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

Abstract

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.

Loss of Polycomb Repressive Complex 2 Function Alters Digestive Organ Homeostasis and Neuronal Differentiation in Zebrafish

“…Oil red-O staining clearly shows a strong accumulation of neutral lipids in the liver of eed −/− larvae à 11 dpf when compared to wild-type larvae (Figure 6a). Such a strong oil red-O staining of the liver was also found in ezh2 −/− homozygous mutants [38], whereas exposure to Ezh1/2 inhibitor PF-06726304 acetate increases lipid accumulation in larval zebrafish [71]. Moreover, hematoxylin and eosin stain on histological sections of zebrafish larvae at 11 dpf showed signs of macrovesicular steatosis in eed −/− larvae (Figures 5b and 6b,c).…”

Epigenetic Transgenerational Inheritance of the Effects of Obesogen Exposure

“…These mechanisms might interact and be preserved across generations and in various types of differentiated cells ( 32 ). In accordance with this hypothesis, exposure of zebrafish to low levels of TBT led to increased lipid accumulation and altered DNA accessibility at 349 chromatin regions at the H3K27me3 loci near genes involved in adipogenesis and metabolism ( 33 ). The epigenetic changes that took place in the chromatin of zebra fish larvae were proposed to prime the chromatin in early development, which led to changes gene expression and lipid accumulation later in life throughout development.…”

KDM6B interacts with TFDP1 to activate P53 signaling in regulating mouse palatogenesis

“…Chromatin accessibility represents the degree to which chromatinized DNA is able to physically interact with nuclear macromolecules such as transcription factors for gene regulation ( Klemm et al, 2019 ). The repressive mark H3K27me3 is usually associated with facultative heterochromatin and results in transcriptional repression due to decreased chromatin accessibility ( Wiles and Selker, 2017 ; Möller et al, 2019 ; den Broeder et al, 2020 ). The methyltransferase EZH2 and demethylases KDM6A/KDM6B can regulate the methylation status of H3K27 to affect gene expression ( Pediconi et al, 2019 ).…”