Limits...
In planta assays involving epigenetically silenced genes reveal inhibition of cytosine methylation by genistein.

Arase S, Kasai M, Kanazawa A - Plant Methods (2012)

Bottom Line: Using these systems, we found that genistein, a major isoflavonoid compound, inhibits cytosine methylation, thus restoring transgene transcription.Genistein also restored the transcription of an epigenetically silenced endogenous gene in Arabidopsis plants.These results suggest a novel role of flavonoids in plant cells and that genistein is useful for modifying the epigenetic state of plant genomes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan. kanazawa@res.agr.hokudai.ac.jp.

ABSTRACT

Background: Cytosine methylation is involved in epigenetic control of gene expression in a wide range of organisms. An increasing number of examples indicate that changing the frequency of cytosine methylation in the genome is a feasible tool to engineer novel traits in plants. Although demethylating effects of compounds have been analyzed in human cultured cells in terms of suppressing cancer, their effect in plant cells has not been analyzed extensively. Here, we developed in planta assay systems to detect inhibition of cytosine methylation using plants that contain a transgene transcriptionally silenced by an epigenetic mechanism.

Results: Seeds of two transgenic plants were used: a petunia line that has been identified as a revertant of the co-suppression of the chalcone synthase-A (CHS-A) gene and contains CHS-A transgenes whose transcription is repressed; Nicotiana benthamiana plants that contain the green fluorescent protein (GFP) reporter gene whose transcription is repressed through virus-induced transcriptional gene silencing. Seeds of these plants were sown on a medium that contained a demethylating agent, either 5-azacytidine or trichostatin A, and the restoration of the transcriptionally active state of the transgene was detected in seedlings. Using these systems, we found that genistein, a major isoflavonoid compound, inhibits cytosine methylation, thus restoring transgene transcription. Genistein also restored the transcription of an epigenetically silenced endogenous gene in Arabidopsis plants.

Conclusions: Our assay systems allowed us to assess the inhibition of cytosine methylation, in particular of maintenance of methylation, by compounds in plant cells. These results suggest a novel role of flavonoids in plant cells and that genistein is useful for modifying the epigenetic state of plant genomes.

No MeSH data available.


Related in: MedlinePlus

Analysis of methylation status of CaMV 35S promoter in genistein-treated N. benthamiana plants by restriction digestion of DNA fragments amplified with PCR from bisulfite-treated DNA. (A) Analysis of cytosine at position -288 (relative to the transcription initiation site) of the promoter using AluI. (B) Analysis of cytosines at positions -130, -119, and -79 of the promoter using MaeII. Note that treatments of PCR-amplified fragments with AluI and MaeII both resulted in lower levels of digestion when DNA isolated from genistein-treated plants was used for the analysis, indicating that genistein-treated plants have a lower frequency of cytosine methylation in the promoter. Sizes of DNA fragments (in bp) predicted by complete or partial digestions are indicated below the maps of the promoter. Arrows indicate primers for PCR. The position of the cis-acting as-1 element, to which binding of protein factor(s) is inhibited by cytosine methylation [60], is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3362751&req=5

Figure 6: Analysis of methylation status of CaMV 35S promoter in genistein-treated N. benthamiana plants by restriction digestion of DNA fragments amplified with PCR from bisulfite-treated DNA. (A) Analysis of cytosine at position -288 (relative to the transcription initiation site) of the promoter using AluI. (B) Analysis of cytosines at positions -130, -119, and -79 of the promoter using MaeII. Note that treatments of PCR-amplified fragments with AluI and MaeII both resulted in lower levels of digestion when DNA isolated from genistein-treated plants was used for the analysis, indicating that genistein-treated plants have a lower frequency of cytosine methylation in the promoter. Sizes of DNA fragments (in bp) predicted by complete or partial digestions are indicated below the maps of the promoter. Arrows indicate primers for PCR. The position of the cis-acting as-1 element, to which binding of protein factor(s) is inhibited by cytosine methylation [60], is shown.

Mentions: Bisulfite sequencing analysis showed that the frequency of cytosine methylation was reduced in the GFP-restored plants grown in a medium supplemented with genistein compared with plants grown in a medium with no supplement (Figure 5). Changes in the frequency of methylcytosine were also analyzed by digesting the DNA fragments amplified by PCR from bisulfite-treated DNA (Figure 6). In this experiment, tolerance of PCR products to digestion indicates lack of methylation of the cytosine at the restriction sites because of the conversion of cytosine by the bisulfite treatment. The results clearly indicated that genistein-treated plants had a lower frequency of methylation at the AluI and MaeII sites in the CaMV 35S promoter than the control plants.


