in 16c, while it was barely detected in S2:208 RED. On the other hand, unlike our expectation, the GFP level differed little between Vein and LwV in the samples.
In parallel with the VITGS experiments, we were creating transgenic Arabidopsis plants to screen for plants that showed TGS of the GFP gene under the 35S promoter; we obtained a transgenic line that contained a direct repeat sequence of the 35S promoter fused to the GFP gene. In the progeny generations (T1 to T5) of the direct repeat line, we noted two types of GFP fluorescence: one showing GFP fluorescence at an early stage, then developed post-transcriptional gene silencing (PTGS) for GFP expression and eventually induced TGS at a late stage (PTGS-TGS plants); the other already had perfect TGS in small seedlings (TGS plants), and the TGS was stably inherited by the next generation. From the results of bisulfite sequencing to analyze the methylation status of the 35S promoter sequences in those plants, we found that CG or CHG methylations at positions − 46, − 66, − 78 and − 82 in the subdomain A1 were higher in the TGS plants than in the PTGS-TGS plants (Additional file 4: Figure S4). Furthermore, the overall CHH methylation was greatly reduced in the TGS plants compared to the PTGS-TGS plants. These observations therefore agree well with the results of VITGS.
CG methylation at a few cytosine residues in the minus-strand of the 105-bp sequence containing the subdomain A1 caused significant reduction in promoter activity
CHH methylation at a few cytosine residues in the subdomain A1 also contributed to reducing promoter activity
Because only three cytosine residues at CHH context (− 33, − 56, and − 84 in plus-sense) were found to be highly methylated within the domain A (− 90 ~ + 1) of the 35S promoter (Fig. 5a, Additional file 2: Figure S2a), we integrated methylated cytosine residues at those three CHH positions; two residues are located in the subdomain A1 and one residue is located in subdomain mp (region from − 45 to + 1). As shown in Fig. 8c, the introduced CHH methylation reduced the luciferase activity down to 1/3 of the control although the specific CG methylation tested was twice more effective (1/6 of the control).
Unstable TGS phenotype in the progeny from VITGS-16c plants
In our analyses of RdDM on the 35S promoter induced by VITGS, we found discrete phenotypes of the TGS patterns against the GFP gene downstream of the 35S promoter. The TGS seemed to depend on the sizes of the 35S promoter sequence inserted in the viral vector. The TGS in the 16c plants inoculated with A1–345 was maintained in the S1 progeny, whereas we observed two phenotypes (RED and ORN) in the S1 progeny plants after inoculation with A1–208. The RED, from chlorophyll autofluorescence, represents a strong GFP TGS, and the ORN seems to be an intermediate TGS between those of 16c and the RED plants. In the S1 progeny from the plants inoculated with A1–116, most of the S1 plants lost the TGS of the GFP gene that had been observed in the virus-inoculated plants. Based on these observations, we considered that by analyzing the 35S promoters in these phenotypes, we might be able to reveal some important factor(s), which can control the robustness of the RdDM-mediated TGS.
We then analyzed the S2:208 plants in detail to obtain a clue about fixing a stable TGS for next generation. As shown in Fig. 4, the S2:208 ORN plants clearly had stronger GFP fluorescence in the stems and leaf veins (Vein) than in the leaf areas without veins (LwV). The GFP mRNA levels in the S2:208 ORN plants were significantly higher than in the S2:208 RED plants. Although we initially expected that the GFP levels in Vein would be higher than those in LwV, GFP expression differed little between the two tissues, perhaps because chlorophyll content in Vein tissues is lower than in LwV and the chlorophyll autofluorescence interfered less with the GFP fluorescence.
