High-resolution Nanopore methylome-maps reveal random hyper-methylation at CpG-poor regions as driver of chemoresistance in leukemias.

Aberrant DNA methylation at CpG dinucleotides is a cancer hallmark that is associated with the emergence of resistance to anti cancer treatment, though molecular mechanisms and biological significance remain elusive. Genome scale methylation maps by currently used methods are based on chemical modification of DNA and are best suited for analyses of methylation at CpG rich regions (CpG islands). We report the first high coverage whole-genome map in cancer using the long read nanopore technology, which allows simultaneous DNA-sequence and -methylation analyses on native DNA. We analyzed clonal epigenomic/genomic evolution in Acute Myeloid Leukemias (AMLs) at diagnosis and relapse, after chemotherapy. Long read sequencing coupled to a novel computational method allowed definition of differential methylation at unprecedented resolution, and showed that the relapse methylome is characterized by hypermethylation at both CpG islands and sparse CpGs regions. Most differentially methylated genes, however, were not differentially expressed nor enriched for chemoresistance genes. A small fraction of under-expressed and hyper-methylated genes at sparse CpGs, in the gene body, was significantly enriched in transcription factors (TFs). Remarkably, these few TFs supported large gene-regulatory networks including 50% of all differentially expressed genes in the relapsed AMLs and highly-enriched in chemoresistance genes. Notably, hypermethylated regions at sparse CpGs were poorly conserved in the relapsed AMLs, under-represented at their genomic positions and showed higher methylation entropy, as compared to CpG islands. Analyses of available datasets confirmed TF binding to their target genes and conservation of the same gene-regulatory networks in large patient cohorts. Relapsed AMLs carried few patient specific structural variants and DNA mutations, apparently not involved in drug resistance. Thus, drug resistance in AMLs can be mainly ascribed to the selection of random epigenetic alterations at sparse CpGs of a few transcription factors, which then induce reprogramming of the relapsing phenotype, independently of clonal genomic evolution.

S-farnesylation is essential for antiviral activity of the long ZAP isoform against RNA viruses with diverse replication strategies.

The zinc finger antiviral protein (ZAP) is a broad inhibitor of virus replication. Its best-characterized function is to bind CpG dinucleotides present in viral RNAs and, through the recruitment of TRIM25, KHNYN and other cofactors, target them for degradation or prevent their translation. The long and short isoforms of ZAP (ZAP-L and ZAP-S) have different intracellular localization and it is unclear how this regulates their antiviral activity against viruses with different sites of replication. Using ZAP-sensitive and ZAP-insensitive human immunodeficiency virus type I (HIV-1), which transcribe the viral RNA in the nucleus and assemble virions at the plasma membrane, we show that the catalytically inactive poly-ADP-ribose polymerase (PARP) domain in ZAP-L is essential for CpG-specific viral restriction. Mutation of a crucial cysteine in the C-terminal CaaX box that mediates S-farnesylation and, to a lesser extent, the residues in place of the catalytic site triad within the PARP domain, disrupted the activity of ZAP-L. Addition of the CaaX box to ZAP-S partly restored antiviral activity, explaining why ZAP-S lacks antiviral activity for CpG-enriched HIV-1 despite conservation of the RNA-binding domain. Confocal microscopy confirmed the CaaX motif mediated localization of ZAP-L to vesicular structures and enhanced physical association with intracellular membranes. Importantly, the PARP domain and CaaX box together jointly modulate the interaction between ZAP-L and its cofactors TRIM25 and KHNYN, implying that its proper subcellular localisation is required to establish an antiviral complex. The essential contribution of the PARP domain and CaaX box to ZAP-L antiviral activity was further confirmed by inhibition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication, which replicates in double-membrane vesicles derived from the endoplasmic reticulum. Thus, compartmentalization of ZAP-L on intracellular membranes provides an essential effector function in ZAP-L-mediated antiviral activity against divergent viruses with different subcellular replication sites.

