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DNA methylation dynamics in cellular commitment and differentiation
Abstract and Figures
DNA methylation is an essential epigenetic modification for mammalian development and is crucial for the establishment and maintenance of cellular identity. Traditionally, DNA methylation has been considered as a permanent repressive epigenetic mark. However, the application of genome-wide approaches has allowed the analysis of DNA methylation in different genomic contexts, revealing a more dynamic regulation than originally thought, as active DNA methylation and demethylation occur during cell fate commitment and terminal differentiation. Recent data provide insights into the contribution of different epigenetic factors, and DNA methylation in particular, to the establishment of cellular memory during embryonic development and the modulation of cell type-specific gene regulation programs to ensure proper differentiation. This review summarizes published data regarding DNA methylation changes along lineage specification and differentiation programs. We also discuss the current knowledge about DNA methylation alterations occurring in physiological and pathological conditions such as aging and cancer.
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... The deposition of 5mC epigenetic mark represses transposable elements and is traditionally associated with gene silencing; however, its more dynamic regulation role has been uncovered. DNA methylation is now considered important for embryonic development through the provision of cellular memory and modulation of gene regulation specificity in different cell types (Suelves, Carrio, Nunez-Alvarez, & Peinado, 2016). DNA methylation is concentrated in CpG islands, which modulate transcriptional activity by changing the binding capacities of transcriptional factors, positioning nucleosomes and linking methyl CpG binding proteins (Suelves et al., 2016). ...
... DNA methylation is now considered important for embryonic development through the provision of cellular memory and modulation of gene regulation specificity in different cell types (Suelves, Carrio, Nunez-Alvarez, & Peinado, 2016). DNA methylation is concentrated in CpG islands, which modulate transcriptional activity by changing the binding capacities of transcriptional factors, positioning nucleosomes and linking methyl CpG binding proteins (Suelves et al., 2016). In addition, DNA methylation occurs in gene bodies where it promotes gene expression, although only (indirectly) by repressing their mechanics. ...
DNA replication in high eukaryotes requires the faithful transmission of both the sequence and its methylation status. DNA methyltransferases are not consistently reliable enzymes for accurately restoring methylation in newly synthesised DNA strands when working in isolation. In this study, we evaluated some of the factors that enhance the fidelity of DNA methyltransferases and measure their local and distant impacts on the neighbouring methylation status. A clear understanding of DNA methylation fidelity during replication is important for advances in epigenetic knowledge in general and the potential development of new diagnostics and therapies for epigenetic diseases. We used the Arabidopsis model system because it is moderately methylated, which is an important advantage over animal models since it reduces the chance of a fully-methylated symmetric methylation to virtually nil while providing sufficient occupancy to ensure reliable interpretations. In this study, we employed hairpin bisulfite and nanopore sequencing (1D and 1D ² ) to test the inter- and intra-strand relationships among the neighbouring 5-methylcytosines (5mC) and evaluate the distribution of hemimethylated sites, as well as the correlations to other cues such as DNA methylation and histone marks. We observed that the presence of a CpG methylation site can predict the occurrence of neighbouring CpG methylation sites, but not necessarily CWG sites, whereas CWG methylation can predict both neighbouring CWG and CpG methylation locations. These predictabilities extend far beyond the size of a nucleosome, and sites 4 kb away still show higher probabilities of methylation compared to the global average.
Graphic abstract
Graphic abstract Fidelity and longitudinal conditional methylation probability of CpG and CWG DNA methylation in Arabidopsis plants
... DNMT3L (DNA methyltransferase 3-like) cooperates with DNMT3A and DNMT3B to stimulate their catalytic activity and is also highly expressed in ESCs [29]. Somatic cells and ESCs display distinct DNA methylation signatures associated with lineage specification [21,30,31]. The chemical inhibition of DNMT3B with adriamycin and azacytidine reduced human TERT (hTERT) expression and, in changing GC to AT, abolished promoter activity through sitedirected mutagenesis in glioma cell lines, demonstrating that DNMT3B and GC islands in the TERT promoter play an important role in the regulation of telomerase expression [12,32]. ...
