Single-cell RNA-seq and single-cell bisulfite-sequencing reveal insights into yak preimplantation embryogenesis.

Extensive epigenetic reprogramming occurs during preimplantation embryonic development. However, the impact of DNA methylation in plateau yak preimplantation embryos and how epigenetic reprogramming contributes to transcriptional regulatory networks are unclear. In this study, we quantified gene expression and DNA methylation in oocytes and a series of yak embryos at different developmental stages and at single-cell resolution using single-cell bisulfite-sequencing and RNA-seq. We characterized embryonic genome activation and maternal transcript degradation and mapped epigenetic reprogramming events critical for embryonic development. Through cross-species transcriptome analysis, we identified 31 conserved maternal hub genes and 39 conserved zygotic hub genes, including SIN3A, PRC1, HDAC1/2, and HSPD1. Notably, by combining single-cell DNA methylation and transcriptome analysis, we identified 43 candidate methylation driver genes, such as AURKA, NUSAP1, CENPF, and PLK1, that may be associated with embryonic development. Finally, using functional approaches, we further determined that the epigenetic modifications associated with the histone deacetylases HDAC1/2 are essential for embryonic development and that the deubiquitinating enzyme USP7 may affect embryonic development by regulating DNA methylation. Our data represent an extensive resource on the transcriptional dynamics of yak embryonic development and DNA methylation remodeling, and provide new insights into strategies for the conservation of germplasm resources, as well as a better understanding of mammalian early embryonic development that can be applied to investigate the causes of early developmental disorders.

Chromosome-scale de novo genome assembly and annotation of three representative Casuarina species: C. equisetifolia, C. glauca, and C. cunninghamiana.

Australian pine (Casuarina spp.) is extensively planted in tropical and subtropical regions for wood production, shelterbelts, environmental protection, and ecological restoration due to their superior biological characteristics, such as rapid growth, wind and salt tolerance, and nitrogen fixation. To analyze the genomic diversity of Casuarina, we sequenced the genomes and constructed de novo genome assemblies of the three most widely planted Casuarina species: C. equisetifolia, C. glauca, and C. cunninghamiana. We generated chromosome-scale genome sequences using both Pacific Biosciences (PacBio) Sequel sequencing and chromosome conformation capture technology (Hi-C). The total genome sizes for C. equisetifolia, C. glauca, and C. cunninghamiana are 268 942 579 bp, 296 631 783 bp, and 293 483 606 bp, respectively, of which 25.91, 27.15, and 27.74% were annotated as repetitive sequences. We annotated 23 162, 24 673, and 24 674 protein-coding genes in C. equisetifolia, C. glauca, and C. cunninghamiana, respectively. We then collected branchlets from male and female individuals for whole-genome bisulfite sequencing (BS-seq) to explore the epigenetic regulation of sex determination in these three species. Transcriptome sequencing (RNA-seq) revealed differential expression of phytohormone-related genes between male and female plants. In summary, we generated three chromosome-level genome assemblies and comprehensive DNA methylation and transcriptome datasets from both male and female material for three Casuarina species, providing a basis for the comprehensive investigation of genomic diversity and functional gene discovery of Casuarina in the future.

Epigenetic inactivation of ERF reactivates γ-globin expression in ß-thalassemia.