The dinucleotide composition of the Zika virus genome is shaped by conflicting evolutionary pressures in mammalian hosts and mosquito vectors.

Most vertebrate RNA viruses show pervasive suppression of CpG and UpA dinucleotides, closely resembling the dinucleotide composition of host cell transcriptomes. In contrast, CpG suppression is absent in both invertebrate mRNA and RNA viruses that exclusively infect arthropods. Arthropod-borne (arbo) viruses are transmitted between vertebrate hosts by invertebrate vectors and thus encounter potentially conflicting evolutionary pressures in the different cytoplasmic environments. Using a newly developed Zika virus (ZIKV) model, we have investigated how demands for CpG suppression in vertebrate cells can be reconciled with potentially quite different compositional requirements in invertebrates and how this affects ZIKV replication and transmission. Mutant viruses with synonymously elevated CpG or UpA dinucleotide frequencies showed attenuated replication in vertebrate cell lines, which was rescued by knockout of the zinc-finger antiviral protein (ZAP). Conversely, in mosquito cells, ZIKV mutants with elevated CpG dinucleotide frequencies showed substantially enhanced replication compared to wild type. Host-driven effects on virus replication attenuation and enhancement were even more apparent in mouse and mosquito models. Infections with CpG- or UpA-high ZIKV mutants in mice did not cause typical ZIKV-induced tissue damage and completely protected mice during subsequent challenge with wild-type virus, which demonstrates their potential as live-attenuated vaccines. In contrast, the CpG-high mutants displayed enhanced replication in Aedes aegypti mosquitoes and a larger proportion of mosquitoes carried infectious virus in their saliva. These findings show that mosquito cells are also capable of discriminating RNA based on dinucleotide composition. However, the evolutionary pressure on the CpG dinucleotides of viral genomes in arthropod vectors directly opposes the pressure present in vertebrate host cells, which provides evidence that an adaptive compromise is required for arbovirus transmission. This suggests that the genome composition of arbo flaviviruses is crucial to maintain the balance between high-level replication in the vertebrate host and persistent replication in the mosquito vector.

B1a and B2 cells are characterized by distinct CpG modification states at DNMT3A-maintained enhancers.

The B1 and B2 lineages of B cells contribute to protection from pathogens in distinct ways. The role of the DNA CpG methylome in specifying these two B-cell fates is still unclear. Here we profile the CpG modifications and transcriptomes of peritoneal B1a and follicular B2 cells, as well as their respective proB cell precursors in the fetal liver and adult bone marrow from wild-type and CD19-Cre Dnmt3a floxed mice lacking DNMT3A in the B lineage. We show that an underlying foundational CpG methylome is stably established during B lineage commitment and is overlaid with a DNMT3A-maintained dynamic methylome that is sculpted in distinct ways in B1a and B2 cells. This dynamic DNMT3A-maintained methylome is composed of novel enhancers that are closely linked to lineage-specific genes. While DNMT3A maintains the methylation state of these enhancers in both B1a and B2 cells, the dynamic methylome undergoes a prominent programmed demethylation event during B1a but not B2 cell development. We propose that the methylation pattern of DNMT3A-maintained enhancers is determined by the coincident recruitment of DNMT3A and TET enzymes, which regulate the developmental expression of B1a and B2 lineage-specific genes.

MGMT Epigenetics: The Influence of Gene Body Methylation and Other Insights Derived from Integrated Methylomic, Transcriptomic, and Chromatin Analyses in Various Cancer Types.