Telomere repeats protect linear chromosomes from degradation, and telomerase has a prominent role in their maintenance. Telomerase has telomere-independent effects on cell proliferation, DNA replication, differentiation, and tumorigenesis. TERT (telomerase reverse transcriptase enzyme), the catalytic subunit of telomerase, is required for enzyme activity. TERT promoter mutation and methylation are strongly associated with increased telomerase activation in cancer cells. TERT levels and telomerase activity are downregulated in stem cells during differentiation. The link between differentiation and telomerase can provide a valuable tool for the study of the epigenetic regulation of TERT. Oxygen levels can affect cellular behaviors including proliferation, metabolic activity, stemness, and differentiation. The role of oxygen in driving TERT promoter modifications in embryonic stem cells (ESCs) is poorly understood. We adopted a monolayer ESC differentiation model to explore the role of physiological oxygen (physoxia) in the epigenetic regulation of telomerase and TERT. We further hypothesized that DNMTs played a role in physoxia-driven epigenetic modification. ESCs were cultured in either air or a 2% O2 environment. Physoxia culture increased the proliferation rate and stemness of the ESCs and induced a slower onset of differentiation than in ambient air. As anticipated, downregulated TERT expression correlated with reduced telomerase activity during differentiation. Consistent with the slower onset of differentiation in physoxia, the TERT expression and telomerase activity were elevated in comparison to the air-oxygen-cultured ESCs. The TERT promoter methylation levels increased during differentiation in ambient air to a greater extent than in physoxia. The chemical inhibition of DNMT3B reduced TERT promoter methylation and was associated with increased TERT gene and telomerase activity during differentiation. DNMT3B ChIP (Chromatin immunoprecipitation) demonstrated that downregulated TERT expression and increased proximal promoter methylation were associated with DNMT3B promoter binding. In conclusion, we have demonstrated that DNMT3B directly associates with TERT promoter, is associated with differentiation-linked TERT downregulation, and displays oxygen sensitivity. Taken together, these findings help identify novel aspects of telomerase regulation that may play a role in better understanding developmental regulation and potential targets for therapeutic intervention.
... Due to the critical role in gene regulation for cell lineage specification [18,19], DNA methylation profiles can be leveraged to estimate cell-type proportions [20][21][22]. The major advantages of DNA methylation cytometry are high accuracy and the ability to work in archival specimens [23][24][25]. ...
Background: Bladder cancer and therapy responses hinge on immune profiles in the tumor microenvironment (TME) and blood, yet studies linking tumor-infiltrating immune cells to peripheral immune profiles are limited. Methods: DNA methylation cytometry quantified TME and matched peripheral blood immune cell proportions. With tumor immune profile data as the input, subjects were grouped by immune infiltration status and consensus clustering. Results: Immune hot and cold groups had different immune compositions in the TME but not in circulating blood. Two clusters of patients identified with consensus clustering had different immune compositions not only in the TME but also in blood. Conclusion: Detailed immune profiling via methylation cytometry reveals the significance of understanding tumor and systemic immune relationships in cancer patients.
... Epigenomics technology has opened the potential to scale this type of data collection without sacrificing resolution. DNA methylation (DNAm) is an epigenetic marker placed on cytosine bases and is used to regulate gene expression and cell differentiation 40 ; cell-type specific genes can be expressed and are hypomethylated, while other genes are repressed with hypermethylation of regulatory gene elements 41 . These durable, intrinsic lineage markers allow researchers to use DNAm data to estimate the composition of a specimen by using cell-type specific DNAm profiles [42][43][44][45] . ...
Parkinson’s disease (PD) is the second most common neurodegenerative disease in the United States. Decades before motor symptoms manifest, non-motor symptoms such as hyposmia and rapid eye movement (REM) sleep behavior disorder are highly predictive of PD. Previous immune profiling studies have identified alterations to the proportions of immune cells in the blood of clinically defined PD patients. However, it remains unclear if these phenotypes manifest before the clinical diagnosis of PD. We utilized longitudinal DNA methylation (DNAm) microarray data from the Parkinson’s Progression Marker’s Initiative (PPMI) to perform immune profiling in clinically defined PD and prodromal PD patients (Prod). We identified previously reported changes in neutrophil, monocyte, and T cell numbers in PD patients. Additionally, we noted previously unrecognized decreases in the naive B cell compartment in the defined PD and Prod patient group. Over time, we observed the proportion of innate immune cells in PD blood increased, but the proportion of adaptive immune cells decreased. We identified decreases in T and B cell subsets associated with REM sleep disturbances and early cognitive decline. Lastly, we identified increases in B memory cells associated with both genetic (LRRK2 genotype) and infectious (cytomegalovirus seropositivity) risk factors of PD. Our analysis shows that the peripheral immune system is dynamic as the disease progresses. The study provides a platform to understand how and when peripheral immune alterations occur in PD and whether intervention at particular stages may be therapeutically advantageous.
