Inhibition of methyltransferase activity of enhancer of zeste 2 leads to enhanced lipid accumulation and altered chromatin status in zebrafish

BACKGROUND: Recent studies indicate that exposure to environmental chemicals may increase susceptibility to developing metabolic diseases. This susceptibility may in part be caused by changes to the epigenetic landscape which consequently affect gene expression and lead to changes in lipid metabolism. The epigenetic modifier enhancer of zeste 2 (Ezh2) is a histone H3K27 methyltransferase implicated to play a role in lipid metabolism and adipogenesis. In this study, we used the zebrafish (Danio rerio) to investigate the role of Ezh2 on lipid metabolism and chromatin status following developmental exposure to the Ezh1/2 inhibitor PF-06726304 acetate. We used the environmental chemical tributyltin (TBT) as a positive control, as this chemical is known to act on lipid metabolism via EZH-mediated pathways in mammals. RESULTS: Zebrafish embryos (0-5 days post-fertilization, dpf) exposed to non-toxic concentrations of PF-06726304 acetate (5 μM) and TBT (1 nM) exhibited increased lipid accumulation. Changes in chromatin were analyzed by the assay for transposase-accessible chromatin sequencing (ATAC-seq) at 50% epiboly (5.5 hpf). We observed 349 altered chromatin regions, predominantly located at H3K27me3 loci and mostly more open chromatin in the exposed samples. Genes associated to these loci were linked to metabolic pathways. In addition, a selection of genes involved in lipid homeostasis, adipogenesis and genes specifically targeted by PF-06726304 acetate via altered chromatin accessibility were differentially expressed after TBT and PF-06726304 acetate exposure at 5 dpf, but not at 50% epiboly stage. One gene, cebpa, did not show a change in chromatin, but did show a change in gene expression at 5 dpf. Interestingly, underlying H3K27me3 marks were significantly decreased at this locus at 50% epiboly. CONCLUSIONS: Here, we show for the first time the applicability of ATAC-seq as a tool to investigate toxicological responses in zebrafish. Our analysis indicates that Ezh2 inhibition leads to a partial primed state of chromatin linked to metabolic pathways which results in gene expression changes later in development, leading to enhanced lipid accumulation. Although ATAC-seq seems promising, our in-depth assessment of the cebpa locus indicates that we need to consider underlying epigenetic marks as well.</p

Dose rate dependent reduction in chromatin accessibility at transcriptional start sites long time after exposure to gamma radiation

Ionizing radiation (IR) impact cellular and molecular processes that require chromatin remodelling relevant for cellular integrity. However, the cellular implications of ionizing radiation (IR) delivered per time unit (dose rate) are still debated. This study investigates whether the dose rate is relevant for inflicting changes to the epigenome, represented by chromatin accessibility, or whether it is the total dose that is decisive. CBA/CaOlaHsd mice were whole-body exposed to either chronic low dose rate (2.5 mGy/h for 54 d) or the higher dose rates (10 mGy/h for 14 d and 100 mGy/h for 30 h) of gamma radiation (60Co, total dose: 3 Gy). Chromatin accessibility was analysed in liver tissue samples using Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-Seq), both one day after and over three months post-radiation (>100 d). The results show that the dose rate contributes to radiation-induced epigenomic changes in the liver at both sampling timepoints. Interestingly, chronic low dose rate exposure to a high total dose (3 Gy) did not inflict long-term changes to the epigenome. In contrast to the acute high dose rate given to the same total dose, reduced accessibility at transcriptional start sites (TSS) was identified in genes relevant for the DNA damage response and transcriptional activity. Our findings link dose rate to essential biological mechanisms that could be relevant for understanding long-term changes after ionizing radiation exposure. However, future studies are needed to comprehend the biological consequence of these findings.publishedVersio

Chromatin accessibility after inhibition of enhancer of zeste homolog 2 (Ezh2) in zebrafish embryos

The thesis is a part of a Centre of Excellence Centre for Environmental Radioactivity (CoE CERAD) project, focusing on trans-generational studies of low-dose gamma-irradiation using zebrafish as a model organism. As a part of the epigenetic toolbox, Assay for Transposase-Accessible Chromatin (ATAC-seq) is established using a known epigenetic inhibitor (Ezh2i), PF-06726304 acetate, in a pilot study with the hypothesis that PF-06726304 acetate would induce a decline in H3K27me3 marks in chromatin, correlated with condensed chromatin and silent genes. Subgoals were to establish methods for manual dechorionation and protocols for enzymatic dechorionation, establish methods for ATAC-seq library generation and clean-up, and establish a bioinformatic pipeline for preliminary ATAC-seq analysis. The ATAC-seq results from the treatment of PF-06726304 were validated with Western Blot and Chromatin Immunoprecipitation. The thesis shows that low-dose exposure to the Ezh2 inhibitor, PF-06726304 acetate, indeed induces chromatin reorganization. Our results suggest that a GUI tool for the Analysis and Visualization of ATAC-seq data (GUAVA) can be used as a primary dataset analysis tool, but that more thorough post-analysis is necessary