BACKGROUND: MGMT (O6-methylguanine-DNA methyltransferase) is primarily responsible for limiting the activity of some widely used chemotherapeutic agents, including temozolomide (TMZ) and carmustine (BCNU). The gene encoding this protein is epigenetically regulated, and assessment of methylation at its promoter region is used to predict glioma patients' response to TMZ. METHODS: In this report, we employed a bioinformatic approach to elucidate MGMT's epigenetic regulation. Integrated for the analysis were genome-wide methylation and transcription datasets for > 8,600 human tissue (representing 31 distinct cancer types) and 500 human cancer cell line samples. Also crucial to the interpretation of results were publicly available data from the ENCODE Project: tracks for histone modifications (via ChIP-seq) and DNase I hypersensitivity (via DNaseseq), as well as methylation and transcription data for representative cell lines (HeLa-S3, HMEC, K562). RESULTS AND DISCUSSION: We were able to validate (perhaps more comprehensively) the contrasting influences of CpG methylation at promoter region and at gene body on MGMT transcription. While the MGMT promoter is populated by CpG sites whose methylation levels displayed high negative correlation (R) with MGMT mRNA counts, the gene body harbors CpG sites exhibiting high positive R values. The promoter CpG sites with very high negative R's across cancer types include cg12981137, cg12434587, and cg00618725. Among the notable gene body CpG sites (high positive R's across cancer types) are cg00198994 (Intron 1), cg04473030 (Intron 2), and cg07367735 (Intron 4). For certain cancer types, such as melanoma, gene body methylation appears to be a better predictor of MGMT transcription (compared to promoter methylation). In general, the CpG methylation v. MGMT expression R values are higher in cell lines relative to tissues. Also, these correlations are noticeably more prominent in certain cancer types such as colorectal, adrenocortical, esophageal, skin, and head and neck cancers, as well as glioblastoma. As expected, hypomethylation at the promoter region is associated with more open chromatin, and enrichment of histone marks H3K4m1, H3K4m2, H3K4m3, and H3K9ac. CONCLUSION: Overall, our analysis illustrated the contrasting influence of promoter and gene body methylation on MGMT expression. These observations may help improve diagnostic assays for MGMT.

Intratumorally CpG immunotherapy with carbon nanotubes inhibits local tumor growth and liver metastasis by suppressing the epithelial-mesenchymal transition of colon cancer cells.

Colon cancer liver metastasis accounts for the major cause of death of colon cancer patients. Previous study reported a carbon nanotubes (CNT)-conjugated CpG complex (CNT-CpG), which displayed a significant antitumor effect in gliomas. However, whether CNT-CpG could limit colon tumor growth and suppress the colon cancer liver metastasis has not been evaluated. In this study, we report CNT enhances CpG uptake in mouse colon cancer cells. Results demonstrated only CpG with CNT conjugation showed significant activation of NF-κB signal. Moreover, intratumorally delivery of CNT-CpG successfully suppressed local xenograft tumor growth and liver metastasis. CNT-CpG treatments cured 75% of mice and inhibited local tumor growth, significantly prolonged survival outcomes and limited liver metastatic tumor nodules from colon cancer cells. Using human colon cancer cell line, HCT116, we observed significantly inhibitory effects of CNT-CpG on cell growth, invasion and migration. Importantly, CNT-CpG treatment blocked the epithelial to mesenchymal transition (EMT). We compared the mRNA levels of EMT markers of colon cancer cells without or with CNT-CpG treatment from in-vitro and in-vivo models. Consistent results demonstrated expression of epithelial marker, E-cadherin was upregulated by CNT-CpG. In contrast, three mesenchymal markers, snail, fibronectin and vimentin were significantly suppressed by CNT-CpG treatment compared with control or free CpG. In summary, our data suggest CNT-CpG is an effective therapeutic approach against local colon tumor and their liver metastasis. This study presents the CNT-CpG complex as a promising therapeutic target for developing novel therapies against both local colon tumors and liver metastatic tumors.

Methylome-wide association study of first-episode schizophrenia reveals a hypermethylated CpG site in the promoter region of the TNIK susceptibility gene.

