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

Abstract

Human mesenchymal stem cells are able to differentiate into chondrocytes, the cell type found in cartilage, making them an accessible system to study gene regulation during this process. Epigenetic mechanisms such as histone modifications and DNA methylation together with transcription factor binding play a role in activating and repressing gene expression. In this study, we investigated the genome-wide histone modification changes during chondrocyte differentiation. Integration of this data with DNA methylation and SOX9 transcription factor ChIP-seq revealed epigenetic changes at gene enhancer elements.Regions of the genome that transition from non-enhancers to enhancers in chondrocytes are enriched for SOX9 transcription factor binding sites. Luciferase reporter assays revealed that enhancer activity may be modulated by manipulating DNA methylation and SOX9 expression.This study has defined important regulatory elements in chondrocytes which could serve as targets for future mechanistic studies. AbstractEpigenetic mechanisms are known to regulate gene expression during chondrogenesis. In this study, we have characterised the epigenome during 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 post-transcriptional modifications (marks) to histone H3 lysines (H3K4me3, H3K4me1, H3K27ac, H3K27me3 and H3K36me3) in both hMSCs and differentiated chondrocytes.Chromatin states were characterised 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 studywith 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 characterised in this study exhibited enhancer activity which may be modulated by inducing DNA methylation and SOX9 overexpression. Altogether, these integrated data illustrate the cross-talk between different epigenetic mechanisms during chondrocyte differentiation.

STAT3 promotes a youthful epigenetic state in articular chondrocytes

“…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 likely reflects the fact that cell type specific enhancers are activated upon chondrocyte differentiation (Cheung et al, 2020). Together, these findings affirm that age‐correlated CpGs are intrinsically tied to distinct chromatin states.…”

Epigenetic Regulators of Mesenchymal Stem/Stromal Cell Lineage Determination

“…A similar study examining chondrogenesis in vitro employed a gene stratification analysis into low, middle and high expression. Histone methylation marks H3K4me3, H3K27Ac, H3K4me1 and H3K36me3 were associated with high expressing genes [69]. Conversely, H3K27me3 was associated with low expressing genes.…”