As development varies greatly across the tree of life, it may seem difficult to suggest a model that proposes a single mechanism for understanding collective cell behaviors and the coordination of tissue formation. Here we propose a mechanism called differentiation waves, which unify many disparate results involving developmental systems from across the tree of life. We demonstrate how a relatively simple model of differentiation proceeds not from function-related molecular mechanisms, but from so-called differentiation waves. A phenotypic model of differentiation waves is introduced, and its relation to molecular mechanisms is proposed. These waves contribute to a differentiation tree, which is an alternate way of viewing cell lineage and local action of the molecular factors. We construct a model of differentiation wave-related molecular mechanisms (genome, epigenome, and proteome) based on C. elegans bioinformatic data. To validate this approach across different modes of development, we evaluate protein expression across different types of development by comparing the nematode Caenorhabditis elegans with several model organisms: fruit flies (Drosophila melanogaster), yeast (Saccharomyces cerevisiae), and mouse (Mus musculus). Inspired by gene regulatory networks, two Models of Interactive Contributions (fully-connected MICs and ordered MICs) are used to suggest potential genomic contributions to differentiation wave-related proteins. This, in turn, provides a framework for understanding differentiation and development.
Obesity, characterised by the accumulation of excess fat, is a complex condition resulting from the combination of genetic and epigenetic factors. Recent studies have found correspondence between DNA methylation and cell differentiation, suggesting a role of the former in cell fate determination. There is a lack of comprehensive understanding concerning the underpinnings of preadipocyte differentiation, specifically when cells are undergoing terminal differentiation (TD). To gain insight into dynamic genome-wide methylation, 3T3 L1 preadipocyte cells were differentiated by a hormone cocktail. The genomic DNA was isolated from undifferentiated cells and 4 hrs (4H), 2 days (2D) post-differentiated cells, and 15 days (15D) TD cells. We employed whole-genome bisulfite sequencing (WGBS) to ascertain global genomic DNA methylation alterations at single base resolution as preadipocyte cells differentiate. The genome-wide distribution of DNA methylation showed similar overall patterns in pre- and post- and terminally differentiated adipocytes, according to WGBS analysis. DNA methylation decreases at 4H after differentiation initiation, followed by methylation gain as cells approach TD. Studies revealed novel differentially methylated regions (DMRs) associated with adipogenesis. DMR analysis suggested that though DNA methylation is global, noticeable changes are observed at specific sites known as ‘hotspots.’ Hotspots are genomic regions rich in transcription factor (TF) binding sites and exhibit methylation-dependent TF binding. Subsequent analysis indicated hotspots as part of DMRs. The gene expression profile of key adipogenic genes in differentiating adipocytes is context-dependent, as we found a direct and inverse relationship between promoter DNA methylation and gene expression.
Prostate cancer (PCa) is associated with widespread promoter hypermethylation. We hypothesized that aberrant DNA methylation also targets gene enhancers, modulating their activity and contributing to disease etiology. A patient discovery set ( n = 37) was used for differential methylation analysis and biomarker identification using the Infinium methylation EPIC array, on high‐risk ( n = 13), low‐risk ( n = 11), and histologically benign ( n = 13) tissues. Enhancers were primarily hypermethylated. However proportionally, hypomethylated enhancers were more prominent in high‐risk ( n = 385, 15%) than low‐risk ( n = 105, 10%) disease, primarily targeting genes involved in development and enriched for oncoprotein binding motifs, including FOXA1. The clinical significance of enhancer methylation was evaluated by identifying a 17 enhancer differentially methylated probe (DMP) signature using a Least Absolute Shrinkage and Selection Operator model in the discovery set. A large external dataset ( n = 746) obtained from four publicly available prostate tissue methylation array studies was used to assess the enhancer signature through logistic regression models trained on a 2/3 training set and tested in a 1/3 test set. This delivered an area under the curve of 0.81 (95% bootstrapped CI 0.78–0.9) for selective detection of high‐risk PCa, achieving a 0.71 sensitivity and 0.76 specificity. Array‐wide aberrant DNA methylation at enhancers highlighted their epigenetic perturbance in high‐risk disease. A clinically significant enhancer signature from this study could be used for detecting high‐risk PCa.