Accumulating evidence suggests that epigenetics plays an important role in the etiology of schizophrenia. Here, we performed a methylome-wide association study (MWAS) of first-onset schizophrenia patients and controls from the Han Chinese population using microarray technology. The DNA methylation profiles revealed 4494 differentially methylated CpG sites. Gene ontology (GO) analysis showed that the functions of differentially methylated genes were primarily involved in enzymatic activity, cytoskeleton organization and cell adhesion, and the TNIK (encoding TRAF2- and NCK-interacting kinase) gene was enriched in most of these terms. By combining the MWAS results with those of previous genome-wide association studies (GWASs), we identified 72 candidate genes located in 49 human genome loci. Among the overlapping genes, the most significantly methylated CpG sites were in the transcriptional start site (TSS) 200 region (cg21413905, Punadjusted = 3.20 × 10-5) of TNIK. TNIK was listed in the top 50 differentially methylated loci. The results of pyrosequencing and TNIK mRNA expression were consistent with those of the microarray study. Bioinformatics analyses, dual-luciferase reporter assays and chromatin immunoprecipitation (ChIP) studies showed that TNIK interacted with genes associated with schizophrenia and NRF1 was identified as a novel transcription factor (TF) that binds to TNIK in its TSS200 region. Thus, the regulatory function of NRF1 may be influenced by the status of the methylated CpG site in this region. In summary, our study provides new insights into the epigenetic mechanisms that regulate schizophrenia. Studies of the functions of TNIK methylation should be performed in vitro and in vivo to provide a better understanding of the pathophysiology of schizophrenia.

Mechanistic insights into recognition of symmetric methylated cytosines in CpG and non-CpG DNA by UHRF1 SRA.

Non-CpG DNA methylation (non-mCpG) is enriched in the genome of brain neurons and germline cells in mammals. Accumulation of non-mCpG during postnatal brain development correlates with gene regulation and inactivation of distal regulatory elements. Recently, UHRF1 has been found to contribute to de novo non-CpG methylation, however, whether UHRF1 could recognize non-mCpG is unknown. Here, we have demonstrated through calorimetric measurements that the UHRF1 SRA can recognize mCpH and fully-mCpHpG, types of non-mCpG. Our ITC binding studies endorse the preferential reading of hemi-mCpG by UHRF1 SRA and also show 6-fold weaker binding for fully-mCpG than hemi-mCpG. Despite presence of symmetrical (5-methyl cytosine) 5mCs, stoichiometry of 1:1 for UHRF1 SRA binding to fully-mCpG indicates that UHRF1 SRA may not form a stable complex with fully-mCpG DNA. Contrarily, UHRF1 SRA recognizes fully-mCpHpG with a stoichiometry of 2:1 protein to DNA duplex with binding affinity higher than fully-mCpG. Our crystal structure of UHRF1 SRA bound to fully-mCpHpG DNA reveals dual flip-out mechanism of 5mC recognition. Metadynamics studies corroborates with ITC data that UHRF1 SRA could not form a stable complex with fully-mCpG DNA. Altogether, this study demonstrates that UHRF1 SRA recognizes non-mCpG DNA and exhibits contrasting mechanisms for hemi-mCpG and fully-mCpHpG DNA recognition.

Equilibrium Binding Model for CpG DNA-Dependent Dimerization of Toll-like Receptor 9 Ectodomain.

Microbial nucleic acids in the extracellular milieu are recognized in vertebrates by Toll-like receptors (TLRs), one of the most important families of innate immune receptors. TLR9 recognizes single-stranded unmethylated CpG DNA in endosomes. DNA binding induces TLR9 dimerization and activation of a potent inflammatory response. To provide insights on how DNA ligands induce TLR9 dimerization, we developed a detailed theoretical framework for equilibrium ligand binding, modeling the binding of the ssDNA at the two main sites on the TLR9 ectodomain. Light scattering and fluorescence anisotropy assays performed with recombinant TLR9 ectodomain and a panel of agonistic and antagonistic DNA ligands provide data that restrain the binding parameters, identify the likely ligand binding intermediates, and suggest cooperative modes of binding. This work brings us one step closer to establishing a rigorous biochemical understanding of how TLRs are activated by their ligands.

