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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.

Veerappan V, Chen N, Reichert AI, Allen RD - BMC Plant Biol. (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

ABSTRACT

Background: 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 (WT LUC ) that was used for mutagenesis harbors two independent transgene loci, Kan R and Kan S . Both loci express luciferase whereas only the Kan R locus confers resistance to kanamycin.

Results: 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 Kan R and Kan S 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.

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

No MeSH data available.


Related in: MedlinePlus

Transcript levels of endogenousGSTF8and transgenes in WTLUCandhsi2mutants carrying eitherKanRorKanStransgene 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).
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Fig2: Transcript levels of endogenousGSTF8and transgenes in WTLUCandhsi2mutants carrying eitherKanRorKanStransgene 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).

Mentions: 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


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

Veerappan V, Chen N, Reichert AI, Allen RD - BMC Plant Biol. (2014)

Transcript levels of endogenousGSTF8and transgenes in WTLUCandhsi2mutants carrying eitherKanRorKanStransgene 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).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4232687&req=5

Fig2: Transcript levels of endogenousGSTF8and transgenes in WTLUCandhsi2mutants carrying eitherKanRorKanStransgene 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).
Mentions: 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

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

ABSTRACT

Background: 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 (WT LUC ) that was used for mutagenesis harbors two independent transgene loci, Kan R and Kan S . Both loci express luciferase whereas only the Kan R locus confers resistance to kanamycin.

Results: 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 Kan R and Kan S 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.

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

No MeSH data available.


Related in: MedlinePlus