ScHiMe: predicting single-cell DNA methylation levels based on single-cell Hi-C data

Most publicly accessible single-cell Hi-C data are sparse and cannot reach a higher resolution. Therefore, learning latent representations (bin-specific embeddings) of sparse single-cell Hi-C matrices would provide us with a novel way of mining valuable information hidden in the limited number of single-cell Hi-C contacts. We present scHiCEmbed, an unsupervised computational method for learning bin-specific embeddings of single-cell Hi-C data, and the computational system is applied to the tasks of 3D structure reconstruction of whole genomes and detection of topologically associating domains (TAD). The only input of scHiCEmbed is a raw or scHiCluster-imputed single-cell Hi-C matrix. The main process of scHiCEmbed is to embed each node/bin in a higher dimensional space using graph auto-encoders. The learned n-by-3 bin-specific embedding/latent matrix is considered the final reconstructed 3D genome structure. For TAD detection, we use constrained hierarchical clustering on the latent matrix to classify bins: S_Dbw is used to determine the optimal number of clusters, and each cluster is considered as one potential TAD. Our reconstructed 3D structures for individual chromatins at different cell stages reveal the expanding process of chromatins during the cell cycle. We observe that the TADs called from single-cell Hi-C data are not shared across individual cells and that the TAD boundaries called from raw or imputed single-cell Hi-C are significantly different from those called from bulk Hi-C, confirming the cell-to-cell variability in terms of TAD definitions. The source code for scHiCEmbed is publicly available, and the URL can be found in the conclusion section.

Background: Global DNA Methylation (GDM), an epigenomic modification has been linked to the development of Cardiovascular Disease and its risk factors. Purpose: The purpose of this study was to determine if there is a significant correlation between GDM and cardiovascular fitness, as well as, measures of body composition. Methodology: 26 apparently healthy, adults (11 males) completed a physical activity and diet questionnaire, as well as, had a small blood sample (600?L) collected via finger prick for the determination of GDM. Body composition was assessed by means of a Dual-Energy X-ray Absorptiometry (DEXA) scan, while cardiovascular fitness was evaluated by the completion of a maximal exertion, graded exercise test (VO2max) on a treadmill. Peasron's "r" value was used to determine the correlation GDM and various variables, while t-tests were used to determine if any differences between high and low value groups for each variable existed. Results: Body Mass Index was significantly correlated (p-value, r value; 0.031, -.556) with GDM while there was a significant difference between high and low level folate groups (p=0.034) as determined by the diet questionnaire. No significant correlations or differences were found in males. Conclusion: The results conclude that as BMI increases, GDM decreases in females. In attempts to further investigate the relationships between GDM and these variables, auxiliary research needs to be conducted with larger subject pools containing additional sedentary participants.

to investigate changes in DNA methylation in bystander and inducer cells during the manifestation ofdirect and rescue bystander effects. Separate and co-cultivation of peripheral blood lymphocytes (PBL) of 10 conditionally healthy individuals; γ-quantum irradiation (IBL-237C emitter); modified comet electrophoresis method (Comet assay) under neutralconditions using the methylation-sensitive restriction enzyme HpaII; fluorescence microscopy with an automatedcomputer software system for analyzing the results; statistical methods. The level of DNA methylation in PBL was quantitatively assessed using DNA migration parameters inagarose gel: the length of the comet tail (in μm), the percentage of DNA in the tail part of the comet, and TailMoment (TM), which simultaneously takes into account both the amount of DNA in the tail part of the comet andthe length of the tail. In separate cultivation of PBL irradiated with γ-quanta (dose 1.0 Gy, power 2.34 Gy/min),a reliable decrease in the average values compared to the non-irradiated control was noted for the length of the«comet» tail ((57.03 ∓ 1.17) μm versus (66.64 ∓ 2.03) μm; p < 0.001) and Tail Moment (67.77 ∓ 1.22 versus85.06 ∓ 2.30; p < 0.001), which may indicate a decrease in the number of restriction sites of the methyl-sensitiverestriction enzyme HpaII and, as a consequence, an increase in the level of global DNA methylation. When thebystander effect is realized, the level of DNA damage in bystander cells increases, while there is a significantdecrease in the average values of the following parameters: the percentage of DNA in the tail part of the «comets»(p<0.001), the length of the tail part (p<0.001) and TailMoment (p<0.001) compared to both the non-irradiatedcontrol and irradiated PBLs in separate cultivation, and indicates an increase in the level of global DNA methylation.As in irradiated lymphocyte cultures cultured separately, in inducer cells a reliable decrease in the mean values oftail length (p < 0.01) and TailMoment (p < 0.001) was observed compared to the control, which may indicate adecrease in the number of restriction sites and an increase in the level of global DNA methylation as a result of irradiation. Between inducer cells and irradiated cells that were cultured separately, no difference was found in themean values of all the studied parameters: the percentage of DNA in the tail part of comets (p > 0.05), tail length(p > 0.05) and TailMoment (p 0.05), which may indicate the absence of changes in the level of DNA methylationwhen a non-irradiated culture is exposed to an irradiated one during co-cultivation. The development of the direct bystander effect is accompanied by epigenetic changes, which are characterized by an increase in the level of DNA methylation in bystander cells. At the same time, in inducer cells,changes in the level of DNA methylation were not determined, which indicates the absence of manifestations of thereverse bystander effect at the epigenetic level.