Plant stress is a significant challenge that affects the development, growth, and productivity of plants and causes an adverse environmental condition that disrupts normal physiological processes and hampers plant survival. Epigenetic regulation is a crucial mechanism for plants to respond and adapt to stress. Several studies have investigated the role of DNA methylation (DM), non-coding RNAs, and histone modifications in plant stress responses. However, there are various limitations or challenges in translating the research findings into practical applications. Hence, this review delves into the recent recovery, implications, and applications of epigenetic regulation in response to plant stress. To better understand plant epigenetic regulation under stress, we reviewed recent studies published in the last 5-10 years that made significant contributions, and we analyzed the novel techniques and technologies that have advanced the field, such as next-generation sequencing and genome-wide profiling of epigenetic modifications. We emphasized the breakthrough findings that have uncovered specific genes or pathways and the potential implications of understanding plant epigenetic regulation in response to stress for agriculture, crop improvement, and environmental sustainability. Finally, we concluded that plant epigenetic regulation in response to stress holds immense significance in agriculture, and understanding its mechanisms in stress tolerance can revolutionize crop breeding and genetic engineering strategies, leading to the evolution of stress-tolerant crops and ensuring sustainable food production in the face of climate change and other environmental challenges. Future research in this field will continue to unveil the intricacies of epigenetic regulation and its potential applications in crop improvement.
Glioblastoma multiforme (GBM) is one of the deadliest tumours. This study aimed to construct radiogenomic prognostic models of glioblastoma overall survival (OS) based on magnetic resonance imaging (MRI) Gd-T1WI images and deoxyribonucleic acid (DNA) methylation-seq and to understand the related biological pathways. The ResNet3D-18 model was used to extract radiomic features, and Lasso-Cox regression analysis was utilized to establish the prognostic models. A nomogram was constructed by combining the radiogenomic features and clinicopathological variables. The DeLong test was performed to compare the area under the curve (AUC) of the models. We screened differentially expressed genes (DEGs) with original ribonucleic acid (RNA)-seq in risk stratification and used Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) annotations for functional enrichment analysis. For the 1-year OS models, the AUCs of the radiogenomic set, methylation set and deep learning set in the training cohort were 0.864, 0.804 and 0.787, and those in the validation cohort were 0.835, 0.768 and 0.651, respectively. The AUCs of the 0.5-, 1- and 2-year nomograms in the training cohort were 0.943, 0.861 and 0.871, and those in the validation cohort were 0.864, 0.885 and 0.805, respectively. A total of 245 DEGs were screened; functional enrichment analysis showed that these DEGs were associated with cell immunity. The survival risk-stratifying radiogenomic models for glioblastoma OS had high predictability and were associated with biological pathways related to cell immunity.
Significant losses of DNA 5-methyldeoxycytidine residues in old age could disrupt cellular gene expression and contribute to the physiological decline of the animal. Thus, the 5-methyldeoxycytidine content of DNAs, isolated from the tissues of two rodent species of various ages, were determined. Mus musculus lost DNA methylation sites at a rate of about 4.7×10(4) (approximately 0.012% of the newborn level)/month. Peromyscus leucopus lost DNA 5-methyldeoxycytidine residues at a rate of only 2.3×10(4) (approximately 0.006% of the newborn level)/month. Since P. leucopus generally live twice as long as M. musculus, the rate of loss of DNA 5-methyldeoxycytidine residues appears to be inversely related to life span. Similar losses in genomic 5-methyldeoxycytidine content were also observed to correlate with donor age in cultured normal human bronchial epithelial cells.
