Epigenomes, Volume 8, Issue 2 (June 2024) – 12 articles

The post-genomic era has ushered in the extensive application of epigenetic editing tools, allowing for precise alterations of gene expression. The use of reprogrammable editors that carry transcriptional corepressors has significant potential for long-term epigenetic silencing for the treatment of human diseases. The ideal scenario involves precise targeting of a specific genomic location by a DNA-binding domain, ensuring there are no off-target effects and that the process yields no genetic remnants aside from specific epigenetic modifications (i.e., DNA methylation). A notable example is a recent study on the mouse Pcsk9 gene, crucial for cholesterol regulation and expressed in hepatocytes, which identified synthetic zinc-finger (ZF) proteins as the most effective DNA-binding editors for silencing Pcsk9 efficiently, specifically, and persistently. This discussion focuses on enhancing the specificity of ZF-array DNA binding by optimizing interactions between specific amino acids and DNA bases across three promoters containing CpG islands. Full article

The development of the nervous system is regulated by numerous intracellular molecules and cellular signals that interact temporally and spatially with the extracellular microenvironment. The three major cell types in the brain, i.e., neurons and two types of glial cells (astrocytes and oligodendrocytes), are generated from common multipotent neural stem cells (NSCs) throughout life. However, NSCs do not have this multipotentiality from the beginning. During cortical development, NSCs sequentially obtain abilities to differentiate into neurons and glial cells in response to combinations of spatiotemporally modulated cell-intrinsic epigenetic alterations and extrinsic factors. After the completion of brain development, a limited population of NSCs remains in the adult brain and continues to produce neurons (adult neurogenesis), thus contributing to learning and memory. Many biological aspects of brain development and adult neurogenesis are regulated by epigenetic changes via behavioral control of NSCs. Epigenetic dysregulation has also been implicated in the pathogenesis of various brain diseases. Here, we present recent advances in the epigenetic regulation of NSC behavior and its dysregulation in brain disorders. Full article

Variant H3.3, along with H2A.Z, is notably enriched at promoter regions and is commonly associated with transcriptional activation. However, the specific molecular mechanisms through which H3.3 influences chromatin dynamics at transcription start sites, and its role in gene regulation, remain elusive. Using a combination of biochemistry and cryo-electron microscopy (cryo-EM), we show that the inclusion of H3.3 alone does not induce discernible changes in nucleosome DNA dynamics. Conversely, the presence of both H3.3 and H2A.Z enhances DNA’s flexibility similarly to H2A.Z alone. Interestingly, our findings suggest that the presence of H3.3 in the H2A.Z nucleosome provides slightly increased protection to DNA at internal sites within the nucleosome. These results imply that while H2A.Z at active promoters promotes the formation of more accessible nucleosomes with increased DNA accessibility to facilitate transcription, the simultaneous presence of H3.3 offers an additional mechanism to fine-tune nucleosome accessibility and the chromatin environment. Full article

Nucleosomes are non-uniformly distributed across eukaryotic genomes, with stretches of ‘open’ chromatin strongly associated with transcriptionally active promoters and enhancers. Understanding chromatin accessibility patterns in normal tissue and how they are altered in pathologies can provide critical insights to development and disease. With the advent of high-throughput sequencing, a variety of strategies have been devised to identify open regions across the genome, including DNase-seq, MNase-seq, FAIRE-seq, ATAC-seq, and NicE-seq. However, the broad application of such methods to FFPE (formalin-fixed paraffin-embedded) tissues has been curtailed by the major technical challenges imposed by highly fixed and often damaged genomic material. Here, we review the most common approaches for mapping open chromatin regions, recent optimizations to overcome the challenges of working with FFPE tissue, and a brief overview of a typical data pipeline with analysis considerations. Full article

We consider the newly developed multinomial mixed-link models for a high-risk intestinal metaplasia (IM) study with DNA methylation data. Different from the traditional multinomial logistic models commonly used for categorical responses, the mixed-link models allow us to select the most appropriate link function for each category. We show that the selected multinomial mixed-link model (Model 1) using the total number of stem cell divisions (TNSC) based on DNA methylation data outperforms the traditional logistic models in terms of cross-entropy loss from ten-fold cross-validations with significant p-values $8.12\times {10}^{-4}$ and $6.94\times {10}^{-5}$. Based on our selected model, the significance of TNSC’s effect in predicting the risk of IM is justified with a p-value less than ${10}^{-6}$. We also select the most appropriate mixed-link models (Models 2 and 3) when an additional covariate, the status of gastric atrophy, is available. When the status is negative, mild, or moderate, we recommend Model 2; otherwise, we prefer Model 3. Both Models 2 and 3 can predict the risk of IM significantly better than Model 1, which justifies that the status of gastric atrophy is informative in predicting the risk of IM. Full article

