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Inhibition of the epigenetic regulator Ezh2 redistributes bivalent domains within transcriptional regulators associated with Wnt and Hedgehog pathways

ABSTRACT: Osteoporosis is characterized by bone loss, reduced bone strength, and increased fracture risk. Studies have shown that epigenetic mechanisms play a role during bone formation and bone-related disorders, including osteoporosis. However, the contribution of bivalent domains, which are chromatin regions that are co-occupied by the gene activating Histone 3 lysine 4 trimethylation (H3K4me3) and the gene suppressive Histone 3 lysine 27 trimethylation (H3K27me3) marks, has not been elucidated during osteogenesis. Previous work demonstrated that the enhancer of zeste homolog 2 (Ezh2), a histone 3 lysine 27 (H3K27) methyltransferase, suppressed differentiation of osteogenic progenitor cells and that a small molecule inhibitor of Ezh2 (GSK126) enhanced osteogenic commitment of osteoblast progenitors in vitro and bone formation in vivo. Tazemetostat (or EPZ6438) is an Ezh2 inhibitor (iEzh2) that is FDA approved for the treatment of epithelioid sarcoma and might be repurposed to mitigate bone loss. The present study aimed to elucidate mechanisms by which Tazemetostat-mediated H3K27me3 reduction regulates bivalent domain associated enhancers critical for bone formation. Tazemetostat enhanced the expression of bone-related genes (e.g., Runx2-p57, Bglap, Ibsp) as measured by qPCR analysis and stimulated extracellular matrix deposition as revealed by alizarin red staining of cell cultures. Western blotting analysis revealed that Tazemetostat reduced H3K27me3 levels and concomitantly enhanced H3K27Ac, as well as increased protein levels of the osteoblastic master regulator Runx2. Integrative analysis of RNA-seq and ChIP-seq datasets revealed that upregulation of group of genes that are associated with bivalent domains. Importantly, gene ontology analysis showed that genes associated with Wnt, Pth and Hh signaling pathways are enriched within these up-regulated genes. Follow-up mechanistic-based studies revealed that the pro-osteogenic effects induced by Ezh2 inhibition are attenuated by inhibition of Hh signaling and enhanced by disruption of Wnt signaling. Our data demonstrate that the genes controlled by bivalent domains are associated with key osteogenic signaling pathways and are induced upon Ezh2 inhibition and subsequent H3K27me3 reduction. Our data also demonstrate that iEzh2-induced osteogenic differentiation is modulated by Wnt and Hh signaling pathways.

ORGANISM(S): Mus musculus

PROVIDER: GSE196583 | GEO | 2023/01/01

REPOSITORIES: GEO

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Project description:High-throughput microRNA sequencing was used to identify microRNAs (miRs) that regulate osteoblast differentiation. We establish that miR-101a, which targets the epigenetic enzyme Ezh2, is up-regulated in differentiated MC3T3 cells and robustly expressed in mouse calvaria. Our previous studies showed that loss of Ezh2 function enhances osteogenesis and bone formation. Thus, we tested the attractive mechanistic model that increased miR-101s expression may suppress Ezh2 to stimulate the osteogenic process. Transient miR-101a over-expression suppresses Ezh2 levels and reduces tri-methylation on lysine 27 of histone 3 (H3K27me3), a heterochromatic mark catalyzed by Ezh2. Importantly, over-expression of miR-101a stimulates osteogenic differentiation of MC3T3 cells as quantified by alizarin red staining. Therefore, we examined skeletal phenotypes of mice transgenic for miR-101a under the control of Col1a1promoer and doxycycline administration. Experimental controls and mir-101 over-expressing mice were exposed to doxycycline during pregnancy and postnatal stages (phenotyping at 8 weeks) to maximize penetrance of skeletal phenotypes. Our analyses revealed that miR-101a over-expressing male mice are bigger as measured by total body weight and exhibit an increase in long bone length. These mice exhibit significant increases in trabecular bone volume fraction, trabecular number, trabecular thickness, as well as a reduction in trabecular spacing based on microCT analysis. Histomorphometric examination established a significant reduction in osteoid volume to bone volume and osteoid surface to bone surface. These findings suggest that the enhancement in trabecular bone may be due to accelerated bone mineralization in miR-101a over-expressing male mice. Remarkably, while female mice exhibit a significant increase in bone length, no significant changes were noted by microCT (trabecular bone parameters) and histomorphometry (osteoid parameters). We conclude that miR-101a upregulation during osteoblast maturation enhances trabecular bone parameters in male mice. Thus, miR-101 and Ezh2 may form part of an intricate epigenetic feed-forward mechanisms to regulate bone formation.

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Inhibition of the epigenetic regulator Ezh2 redistributes bivalent domains within transcriptional regulators associated with Wnt and Hedgehog pathways

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