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

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.

Epigenetic therapy targeting bone marrow mesenchymal stem cells for age-related bone diseases

“…In the process of cartilage formation, methylation changes are distinctive in BMSCs. Especially in the enhancers marked by characteristic histone modifications (H3K4me1 and H3K27ac), abundant DNA demethylation occurs, while the CpGs of these enhancers are highly methylated in other tissues [ 130 , 131 ]. Researchers have also discovered new clues between epigenetic pathophysiology and OA.…”

Genome-wide DNA methylation and multi-omics study of human chondrocyte ontogeny and an epigenetic clock analysis of adult chondrocytes

“…Besides CpGs in adult chondrocytes, which lose methylation with age are most enriched for the active enhancer chromatin state suggestive of transcriptional regulation from these regions. Of note, gain or loss of methylation in CpGs correlated with age in both fetal and adult chondrocytes show enrichment for chromatin states associated with enhancers (marked by H3K27ac) which might indicate the previously known fact that chondrocytes acquire cell-type-specific enhancers upon differentiation 32 . We further investigated the chromatin state for the chondrogenic genes mentioned previously in Fig 1e and closer inspection of these loci demonstrate presence of active histone modifications characterized by presence of H3K27ac while H3K27me3 repressive mark is mostly absent ( Fig 2c ).…”

Histone Modifications and Chondrocyte Fate: Regulation and Therapeutic Implications

“…H3K4me3 and histone 3 lysine 27 acetylation (H3K27ac) are enriched in highly expressed genes, while H3K27me3 shows a greater enrichment in genes with low expression levels in different developmental stages of chondrocyte. Histone modifications around the enhancer and promoter of the key chondrocyte marker genes SOX9 and collagen type II (COL2A1) switch from repression in human MSCs to transcriptionally active in differentiated chondrocytes (Wang et al, 2012;Nishimura et al, 2013;Cheung et al, 2020). Therefore, a comprehensive understanding of the histone modifications associated with cartilage development and…”