In planta assays involving epigenetically silenced genes reveal inhibition of cytosine methylation by genistein.

Arase S, Kasai M, Kanazawa A - Plant Methods (2012)

Analysis of methylation status of CaMV 35S promoter in genistein-treated N. benthamiana plants by restriction digestion of DNA fragments amplified with PCR from bisulfite-treated DNA. (A) Analysis of cytosine at position -288 (relative to the transcription initiation site) of the promoter using AluI. (B) Analysis of cytosines at positions -130, -119, and -79 of the promoter using MaeII. Note that treatments of PCR-amplified fragments with AluI and MaeII both resulted in lower levels of digestion when DNA isolated from genistein-treated plants was used for the analysis, indicating that genistein-treated plants have a lower frequency of cytosine methylation in the promoter. Sizes of DNA fragments (in bp) predicted by complete or partial digestions are indicated below the maps of the promoter. Arrows indicate primers for PCR. The position of the cis-acting as-1 element, to which binding of protein factor(s) is inhibited by cytosine methylation [60], is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3362751&req=5

Figure 6: Analysis of methylation status of CaMV 35S promoter in genistein-treated N. benthamiana plants by restriction digestion of DNA fragments amplified with PCR from bisulfite-treated DNA. (A) Analysis of cytosine at position -288 (relative to the transcription initiation site) of the promoter using AluI. (B) Analysis of cytosines at positions -130, -119, and -79 of the promoter using MaeII. Note that treatments of PCR-amplified fragments with AluI and MaeII both resulted in lower levels of digestion when DNA isolated from genistein-treated plants was used for the analysis, indicating that genistein-treated plants have a lower frequency of cytosine methylation in the promoter. Sizes of DNA fragments (in bp) predicted by complete or partial digestions are indicated below the maps of the promoter. Arrows indicate primers for PCR. The position of the cis-acting as-1 element, to which binding of protein factor(s) is inhibited by cytosine methylation [60], is shown.
Mentions: Bisulfite sequencing analysis showed that the frequency of cytosine methylation was reduced in the GFP-restored plants grown in a medium supplemented with genistein compared with plants grown in a medium with no supplement (Figure 5). Changes in the frequency of methylcytosine were also analyzed by digesting the DNA fragments amplified by PCR from bisulfite-treated DNA (Figure 6). In this experiment, tolerance of PCR products to digestion indicates lack of methylation of the cytosine at the restriction sites because of the conversion of cytosine by the bisulfite treatment. The results clearly indicated that genistein-treated plants had a lower frequency of methylation at the AluI and MaeII sites in the CaMV 35S promoter than the control plants.

Bottom Line: Using these systems, we found that genistein, a major isoflavonoid compound, inhibits cytosine methylation, thus restoring transgene transcription.Genistein also restored the transcription of an epigenetically silenced endogenous gene in Arabidopsis plants.These results suggest a novel role of flavonoids in plant cells and that genistein is useful for modifying the epigenetic state of plant genomes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan. kanazawa@res.agr.hokudai.ac.jp.

ABSTRACT

Background: Cytosine methylation is involved in epigenetic control of gene expression in a wide range of organisms. An increasing number of examples indicate that changing the frequency of cytosine methylation in the genome is a feasible tool to engineer novel traits in plants. Although demethylating effects of compounds have been analyzed in human cultured cells in terms of suppressing cancer, their effect in plant cells has not been analyzed extensively. Here, we developed in planta assay systems to detect inhibition of cytosine methylation using plants that contain a transgene transcriptionally silenced by an epigenetic mechanism.

Results: Seeds of two transgenic plants were used: a petunia line that has been identified as a revertant of the co-suppression of the chalcone synthase-A (CHS-A) gene and contains CHS-A transgenes whose transcription is repressed; Nicotiana benthamiana plants that contain the green fluorescent protein (GFP) reporter gene whose transcription is repressed through virus-induced transcriptional gene silencing. Seeds of these plants were sown on a medium that contained a demethylating agent, either 5-azacytidine or trichostatin A, and the restoration of the transcriptionally active state of the transgene was detected in seedlings. Using these systems, we found that genistein, a major isoflavonoid compound, inhibits cytosine methylation, thus restoring transgene transcription. Genistein also restored the transcription of an epigenetically silenced endogenous gene in Arabidopsis plants.

Conclusions: Our assay systems allowed us to assess the inhibition of cytosine methylation, in particular of maintenance of methylation, by compounds in plant cells. These results suggest a novel role of flavonoids in plant cells and that genistein is useful for modifying the epigenetic state of plant genomes.

No MeSH data available.


Related in: MedlinePlus