The early stages of mammalian embryonic development involve the participation and cooperation of numerous complex processes, including nutritional, genetic, and epigenetic mechanisms. However, in embryos cultured in vitro, a developmental block occurs that affects embryo development and the efficiency of culture. Although the block period is reported to involve the transcriptional repression of maternal genes and transcriptional activation of zygotic genes, how epigenetic factors regulate developmental block is still unclear. In this study, we systematically analyzed whole-genome methylation levels during five stages of sheep oocyte and preimplantation embryo development using single-cell level whole genome bisulphite sequencing (SC-WGBS) technology. Then, we examined several million CpG sites in individual cells at each evaluated developmental stage to identify the methylation changes that take place during the development of sheep preimplantation embryos. Our results showed that two strong waves of methylation changes occurred, namely, demethylation at the 8-cell to 16-cell stage and methylation at the 16-cell to 32-cell stage. Analysis of DNA methylation patterns in different functional regions revealed a stable hypermethylation status in 3′UTRs and gene bodies; however, significant differences were observed in intergenic and promoter regions at different developmental stages. Changes in methylation at different stages of preimplantation embryo development were also compared to investigate the molecular mechanisms involved in sheep embryo development at the methylation level. In conclusion, we report a detailed analysis of the DNA methylation dynamics during the development of sheep preimplantation embryos. Our results provide an explanation for the complex regulatory mechanisms underlying the embryo developmental block based on changes in DNA methylation levels.

The early stages of mammalian embryonic development involve the participation and cooperation of numerous complex processes, including nutritional, genetic, and epigenetic mechanisms. However, in embryos cultured in vitro, a developmental block occurs that affects embryo development and the efficiency of culture. Although the block period is reported to involve the transcriptional repression of maternal genes and transcriptional activation of zygotic genes, how epigenetic factors regulate developmental block is still unclear. In this study, we systematically analyzed whole-genome methylation levels during five stages of sheep oocyte and preimplantation embryo development using single-cell level whole genome bisulphite sequencing (SC-WGBS) technology. Then, we examined several million CpG sites in individual cells at each evaluated developmental stage to identify the methylation changes that take place during the development of sheep preimplantation embryos. Our results showed that two strong waves of methylation changes occurred, namely, demethylation at the 8-cell to 16-cell stage and methylation at the 16-cell to 32-cell stage. Analysis of DNA methylation patterns in different functional regions revealed a stable hypermethylation status in 3'UTRs and gene bodies; however, significant differences were observed in intergenic and promoter regions at different developmental stages. Changes in methylation at different stages of preimplantation embryo development were also compared to investigate the molecular mechanisms involved in sheep embryo development at the methylation level. In conclusion, we report a detailed analysis of the DNA methylation dynamics during the development of sheep preimplantation embryos. Our results provide an explanation for the complex regulatory mechanisms underlying the embryo developmental block based on changes in DNA methylation levels.

We describe an approach for reconstructing three-dimensional (3D) structures from single-cell Hi-C data. This approach has been inspired by a method of recurrence plots and visualization tools for nonlinear time series data. Some examples are also presented.