The fetal-to-adult hemoglobin switch is regulated in a developmental stage-specific manner and reactivation of fetal hemoglobin (HbF) has therapeutic implications for treatment of ß-thalassemia and sickle cell anemia, two major global health problems. Although significant progress has been made in our understanding of the molecular mechanism of the fetal-to-adult hemoglobin switch, the mechanism of epigenetic regulation of HbF silencing remains to be fully defined. Here, we performed whole-genome bisulfite sequencing and RNA sequencing analysis of the bone marrow-derived GYPA+ erythroid cells from ß-thalassemia-affected individuals with widely varying levels of HbF groups (HbF ≥ 95th percentile or HbF ≤ 5th percentile) to screen epigenetic modulators of HbF and phenotypic diversity of ß-thalassemia. We identified an ETS2 repressor factor encoded by ERF, whose promoter hypermethylation and mRNA downregulation are associated with high HbF levels in ß-thalassemia. We further observed that hypermethylation of the ERF promoter mediated by enrichment of DNMT3A leads to demethylation of γ-globin genes and attenuation of binding of ERF on the HBG promoter and eventually re-activation of HbF in ß-thalassemia. We demonstrated that ERF depletion markedly increased HbF production in human CD34+ erythroid progenitor cells, HUDEP-2 cell lines, and transplanted NCG-Kit-V831M mice. ERF represses γ-globin expression by directly binding to two consensus motifs regulating γ-globin gene expression. Importantly, ERF depletion did not affect maturation of erythroid cells. Identification of alterations in DNA methylation of ERF as a modulator of HbF synthesis opens up therapeutic targets for ß-hemoglobinopathies.

Comethyl: a network-based methylome approach to investigate the multivariate nature of health and disease.

Health outcomes are frequently shaped by difficult to dissect inter-relationships between biological, behavioral, social and environmental factors. DNA methylation patterns reflect such multivariate intersections, providing a rich source of novel biomarkers and insight into disease etiologies. Recent advances in whole-genome bisulfite sequencing enable investigation of DNA methylation over all genomic CpGs, but existing bioinformatic approaches lack accessible system-level tools. Here, we develop the R package Comethyl, for weighted gene correlation network analysis of user-defined genomic regions that generates modules of comethylated regions, which are then tested for correlations with multivariate sample traits. First, regions are defined by CpG genomic location or regulatory annotation and filtered based on CpG count, sequencing depth and variability. Next, correlation networks are used to find modules of interconnected nodes using methylation values within the selected regions. Each module containing multiple comethylated regions is reduced in complexity to a single eigennode value, which is then tested for correlations with experimental metadata. Comethyl has the ability to cover the noncoding regulatory regions of the genome with high relevance to interpretation of genome-wide association studies and integration with other types of epigenomic data. We demonstrate the utility of Comethyl on a dataset of male cord blood samples from newborns later diagnosed with autism spectrum disorder (ASD) versus typical development. Comethyl successfully identified an ASD-associated module containing regions mapped to genes enriched for brain glial functions. Comethyl is expected to be useful in uncovering the multivariate nature of health disparities for a variety of common disorders. Comethyl is available at github.com/cemordaunt/comethyl with complete documentation and example analyses.

High-resolution methylome analysis uncovers stress-responsive genomic hotspots and drought-sensitive transposable element superfamilies in the clonal Lombardy poplar.

DNA methylation is environment-sensitive and can mediate stress responses. In trees, changes in the environment might cumulatively shape the methylome landscape over time. However, because high-resolution methylome studies usually focus on single environmental cues, the stress-specificity and long-term stability of methylation responses remain unclear. Here, we studied the methylome plasticity of a Populus nigra cv. 'Italica' clone widely distributed across Europe. Adult trees from different geographic locations were clonally propagated in a common garden experiment and exposed to cold, heat, drought, herbivory, rust infection, and salicylic acid treatments. Whole-genome bisulfite sequencing revealed stress-induced and naturally occurring DNA methylation variants. In CG/CHG contexts, the same genomic regions were often affected by multiple stresses, suggesting a generic methylome response. Moreover, these variants showed striking overlap with naturally occurring methylation variants between trees from different locations. Drought treatment triggered CHH hypermethylation of transposable elements, affecting entire superfamilies near drought-responsive genes. Thus, we revealed genomic hotspots of methylation change that are not stress-specific and that contribute to natural DNA methylation variation, and identified stress-specific hypermethylation of entire transposon superfamilies with possible functional consequences. Our results underscore the importance of studying multiple stressors in a single experiment for recognizing general versus stress-specific methylome responses.

Non-CG DNA methylation marks the transition from pupa to adult in Helicoverpa armigera.

