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CUT&RUN reveals STAT3-regulated DNA Methylation in Cartilage Development and Disease

By Stuart P. Atkinson, Ph.D.

October 10, 2024

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Introduction: Does STAT3-Mediated DNA Methylation Regulate Chondrocyte Function During Aging?

A better understanding of the cellular and molecular mechanisms that regulate the functional alterations and decline in regenerative potential in articular chondrocytes – cells that support the maintenance of cartilage tissues within joints – during normal human aging may support research into conditions such as osteoarthritis (Martel-Pelletier et al.). The implication of the STAT3 transcriptional factor (Burdon et al. and Moresi et al.) in osteoarthritis (Wang et al.) and tissue regeneration (Nakao et al.) has suggested that STAT3 may play differing roles during different epochs ofaging. Early postnatal STAT3 deletion in mouse chondrocytes suppresses proliferation and prompts the degradation of the growth plate, while STAT3 overexpression in postnatal mouse chondrocytes prompts hyperproliferation (Liu et al.). Furthermore, a role for STAT3 may suggest the general importance of epigenetic regulation, given that STAT3 regulates chromatin accessibility via DNA methylation (Zhang et al.), a known regulator of early cartilage development (Fernandez-Tajes et al. and Sanchez-Fernandez et al.). Does STAT3-mediated DNA methylation regulate chondrocyte function during aging?

Researchers guided by Steve Horvath (University of California, Los Angeles) and Denis Evseenko (University of Southern California) recently profiled DNA methylation in human chondrocytes and constructed an "epigenetic clock" to establish an association between DNA methylation at specific CpGs and age. Alongside this advance, their recently published findings now define a role for STAT3 in regulating DNA methylation in cartilage development and disease, thanks partly to the application of the cleavage under targets and release using nuclease (CUT&RUN) assay (Sarkar et al.).

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DNA Methylation and Chromatin State Analysis in Human Chondrocytes

Initial DNA methylation analysis in human fetal and adult chondrocytes revealed a correlation between CpG methylation and age. Genes losing DNA methylation with age with downregulated expression in fetal compared to adult chondrocytes displayed enrichment for functions that indicated a correlation between aging and an increase in destructive responses. Meanwhile, genes gaining DNA methylation with age with upregulated expression in fetal compared to adult chondrocytes displayed enrichment for functions associated with developing chondrocyte homeostasis/anabolism.

Do differing chromatin states associate with these age-correlated CpGs in fetal and adult chondrocytes? CpGs in fetal chondrocytes gaining DNA methylation with age tended to display an enrichment for poised promoters/bivalent states (co-existing H3K4me3 and H3K27me3). In contrast, CpGs in adult chondrocytes losing DNA methylation with age tended to display an active enhancer-associated chromatin state, suggesting transcriptional regulation from these regions. Additionally, gain or loss of DNA methylation at CpGs correlated with age in fetal and adult chondrocytes displayed enrichment for chromatin states reflecting cell type-specific enhancer activation during chondrocyte differentiation.

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A First Epigenetic Clock for Human Adult Chondrocytes

Sarkar et al. next employed their DNA methylation data to construct a human adult chondrocyte epigenetic clock and accurately predict the epigenetic age of adult chondrocytes after treatment with a STAT3 agonist. STAT3 activation in aged adult chondrocytes (low basal STAT3 signaling levels) decreased DNA methylation levels and biological age, which may provide the basis for specific therapeutic approaches for lost function in aged articular chondrocytes. A subsequent analysis of how reduced STAT3 function affected DNA methylation in fetal chondrocytes (high basal STAT3 signaling levels) (Shkhyan et al.) employed short hairpin RNA and revealed a global gain in DNA methylation that could accelerate epigenetic aging. The team next determined whether the gain in DNA methylation in STAT3-ablated fetal chondrocytes occurred at the same CpGs that lose DNA methylation upon STAT3 activation in adult chondrocytes. Overall, the functional enrichment analysis of genes associated with these CpGs indicated that gain/loss of DNA methylation occurs mainly at genes related to critical age-related processes, including cartilage and skeletal system development and chromatin organization.

