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Ectopic hbox12 Expression Evoked by Histone Deacetylase Inhibition Disrupts Axial Specification of the Sea Urchin Embryo.

Cavalieri V, Spinelli G - PLoS ONE (2015)

Bottom Line: Transcription of nodal concomitantly drops, prejudicing dorsal/ventral polarity of the resulting larvae.Remarkably, impairing hbox12 function, either in a spatially-restricted sector or in the whole embryo, specifically rescues nodal transcription in Trichostatin-A-treated larvae.Beyond strengthen the notion that nodal expression is not allowed in the presence of functional Hbox12 in the same cells, these results highlight a critical role of histone deacetylases in regulating the spatial expression of hbox12.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Italy.

ABSTRACT
Dorsal/ventral patterning of the sea urchin embryo depends upon the establishment of a Nodal-expressing ventral organizer. Recently, we showed that spatial positioning of this organizer relies on the dorsal-specific transcription of the Hbox12 repressor. Building on these findings, we determined the influence of the epigenetic milieu on the expression of hbox12 and nodal genes. We find that Trichostatin-A, a potent and selective histone-deacetylases inhibitor, induces histone hyperacetylation in hbox12 chromatin, evoking broad ectopic expression of the gene. Transcription of nodal concomitantly drops, prejudicing dorsal/ventral polarity of the resulting larvae. Remarkably, impairing hbox12 function, either in a spatially-restricted sector or in the whole embryo, specifically rescues nodal transcription in Trichostatin-A-treated larvae. Beyond strengthen the notion that nodal expression is not allowed in the presence of functional Hbox12 in the same cells, these results highlight a critical role of histone deacetylases in regulating the spatial expression of hbox12.

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Impact of TSA on acetylation of H3K9 and hbox12 promoter activity.(A) Western blot analysis using the anti-H3K9ac antibody. Nuclear extracts from control and 50 nM TSA-treated blastula stage embryos were fractioned by SDS-PAGE (on the left is shown the gel staining), blotted on PVDF membrane and incubated with anti-H3K9ac antibody. (B and C) ChIP-qPCR analysis of the hbox12 promoter occupancy by H3K9ac and HDAC-1. ChIP assays were performed on chromatin extracted from control and TSA-treated embryos at the mesenchyme blastula stage and precipitated with commercial antiserum against H3K9ac or HDAC-1, or incubated without adding antibodies, followed by qPCR amplification of an hbox12 promoter fragment. Data are normalized according to the percent of input method. Bars are as in Fig 1C. (D) Zygotes were injected with the phbox12-GFP transgene, the resulting embryos were raised in the presence of TSA 50 nM, and observed at the indicated stages. Images for each embryo are shown under DIC optic superimposed on epifluorescence field. (E) qPCR measurements of gfp transcript abundance in blastulae treated with 50 nM TSA. Data are normalized and indicated as in Fig 1C.
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pone.0143860.g002: Impact of TSA on acetylation of H3K9 and hbox12 promoter activity.(A) Western blot analysis using the anti-H3K9ac antibody. Nuclear extracts from control and 50 nM TSA-treated blastula stage embryos were fractioned by SDS-PAGE (on the left is shown the gel staining), blotted on PVDF membrane and incubated with anti-H3K9ac antibody. (B and C) ChIP-qPCR analysis of the hbox12 promoter occupancy by H3K9ac and HDAC-1. ChIP assays were performed on chromatin extracted from control and TSA-treated embryos at the mesenchyme blastula stage and precipitated with commercial antiserum against H3K9ac or HDAC-1, or incubated without adding antibodies, followed by qPCR amplification of an hbox12 promoter fragment. Data are normalized according to the percent of input method. Bars are as in Fig 1C. (D) Zygotes were injected with the phbox12-GFP transgene, the resulting embryos were raised in the presence of TSA 50 nM, and observed at the indicated stages. Images for each embryo are shown under DIC optic superimposed on epifluorescence field. (E) qPCR measurements of gfp transcript abundance in blastulae treated with 50 nM TSA. Data are normalized and indicated as in Fig 1C.

Mentions: First, by western blot analysis carried out using a specific antibody that recognizes H3K9ac, we ascertained that mesenchyme blastulae treated with 50 nM TSA accumulated a global increase in the acetylated H3 level compared to that of control embryos at the same stage (Fig 2A). Next, quantitative ChIP assays with the anti-acetyl-H3K9 antibody were performed on chromatin purified from mesenchyme blastulae treated or untreated with TSA. As expected, no or faint amplification was detected for the chromatin samples incubated without antibody, used as a negative control (Fig 2B). By contrast, the amplification of a specific DNA segment from the hbox12 promoter clearly demonstrated that it was heavily enriched in acetylated histone H3 in TSA-treated embryos (Fig 2B). Thus, TSA likely exerted a direct effect on histone acetylation at the hbox12 promoter.