Background
The role of cytokines in establishing specific transcriptional programmes in innate immune cells has long been recognized. However, little is known about how these extracellular factors instruct innate immune cell epigenomes to engage specific differentiation states. Human monocytes differentiate under inflammatory conditions into effector cells with non-redundant functions, such as dendritic cells and macrophages. In this context, interleukin 4 (IL-4) and granulocyte macrophage colony-stimulating factor (GM-CSF) drive dendritic cell differentiation, whereas GM-CSF alone leads to macrophage differentiation.
Results
Here, we investigate the role of IL-4 in directing functionally relevant dendritic-cell-specific DNA methylation changes. A comparison of DNA methylome dynamics during differentiation from human monocytes to dendritic cells and macrophages identified gene sets undergoing dendritic-cell-specific or macrophage-specific demethylation. Demethylation is TET2-dependent and is essential for acquiring proper dendritic cell and macrophage identity. Most importantly, activation of the JAK3-STAT6 pathway, downstream of IL-4, is required for the acquisition of the dendritic-cell-specific demethylation and expression signature, following STAT6 binding. A constitutively activated form of STAT6 is able to bypass IL-4 upstream signalling and instruct dendritic-cell-specific functional DNA methylation changes.
Conclusions
Our study is the first description of a cytokine-mediated sequence of events leading to direct gene-specific demethylation in innate immune cell differentiation.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-015-0863-2) contains supplementary material, which is available to authorized users.
DNA methylation is a key epigenetic mark that is critical for gene regulation in multicellular eukaryotes. Although various human cell types may have the same genome, these cells have different methylomes. The systematic identification and characterization of methylation marks across cell types are crucial to understand the complex regulatory network for cell fate determination. In this study, we proposed an entropy-based framework termed SMART to integrate the whole genome bisulfite sequencing methylomes across 42 human tissues/cells and identified 757 887 genome segments. Nearly 75% of the segments showed uniform methylation across all cell types. From the remaining 25% of the segments, we identified cell type-specific hypo/hypermethylation marks that were specifically hypo/hypermethylated in a minority of cell types using a statistical approach and presented an atlas of the human methylation marks. Further analysis revealed that the cell type-specific hypomethylation marks were enriched through H3K27ac and transcription factor binding sites in cell type-specific manner. In particular, we observed that the cell type-specific hypomethylation marks are associated with the cell type-specific super-enhancers that drive the expression of cell identity genes. This framework provides a complementary, functional annotation of the human genome and helps to elucidate the critical features and functions of cell type-specific hypomethylation.
It has been a controversial issue as to how many DNA cytosine methyltransferase mammalian cells have and whether de novo methylation and maintenance methylation activities are encoded by a single gene or two different genes. To address these questions, we have generated a null mutation of the only known mammalian DNA methyltransferase gene through homologous recombination in mouse embryonic stem cells and found that the development of the homozygous embryos is arrested prior to the 8-somite stage. Surprisingly, the null mutant embryonic stem cells are viable and contain low but stable levels of methyl cytosine and methyltransferase activity, suggesting the existence of a second DNA methyltransferase in mammalian cells. Further studies indicate that de novo methylation activity is not impaired by the mutation as integrated provirus DNA in MoMuLV-infected homozygous embryonic stem cells become methylated at a similar rate as in wild-type cells. Differentiation of mutant cells results in further reduction of methyl cytosine levels, consistent with the de novo methylation activity being down regulated in differentiated cells. These results provide the first evidence that an independently encoded DNA methyltransferase is present in mammalian cells which is capable of de novo methylating cellular and viral DNA in vivo.
Aging in mammals is known to be accompanied by a progressive loss of methylated cytosines from DNA. This loss is tissue-specific to a certain extent and affects mainly repeated sequences, transposable elements, and intergenic genome parts. Age-dependent DNA hypomethylation is correlated with and perhaps partly caused by a diminished activity of DNA methyltransferases. Along with the global DNA demethylation during aging, hypermethylation of certain genes occurs. On the whole-genome scale, an age-dependent hypermethylation is typical for genes associated with promoter CG islands, whereas hypomethylation mostly affects CG-poor genes, besides the repeated sequences, transposable elements, and intergenic genome parts mentioned above. The methylation levels of certain CG sites display strict correlation to age and thus could be used as a molecular marker to predict biological age of cells, tissues, and organisms. Epigenetic cell reprogramming, such as induced pluripotent stem cell production, leads to complete resetting of their epigenetic age.