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CUT&RUN Explores STAT3 Targets and Highlights DNMT3B

The epigenetic findings of this study suggested that STAT3 may possess different context-specific targets in development and disease; therefore, the authors performed CUT&RUN profiling on fetal, adult, and osteoarthritic chondrocytes to evaluate STAT3 binding patterns. The CUT&RUN results suggested that STAT3 regulated putative target gene expression by binding to distal regulatory elements and that fetal, adult, and osteoarthritic chondrocytes displayed a distinct set of target binding motifs; however, they detected an overlap of putative target genes shared in development and disease. While most associated with extracellular matrix organization and skeletal system development, the team also observed genes involved in DNA methylation and chromatin remodeling, suggesting that STAT3 mediates context-specific roles via epigenetic regulation. Indeed, the gene encoding the DNMT3B de novo DNA methyltransferase represented one such shared target; additional research indicated that STAT3 binding repressed DNMT3B transcription in fetal chondrocytes while activation of STAT3 signaling in adult chondrocytes significantly decreased DNMT3B levels and DNA methylation. The authors proposed a model in which DNMT3B mediates the global DNA methylation changes induced after STAT3 knockdown in fetal chondrocytes/pharmacological activation in adult chondrocytes, leading to epigenetic age alterations.

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STAT3 Induces an Immature Chondrocytes Program in the Battle Against Osteoarthritis

Finally, the authors explored whether higher STAT3 expression in osteoarthritic chondrocytes induced a fetal-like immature program as an attempt to regenerate lost tissue in response to injury. Interestingly, immature/proliferative gene expression profiling (Ferguson et al.) across fetal, adult, and osteoarthritic chondrocytes revealed higher expression in fetal and osteoarthritic adult chondrocytes than in normal adult chondrocytes. In vivo studies applying a post-traumatic osteoarthritic mouse model (Shkhyan et al.) revealed that Stat3 deletion in chondrocytes induced prominent osteoarthritic progression and a significant increase in DNMT3B expression. These data suggest that osteoarthritic chondrocytes may undergo epigenetic-age reversal in the hope of activating a progenitor-like phenotype driven by high STAT3 expression; however, the chronic activation of this pathway may prove detrimental and prompt tissue degeneration (Pickert et al.).

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STAT3-regulated DNA Methylation in Cartilage Development and Disease: What is Next?

With the help of the always helpful CUT&RUN assay, these data reveal a context-specific role for STAT3-mediated DNA methylation regulation in cartilage development and disease and suggest that STAT3 activation may reduce epigenetic age in adult cells and form a component of an inflammation-induced regenerative mechanism. The authors hoped that these findings would represent the basis for future research into the regulation of chondrocyte aging and the development of effective new treatments for age-related conditions such as osteoarthritis.

For more on how CUT&RUN helped to reveal the importance of STAT3-regulated DNA methylation in cartilage development and disease, see Aging Cell, January 2023.

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About the author

Stuart P. Atkinson, Ph.D.

Stuart was born and grew up in the idyllic town of Lanark (Scotland). He later studied biochemistry at the University of Strathclyde in Glasgow (Scotland) before gaining his Ph.D. in medical oncology; his thesis described the epigenetic regulation of the telomerase gene promoters in cancer cells. Following Post-doctoral stays in Newcastle (England) and Valencia (Spain) where his varied research aims included the exploration of epigenetics in embryonic and induced pluripotent stem cells, Stuart moved into project management and scientific writing/editing where his current interests include polymer chemistry, cancer research, regenerative medicine, and epigenetics. While not glued to his laptop, Stuart enjoys exploring the Spanish mountains and coastlines (and everywhere in between) and the food and drink that it provides!

Contact Stuart on Twitter with any questions

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