DNA methylation is an important regulator of gene expression that can influence tumor heterogeneity and shows weak and varying expression levels among different genes. Gastric cancer (GC) is a highly heterogeneous cancer of the digestive system with a high mortality rate worldwide. The heterogeneous subtypes of GC lead to different prognoses. In this study, we explored the relationships between DNA methylation and gene expression levels by introducing a sparse low-rank regression model based on a GC dataset with 375 tumor samples and 32 normal samples from The Cancer Genome Atlas database. Differences in the DNA methylation levels and sites were found to be associated with differences in the expressed genes related to GC development. Overall, 29 methylation-driven genes were found to be related to the GC subtypes, and in the prognostic model, we explored five prognoses related to the methylation sites. Finally, based on a low-rank matrix, seven subgroups were identified with different methylation statuses. These specific classifications based on DNA methylation levels may help to account for heterogeneity and aid in personalized treatments.

Ultraviolet (UV) radiation has variety of deleterious effects on mammalian skin, and triggers several causative factors such as DNA damage, inflammation, immune suppression and genetic and epigenetic alterations which leads to the development of skin cancer. Earlier, we have shown that the deficiency of interleukin (IL)-12, an immunoregulatory cytokine, enhances photocarcinogenesis in mice through inhibition of repair of damaged DNA. In further studies we have tried to explore and examine the effect of IL-12 on epigenetic regulators in UV-exposed skin and UV-induced skin tumors using IL-12 knock out (IL-12 KO) mice as an in vivo animal model. The exposure of IL-12 KO mice with UVB (200 mJ/cm2) radiation resulted in higher levels of DNA methylation, DNA methyltransferase (Dnmt) activity, Ten-eleven translocations (TETs), a family of methylcytosine dioxygenase, histone deacetylases (HDAC), and lower levels of histone acetylase (HAT) activity than their wild type counterparts. To verify that IL-12 deficiency is associated with DNA hypermethylation and higher HDAC activity in UVB exposed skin, IL-12-KO mice were treated with endotoxin-free murine rIL-12 (50 ng/mouse/100 μl PBS; subcutaneous injection) 3 h before UVB exposure. Our results indicate that significant reduction in the levels of global DNA methylation, Dnmt and HDAC activity were observed compared with non-rIL-12 treated mice. Conversely, UVB exposed IL-12 wild type mice were treated with anti-IL-12 antibody (500 ng/mouse/100 μl PBS; subcutaneous injection). This treatment resulted in increased levels of DNA methylation, Dnmt and HDAC activities. These observations were also noted and compared between skin tumors from IL-12 KO mice and their wild type counterparts. To further explore the role of IL-12 deficiency on epigenetic modification-mediated photocarcinogenesis, we determined its effect on methylated and unmethylated levels of tumor suppressor genes, such as p16INK4a and RASSF1A, in UV-exposed skin and UV-induced skin tumors. It was observed that the levels of methylated tumor suppressor genes were higher in IL-12 KO mice skin than their wild type counterparts. Together, these findings indicate that IL-12 deficiency promotes: (i) DNA hypermethylation, (ii) HDAC activity and (iii) silencing of tumor suppressor genes in UV exposed mice and that makes the mice more susceptible to UV radiation-induced skin cancer risk. Together, these observations suggest that augmentation of IL-12 should be considered as a strategy for the prevention and treatment of UV radiation-induced skin cancer risk. Citation Format: Ram Prasad, Tripti Singh, Santosh K. Katiyar. Interleukin-12 deficiency increases modulation of epigenetic regulators and silencing of tumor suppressor genes in UVB-exposed mouse skin and skin tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4055.

