Histone ChIP‐Seq identifies differential enhancer usage during chondrogenesis as critical for defining cell‐type specificity

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.