In living cells, some reactions can be conducted by more than one enzyme and sometimes it is difficult to establish which enzyme is responsible. Such is the case with proteins from the TET family, capable of converting 5-methyl-2’-deoxycytidine (5-$mdC$) in DNA to 5-(hydroxymethyl)-2’-deoxycytidine (5-$hmdC$) and further to 5-formyl-2’-deoxycytidine (5-$fdC$) and 5-carboxy-2’-deoxycytidine (5-$cadC$). The estimation of the efficiency of particular TETs in particular oxidative reactions and different cell types is important but experimentally difficult. Here, we propose an approach with mathematical modeling in which methylation and known deoxycytidine modification pathways are presented by 343 possible model versions with assumed different combinations of TET1, 2, and 3 activities in different pathways. Model parameters were calculated on the basis of 5-$mdC$, 5-$hmdC$, 5-$fdC$, 5-$cadC$, and 5-$hmdU$ levels experimentally assessed in five human cultured cell lines and previously published. Selection of the model versions that give in simulations the best average fit to experimental data suggested that not all TET proteins participate in all modification reactions and that TET3 activity may be especially important in the reaction of 5-$fdC$ removal. Full article

The treatment of metastatic melanoma has been revolutionised by immunotherapy, yet a significant number of patients do not respond, and many experience autoimmune adverse events. Associations have been reported between patient outcome and monocyte subsets, whereas vitamin C (ascorbate) has been shown to mediate changes in cancer-stimulated monocytes in vitro. We therefore investigated the relationship of ascorbate with monocyte subsets and epigenetic modifications in patients with metastatic melanoma receiving immunotherapy. Patients receiving immunotherapy were compared to other cancer cohorts and age-matched healthy controls. Ascorbate levels in plasma and peripheral blood-derived mononuclear cells (PBMCs), monocyte subtype and epigenetic markers were measured, and adverse events, tumour response and survival were recorded. A quarter of the immunotherapy cohort had hypovitaminosis C, with plasma and PBMC ascorbate levels significantly lower than those from other cancer patients or healthy controls. PBMCs from the immunotherapy cohort contained similar frequencies of non-classical and classical monocytes. DNA methylation markers and intracellular ascorbate concentration were correlated with monocyte subset frequency in healthy controls, but correlation was lost in immunotherapy patients. No associations between ascorbate status and immune-related adverse events or tumour response or overall survival were apparent. Full article

Queuosine (Q) is a modification of the wobble base of tRNA harboring GUN anticodons with roles in decoding accuracy and efficiency. Its synthesis is complex with multiple enzymatic steps, and several pathway intermediates can be salvaged. The only two transporter families known to salvage Q precursors are QPTR/COG1738 and QrtT/QueT. Analyses of the distribution of known Q synthesis and salvage genes in human gut and oral microbiota genomes have suggested that more transporter families remain to be found and that Q precursor exchanges must occur within the structured microenvironments of the mammalian host. Using physical clustering and fusion-based association with Q salvage genes, candidate genes for missing transporters were identified and five were tested experimentally by complementation assays in Escherichia coli. Three genes encoding transporters from three different Pfam families, a ureide permease (PF07168) from Acidobacteriota bacterium, a hemolysin III family protein (PF03006) from Bifidobacterium breve, and a Major Facilitator Superfamily protein (PF07690) from Bartonella henselae, were found to allow the transport of both preQ₀ and preQ₁ in this heterologous system. This work suggests that many transporter families can evolve to transport Q precursors, reinforcing the concept of transporter plasticity. Full article

Background: One of the mechanisms regulating the biological activity of tumor necrosis factor (TNF) in cells is the co-expression of TNFR1/TNFR2 receptors. A model with a differential level of receptor expression is required to evaluate the contribution of these mechanisms. Aim: The development of a cellular model to compare the effects of TNF on cells depending on the presence of both receptors and TNFR2 alone. Methods: TNFR1 absence modifications of ZR-75/1 and K-562 cell lines were obtained by TNFR1 knockout. The presence of deletions was confirmed by Sanger sequencing, and the absence of cell membrane receptor expression was confirmed by flow cytometry. The dose-dependent effect of TNF on intact and knockout cells was comparatively evaluated by the effect on the cell cycle, the type of cell death, and the profile of expressed genes. Results: Knockout of TNFR1 resulted in a redistribution of TNFR2 receptors with an increased proportion of TNFR2+ cells in both lines and a multidirectional change in the density of expression in the lines (increased in K562 and decreased in ZR75/1). The presence of a large number of cells with high TNFR2 density in the absence of TNFR1 in the K562 cells was associated with greater sensitivity to TNF-stimulating doses and increased proliferation but did not result in a significant change in cell death parameters. A twofold increase in TNFR2+ cell distribution in this cell line at a reduced expression density in ZR75/1 cells was associated with a change in sensitivity to low cytokine concentrations in terms of proliferation; an overall increase in cell death, most pronounced at standard stimulating concentrations; and increased expression of the lymphocyte-activation gene groups, host–pathogen interaction, and innate immunity. Conclusions: The absence of TNFR1 leads to different variants of compensatory redistribution of TNFR2 in cellular models, which affects the type of cell response and the threshold level of sensitivity. The directionality of cytokine action modulation and sensitivity to TNF levels depends not only on the fraction of cells expressing TNFR2 but also on the density of expression. Full article