IFNs Drive Development of Novel IL-15-Responsive Macrophages.

Disruption in homeostasis of IL-15 is linked to poor maternal and fetal outcomes during pregnancy. The only cells described to respond to IL-15 at the early maternal-fetal interface have been NK cells. We now show a novel population of macrophages, evident in several organs but enriched in the uterus of mice and humans, expressing the ß-chain of the IL-15R complex (CD122) and responding to IL-15. CD122+ macrophages (CD122+Macs) are morphologic, phenotypic, and transcriptomic macrophages that can derive from bone marrow monocytes. CD122+Macs develop in the uterus and placenta with kinetics that mirror IFN activity at the maternal-fetal interface. M-CSF permits macrophages to express CD122, and IFNs are sufficient to drive expression of CD122 on macrophages. Neither type I nor type II IFNs are required to generate CD122+Macs, however. In response to IL-15, CD122+Macs activate the ERK signaling cascade and enhance production of proinflammatory cytokines after stimulation with the TLR9 agonist CpG. Finally, we provide evidence of human cells that phenocopy murine CD122+Macs in secretory phase endometrium during the implantation window and in first-trimester uterine decidua. Our data support a model wherein IFNs local to the maternal-fetal interface direct novel IL-15-responsive macrophages with the potential to mediate IL-15 signals critical for optimal outcomes of pregnancy.

Zika Virus with Increased CpG Dinucleotide Frequencies Shows Oncolytic Activity in Glioblastoma Stem Cells.

We studied whether cytosine phosphate-guanine (CpG) recoding in a viral genome may provide oncolytic candidates with reduced infection kinetics in nonmalignant brain cells, but with high virulence in glioblastoma stem cells (GSCs). As a model, we used well-characterized CpG-recoded Zika virus vaccine candidates that previously showed genetic stability and safety in animal models. In vitro, one of the CpG-recoded Zika virus variants had reduced infection kinetics in nonmalignant brain cells but high infectivity and oncolytic activity in GSCs as represented by reduced cell proliferation. The recoded virus also efficiently replicated in GSC-derived tumors in ovo with a significant reduction of tumor growth. We also showed that some GSCs may be resistant to Zika virus oncolytic activity, emphasizing the need for personalized oncolytic therapy or a strategy to overcome resistance in GSCs. Collectively, we demonstrated the potential of the CpG recoding approach for oncolytic virus development that encourages further research towards a better understanding of host-tumor-CpG-recoded virus interactions.

Regulation of interleukin 6 by a polymorphic CpG within the frontal cortex in Alzheimer's disease.

The cytokine interleukin 6 (IL-6) has been linked to the pathogenesis of Alzheimer's disease (AD). This is the first study to investigate the genetic and epigenetic interactions in the control of IL-6 in human brain and its relation to AD neuropathology in prefrontal cortex tissues from AD and controls genotyped for the SNP -174 C/G rs1800795, a polymorphic CpG in which the G allele creates a CpG site. Within CC homozygotes there were significantly higher brain levels of IL-6 protein compared to G allele carriers. The C allele that resulted in an absence of methylation at a CpG was also associated with significant changes in methylation at neighboring CpGs. Furthermore, there were significant differences in methylation between CC and CG/GG at CpG sites in the AD and control groups. That DNA methylation was altered in the brains by the presence of rs1800795, which further correlated with protein levels suggests the presence of a polymorphic CpG and genetic-epigenetic interactions in the regulation of IL-6 in the prefrontal cortex within AD brains.

Penalized logistic regression based on L1/2 penalty for high-dimensional DNA methylation data.

