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Histone acetylation accompanied with promoter sequences displaying differential expression profiles of B-class MADS-box genes for phalaenopsis floral morphogenesis.

Hsu CC, Wu PS, Chen TC, Yu CW, Tsai WC, Wu K, Wu WL, Chen WH, Chen HH - PLoS ONE (2014)

Bottom Line: The amplified promoter sequences of PeMADS2∼6 could drive beta-glucuronidase (GUS) gene expression in all floral organs, similar to their expression at the floral primordia stage.The promoter sequence of PeMADS4, exclusively expressed in lip and column, showed a 1.6∼3-fold higher expression in lip/column than in sepal/petal.Furthermore, we noted a 4.9-fold increase in histone acetylation (H3K9K14ac) in the translation start region of PeMADS4 in lip as compared in petal.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, National Cheng Kung University, Tainan, Taiwan.

ABSTRACT
Five B-class MADS-box genes, including four APETALA3 (AP3)-like PeMADS2∼5 and one PISTILLATA (PI)-like PeMADS6, specify the spectacular flower morphology in orchids. The PI-like PeMADS6 ubiquitously expresses in all floral organs. The four AP3-like genes, resulted from two duplication events, express ubiquitously at floral primordia and early floral organ stages, but show distinct expression profiles at late floral organ primordia and floral bud stages. Here, we isolated the upstream sequences of PeMADS2∼6 and studied the regulatory mechanism for their distinct gene expression. Phylogenetic footprinting analysis of the 1.3-kb upstream sequences of AP3-like PeMADS2∼5 showed that their promoter regions have sufficiently diverged and contributed to their subfunctionalization. The amplified promoter sequences of PeMADS2∼6 could drive beta-glucuronidase (GUS) gene expression in all floral organs, similar to their expression at the floral primordia stage. The promoter sequence of PeMADS4, exclusively expressed in lip and column, showed a 1.6∼3-fold higher expression in lip/column than in sepal/petal. Furthermore, we noted a 4.9-fold increase in histone acetylation (H3K9K14ac) in the translation start region of PeMADS4 in lip as compared in petal. All these results suggest that the regulation via the upstream sequences and increased H3K9K14ac level may act synergistically to display distinct expression profiles of the AP3-like genes at late floral organ primordia stage for Phalaenopsis floral morphogenesis.

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Histone modification on the ATG and 5th intron regions of PeMADS4.(A) Locations of the primers used in ChIP assay. Gray, black, and white boxes indicate the promoter, exon, and intron regions of PeMADS4 gene, respectively. The rhombus and black triangles are the predicted CArG boxes. ChIP assay of histone modification of dimethyl-H3K9 (H3K9me2), trimethyl-H3K4 (H3K4me3), acetyl-H3K9 and H3K14 (H3Ac) was analyzed on the ATG (B) and 5th intron regions (C) of PeMADS4 in the petal and lip of P. equestris. The amount of DNA after ChIP was quantified and normalized to an internal control ACTIN2 for H3K4me3 and H3K9K14ac or Ta3 for H3K9me2. Data are mean ± SD calculated from three technological and two biological replicates. X =  fold.
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pone-0106033-g008: Histone modification on the ATG and 5th intron regions of PeMADS4.(A) Locations of the primers used in ChIP assay. Gray, black, and white boxes indicate the promoter, exon, and intron regions of PeMADS4 gene, respectively. The rhombus and black triangles are the predicted CArG boxes. ChIP assay of histone modification of dimethyl-H3K9 (H3K9me2), trimethyl-H3K4 (H3K4me3), acetyl-H3K9 and H3K14 (H3Ac) was analyzed on the ATG (B) and 5th intron regions (C) of PeMADS4 in the petal and lip of P. equestris. The amount of DNA after ChIP was quantified and normalized to an internal control ACTIN2 for H3K4me3 and H3K9K14ac or Ta3 for H3K9me2. Data are mean ± SD calculated from three technological and two biological replicates. X =  fold.

Mentions: It is possible that the tissue-specific expression profiles of PeMADS genes not only reside in the DNA-level promoter sequences, but also in the protein-level histone modification. To address this, various histone modifications were analyzed by ChIP assay with antibodies against the gene repression marker H3K9me2 and gene activation markers H3K4me3 and H3K9K14ac. The precipitated DNA samples from both petal and lip were analyzed by real-time PCR with the primer sequences located at the translation start site (ATG) and the 5th intron regions of PeMADS4 (Fig. 8A). Notably, we detected a 4.9-fold higher H3K9K14ac at the translation start site in lip than in petal (Fig. 8B). In contrast, no differential levels of H3K9me2 and H3K4me3 were detected in the translation start site in both petal and lip (Fig. 8B). Furthermore, no substantial differential levels of H3K9me2, H3K4me3, and H3K9K14ac within the 5th intron region of PeMADS4 were detected in petal and lip (Fig. 8C). Thus, the increased level of H3K9K14ac on the translation start site of PeMADS4 gene may allow for more access of the transcription factor and the further increased gene expression in lip, thus leading to its optimized expression in Phalaenopsis.


