Transcriptional deregulation during osteoclastic differentiation due to diuron exposure affects preferentially super-enhancers regulated genes

Osteoclasts are major actors in the maintenance of bone homeostasis. The full functional maturation of osteoclasts from monocyte lineage cells is essential for the degradation of old/damaged bone matrix. To better characterize the differentiation and maturation of CD14+ monocytes into functionally mature osteoclasts, we performed Chromatin Immunoprecipitation against H3K27ac followed by Sequencing (ChIP-Seq) and RNA-Sequencing at different stages of an in vitro differentiation model. The combinatorial study of the epigenetic and transcriptional dynamics taking place during the course of differentiation has allowed us to reveal a very dynamic epigenetic profile that supports the expression of genes vital for osteoclasts differentiation and function. In total, we identified 122 genes induced by dynamic super-enhancers at late days. Since diuron, a commonly detected herbicide in our environment, is suspected to affect bone development, we then sought to evaluate the potential impact of diuron exposure on bone homeostasis. We performed RNA-Seq and functional test during in vitro osteoblastic and osteoclastic differentiation. First of all, our data suggest that high-dose of diuron (50 µM) affects viability of mesenchymal stem cells and thus inhibits bone mineralization. We observed at lower dose (1 µM) an inhibitory effect of this herbicide on number of osteoclasts derived from CD14+ monocytes without affecting cell viability. RNA-Seq data analysis revealed that diuron is specifically affecting the transcriptional network dedicated to osteoclasts differentiation and activity. Among the diuron affected genes, our analysis suggests a significant enrichment of genes targeted by gained super-enhancers along the differentiation process, with an odds ratio of 5.12 (ρ = 2.59 x 10-5). Taken together our results suggest that diuron exposure disrupts osteoclasts maturation by impairing the expression of cell-identity determining genes. Diuron dataset: CD14+ pre-monocytes were differentiated into osteoclasts using RANK+M-CSF treatment in the presence of either non-cytotoxic concentration of diuron or DMSO and RNA-Sequencing was performed at early stage (day3) or late stage of differentiation (day 10). Differentiation RNA-Seq data: CD14+ pre-monocytes were cultered with or withour differentiation medium and RNA-Sequencing was performed at early stage (day3), intermediate stage (day7) or late stage of differentiation (day 10). Differentiation ChIP-Seq data: In order to track epigenetic modifications within enhancers and super-enhancers H3K27ac ChIP-Seq was performed on cells cultered for 3 days without differentiation medium, cultered for 7 days or 10 days with differentiation medium.