This study investigates the dynamic changes in DNA methylation during the differentiation of human embryonic stem cells (hESCs) into fibroblasts and neonatal fibroblasts. Using whole-genome bisulfite sequencing, the researchers compared DNA methylation profiles across these cell types, as well as with a methylome map of fully differentiated adult cells, mature peripheral blood mononuclear cells (monocytes). Key findings include: 1. **Global and Non-CpG Methylation**: hESCs exhibited the highest global methylation levels and the greatest frequency of non-CpG methylation. Non-CpG methylation, particularly CpA methylation, was highly conserved among differentiated cell types, suggesting a role in developmental regulation. 2. **Promoter Hypomethylation and Gene Body Methylation**: Promoter regions showed hypomethylation (both CG and CA), while gene bodies had higher methylation levels. This pattern was positively correlated with transcription in all cell types. 3. **Exon-Intron Boundaries**: Sharp transitions in methylation occurred at exon-intron boundaries, indicating a potential role for differential methylation in transcript splicing. 4. **Developmental Stage and Methylation Levels**: The level of global methylation and the extent of non-CpG methylation were reflective of the developmental stage, with hESCs having the highest methylation levels and monocytes the lowest. 5. **Differentiation-Associated Differentially Methylated Regions (DMRs)**: DMRs were identified in hESCs and fibroblasts, with many associated with pluripotency and differentiation-associated genes. These DMRs were preferentially located in promoter, gene body, and TSS regions. 6. **Correlation with Gene Expression**: DNA methylation around the TSS was negatively correlated with gene expression, while methylation levels in the gene body and TTS regions were positively correlated. 7. **Histone Modifications**: A strong anti-correlation was observed between DNA methylation and H3K4me3, a mark of active transcription, suggesting that DNA methylation and histone modifications work in concert to regulate gene expression. The study highlights the importance of high-resolution methylation maps in understanding the complex regulatory mechanisms involved in cellular differentiation and development.This study investigates the dynamic changes in DNA methylation during the differentiation of human embryonic stem cells (hESCs) into fibroblasts and neonatal fibroblasts. Using whole-genome bisulfite sequencing, the researchers compared DNA methylation profiles across these cell types, as well as with a methylome map of fully differentiated adult cells, mature peripheral blood mononuclear cells (monocytes). Key findings include: 1. **Global and Non-CpG Methylation**: hESCs exhibited the highest global methylation levels and the greatest frequency of non-CpG methylation. Non-CpG methylation, particularly CpA methylation, was highly conserved among differentiated cell types, suggesting a role in developmental regulation. 2. **Promoter Hypomethylation and Gene Body Methylation**: Promoter regions showed hypomethylation (both CG and CA), while gene bodies had higher methylation levels. This pattern was positively correlated with transcription in all cell types. 3. **Exon-Intron Boundaries**: Sharp transitions in methylation occurred at exon-intron boundaries, indicating a potential role for differential methylation in transcript splicing. 4. **Developmental Stage and Methylation Levels**: The level of global methylation and the extent of non-CpG methylation were reflective of the developmental stage, with hESCs having the highest methylation levels and monocytes the lowest. 5. **Differentiation-Associated Differentially Methylated Regions (DMRs)**: DMRs were identified in hESCs and fibroblasts, with many associated with pluripotency and differentiation-associated genes. These DMRs were preferentially located in promoter, gene body, and TSS regions. 6. **Correlation with Gene Expression**: DNA methylation around the TSS was negatively correlated with gene expression, while methylation levels in the gene body and TTS regions were positively correlated. 7. **Histone Modifications**: A strong anti-correlation was observed between DNA methylation and H3K4me3, a mark of active transcription, suggesting that DNA methylation and histone modifications work in concert to regulate gene expression. The study highlights the importance of high-resolution methylation maps in understanding the complex regulatory mechanisms involved in cellular differentiation and development.