Effect of cytosine methylation in the 35S promoter in a TSM assay on downstream gene expression
Here, we successfully developed an assay by which we can analyze the effect of specific cytosine methylation in a promoter sequence on the downstream gene expression. In addition, we can even discriminate DNA strand-sense when specific methylation is introduced into a target promoter. We here call this method strand-sense-specific methylation (triple S methylation, TSM) assay (Fig. 7). Previously, the promoter sequences that are important for TGS had been identified mainly using point-mutation and subdomain-deletion strategies [40, 41, 42, 43, 44, 45, 46]. However, without introducing actual methylation to specific cytosine residues, we can never conclude that methylation of the predicted sites is really responsible for the influence on the expression of downstream gene. Because we can directly manipulate cytosine methylation in a target promoter with a TSM assay, various promoter sequences can be analyzed to elucidate a link between specific cytosine methylation and promoter activities. The results of our TSM assays demonstrated that TGS levels of the 35S promoter might be differentially regulated depending on the heterogeneity of cytosine methylation in and between the plus- and minus-strands corresponding to the target domain sequences. Especially cytosine methylation at positions close to the as-1 element (− 83 to − 63) is very important for stable, inherited TGS, which agrees well with the previously predicted cytosine residues for the promoter activity [40, 43, 45]. To be more specific, we found that TGS of the 35S promoter occurred efficiently when the cytosine residues (positions − 78, − 66 and − 46) on the minus-strand were methylated. On the other hand, surprisingly, the methylation of the corresponding sites on the plus-strand had little effect on the TGS, but the maximum TGS was observed when both strands were methylated at the specific cytosine residues. Our TSM assay also indicated that CHH methylation in the subdomain A1 significantly reduced promoter activity. However, considering that CHH methylation is not maintained through the next generation, the stable inherited TGS in the RED phenotype will be driven mainly by some specific symmetric methylations. No report has previously discussed the effect of heterogeneity of DNA methylation in and between the plus- and minus-DNA strands in relation to TGS. Although we have always assumed that both strands would be simultaneously and uniformly methylated in a model for RdDM , we should rather consider that the two strands may not be necessarily uniformly methylated in nature. Whether our observation for the 35S promoter can be generally applied to other promoters or not should be independently tested using a similar assay. Although in this study, we focused only on methylation of the subdomain A1, subdomains B2 and B4 in the 35S promoter have also been proved to be essential for promoter activity . It would be worth evaluating the cytosine methylation on the other elements in the 35S promoter using the TSM assay.
Link between symmetric and asymmetric methylation in inherited DNA methylation
In our bisulfite sequencing analyses, we noticed an interesting phenomenon that was always associated with the stable, inherited TGS in many replicates. In the RED plants in S2:208, we found that the overall levels of asymmetric (CHH) methylation were significantly reduced in both plus- and minus-strands, while the levels of symmetric methylation (CG and CHG) were conversely increased (Fig. 5 and Additional file 2: Figure S2). Most of the S1 plants generated from A1–116-inoculated plants lost TGS of the GFP gene. This unstable TGS in next generation may be associated with a decrease in symmetric methylation and an increase of asymmetric methylation in the target sequence. For cytosine methylation of the 35S promoter in the TGS-induced progeny plants (S1 to S4 generations), similar observations have also been shown in the study of the ALSV vector . To understand the involvement of methylation status in TGS induction, we can test how the level of asymmetric methylation in the 35S promoter can affect TGS by a method like our TSM assay. Although we do not know the exact reason for a decrease in asymmetric methylation at this moment, it is conceivable that some factor during reproduction may have effects in the association between RdDM and inherited methylation. Considering the results together, we infer that the generation of the RED and ORN plants observed among the VITGS progeny plants may depend on the accumulation levels of symmetric methylation at some specific cytosine sites and a decrease in asymmetric methylation in the progeny plants.