DNA methylation in insects is generally low in abundance, and its role is not well understood. It is often localised in protein coding regions and associated with the expression of 'housekeeping' genes. Few studies have explored DNA methylation dynamics during lifecycle stage transitions in holometabolous (metamorphosing) insects. Using targeted mass spectrometry, we have found a significant difference in global DNA methylation levels between larvae, pupae and adults of Helicoverpa armigera (Lepidoptera: Noctuidae) Hübner, a polyphagous pest of agricultural importance. Whole-genome bisulfite sequencing confirmed these observations and pointed to non-CG context being the primary explanation for the difference observed between pupa and adult. Non-CG methylation was enriched in genes specific to various signalling pathways (Hippo signalling, Hedgehog signalling and mitogen-activated protein kinase (MAPK) signalling) and ATP-dependent chromatin remodelling. Understanding the function of this epigenetic mark could be a target in future studies focusing on integrated pest management.

Epigenomic signature of major congenital heart defects in newborns with Down syndrome.

BACKGROUND: Congenital heart defects (CHDs) affect approximately half of individuals with Down syndrome (DS), but the molecular reasons for incomplete penetrance are unknown. Previous studies have largely focused on identifying genetic risk factors associated with CHDs in individuals with DS, but comprehensive studies of the contribution of epigenetic marks are lacking. We aimed to identify and characterize DNA methylation differences from newborn dried blood spots (NDBS) of DS individuals with major CHDs compared to DS individuals without CHDs. METHODS: We used the Illumina EPIC array and whole-genome bisulfite sequencing (WGBS) to quantitate DNA methylation for 86 NDBS samples from the California Biobank Program: (1) 45 DS-CHD (27 female, 18 male) and (2) 41 DS non-CHD (27 female, 14 male). We analyzed global CpG methylation and identified differentially methylated regions (DMRs) in DS-CHD versus DS non-CHD comparisons (both sex-combined and sex-stratified) corrected for sex, age of blood collection, and cell-type proportions. CHD DMRs were analyzed for enrichment in CpG and genic contexts, chromatin states, and histone modifications by genomic coordinates and for gene ontology enrichment by gene mapping. DMRs were also tested in a replication dataset and compared to methylation levels in DS versus typical development (TD) WGBS NDBS samples. RESULTS: We found global CpG hypomethylation in DS-CHD males compared to DS non-CHD males, which was attributable to elevated levels of nucleated red blood cells and not seen in females. At a regional level, we identified 58, 341, and 3938 CHD-associated DMRs in the Sex Combined, Females Only, and Males Only groups, respectively, and used machine learning algorithms to select 19 Males Only loci that could distinguish CHD from non-CHD. DMRs in all comparisons were enriched for gene exons, CpG islands, and bivalent chromatin and mapped to genes enriched for terms related to cardiac and immune functions. Lastly, a greater percentage of CHD-associated DMRs than background regions were differentially methylated in DS versus TD samples. CONCLUSIONS: A sex-specific signature of DNA methylation was detected in NDBS of DS-CHD compared to DS non-CHD individuals. This supports the hypothesis that epigenetics can reflect the variability of phenotypes in DS, particularly CHDs.

Pregnancy induced hypertension and umbilical cord blood DNA methylation in newborns: an epigenome-wide DNA methylation study.

