Abstract
Epigenetic mechanisms are known to regulate gene expression during chondrogenesis. In this study, we have characterized the epigenome during the in vitro differentiation of human mesenchymal stem cells (hMSCs) into chondrocytes. Chromatin immunoprecipitation followed by next‐generation sequencing (ChIP‐seq) was used to assess a range of N‐terminal posttranscriptional modifications (marks) to histone H3 lysines (H3K4me3, H3K4me1, H3K27ac, H3K27me3, and H3K36me3) in both hMSCs and differentiated chondrocytes. Chromatin states were characterized using histone ChIP‐seq and cis‐regulatory elements were identified in chondrocytes. Chondrocyte enhancers were associated with chondrogenesis‐related gene ontology (GO) terms. In silico analysis and integration of DNA methylation data with chondrogenesis chromatin states revealed that enhancers marked by histone marks H3K4me1 and H3K27ac were de‐methylated during in vitro chondrogenesis. Similarity analysis between hMSC and chondrocyte chromatin states defined in this study with epigenomes of cell‐types defined by the Roadmap Epigenomics project revealed that enhancers are more distinct between cell‐types compared to other chromatin states. Motif analysis revealed that the transcription factor SOX9 is enriched in chondrocyte enhancers. Luciferase reporter assays confirmed that chondrocyte enhancers characterized in this study exhibited enhancer activity which may be modulated by DNA methylation and SOX9 overexpression. Altogether, these integrated data illustrate the cross‐talk between different epigenetic mechanisms during chondrocyte differentiation.
Highlights
Chondrogenesis is the process of differentiation of mesenchymal progenitors into chondrocytes
Hypertrophic chondrocytes bound for ossification have high expression of COL10A1 and osteoblast markers such as runt-related transcription factor 2 (RUNX2), and low expression of cartilage-specific genes such as collagen type II alpha chain (COL2A1) and sex determining region Y box 9 (SOX9).1,2 Chondrogenesis is a multi-step tightly regulated process mediated by growth and transcription factors, with the SOX9 transcription factor instrumental to the progression of chondrogenic differentiation 3 not initiation.[4]
Mesenchymal stem cells (MSCs) are able to differentiate into chondrocytes and have been used to study chondrogenesis in vitro
Summary
Chondrogenesis is the process of differentiation of mesenchymal progenitors into chondrocytes. Deletions in a distal regulatory region of the SOX9 transcription factor gene and within the SOX9 gene itself both lead to campomelic dysplasia in humans.[11,12] Mutations in enhancers of collagen genes are associated with chondrodysplasias.[13,14] Osteoarthritis (OA), an age-related cartilage degenerative disease, has a strong genetic component and to date, the vast majority of polymorphisms that confer an increased risk are located in non-coding regions of the genome, including enhancers.[15,16] There is evidence that the OA phenotype may be linked to the reactivation of developmental pathways.[17] Articular cartilage affected by OA shows gene expression changes reminiscent of hypertrophic chondrocytes.[1,18] These studies demonstrate that epigenetic mechanisms regulate gene expression in numerous biological processes. Our study provides a comprehensive analysis of the global epigenetic changes during MSC chondrogenesis and highlights the role of enhancers in defining cell-type specificity
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