BACKGROUND: DNA methylation is a molecular modification of DNA that is vital and occurs in gene expression. In cancer tissues, the 5'-C-phosphate-G-3'(CpG) rich regions are abnormally hypermethylated or hypomethylated. Therefore, it is useful to find out the diseased CpG sites by employing specific methods. CpG sites are highly correlated with each other within the same gene or the same CpG island. OBJECTIVE: Based on this group effect, we proposed an efficient and accurate method for selecting pathogenic CpG sites. METHODS: Our method aimed to combine a L1/2 regularized solver and a central node fully connected network to penalize group constrained logistic regression model. Consequently, both sparsity and group effect were brought in with respect to the correlated regression coefficients. RESULTS: Extensive simulation studies were used to compare our proposed approach with existing mainstream regularization in respect of classification accuracy and stability. The simulation results show that a greater predictive accuracy was attained in comparison to previous methods. Furthermore, our method was applied to over 20000 CpG sites and verified using the ovarian cancer data generated from Illumina Infinium HumanMethylation 27K Beadchip. In the result of the real dataset, not only the indicators of predictive accuracy are higher than the previous methods, but also more CpG sites containing genes are confirmed pathogenic. Additionally, the total number of CpG sites chosen is less than other methods and the results show higher accuracy rates in comparison to other methods in simulation and DNA methylation data. CONCLUSION: The proposed method offers an advanced tool to researchers in DNA methylation and can be a powerful tool for recognizing pathogenic CpG sites.

Expression Profiles of dmrt1 in Schizothorax kozlovi, and Their Relation to CpG Methylation of Its Promoter and Temperature.

To elucidate the role of dmrt1 in sex differentiation of a teleost fish Schizothorax kozlovi, the full-length sequences of its cDNA and promoter were cloned by rapid amplification of cDNA ends (RACE) and genome walking. The relative mRNA expression levels were determined by quantitative real-time PCR (RT-PCR). The 1095-bp dmrt1 cDNA was predicted to encode a protein of 264 amino acids. It was expressed only in the gonads, and the expression was 17-times higher in the testis than in the ovary. The 1215-bp promoter sequence of dmrt1 was cloned and analyzed to detect sex-related differences in its methylation levels. A significant negative relationship between the dmrt1 expression and CpG methylation of its promoter were found in the testes and ovaries of S. kozlovi. Significant differences in dmrt1 expression levels were also found between the larval and juvenile stages. No significant differences in expression were found during the entire larval stage, and in the individuals among three different temperature groups (10°C, 14°C, and 18°C). Considering that the sex of sampled larval fish cannot be distinguished, correlations between dmrt1 expression and effects of temperature on sex differentiation in S. kozlovi need further study.

Changes of DNA methylation are associated with changes in lung function during adolescence.

BACKGROUND: Adolescence is a significant period for the gender-dependent development of lung function. Prior studies have shown that DNA methylation (DNA-M) is associated with lung function and DNA-M at some cytosine-phosphate-guanine dinucleotide sites (CpGs) changes over time. This study examined whether changes of DNA-M at lung-function-related CpGs are associated with changes in lung function during adolescence for each gender, and if so, the biological significance of the detected CpGs. METHODS: Genome-scale DNA-M was measured in peripheral blood samples at ages 10 (n = 330) and 18 years (n = 476) from the Isle of Wight (IOW) birth cohort in United Kingdom, using Illumina Infinium arrays (450 K and EPIC). Spirometry was conducted at both ages. A training and testing method was used to screen 402,714 CpGs for their potential associations with lung function. Linear regressions were applied to assess the association of changes in lung function with changes of DNA-M at those CpGs potentially related to lung function. Adolescence-related and personal and family-related confounders were included in the model. The analyses were stratified by gender. Multiple testing was adjusted by controlling false discovery rate of 0.05. Findings were further examined in two independent birth cohorts, the Avon Longitudinal Study of Children and Parents (ALSPAC) and the Children, Allergy, Milieu, Stockholm, Epidemiology (BAMSE) cohort. Pathway analyses were performed on genes to which the identified CpGs were mapped. RESULTS: For females, 42 CpGs showed statistically significant associations with change in FEV1/FVC, but none for change in FEV1 or FVC. No CpGs were identified for males. In replication analyses, 16 and 21 of the 42 CpGs showed the same direction of associations among the females in the ALSPAC and BAMSE cohorts, respectively, with 11 CpGs overlapping across all the three cohorts. Through pathway analyses, significant biological processes were identified that have previously been related to lung function development. CONCLUSIONS: The detected 11 CpGs in all three cohorts have the potential to serve as the candidate epigenetic markers for changes in lung function during adolescence in females.