OBJECTIVIES: Pregnancy induced hypertension (PIH) syndrome is a disease that unique to pregnant women and is associated with elevated risk of offspring cardiovascular diseases (CVDs) and neurodevelopmental disorders in their kids. Previous research on cord blood utilizing the Human Methylation BeadChip or EPIC array revealed that PIH is associated with specific DNA methylation site. Here, we investigate the whole genome DNA methylation landscape of cord blood from newborns of PIH mother. METHODS: Whole-genome bisulfite sequencing (WGBS) was used to examine the changes in whole genome DNA methylation in the umbilical cord blood of three healthy (NC) and four PIH individuals. Using methylKit, we discovered Hypo- and hyper- differentially methylated probes (DMPs) or methylated regions (DMRs) in the PIH patients' cord blood DNA. Pathway enrichments were assessed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment assays. DMPs or DMRs relevant to the immunological, neurological, and circulatory systems were also employed for enrichment assay, Metascape analysis and PPI network analysis. RESULTS: 520 hyper- and 224 hypo-DMPs, and 374 hyper- and 186 hypo-DMRs between NC and PIH group, respectively. Both DMPs and DMRs have enhanced pathways for cardiovascular, neurological system, and immune system development. Further investigation of DMPs or DMRs related to immunological, neurological, and circulatory system development revealed that TBK1 served as a hub gene for all three developmental pathways. CONCLUSION: PIH-associated DMPs or DMRs in umbilical cord blood DNA may play a role in immunological, neurological, and circulatory system development. Abnormal DNA methylation in the immune system may also contribute to the development of CVDs and neurodevelopment disorders.

Single-Base Methylome Analysis of Sweet Cherry (Prunus avium L.) on Dwarfing Rootstocks Reveals Epigenomic Differences Associated with Scion Dwarfing Conferred by Grafting.

Plant grafting using dwarfing rootstocks is one of the important cultivation measures in the sweet cherry (Prunus avium) industry. In this work, we aimed to explore the effects of the dwarfing rootstock "Pd1" (Prunus tomentosa) on sweet cherry 'Shuguang2' scions by performing morphological observations using the paraffin slice technique, detecting GA (gibberellin) and IAA (auxin) contents using UPLC-QTRAP-MS (ultra-performance liquid chromatography coupled with a hybrid triple quadrupole-linear ion trap mass spectrometer), and implementing integration analyses of the epigenome and transcriptome using whole-genome bisulfite sequencing and transcriptome sequencing. Anatomical analysis indicated that the cell division ability of the SAM (shoot apical meristem) in dwarfing plants was reduced. Pd1 rootstock significantly decreased the levels of GAs and IAA in sweet cherry scions. Methylome analysis showed that the sweet cherry genome presented 15.2~18.6%, 59.88~61.55%, 28.09~33.78%, and 2.99~5.28% methylation at total C, CG, CHG, and CHH sites, respectively. Shoot tips from dwarfing plants exhibited a hypermethylated pattern mostly due to increased CHH methylation, while leaves exhibited a hypomethylated pattern. According to GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, DMGs (differentially methylated genes) and DEGs (differentially expressed genes) were enriched in hormone-related GO terms and KEGG pathways. Global correlation analysis between methylation and transcription revealed that mCpG in the gene body region enhanced gene expression and mCHH in the region near the TSS (transcription start site) was positively correlated with gene expression. Next, we found some hormone-related genes and TFs with significant changes in methylation and transcription, including SAURs, ARF, GA2ox, ABS1, bZIP, MYB, and NAC. This study presents a methylome map of the sweet cherry genome, revealed widespread DNA methylation alterations in scions caused by dwarfing rootstock, and obtained abundant genes with methylation and transcription alterations that are potentially involved in rootstock-induced growth changes in sweet cherry scions. Our findings can lay a good basis for further epigenetic studies on sweet cherry dwarfing and provide valuable new insight into understanding rootstock-scion interactions.

The Effects of DNA Methylation on Cytoplasmic Male Sterility in Sugar Beet.

DNA methylation is widely found in higher plants and can control gene expression by regulation without changing the DNA sequence. In this study, the whole-genome methylation map of sugar beet was constructed by WGBS (whole-genome bisulfite sequencing) technology, and the results of WGBS were verified by bisulfite transformation, indicating that the results of WGBS technology were reliable. In addition, 12 differential methylation genes (DMGs) were identified, which were related to carbohydrate and energy metabolism, pollen wall development, and endogenous hormone regulation. Quantitative real-time PCR (qRT-PCR) showed that 75% of DMG expression levels showed negative feedback with methylation level, indicating that DNA methylation can affect gene expression to a certain extent. In addition, we found hypermethylation inhibited gene expression, which laid a foundation for further study on the molecular mechanism of DNA methylation at the epigenetic level in sugar beet male sterility.