DNA methylation is a critical epigenetic regulator in the occurrence and development of diseases and is closely related to various functional responses in relation to spinal cord injury. To investigate the role of DNA methylation in spinal cord injury, we constructed a library with reduced-representation bisulfite sequencing data obtained at various time points (day 0-42) after spinal cord injury in mice. Global DNA methylation levels, specifically non-CpG (CHG and CHH) methylation levels, decreased modestly following spinal cord injury. Stages post-spinal cord injury were classified as early (day 0-3), intermediate (day 7-14), and late (day 28-42) based on similarity and hierarchical clustering of global DNA methylation patterns. The non-CpG methylation level, which included CHG and CHH methylation levels, was markedly reduced despite accounting for a minor proportion of total methylation abundance. At multiple genomic sites, including the 5' untranslated regions, promoter, exon, intron, and 3' untranslated regions, the non-CpG methylation level was markedly decreased following spinal cord injury, whereas the CpG methylation level remained unchanged at these locations. Approximately one-half of the differentially methylated regions were located in intergenic areas; the other differentially methylated regions in both CpG and non-CpG regions were clustered in intron regions, where the DNA methylation level was highest. The function of genes associated with differentially methylated regions in promoter regions was also investigated. From Gene Ontology analysis results, DNA methylation was implicated in a number of essential functional responses to spinal cord injury, including neuronal synaptic connection creation and axon regeneration. Notably, neither CpG methylation nor non-CpG methylation was implicated in the functional response of glial or inflammatory cells. In summary, our work elucidated the dynamic pattern of DNA methylation in the spinal cord following injury and identified reduced non-CpG methylation as an epigenetic target after spinal cord injury in mice.

Placental lipoprotein lipase (LPL) is crucial for placental lipid transfer. Impaired LPL gene expression and activity were reported in pregnancies complicated by gestational diabetes mellitus (GDM) and intra-uterine growth restriction. We hypothesized that placental LPL DNA methylation is altered by maternal metabolic status and could contribute to fetal programming. The objective of this study was thus to assess whether placental LPL DNA methylation is associated with GDM and both maternal and newborn lipid profiles. Placenta biopsies were sampled at delivery from 126 women including 27 women with GDM diagnosed following a post 75 g-oral glucose tolerance test (OGTT) between weeks 24 and 28 of gestation. Placental LPL DNA methylation and expression levels were determined using bisulfite pyrosequencing and quantitative real-time PCR, respectively. DNA methylation levels within LPL proximal promoter region (CpG1) and intron 1 CpG island (CpGs 2 and 3) were lower in placenta of women with GDM. DNA methylation levels at LPL-CpG1 and CpG3 were also negatively correlated with maternal glucose (2-h post OGTT; r=-0.22; P=0.02) and HDL-cholesterol levels (third trimester of pregnancy; r=-0.20; p=0.03), respectively. Moreover, we report correlation between LPL-CpG2 DNA methylation and cord blood lipid profile. DNA methylation levels within intron 1 CpG island explained up to 26% (r⩽-0.51; P<0.001) of placental LPL mRNA expression variance. Overall, we showed that maternal metabolic profile is associated with placental LPL DNA methylation dysregulation. Our results suggest that site-specific LPL epipolymorphisms in the placenta are possibly functional and could potentially be involved in determining the future metabolic health of the newborn.

Robust validation of methylation levels association at CPT1A locus with lipid plasma levels

Assessment of TET1 gene expression, DNA methylation and H3K27me3 level of its promoter region in eutopic endometrium of women with endometriosis and infertility.

BackgroundEpigenetic modifications play important roles in plant and animal development. DNA methylation impacts the transposable element (TE) silencing, gene imprinting and expression regulation.ResultsThrough a genome-wide analysis, DNA methylation peaks were characterized and mapped in maize embryo and endosperm genome, respectively. Distinct methylation level was observed across maize embryo and endosperm. The maize embryo genome contained more DNA methylation than endosperm. Totally, 985,478 CG islands (CGIs) were identified and most of them were unmethylated. More CGI shores were methylated than CGIs in maize suggested that DNA methylation level was not positively correlated with CpG density. The promoter sequence and transcriptional termination region (TTR) were more methylated than the gene body (intron and exon) region based on peak number and methylated depth. Result showed that 99% TEs were methylated in maize embryo, but a large portion of them (34.8%) were not methylated in endosperm. Maize embryo and endosperm exhibit distinct pattern/level of methylation. The most differentially methylated region between embryo and endosperm are CGI shores. Our results indicated that DNA methylation is associated with both gene silencing and gene activation in maize. Many genes involved in embryogenesis and seed development were found differentially methylated in embryo and endosperm. We found 41.5% imprinting genes were similarly methylated and 58.5% imprinting genes were differentially methylated between embryo and endosperm. Methylation level was associated with allelic silencing of only a small number of imprinting genes. The expression of maize DEMETER-like (DME-like) gene and MBD101 gene (MBD4 homolog) were higher in endosperm than in embryo. These two genes may be associated with distinct methylation levels across maize embryo and endosperm.ConclusionsThrough MeDIP-seq we systematically analyzed the methylomes of maize embryo and endosperm and results indicated that the global methylation status of embryo was more than that of the endosperm. Differences could be observed at the total number of methylation peaks, DMRs and specific methylated genes which were tightly associated with development of embryo and endosperm. Our results also revealed that many DNA methylation regions didn’t affect transcription of the corresponding genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-014-1204-7) contains supplementary material, which is available to authorized users.

