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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

Chromatin immunoprecipitation (ChIP) and quantitative PCR (qPCR) analyses of H3K4me3, H3K9me2, H3K27me3 and H3K36me3 levels on endogenousGSTF8promoter and transgene chromatin in WTLUCandhsi2-4LUCmutant. 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).
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Fig5: Chromatin immunoprecipitation (ChIP) and quantitative PCR (qPCR) analyses of H3K4me3, H3K9me2, H3K27me3 and H3K36me3 levels on endogenousGSTF8promoter and transgene chromatin in WTLUCandhsi2-4LUCmutant. 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).

Mentions: 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


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)

Chromatin immunoprecipitation (ChIP) and quantitative PCR (qPCR) analyses of H3K4me3, H3K9me2, H3K27me3 and H3K36me3 levels on endogenousGSTF8promoter and transgene chromatin in WTLUCandhsi2-4LUCmutant. 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).
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getmorefigures.php?uid=PMC4232687&req=5

Fig5: Chromatin immunoprecipitation (ChIP) and quantitative PCR (qPCR) analyses of H3K4me3, H3K9me2, H3K27me3 and H3K36me3 levels on endogenousGSTF8promoter and transgene chromatin in WTLUCandhsi2-4LUCmutant. 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).
Mentions: 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

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