Physical Activity and Genome-wide DNA Methylation: The REgistre GIroní del COR Study.

INTRODUCTION: DNA methylation may be one of the biological mechanisms underlying the health benefits of physical activity (PA). Our objective was to determine the association between PA and genome-wide DNA methylation at CpG level. METHODS: We designed a two-stage epigenome wide association study. In the discovery stage, we used 619 individuals from the REgistre GIroní del COR cohort. Next, we validated the CpG suggestively associated with PA (P < 10) in two independent populations (n = 1735 and 190, respectively). Physical activity was assessed with validated questionnaires and classified as light PA (LPA), moderate PA, vigorous PA, moderate-vigorous PA (MVPA) and total PA. We examined linear and nonlinear associations and meta-analyzed the results in the three populations. The linear associations were meta-analyzed with a fixed-effects model and the P values of the nonlinear associations with the Stouffer and Fisher methods. We established a P value threshold that fulfilled Bonferroni criteria over the number of CpG analyzed (0.05/421,940 = 1.185 × 10). RESULTS: In the meta-analyses, two CpG sites had a statistically significant nonlinear association with MVPA. cg24155427 (P = 1.19 × 10), located in an intergenic region in chromosome 1, has been previously associated with smoking, lupus, and aging. cg09565397 (P = 1.59 × 10), located within DGAT1 in chromosome 8, which encodes an enzyme involved in triacylglycerol synthesis. CONCLUSIONS: This population-based study identified two new, differentially methylated CpG sites with a nonlinear dose-response relationship to MVPA. These associations must be additionally validated and may be considered for further research on the biological mechanisms underlying health benefits of PA.

A genomic predictor of lifespan in vertebrates.

Biological ageing and its mechanistic underpinnings are of immense biomedical and ecological significance. Ageing involves the decline of diverse biological functions and places a limit on a species' maximum lifespan. Ageing is associated with epigenetic changes involving DNA methylation. Furthermore, an analysis of mammals showed that the density of CpG sites in gene promoters, which are targets for DNA methylation, is correlated with lifespan. Using 252 whole genomes and databases of animal age and promotor sequences, we show a pattern across vertebrates. We also derive a predictive lifespan clock based on CpG density in a selected set of promoters. The lifespan clock accurately predicts maximum lifespan in vertebrates (R2 = 0.76) from the density of CpG sites within only 42 selected promoters. Our lifespan clock provides a wholly new method for accurately estimating lifespan using genome sequences alone and enables estimation of this challenging parameter for both poorly understood and extinct species.

Placental CpG Methylation of Inflammation, Angiogenic, and Neurotrophic Genes and Retinopathy of Prematurity.

Purpose: Extremely preterm infants are at increased risk for retinopathy of prematurity (ROP). We previously identified several inflammatory proteins that were expressed early in life and are associated with an increased risk of ROP and several angiogenic and neurotrophic growth factors in the neonatal systemic circulation that are associated with a lower risk of ROP. In this paper, we report the results of a set of analyses designed to test the hypothesis that placental CpG methylation levels of 12 inflammation-, angiogenic-, and neurotrophic-associated genes predict the occurrence of prethreshold ROP in extremely preterm newborns. Methods: We used placental CpG methylation data from 395 newborns from the Extremely Low Gestational Age Newborns study. Results: Multivariable regression models revealed that placental DNA methylation of 16 CpG sites representing 8 genes were associated with prethreshold ROP. Specifically, CpG methylation in the serum amyloid A SAA1 and SAA2, brain-derived neurotrophic factor (BDNF), myeloperoxidase (MPO), C-reactive protein (CRP), angiopoietin 1 (ANGPT1), and tumor necrosis factor receptor superfamily member 1B (TNFRSF1B) genes was associated with a lower risk of prethreshold ROP. Conversely, CpG methylation at three probes within tumor necrosis factor receptor superfamily member 1A (TNFRSF1A) and in two alternative probes within the BDNF and ANGPT1 genes was associated with an increased risk of ROP. Conclusions: CpG methylation may be a useful marker for improving ROP prediction, opening the opportunity for early intervention to lessen disease severity.

