Chromatin Changes Precede Gene Expression in Pig Stem Cell Bone Formation

Researchers at the Research Institute for Farm Animal Biology in Germany have mapped the dynamic changes in chromatin accessibility and gene expression within porcine synovium-derived mesenchymal stem cells (SMSCs) during in vitro osteogenic induction. Published in Epigenetics & Chromatin, the study reveals that chromatin accessibility captures lineage-specific programs prior to overt gene expression changes, highlighting the precedence of epigenetic remodeling in bone formation. The team isolated SMSCs from two distinct pig breeds, German Saddleback (GS) and German Landrace (GL), to investigate how donor-specific metabolic characteristics influence the transcription factors and cis-regulatory elements that orchestrate osteogenic commitment.

The study design involved profiling the dynamic chromatin accessibility and transcriptomes of these cells over a 21-day induction period. The researchers utilized Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) to measure chromatin accessibility and RNA sequencing (RNA-seq) to measure gene expression. Cells were collected at days 0, 7, 14, and 21 for both the osteogenic differentiation group and the control group. In total, 28 samples were analyzed, comprising 16 non-differentiated control samples and 12 differentiating samples across the two breeds and technical replicates. This comprehensive approach allowed the team to reconstruct gene regulatory networks and perform footprinting analyses to prioritize transcription factors driving osteogenic transitions.

The principal component analysis of the RNA-seq data showed a clear separation between osteogenic-induced and control SMSCs along the first principal component, while cells from different pig breeds did not form distinct clusters. A total of 5,207, 4,988, and 5,754 differentially expressed genes (DEGs) were detected at days 7, 14, and 21, respectively, when comparing osteogenic-induced and control SMSCs. Genes such as FKBP5, PCSK6, NALF2, and ARRDC2 were consistently ranked among the top upregulated genes, while PRSS23 was consistently among the top downregulated genes. These transcriptional changes reflect the extensive molecular reprogramming required for the cells to commit to the osteogenic lineage.

Concurrently, the ATAC-seq data revealed a dynamic chromatin accessibility landscape. The study identified 144,288 consensus peaks across all samples, with approximately 15.67% annotated to promoter regions and 39.97% to intergenic regions. Differential analysis identified 44,656, 34,549, and 38,013 differentially accessible regions (DARs) at days 7, 14, and 21, respectively. A key finding was that GO terms related to ossification and mesenchymal cell differentiation emerged earlier in the chromatin landscape at day 7 than at the transcriptional level, which showed these terms at day 21. This indicates that changes in the chromatin accessibility landscape capture lineage-specific programs ahead of detectable transcriptional activation.

Footprinting analysis provided insights into the binding activities of specific transcription factors. The analysis showed stronger binding affinity of C/EBP family members in osteogenic-induced SMSCs, whereas the FOS::JUN heterodimer exhibited greater occupancy in control cells. Interestingly, RUNX2 footprints displayed a slight decrease from day 0 to day 21 despite its established role in osteogenesis. The stable expression of RUNX2 suggests it is functionally redirecting, leading to its binding at genomic regions that become more compact with induction, potentially participating in targeted gene repression. De novo motif analysis further revealed that RUNX2/RUNX motifs were enriched in regions of reduced accessibility, while CEBP motifs were enriched in regions of increased accessibility.

The integration of RNA-seq and ATAC-seq results demonstrated a significant positive correlation between differentially expressed genes and differentially accessible regions across all three time points, with Pearson’s r ranging from 0.514 to 0.526. This coordinated pattern suggests that chromatin state shifts and transcriptional changes are tightly coupled during osteogenic differentiation. The study also identified 3,841 significant gene-peak pairs, of which 3,051 showed a positive correlation between gene expression and promoter accessibility. Genes such as MEGF6, FKBP5, and ALDH1L1 exhibited strong positive correlations, indicating that promoter accessibility is a key driver of gene expression in this context.

Using the ANANSE algorithm, the researchers reconstructed directed gene regulatory networks to identify master regulators of osteogenic induction. Six transcription factors—KLF9, FOXO3, HOXA5, RXRA, FOXO1, and HLX—were identified as critical master regulators due to their consistently high regulatory impact across all time points. Notably, CEBPB consistently exhibited the largest number of direct target genes, underscoring the broad gene-regulatory effects of the CEBP family throughout the course of osteogenic induction. These networks highlight the complex interplay between transcription factors and their target genes in guiding cell fate decisions.

The study also examined donor-specific differences between the GS and GL pig breeds. At baseline (day 0), chromatin accessibility profiles were nearly indistinguishable between breeds, with only one differentially accessible region identified. However, differences became evident early after induction, with 308 DARs identified at day 7, before diminishing to 156 and 174 at days 14 and 21, respectively. This transient divergence suggests that breed-specific differences reflect variation in early chromatin remodeling responses rather than stable baseline differences. The magnitude of these differences remained small relative to the dominant effects of induction state and time, indicating that the core osteogenic program is largely conserved between breeds.

