DNA hypomethylation during MSC chondrogenesis occurs predominantly at enhancer regions

Barter, M.J.; Bui, Catherine; Cheung, Kathleen; Gómez, Rodolfo; Skelton, Andrew; Elliott, Hannah R; Reynard, Louise N.; Young, David A.

doi:10.1101/625954

Cited by 4 publications

(5 citation statements)

References 53 publications

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“…There was also a significantly higher level of global methylation in GD13 than in GD14 cultivated limb buds. This all agrees with the theory that global levels of DNA methylation increase during differentiation, although the expression of genes specific for a cell line, e.g., chondrocytes, is associated with hypomethylation of genes typical of cartilage, mostly in enhancer regions [5].…”

Section: Discussionsupporting

confidence: 89%

“…At a gene promoter or enhancer sequences, DNA methylation is frequently associated with gene repression, correlating with the presence of inhibitory histone modifications that prevent the binding of transcription factors. Passive DNA demethylation may occur during cell replication, while active DNA demethylation is achieved by ten-eleven translocation proteins (TET) and oxidative intermediates [5,6]. DNA methylation marks are mostly posed on cytosines of the promoter and enhancer regions but also on gene bodies or repetitive sequences.…”

Section: Introductionmentioning

confidence: 99%

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Global DNA methylation and chondrogenesis of rat limb buds in a three-dimensional organ culture system

Radović¹,

Bunoza²,

Marić³

et al. 2022

Bosn J of Basic Med Sci

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Although DNA methylation epigenetically regulates development, data on global DNA methylation during development of limb buds (LBs) are scarce. We aimed to investigate the global DNA methylation developmental dynamics in rat LBs cultivated in a serum-supplemented (SS) and in chemically defined serum- and protein-free (SF) three-dimensional organ culture. Fischer rat front- and hind-LBs at 13th and 14th gestation days (GD) were cultivated at the air-liquid interface in Eagle's Minimal Essential Medium (MEM) or MEM with 50% rat serum for 14 days, as SF and SS conditions, respectively. The methylation of repetitive DNA sequences (SINE rat ID elements) was assessed by pyrosequencing. Development was evaluated by light microscopy and extracellular matrix glycosaminoglycans staining by Safranin O. Upon isolation, weak Safranin O staining was present only in more developed GD14 front-LBs. Chondrogenesis proceeded well in all cultures towards day 14, except in the SF-cultivated GD13 hind-LBs, where Safranin O staining was almost absent on day 3. That was associated with a higher percentage of DNA methylation than in SF-cultivated GD13 front-LBs on day three. In SF-cultivated front-LBs, a significant methylation increase between the 3rd and 14th day was detected. In SS-cultivated GD13 front-LBs, methylation increased significantly on day three and then decreased. In older GD14 SS-cultivated LBs, there was no increase of DNA methylation, but they were significantly hypomethylated relative to the SS-cultivated GD13 at days 3 and 14. We confirmed that the global DNA methylation increase is associated with less developed limb organ primordia that strive towards differentiation in vitro, which is of importance for regenerative medicine strategies.

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Section: Discussionsupporting

confidence: 89%

Section: Introductionmentioning

confidence: 99%

Global DNA methylation and chondrogenesis of rat limb buds in a three-dimensional organ culture system

Radović¹,

Bunoza²,

Marić³

et al. 2022

Bosn J of Basic Med Sci

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“…An Illumina Infinium HumanMethylation450K BeadChip array was used to measure DNA methylation. DNA methylation changes during the in vitro Transwell model of chondrogenesis were largely de‐methylation events that were associated with chondrogenesis‐related GO terms . We integrated the DNA methylation and ChIP‐seq data in order to investigate the DNA methylation changes in chromatin states during MSC chondrogenesis, focusing on the hypomethylated CpGs (94% of the significantly differentially methylated loci during chondrogenesis) since this is linked to gene transcription activation.…”

Section: Resultsmentioning

confidence: 97%

“…An Infinium HumanMethylation450 BeadChip array was used to quantify DNA methylation in the Transwell model of chondrogenesis, GEO dataset http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE129266. CpG probes from the 450K methylation array were based on human reference genome hg19; therefore, CpG coordinates from the array were first converted to hg38 and intersected with chromatin state coordinates from hMSC and differentiated chondrocytes.…”

Section: Methodsmentioning

confidence: 99%

“…DNA methylation changes during the in vitro Transwell model of chondrogenesis were largely demethylation events that were associated with chondrogenesisrelated GO terms. 36 We integrated the DNA methylation and ChIP-seq data in order to investigate the DNA methylation changes in chromatin states during MSC chondrogenesis, focusing on the hypomethylated CpGs (94% of the significantly differentially methylated loci during chondrogenesis) since this is linked to gene transcription activation. Global methylation patterns reflect known trends (Figure 5A,B), for example, gene promoters tend to have low percentage methylation relative to the rest of the genome.…”

Section: Dna Methylation At Gene Enhancersmentioning

confidence: 99%

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

et al. 2020

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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.

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

Cheung

Barter

Falk

et al. 2019

Preprint

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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.

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DNA hypomethylation during MSC chondrogenesis occurs predominantly at enhancer regions

Cited by 4 publications

References 53 publications

Global DNA methylation and chondrogenesis of rat limb buds in a three-dimensional organ culture system

Global DNA methylation and chondrogenesis of rat limb buds in a three-dimensional organ culture system

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

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

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