Epigenome-wide exploratory study of monozygotic twins suggests differentially methylated regions to associate with hand grip strength.

Hand grip strength is a measure of muscular strength and is used to study age-related loss of physical capacity. In order to explore the biological mechanisms that influence hand grip strength variation, an epigenome-wide association study (EWAS) of hand grip strength in 672 middle-aged and elderly monozygotic twins (age 55-90 years) was performed, using both individual and twin pair level analyses, the latter controlling the influence of genetic variation. Moreover, as measurements of hand grip strength performed over 8 years were available in the elderly twins (age 73-90 at intake), a longitudinal EWAS was conducted for this subsample. No genome-wide significant CpG sites or pathways were found, however two of the suggestive top CpG sites were mapped to the COL6A1 and CACNA1B genes, known to be related to muscular dysfunction. By investigating genomic regions using the comb-p algorithm, several differentially methylated regions in regulatory domains were identified as significantly associated to hand grip strength, and pathway analyses of these regions revealed significant pathways related to the immune system, autoimmune disorders, including diabetes type 1 and viral myocarditis, as well as negative regulation of cell differentiation. The genes contributing to the immunological pathways were HLA-B, HLA-C, HLA-DMA, HLA-DPB1, MYH10, ERAP1 and IRF8, while the genes implicated in the negative regulation of cell differentiation were IRF8, CEBPD, ID2 and BRCA1. In conclusion, this exploratory study suggests hand grip strength to associate with differentially methylated regions enriched in immunological and cell differentiation pathways, and hence merits further investigations.

Genome-wide DNA methylation analysis of pituitaries during the initiation of puberty in gilts.

It has been widely recognized that the early or delayed puberty appears to display harmful effects on adult health outcomes. During the timing of puberty, pituitaries responds to the hypothalamus and then introduce the following response of ovaries in hypothalamic-pituitary-gonadal axis. DNA methylation has been recently suggested to regulate the onset of puberty in female mammals. However, to date, the changes of DNA methylation in pituitaries have not been investigated during pubertal transition. In this study, using gilts as the pubertal model, the genome-scale DNA methylation of pituitaries was profiled and compared across Pre-, In- and Post-puberty by using the reduced representation bisulfite sequencing. We found that average methylation levels of each genomic feature in Post- were lower than Pre- and In-pubertal stage in CpG context, but they were higher in In- than that in Pre- and Post-pubertal stage in CpH (where H = A, T, or C) context. The methylation patterns of CpHs were more dynamic than that of CpGs at the location of high CpG content, low CpG content promoter genes, and differently genomic CGIs. Furthermore, the differently genomic CGIs were likely to show in a similar manner in CpG context but display in a stage-specific manner in the CpH context across the Pre-, In- and Post-pubertal stage. Among these pubertal stages, 5 kb upstream regions of the transcription start sites were protected from both CpG and CpH methylation changes. 12.65% of detected CpGs were identified as the differentially methylated CpGs, regarding 4301 genes which were involved in the fundamental functions of pituitaries. 0.35% of detected CpHs were identified as differentially methylated CpHs, regarding 3691 genes which were involved in the biological functions of releasing gonadotropin hormones. These observations and analyses would provide valuable insights into epigenetic mechanism of the initiation of puberty in pituitary level.