Geminiviruses are important plant pathogens characterized by circular, single-stranded DNA (ssDNA) genomes. However, in the nuclei of infected cells, viral double-stranded DNA (dsDNA) associates with host histones to form a minichromosome. In phloem-limited geminiviruses, the characterization of viral minichromosomes is hindered by the low concentration of recovered complexes due to the small number of infected cells. Nevertheless, geminiviruses are both inducers and targets of the host posttranscriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) machinery. We have previously characterized a "recovery" phenomenon observed in pepper plants infected with pepper golden mosaic virus (PepGMV) that is associated with a reduction of viral DNA and RNA levels, the presence of virus-related siRNAs, and an increase in the levels of viral DNA methylation. Initial micrococcal nuclease-based assays pinpointed the presence of different viral chromatin complexes in symptomatic and recovered tissues. Using the pepper-PepGMV system, we developed a methodology to obtain a viral minichromosome-enriched fraction that does not disturb the basic chromatin structural integrity, as evaluated by the detection of core histones. Using this procedure, we have further characterized two populations of viral minichromosomes in PepGMV-infected plants. After further purification using sucrose gradient sedimentation, we also observed that minichromosomes isolated from symptomatic tissue showed a relaxed conformation (based on their sedimentation rate), are associated with a chromatin activation marker (H3K4me3), and present a low level of DNA methylation. The minichromosome population obtained from recovered tissue, on the other hand, sedimented as a compact structure, is associated with a chromatin-repressive marker (H3K9me2), and presents a high level of DNA methylation. Viral minichromosomes have been reported in several animal and plant models. However, in the case of geminiviruses, there has been some recent discussion about the importance of this structure and the significance of the epigenetic modifications that it can undergo during the infective cycle. Major problems in this type of studies are the low concentration of these complexes in an infected plant and the asynchronicity of infected cells along the process; therefore, the complexes isolated in a given moment usually represent a mixture of cells at different infection stages. The recovery process observed in PepGMV-infected plants and the isolation procedure described here provide two distinct populations of minichromosomes that will allow a more precise characterization of the modifications of viral DNA and its host proteins associated along the infective cycle. This structure could be also an interesting model to study several processes involving plant chromatin.

Transient Receptor Ankyrin Member 1 (TRPA1) is an ion channel family protein that regulates pain sensation through sensory neurons' activity. This study's purpose to analyzes the DNA methylation and mRNA expression level of the TRPA1 gene in endometriosis and its correlation with pain level. Twenty samples of peritoneal endometriosis and endometrial samples were obtained from women with endometriosis, which was subsequently compared to 20 endometrial samples of women without endometriosis. The DNA methylation level of TRPA1 was analyzed using Methylation-specific PCR (MS-PCR) and ImageJ software, while the mRNA expression of TRPA1 was analyzed using qRT-PCR. Furthermore, the pain level was measured using the numeric rating scale (NRS) by interviewing all the women. This study showed that there was a significant difference in the mRNA expression of TRPA1 in peritoneal endometriosis. The TRPA1 was unmethylated in both peritoneal and endometrial samples in endometriosis. However, DNA Methylation level of TRPA1 in peritoneal and endometrial of endometriosis compared to normal endometrial were no significant difference. Additionally, there was no correlation between DNA methylation level and mRNA expression level of TRPA1 in all samples, along with the endometriosis-associated pain.