We induced TGS against the GFP gene under the 35S promoter through RdDM by a viral vector containing various sizes of the promoter sequence. In the first self-fertilized generation, the stable TGS seemed to be correlated with high levels of methylation on the promoter. However, we observed an unstable TGS in the second generation; S2 progenies were segregated into RED and ORN. Our bisulfite sequencing analyses suggested that specific methylation at a few cytosine residues in the subdomain A1 and a decrease in frequency of asymmetric methylation might be important for efficient induction of TGS through RdDM. To probe the importance of the methylation at specific cytosine residues, we developed a method by which we can analyze a direct effect of methylated cytosine residues on TGS in a strand-specific manner. In this assay, we found that cytosine methylation on either or both of the plus- and minus-sense promoter sequences could differentially drive gene expression downstream of the promoter. We therefore infer that robust TGS may be determined by the heterogeneity of DNA methylation at specific sites in and between the plus- and minus-strands.
Nicotiana benthamiana line 16c was obtained from Dr. David Baulcombe. The plants were grown with 16-h light (8-h dark) at 24 °C.
Inoculation with virus and GFP observation
Fragments from the 35S promoter were amplified by PCR and inserted into the CMV-A1 vector. N. benthamiana line 16c plants were inoculated with in vitro transcripts of CMV as previously described by Otagaki et al. . Total RNA was isolated 15 days postinoculation from the infected tissues. Viral cDNAs were PCR-amplified using a primer pair of 2b-5up and R2–2814-R2 (Additional file 5: Table S1) and the TaKaRa One Step RNA PCR Kit AMV (TaKaRa) kit according to the supplier’s instruction. To confirm that the viruses did not have serious mutations during their replication, the amplified viral cDNAs were directly sequenced: the sequencing chromatograms were shown in Additional file 6: Figure S5. GFP fluorescence was examined with a Keyence Microscope VB-7000 with the GFP filter (OP-42313).
DNA methylation assay
DNA was extracted using the Illusta DNA extraction Kit PhytoPure (GE Healthcare) according to the supplier’s instructions. Bisulfite treatment of DNA was performed by using EZ DNA Methylation-Lightning Kit (Zymo Research) according to the supplier’s protocol. To amplify the target sequences, primer pair 35S-346F-bisuT/35S + 1A-bisuA were used for the plus-strand, and primer pair 35S (−)-5-BS/35S (−)-3-BS were used for the minus-strand (Additional file 5: Table S1). The primers were initially designed by the program, MethPrimer (http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi). To amplify the entire (~ 345 bp) core sequence of the 35S promoter, we modified the designed primer so that they contained cytosine residues as less as possible, and synthesized degenerated primers. The PCR was conducted by Takara Epi-taq (TaKaRa) and the PCR conditions were as follows, 40 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s. The PCR products were then cloned into the pTAC1 vector using the Dyna Express TA PCR Cloning Kit (Bio Dynamics Laboratory). For clone-based bisulfite sequencing, we used 22–24 clones and the sequencing data were aligned using MEGA version 6 . The graphs were created in Microsoft Excel (Additional file 7: Table S2).
Quantitative RT-PCR analysis
Total RNA was isolated using TRIzol reagent (Invitrogen) according to the supplier’s instructions. For the DNA and RNA extraction in the S2 progenies, we separated class I and class II veins (Vein)  and leaf tissues without those veins (LwV). Extracted RNA was treated by DNase I recombinant RNase-free (Roche Applied Science), and then cDNA was synthesized from 0.2 μg of RNA using the PrimeScript RT reagent Kit (TaKaRa) according to the supplier’s instruction. qRT-PCR analysis was carried out by the Applied Biosystems StepOnePlus Real-time PCR system using the PowerUp SYBR Green Master Mix (Applied Biosystems). The PCR cycling conditions were 40 cycles of 95 °C for 15 s, 55 °C for 30 s and 72 °C for 30 s. The GFP expression levels were calculated using the comparative Ct method and normalized by the expression of the L23 gene. Target sequences were amplified using primer pair mGFP-5-160/mGFP3–160 for the GFP gene and primer pair Nb-L23–5-110/Nb-L23–3-110 for the internal control.