A pipeline for sample tagging of whole genome bisulfite sequencing data using genotypes of whole genome sequencing.

BACKGROUND: In large-scale high-throughput sequencing projects and biobank construction, sample tagging is essential to prevent sample mix-ups. Despite the availability of fingerprint panels for DNA data, little research has been conducted on sample tagging of whole genome bisulfite sequencing (WGBS) data. This study aims to construct a pipeline and identify applicable fingerprint panels to address this problem. RESULTS: Using autosome-wide A/T polymorphic single nucleotide variants (SNVs) obtained from whole genome sequencing (WGS) and WGBS of individuals from the Third China National Stroke Registry, we designed a fingerprint panel and constructed an optimized pipeline for tagging WGBS data. This pipeline used Bis-SNP to call genotypes from the WGBS data, and optimized genotype comparison by eliminating wildtype homozygous and missing genotypes, and retaining variants with identical genomic coordinates and reference/alternative alleles. WGS-based and WGBS-based genotypes called from identical or different samples were extensively compared using hap.py. In the first batch of 94 samples, the genotype consistency rates were between 71.01%-84.23% and 51.43%-60.50% for the matched and mismatched WGS and WGBS data using the autosome-wide A/T polymorphic SNV panel. This capability to tag WGBS data was validated among the second batch of 240 samples, with genotype consistency rates ranging from 70.61%-84.65% to 49.58%-61.42% for the matched and mismatched data, respectively. We also determined that the number of genetic variants required to correctly tag WGBS data was on the order of thousands through testing six fingerprint panels with different orders for the number of variants. Additionally, we affirmed this result with two self-designed panels of 1351 and 1278 SNVs, respectively. Furthermore, this study confirmed that using the number of genetic variants with identical coordinates and ref/alt alleles, or identical genotypes could not correctly tag WGBS data. CONCLUSION: This study proposed an optimized pipeline, applicable fingerprint panels, and a lower boundary for the number of fingerprint genetic variants needed for correct sample tagging of WGBS data, which are valuable for tagging WGBS data and integrating multi-omics data for biobanks.

Analysis of DNA Methylation Differences during the JIII Formation of Bursaphelenchus xylophilus.

DNA methylation is a pivotal process that regulates gene expression and facilitates rapid adaptation to challenging environments. The pinewood nematode (PWN; Bursaphelenchus xylophilus), the causative agent of pine wilt disease, survives at low temperatures through third-stage dispersal juvenile, making it a major pathogen for pines in Asia. To comprehend the impact of DNA methylation on the formation and environmental adaptation of third-stage dispersal juvenile, we conducted whole-genome bisulfite sequencing and transcriptional sequencing on both the third-stage dispersal juvenile and three other propagative juvenile stages of PWN. Our findings revealed that the average methylation rate of cytosine in the samples ranged from 0.89% to 0.99%. Moreover, we observed significant DNA methylation changes in the third-stage dispersal juvenile and the second-stage propagative juvenile of PWN, including differentially methylated cytosine (DMCs, n = 435) and regions (DMRs, n = 72). In the joint analysis of methylation-associated transcription, we observed that 23 genes exhibited overlap between differentially methylated regions and differential gene expression during the formation of the third-stage dispersal juvenile of PWN. Further functional analysis of these genes revealed enrichment in processes related to lipid metabolism and fatty acid synthesis. These findings emphasize the significance of DNA methylation in the development of third-stage dispersal juvenile of PWN, as it regulates transcription to enhance the probability of rapid expansion in PWN.

Comprehensive benchmarking of software for mapping whole genome bisulfite data: from read alignment to DNA methylation analysis.