Histone acetylation accompanied with promoter sequences displaying differential expression profiles of B-class MADS-box genes for phalaenopsis floral morphogenesis.

Hsu CC, Wu PS, Chen TC, Yu CW, Tsai WC, Wu K, Wu WL, Chen WH, Chen HH - PLoS ONE (2014)

Histone modification on the ATG and 5th intron regions of PeMADS4.(A) Locations of the primers used in ChIP assay. Gray, black, and white boxes indicate the promoter, exon, and intron regions of PeMADS4 gene, respectively. The rhombus and black triangles are the predicted CArG boxes. ChIP assay of histone modification of dimethyl-H3K9 (H3K9me2), trimethyl-H3K4 (H3K4me3), acetyl-H3K9 and H3K14 (H3Ac) was analyzed on the ATG (B) and 5th intron regions (C) of PeMADS4 in the petal and lip of P. equestris. The amount of DNA after ChIP was quantified and normalized to an internal control ACTIN2 for H3K4me3 and H3K9K14ac or Ta3 for H3K9me2. Data are mean ± SD calculated from three technological and two biological replicates. X =  fold.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263434&req=5

pone-0106033-g008: Histone modification on the ATG and 5th intron regions of PeMADS4.(A) Locations of the primers used in ChIP assay. Gray, black, and white boxes indicate the promoter, exon, and intron regions of PeMADS4 gene, respectively. The rhombus and black triangles are the predicted CArG boxes. ChIP assay of histone modification of dimethyl-H3K9 (H3K9me2), trimethyl-H3K4 (H3K4me3), acetyl-H3K9 and H3K14 (H3Ac) was analyzed on the ATG (B) and 5th intron regions (C) of PeMADS4 in the petal and lip of P. equestris. The amount of DNA after ChIP was quantified and normalized to an internal control ACTIN2 for H3K4me3 and H3K9K14ac or Ta3 for H3K9me2. Data are mean ± SD calculated from three technological and two biological replicates. X =  fold.
Mentions: It is possible that the tissue-specific expression profiles of PeMADS genes not only reside in the DNA-level promoter sequences, but also in the protein-level histone modification. To address this, various histone modifications were analyzed by ChIP assay with antibodies against the gene repression marker H3K9me2 and gene activation markers H3K4me3 and H3K9K14ac. The precipitated DNA samples from both petal and lip were analyzed by real-time PCR with the primer sequences located at the translation start site (ATG) and the 5th intron regions of PeMADS4 (Fig. 8A). Notably, we detected a 4.9-fold higher H3K9K14ac at the translation start site in lip than in petal (Fig. 8B). In contrast, no differential levels of H3K9me2 and H3K4me3 were detected in the translation start site in both petal and lip (Fig. 8B). Furthermore, no substantial differential levels of H3K9me2, H3K4me3, and H3K9K14ac within the 5th intron region of PeMADS4 were detected in petal and lip (Fig. 8C). Thus, the increased level of H3K9K14ac on the translation start site of PeMADS4 gene may allow for more access of the transcription factor and the further increased gene expression in lip, thus leading to its optimized expression in Phalaenopsis.

Bottom Line: The amplified promoter sequences of PeMADS2∼6 could drive beta-glucuronidase (GUS) gene expression in all floral organs, similar to their expression at the floral primordia stage.The promoter sequence of PeMADS4, exclusively expressed in lip and column, showed a 1.6∼3-fold higher expression in lip/column than in sepal/petal.Furthermore, we noted a 4.9-fold increase in histone acetylation (H3K9K14ac) in the translation start region of PeMADS4 in lip as compared in petal.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, National Cheng Kung University, Tainan, Taiwan.

ABSTRACT
Five B-class MADS-box genes, including four APETALA3 (AP3)-like PeMADS2∼5 and one PISTILLATA (PI)-like PeMADS6, specify the spectacular flower morphology in orchids. The PI-like PeMADS6 ubiquitously expresses in all floral organs. The four AP3-like genes, resulted from two duplication events, express ubiquitously at floral primordia and early floral organ stages, but show distinct expression profiles at late floral organ primordia and floral bud stages. Here, we isolated the upstream sequences of PeMADS2∼6 and studied the regulatory mechanism for their distinct gene expression. Phylogenetic footprinting analysis of the 1.3-kb upstream sequences of AP3-like PeMADS2∼5 showed that their promoter regions have sufficiently diverged and contributed to their subfunctionalization. The amplified promoter sequences of PeMADS2∼6 could drive beta-glucuronidase (GUS) gene expression in all floral organs, similar to their expression at the floral primordia stage. The promoter sequence of PeMADS4, exclusively expressed in lip and column, showed a 1.6∼3-fold higher expression in lip/column than in sepal/petal. Furthermore, we noted a 4.9-fold increase in histone acetylation (H3K9K14ac) in the translation start region of PeMADS4 in lip as compared in petal. All these results suggest that the regulation via the upstream sequences and increased H3K9K14ac level may act synergistically to display distinct expression profiles of the AP3-like genes at late floral organ primordia stage for Phalaenopsis floral morphogenesis.

Show MeSH
Related in: MedlinePlus