Functional enrichment analysis revealed the involvement of several key signaling pathways in the osteogenic process. The AMPK and Wnt pathways were consistently active throughout the induction period, with the AMPK pathway implicated in maintaining cellular energy homeostasis and promoting bone nodule formation. The Wnt pathway, crucial for skeletal development, showed continuous upregulation of WNT5B and WNT11, along with downregulation of inhibitors SFRP2 and SFRP4. Additionally, the cGMP-PKG pathway was enriched in ATAC-seq data but not RNA-seq data, suggesting a primary dominance at the regulatory level. These findings highlight the dynamic regulatory roles of various signaling pathways in transcription and chromatin remodeling during osteogenesis.

The study concludes that chromatin accessibility dynamics in SMSCs during osteogenic differentiation are driven mainly by differentiation state and time rather than donor metabolic differences. The integrated ATAC-seq and RNA-seq analysis highlights key transcription factors and networks guiding osteogenic commitment, supporting porcine SMSCs as a valuable translational model for bone regeneration. By mapping the dynamic chromatin accessibility landscape, the researchers provide a comprehensive understanding of the epigenetic mechanisms underlying osteogenic differentiation and cell fate decisions in a physiologically relevant animal model.

The experimental design accounted for individual variation by pooling cells from three male pigs of each breed, all 59 days of age, after two passages. The osteogenic differentiation protocol was executed in two independent technical replicates to ensure robustness. Cells in the osteogenic group were incubated with specific differentiation medium, while the control group remained in complete culture medium, with half-changes every three to four days. This setup allowed the researchers to distinguish between the effects of the induction process and the inherent characteristics of the cell lines. The study utilized the Sscrofa 11.1 reference genome for alignment, ensuring accurate mapping of the sequencing data. The high quality of the data was confirmed by an average alignment rate of 97.76% for RNA-seq and a Fraction of Reads in Peaks score of 0.45 for ATAC-seq, indicating that the observed signals were not artifacts of low-quality sequencing.

Functional enrichment analysis provided further context on the biological processes driving differentiation. In the osteogenic-induced timeline, Reactome pathway analysis highlighted the dynamic contribution of epigenetic mechanisms, specifically pathways related to histone methyltransferases such as MLL3, MLL4, and WDR-5. This suggests that chromatin remodeling is not merely a passive consequence of gene expression but an active driver of the osteogenic program. Additionally, the continuous participation of extracellular matrix organization and collagen synthesis pathways was evident, aligning with the observed increase in collagen deposition seen in Sirius red staining. In contrast, the control cells showed enrichment for adipogenesis-related pathways over time, reflecting the propensity for spontaneous adipogenesis in in vitro culture. This divergence underscores the lineage commitment of the induced cells toward osteogenesis rather than adipogenesis.

The study also examined the role of specific non-collagenous proteins in the biomineralization process. The expression of SPARC and SPP1, two major non-collagenous proteins, was upregulated at different time points during osteogenesis. Furthermore, the promoter region of SPARC exhibited increased chromatin accessibility in osteogenic-induced SMSCs compared to controls, indicating a coordinated regulation of gene expression and chromatin state. The gene SPARCL1 was upregulated at days 14 and 21, with its promoter region maintaining sustained high chromatin accessibility throughout the time course. This gene is considered a key target of osteogenic transcription factor regulation and acts as a negative regulator of adipogenic differentiation. The decreased chromatin accessibility at the MMP1 promoter during differentiation is consistent with the suppression of matrix metalloproteinase-1 expression, which reduces extracellular matrix remodeling activity as cells transition to a more differentiated state.

Limitations of the study include the in vitro nature of the experiments, which may not fully capture the complexity of the in vivo microenvironment. The study focused on two specific pig breeds, and while the differences between them were transient, the generalizability of these findings to other breeds or species requires further investigation. The footprinting analysis, while informative, has inherent limitations in identifying the exact transcription factors involved due to the high sequence similarity among certain motifs. Additionally, the study did not perform functional validation of the identified transcription factors, such as knockdown or overexpression experiments, to confirm their causal roles in osteogenic differentiation. The data are available in the ArrayExpress database, allowing for further analysis and validation by the research community. The study provides a valuable resource for understanding the epigenetic regulation of osteogenesis in a porcine model, which is physiologically and genomically similar to humans.

Subject of Research: Chromatin accessibility and gene expression dynamics in porcine synovium-derived mesenchymal stem cells during osteogenic induction

Article Title: Time-course ATAC-seq and RNA-seq analysis of porcine synovium-derived mesenchymal stem cells under in vitro osteogenic induction

Article References: Li, S., Siengdee, P., Hadlich, F., Trakooljul, N., Oster, M., Reyer, H., Wimmers, K., & Ponsuksili, S. (2026). Time-course ATAC-seq and RNA-seq analysis of porcine synovium-derived mesenchymal stem cells under in vitro osteogenic induction. Epigenetics & Chromatin, 19(1), Article 17. https://doi.org/10.1186/s13072-026-00668-z

Image Credits: AI Generated

DOI: 10.1186/s13072-026-00668-z

Keywords: ATAC-seq, RNA-seq, osteogenic differentiation, mesenchymal stem cells, chromatin accessibility, transcription factors, porcine model, epigenetics, bone regeneration, gene regulatory networks, Time-course, analysis