Whole genome bisulfite sequencing is currently at the forefront of epigenetic analysis, facilitating the nucleotide-level resolution of 5-methylcytosine (5mC) on a genome-wide scale. Specialized software have been developed to accommodate the unique difficulties in aligning such sequencing reads to a given reference, building on the knowledge acquired from model organisms such as human, or Arabidopsis thaliana. As the field of epigenetics expands its purview to non-model plant species, new challenges arise which bring into question the suitability of previously established tools. Herein, nine short-read aligners are evaluated: Bismark, BS-Seeker2, BSMAP, BWA-meth, ERNE-BS5, GEM3, GSNAP, Last and segemehl. Precision-recall of simulated alignments, in comparison to real sequencing data obtained from three natural accessions, reveals on-balance that BWA-meth and BSMAP are able to make the best use of the data during mapping. The influence of difficult-to-map regions, characterized by deviations in sequencing depth over repeat annotations, is evaluated in terms of the mean absolute deviation of the resulting methylation calls in comparison to a realistic methylome. Downstream methylation analysis is responsive to the handling of multi-mapping reads relative to mapping quality (MAPQ), and potentially susceptible to bias arising from the increased sequence complexity of densely methylated reads.

Long-term storage does not affect the DNA methylation profiles of vitrified-warmed human embryos.

With the widespread application of embryo cryopreservation in assisted reproductive techniques, it is necessary to assess the safety of long-term cryopreservation of human embryos and it is unclear whether storage time has an impact on the DNA methylation profiles of human embryos. Nine women who received IVF treatment were recruited for this study. The retrieved eight-cell human embryos were classified into three groups including fresh embryos, cryopreserved embryos stored for 3 years, and cryopreserved embryos stored for 8 years. Single-cell whole-genome bisulfite sequencing (scWGBS) was conducted. The genome-wide methylation pattern of the fresh and two cryopreserved groups were similar. In addition, the methylation level in different genomic regions showed comparable patterns and no significant differences were observed in the methylation level of imprinted genes among the three groups. A total of 587 differentially methylated regions (DMRs) in the 3-year group and 540 DMRs in the 8-year group were identified comparing to fresh group. However, they were not enriched in promoters and had a similar genome-wide distributions, suggesting that these DMRs may not contribute to the changes in corresponding gene expressions. Our study illustrated that long-term cryopreservation will not affect the DNA methylation profiles of human eight-cell embryos at single-cell level.

Effects of In Utero PFOS Exposure on Epigenetics and Metabolism in Mouse Fetal Livers.

Prenatal exposure to perfluorooctanesulfonate (PFOS) increases fetus' metabolic risk; however, the investigation of the underlying mechanism is limited. In this study, pregnant mice in the gestational days (GD, 4.5-17.5) were exposed to PFOS (0.3 and 3 µg/g of body weight). At GD 17.5, PFOS perturbed maternal lipid metabolism and upregulated metabolism-regulating hepatokines (Angptl4, Angptl8, and Selenop). Mass-spectrometry imaging and whole-genome bisulfite sequencing revealed, respectively, selective PFOS localization and deregulation of gene methylation in fetal livers, involved in inflammation, glucose, and fatty acid metabolism. PCR and Western blot analysis of lipid-laden fetal livers showed activation of AMPK signaling, accompanied by significant increases in the expression of glucose transporters (Glut2/4), hexose-phosphate sensors (Retsat and ChREBP), and the key glycolytic enzyme, pyruvate kinase (Pk) for glucose catabolism. Additionally, PFOS modulated the expression levels of PPARα and PPARγ downstream target genes, which simultaneously stimulated fatty acid oxidation (Cyp4a14, Acot, and Acox) and lipogenesis (Srebp1c, Acaca, and Fasn). Using human normal hepatocyte (MIHA) cells, the underlying mechanism of PFOS-elicited nuclear translocation of ChREBP, associated with a fatty acid synthesizing pathway, was revealed. Our finding implies that in utero PFOS exposure altered the epigenetic landscape associated with dysregulation of fetal liver metabolism, predisposing postnatal susceptibility to metabolic challenges.

The global dynamic of DNA methylation in response to heat stress revealed epigenetic mechanism of heat acclimation in Saccharina japonica.