Kromatintilgjengelighet etter inhibering av enhancer of zeste homolog 2 (Ezh2) i sebrafish embryo

The thesis is a part of a Centre of Excellence Centre for Environmental Radioactivity (CoE CERAD) project, focusing on trans-generational studies of low-dose gamma-irradiation using zebrafish as a model organism. As a part of the epigenetic toolbox, Assay for Transposase-Accessible Chromatin (ATAC-seq) is established using a known epigenetic inhibitor (Ezh2i), PF-06726304 acetate, in a pilot study with the hypothesis that PF-06726304 acetate would induce a decline in H3K27me3 marks in chromatin, correlated with condensed chromatin and silent genes. Subgoals were to establish methods for manual dechorionation and protocols for enzymatic dechorionation, establish methods for ATAC-seq library generation and clean-up, and establish a bioinformatic pipeline for preliminary ATAC-seq analysis. The ATAC-seq results from the treatment of PF-06726304 were validated with Western Blot and Chromatin Immunoprecipitation. The thesis shows that low-dose exposure to the Ezh2 inhibitor, PF-06726304 acetate, indeed induces chromatin reorganization. Our results suggest that a GUI tool for the Analysis and Visualization of ATAC-seq data (GUAVA) can be used as a primary dataset analysis tool, but that more thorough post-analysis is necessary.Denne oppgaven er en del av et prosjekt under Senter for radioaktivitet, mennesker og miljø (SFF CERAD), med fokus på transgenerasjonsstudier av lavdose gamma-bestråling ved bruk av sebrafisk som modellorganisme. Som en del av den epigenetiske verktøykassen er metoden, «Assay for Transposase-Accessible Chromatin (ATAC-seq)» etablert ved bruk av en kjent epigenetisk inhibitor (Ezh2i), PF-06726304 acetat, i en pilotstudie med hypotesen om at PF-06726304 acetat ville indusere en nedgang i H3K27me3 i kromatin, som har sammenheng med pakket sammen kromatin og inaktive gener. Delmål for oppgaven var å etablere en metode for manuell dechorionering, samt å etablere en metode for mer robust enzymatisk dechorionering, etablere metoder for rensning av ATAC-seq bibliotek, og etablere bioinformatisk nedstrømsanalyse for ATAC-seq analyse. ATAC-seq resultatene etter eksponering med PF-06726304 ble validert med Western Blot and Kromatin Immunopresipitering. Oppgaven viser at eksponering med lav dose til Ezh2-inhibitoren, PF-06726304 acetat, faktisk promoterer kromatinomorganisering. Resultatene tyder på at et GUAVA, som er et verktøy med grafisk brukergrensesnitt for analyse og visualisering av ATAC-seq-data (GUAVA) kan brukes som et foreløpig analyseverktøy, men at en grundigere analyse i etterkant er nødvendig.M-BIOTE

Kromatintilgjengelighet etter inhibering av enhancer of zeste homolog 2 (Ezh2) i sebrafish embryo

The thesis is a part of a Centre of Excellence Centre for Environmental Radioactivity (CoE CERAD) project, focusing on trans-generational studies of low-dose gamma-irradiation using zebrafish as a model organism. As a part of the epigenetic toolbox, Assay for Transposase-Accessible Chromatin (ATAC-seq) is established using a known epigenetic inhibitor (Ezh2i), PF-06726304 acetate, in a pilot study with the hypothesis that PF-06726304 acetate would induce a decline in H3K27me3 marks in chromatin, correlated with condensed chromatin and silent genes. Subgoals were to establish methods for manual dechorionation and protocols for enzymatic dechorionation, establish methods for ATAC-seq library generation and clean-up, and establish a bioinformatic pipeline for preliminary ATAC-seq analysis. The ATAC-seq results from the treatment of PF-06726304 were validated with Western Blot and Chromatin Immunoprecipitation. The thesis shows that low-dose exposure to the Ezh2 inhibitor, PF-06726304 acetate, indeed induces chromatin reorganization. Our results suggest that a GUI tool for the Analysis and Visualization of ATAC-seq data (GUAVA) can be used as a primary dataset analysis tool, but that more thorough post-analysis is necessary.Denne oppgaven er en del av et prosjekt under Senter for radioaktivitet, mennesker og miljø (SFF CERAD), med fokus på transgenerasjonsstudier av lavdose gamma-bestråling ved bruk av sebrafisk som modellorganisme. Som en del av den epigenetiske verktøykassen er metoden, «Assay for Transposase-Accessible Chromatin (ATAC-seq)» etablert ved bruk av en kjent epigenetisk inhibitor (Ezh2i), PF-06726304 acetat, i en pilotstudie med hypotesen om at PF-06726304 acetat ville indusere en nedgang i H3K27me3 i kromatin, som har sammenheng med pakket sammen kromatin og inaktive gener. Delmål for oppgaven var å etablere en metode for manuell dechorionering, samt å etablere en metode for mer robust enzymatisk dechorionering, etablere metoder for rensning av ATAC-seq bibliotek, og etablere bioinformatisk nedstrømsanalyse for ATAC-seq analyse. ATAC-seq resultatene etter eksponering med PF-06726304 ble validert med Western Blot and Kromatin Immunopresipitering. Oppgaven viser at eksponering med lav dose til Ezh2-inhibitoren, PF-06726304 acetat, faktisk promoterer kromatinomorganisering. Resultatene tyder på at et GUAVA, som er et verktøy med grafisk brukergrensesnitt for analyse og visualisering av ATAC-seq-data (GUAVA) kan brukes som et foreløpig analyseverktøy, men at en grundigere analyse i etterkant er nødvendig.M-BIOTE