Ectopic hbox12 Expression Evoked by Histone Deacetylase Inhibition Disrupts Axial Specification of the Sea Urchin Embryo.

Cavalieri V, Spinelli G - PLoS ONE (2015)

Impact of TSA on acetylation of H3K9 and hbox12 promoter activity.(A) Western blot analysis using the anti-H3K9ac antibody. Nuclear extracts from control and 50 nM TSA-treated blastula stage embryos were fractioned by SDS-PAGE (on the left is shown the gel staining), blotted on PVDF membrane and incubated with anti-H3K9ac antibody. (B and C) ChIP-qPCR analysis of the hbox12 promoter occupancy by H3K9ac and HDAC-1. ChIP assays were performed on chromatin extracted from control and TSA-treated embryos at the mesenchyme blastula stage and precipitated with commercial antiserum against H3K9ac or HDAC-1, or incubated without adding antibodies, followed by qPCR amplification of an hbox12 promoter fragment. Data are normalized according to the percent of input method. Bars are as in Fig 1C. (D) Zygotes were injected with the phbox12-GFP transgene, the resulting embryos were raised in the presence of TSA 50 nM, and observed at the indicated stages. Images for each embryo are shown under DIC optic superimposed on epifluorescence field. (E) qPCR measurements of gfp transcript abundance in blastulae treated with 50 nM TSA. Data are normalized and indicated as in Fig 1C.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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pone.0143860.g002: Impact of TSA on acetylation of H3K9 and hbox12 promoter activity.(A) Western blot analysis using the anti-H3K9ac antibody. Nuclear extracts from control and 50 nM TSA-treated blastula stage embryos were fractioned by SDS-PAGE (on the left is shown the gel staining), blotted on PVDF membrane and incubated with anti-H3K9ac antibody. (B and C) ChIP-qPCR analysis of the hbox12 promoter occupancy by H3K9ac and HDAC-1. ChIP assays were performed on chromatin extracted from control and TSA-treated embryos at the mesenchyme blastula stage and precipitated with commercial antiserum against H3K9ac or HDAC-1, or incubated without adding antibodies, followed by qPCR amplification of an hbox12 promoter fragment. Data are normalized according to the percent of input method. Bars are as in Fig 1C. (D) Zygotes were injected with the phbox12-GFP transgene, the resulting embryos were raised in the presence of TSA 50 nM, and observed at the indicated stages. Images for each embryo are shown under DIC optic superimposed on epifluorescence field. (E) qPCR measurements of gfp transcript abundance in blastulae treated with 50 nM TSA. Data are normalized and indicated as in Fig 1C.
Mentions: First, by western blot analysis carried out using a specific antibody that recognizes H3K9ac, we ascertained that mesenchyme blastulae treated with 50 nM TSA accumulated a global increase in the acetylated H3 level compared to that of control embryos at the same stage (Fig 2A). Next, quantitative ChIP assays with the anti-acetyl-H3K9 antibody were performed on chromatin purified from mesenchyme blastulae treated or untreated with TSA. As expected, no or faint amplification was detected for the chromatin samples incubated without antibody, used as a negative control (Fig 2B). By contrast, the amplification of a specific DNA segment from the hbox12 promoter clearly demonstrated that it was heavily enriched in acetylated histone H3 in TSA-treated embryos (Fig 2B). Thus, TSA likely exerted a direct effect on histone acetylation at the hbox12 promoter.

Bottom Line: Transcription of nodal concomitantly drops, prejudicing dorsal/ventral polarity of the resulting larvae.Remarkably, impairing hbox12 function, either in a spatially-restricted sector or in the whole embryo, specifically rescues nodal transcription in Trichostatin-A-treated larvae.Beyond strengthen the notion that nodal expression is not allowed in the presence of functional Hbox12 in the same cells, these results highlight a critical role of histone deacetylases in regulating the spatial expression of hbox12.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Italy.

ABSTRACT
Dorsal/ventral patterning of the sea urchin embryo depends upon the establishment of a Nodal-expressing ventral organizer. Recently, we showed that spatial positioning of this organizer relies on the dorsal-specific transcription of the Hbox12 repressor. Building on these findings, we determined the influence of the epigenetic milieu on the expression of hbox12 and nodal genes. We find that Trichostatin-A, a potent and selective histone-deacetylases inhibitor, induces histone hyperacetylation in hbox12 chromatin, evoking broad ectopic expression of the gene. Transcription of nodal concomitantly drops, prejudicing dorsal/ventral polarity of the resulting larvae. Remarkably, impairing hbox12 function, either in a spatially-restricted sector or in the whole embryo, specifically rescues nodal transcription in Trichostatin-A-treated larvae. Beyond strengthen the notion that nodal expression is not allowed in the presence of functional Hbox12 in the same cells, these results highlight a critical role of histone deacetylases in regulating the spatial expression of hbox12.

Show MeSH
Related in: MedlinePlus