Alzheimer’s Disease (AD) is a complex disease and the leading cause of dementia in older people. We aimed to uncover aspects of AD’s pathogenesis that may contribute to drug repurposing efforts by integrating DNA methylation and genetic data. Implementing the network-based tool, a dense module search of genome-wide association studies (dmGWAS), we integrated a large-scale GWAS dataset with DNA methylation data to identify gene network modules associated with AD. Our analysis yielded 286 significant gene network modules. Notably, the foremost module included the BIN1 gene, showing the largest GWAS signal, and the GNAS gene, the most significantly hypermethylated. We conducted Web-based Cell-type-Specific Enrichment Analysis (WebCSEA) on genes within the top 10% of dmGWAS modules, highlighting monocyte as the most significant cell type (p < 5 × 10⁻¹²). Functional enrichment analysis revealed Gene Ontology Biological Process terms relevant to AD pathology (adjusted p < 0.05). Additionally, drug target enrichment identified five FDA-approved targets (p-value = 0.03) for further research. In summary, dmGWAS integration of genetic and epigenetic signals unveiled new gene interactions related to AD, offering promising avenues for future studies. Full article

The effects of PM_2.5 on human health fluctuate greatly among various age groups, influenced by a range of physiological and immunological reactions. This paper compares the pathogenesis of the disease caused by PM_2.5 in people of different ages, focusing on how children, adults, and the elderly are each susceptible to it because of differences in their bodies. Regarding children, exposure to PM_2.5 is linked to many negative consequences. These factors consist of inflammation, oxidative stress, and respiratory problems, which might worsen pre-existing conditions and potentially cause neurotoxicity and developmental issues. Epigenetic changes can affect the immune system and make people more likely to get respiratory diseases. On the other hand, exposures during pregnancy can change how the cardiovascular and central nervous systems develop. In adults, the inhalation of PM_2.5 is associated with a wide range of health problems. These include respiratory difficulties, reduced pulmonary function, and an increased susceptibility to illnesses such as asthma, chronic obstructive pulmonary disease (COPD), and lung cancer. In addition, exposure to PM_2.5 induces systemic inflammation, cardiovascular diseases, insulin resistance, and neurotoxic consequences. Evident disturbances in the immune system and cognitive function demonstrate the broad impact of PM_2.5. The elderly population is prone to developing respiratory and cardiovascular difficulties, which worsen their pre-existing health issues and raise the risk of cognitive decline and neurological illnesses. Having additional medical conditions, such as peptic ulcer disease, significantly increases the likelihood of being admitted to hospital. Full article

The association between newborn DNA methylation (DNAm) and asthma acquisition (AA) during adolescence has been suggested. Lung function (LF) has been shown to be associated with asthma risk and its severity. However, the role of LF in the associations between DNAm and AA is unclear, and it is also unknown whether the association between DNAm and AA is consistent with that between DNAm and LF. We address this question through assessing newborn epigenetic features of preadolescence LF and of AA during adolescence, along with their biological pathways and processes. Our study’s primary medical significance lies in advancing the understanding of asthma’s early life origins. By investigating epigenetic markers in newborns and their association with lung function in preadolescence, we aim to uncover potential early biomarkers of asthma risk. This could facilitate earlier detection and intervention strategies. Additionally, exploring biological pathways linking early lung function to later asthma development can offer insights into the disease’s pathogenesis, potentially leading to novel therapeutic targets. Methods: The study was based on the Isle of Wight Birth cohort (IOWBC). Female subjects with DNAm data at birth and with no asthma at age 10 years were included (n = 249). The R package ttScreening was applied to identify CpGs potentially associated with AA from 10 to 18 years and with LF at age 10 (FEV1, FVC, and FEV1/FVC), respectively. Agreement in identified CpGs between AA and LF was examined, along with their biological pathways and processes via the R function gometh. We tested the findings in an independent cohort, the Avon Longitudinal Study of Parents and Children (ALSPAC), to examine overall replicability. Results: In IOWBC, 292 CpGs were detected with DNAm associated with AA and 1517 unique CpGs for LF (514 for FEV1, 436 for FVC, 408 for FEV1/FVC), with one overlapping CpG, cg23642632 (NCKAP1) between AA and LF. Among the IOWBC-identified CpGs, we further tested in ALSPAC and observed the highest agreement between the two cohorts in FVC with respect to the direction of association and statistical significance. Epigenetic enrichment analyses indicated non-specific connections in the biological pathways and processes between AA and LF. Conclusions: The present study suggests that FEV1, FVC, and FEV1/FVC (as objective measures of LF) and AA (incidence of asthma) are likely to have their own specific epigenetic features and biological pathways at birth. More replications are desirable to fully understand the complexity between DNAm, lung function, and asthma acquisition. Full article