Golden gate cloning and TSM assay
All oligonucleotides used in this study are listed in Additional file 5: Table S1. The unmethylated 35S promoter fragment was amplified from pBI221 (Clontech) by PCR. The target sequences were amplified using primer pair 35S-GGC-5/35S-GGC-3. The Fluc PCR product was also amplified by PCR using primer pair LUC-GGC-5/LUC-GGC-3. The custom-synthesized oligonucleotides (oligo DNAs) with specific methylation at three CG sites (positions − 78, − 66 and − 46) and at three CHH sites (positions − 33, − 56 and − 84) were prepared by the Custom DNA Synthesis Service (Hokkaido System Science). The oligo DNAs (100 μM) were annealed in oligo annealing buffer (10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl) with gradually cooling after incubation at 95 °C for 4 min. To make a DNA fragment where either strand was methylated, 35S-CG-DNA (+), 35S-CG-DNA (−) and 35S-CHH-DNA (+) were annealed with either unmethylated oligonucleotide 35S-3-120 PM or 35S-5-120 PM and then amplified by PCR. The PCR cycling conditions were 40 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s. All four types of DNA constructs were first digested by BsaI and then ligated to the firefly luciferase reporter (Fluc) gene as essentially described as golden gate cloning by Engler et al. . We then analyzed the promoter activity in protoplasts. We call this method strand-sense-specific methylation (triple S methylation, TSM) assay.
Protoplast transfection and luciferase assay
Protoplasts were prepared from the leaves of N. benthamiana as described by Shimura et al. . Prepared Fluc-fused 35S promoter fragments (0.5 μg or 1 μg) were directly transfected to protoplasts together with pE-Rluc (0.45 μg) as an internal control in the presence of polyethylene glycol. After 18 h incubation, luciferase activities (Fluc and Rluc) were assayed using the Dual-Luciferase Reporter Assay System (Promega) and a fluorometer (Wallac 1420 ARVO MX) as previously described by Shimura et al. .
We thank David Baulcombe (University of Cambridge, UK) for kindly providing Nicotiana benthamiana 16c. We are also grateful to Peter Palukaitis (Seoul Women’s University, Korea) for reviewing the manuscript.
This work was supported by the promotion services of the New Energy and Industrial Technology Development Organization (NEDO) (project code: P16009). The funding body was used for the design of the study; collection, analysis, and interpretation of data; or in writing the manuscript.
Availability of data and materials
All data generated and analyzed during this study are included in this published article (Additional file 1: Figure S1, Additional file 2: Figure S2, Additional file 3: Figure S3, Additional file 4: Figure S4, Additional file 5: Table S1, Additional file 6: Figure S5 and Additional file 7: Table S2).
CM and TM designed and coordinated the study. HS directed the protoplast assay. TI, SS and RI designed and performed the experiments using the transgenic Arabidopsis plants. WM performed the bisulfite sequencing analyses. WM and CM mainly wrote the manuscript. All authors contributed to the analysis of results, manuscript revisions and final approval for publication.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
- 5.Zhang Y, Harris CJ, Liu Q, Liu W, Ausin I, Long Y, Xiao L, Feng L, Chen X, Xie Y, Chen X, Zhan L, Feng S, Li JJ, Wang H, Zhai J, Jacobsen SE. Large-scale comparative epigenomics reveals hierarchical regulation of non-CG methylation in Arabidopsis. Proc Natl Acad Sci U S A. 2018;115:E1069–74.CrossRefGoogle Scholar
- 34.Nagamatsu A, Masuta C, Matsuura H, Kitamura K, Abe J, Kanazawa A. Downregulation of flavonoid 3′-hydroxylase gene expression by virus-induced gene silencing in soybean reveals the presence of a threshold mRNA level associated with pigmentation in pubescence. J Plant Physiol. 2009;166:32–9.CrossRefGoogle Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.