Inhibition of methyltransferase activity of enhancer of zeste 2 leads to enhanced lipid accumulation and altered chromatin status in zebrafish

publishedVersio

Inhibition of Methyltransferase Activity of Enhancer of Zeste 2 Leads to Enhanced Lipid Accumulation and Altered Chromatin Status in Zebrafish

Recent studies indicate that exposure to environmental chemicals may increase susceptibility to developing metabolic diseases. This susceptibility might in part be caused by changes to the epigenetic landscape which consequently affect gene expression and lead to changes in lipid metabolism. These changes might be mediated by epigenetic markers such as Enhancer of Zeste2 (Ezh2), a histone H3K27 methyltransferase that has been implicated to play a role in lipid metabolismand adipogenesis. In this study, we used the zebrafish (Danio rerio) to investigate the role of Ezh2 on lipid metabolism and chromatin status following developmental exposure to the Ezh1/2 inhibitor PF-06726304 acetate

Inhibition of Methyltransferase Activity of Enhancer of Zeste 2 Leads to Enhanced Lipid Accumulation and Altered Chromatin Status in Zebrafish

Recent studies indicate that exposure to environmental chemicals may increase susceptibility to developing metabolic diseases. This susceptibility might in part be caused by changes to the epigenetic landscape which consequently affect gene expression and lead to changes in lipid metabolism. These changes might be mediated by epigenetic markers such as Enhancer of Zeste2 (Ezh2), a histone H3K27 methyltransferase that has been implicated to play a role in lipid metabolismand adipogenesis. In this study, we used the zebrafish (Danio rerio) to investigate the role of Ezh2 on lipid metabolism and chromatin status following developmental exposure to the Ezh1/2 inhibitor PF-06726304 acetate

Inhibition of methyltransferase activity of enhancer of zeste 2 leads to enhanced lipid accumulation and altered chromatin status in zebrafish

BACKGROUND: Recent studies indicate that exposure to environmental chemicals may increase susceptibility to developing metabolic diseases. This susceptibility may in part be caused by changes to the epigenetic landscape which consequently affect gene expression and lead to changes in lipid metabolism. The epigenetic modifier enhancer of zeste 2 (Ezh2) is a histone H3K27 methyltransferase implicated to play a role in lipid metabolism and adipogenesis. In this study, we used the zebrafish (Danio rerio) to investigate the role of Ezh2 on lipid metabolism and chromatin status following developmental exposure to the Ezh1/2 inhibitor PF-06726304 acetate. We used the environmental chemical tributyltin (TBT) as a positive control, as this chemical is known to act on lipid metabolism via EZH-mediated pathways in mammals. RESULTS: Zebrafish embryos (0-5 days post-fertilization, dpf) exposed to non-toxic concentrations of PF-06726304 acetate (5 μM) and TBT (1 nM) exhibited increased lipid accumulation. Changes in chromatin were analyzed by the assay for transposase-accessible chromatin sequencing (ATAC-seq) at 50% epiboly (5.5 hpf). We observed 349 altered chromatin regions, predominantly located at H3K27me3 loci and mostly more open chromatin in the exposed samples. Genes associated to these loci were linked to metabolic pathways. In addition, a selection of genes involved in lipid homeostasis, adipogenesis and genes specifically targeted by PF-06726304 acetate via altered chromatin accessibility were differentially expressed after TBT and PF-06726304 acetate exposure at 5 dpf, but not at 50% epiboly stage. One gene, cebpa, did not show a change in chromatin, but did show a change in gene expression at 5 dpf. Interestingly, underlying H3K27me3 marks were significantly decreased at this locus at 50% epiboly. CONCLUSIONS: Here, we show for the first time the applicability of ATAC-seq as a tool to investigate toxicological responses in zebrafish. Our analysis indicates that Ezh2 inhibition leads to a partial primed state of chromatin linked to metabolic pathways which results in gene expression changes later in development, leading to enhanced lipid accumulation. Although ATAC-seq seems promising, our in-depth assessment of the cebpa locus indicates that we need to consider underlying epigenetic marks as well