Saccharina japonica is an ecologically and economically important kelp in cold-temperate regions. When it is cultivated on a large scale in the temperate and even subtropical zones, heat stress is a frequent abiotic stress. This study is the first attempt to reveal the regulatory mechanism of the response to heat stress from the perspective of DNA methylation in S. japonica. We firstly obtained the characteristics of variation in the methylome under heat stress, and observed that heat stress caused a slight increase in the overall methylation level and methylation rate, especially in the non-coding regions of the genome. Secondly, we noted that methylation was probably one of factors affecting the expression of genes, and that methylation within the gene body was positively correlated with the gene expression (rho = 0.0784). Moreover, it was found that among the differentially expressed genes regulated by methylation, many genes were related to heat stress response, such as HSP gene family, genes of antioxidant enzymes, genes related to proteasome-ubiquitination pathway, and plant cell signaling pathways. This study demonstrated that DNA methylation is involved in regulating the response to heat stress, laying a foundation for studying the acclimation and adaptation of S. japonica to heat stress from an epigenetic perspective.

Networks of placental DNA methylation correlate with maternal serum PCB concentrations and child neurodevelopment.

BACKGROUND: Gestational exposure to polychlorinated biphenyls (PCBs) has been associated with elevated risk for neurodevelopmental disorders. Placental epigenetics may serve as a potential mechanism of risk or marker of altered placental function. Prior studies have associated differential placental DNA methylation with maternal PCB exposure or with increased risk of autism spectrum disorder (ASD). However, sequencing-based placental methylomes have not previously been tested for simultaneous associations with maternal PCB levels and child neurodevelopmental outcomes. OBJECTIVES: We aimed to identify placental DNA methylation patterns associated with maternal PCB levels and child neurodevelopmental outcomes in the high-risk ASD MARBLES cohort. METHODS: We measured 209 PCB congeners in 104 maternal serum samples collected at delivery. We identified networks of DNA methylation from 147 placenta samples using the Comethyl R package, which performs weighted gene correlation network analysis for whole genome bisulfite sequencing data. We tested placental DNA methylation modules for association with maternal serum PCB levels, child neurodevelopment, and other participant traits. RESULTS: PCBs 153 + 168, 170, 180 + 193, and 187 were detected in over 50% of maternal serum samples and were highly correlated with one another. Consistent with previous findings, maternal age was the strongest predictor of serum PCB levels, alongside year of sample collection, pre-pregnancy BMI, and polyunsaturated fatty acid levels. Twenty seven modules of placental DNA methylation were identified, including five which significantly correlated with one or more PCBs, and four which correlated with child neurodevelopment. Two modules associated with maternal PCB levels as well as child neurodevelopment, and mapped to CSMD1 and AUTS2, genes previously implicated in ASD and identified as differentially methylated regions in mouse brain and placenta following gestational PCB exposure. CONCLUSIONS: Placental DNA co-methylation modules were associated with maternal PCBs and child neurodevelopment. Methylation of CSMD1 and AUTS2 could be markers of altered placental function and/or ASD risk following maternal PCB exposure.

Comparison of DNA methylation profiles of human embryos cultured in either uninterrupted or interrupted incubators.

PURPOSE: We aimed to compare the DNA methylation profiles of human embryos cultured in uninterrupted or interrupted incubators. METHODS: This study included 9 women, ≤ 30 years old (range: 20-30 years), without a history of genetic diseases or smoking, undergoing ICSI treatment, and each woman donated one oocyte. Embryos were randomly assigned to culture in either time-lapse imaging or standard incubators after ICSI. We compared the DNA methylation profiles of human eight-cell embryos cultured in uninterrupted condition using time-lapse imaging (TLI) incubator (EmbryoScope) to those cultured in interrupted culture model using standard incubators (SI, G185 K-System). Nine single-cell whole-genome bisulfite sequencing (WGBS) datasets were analyzed, including four SI-cultured embryos and five TLI-cultured embryos at the eight-cell stage. RESULTS: A total of 581,140,020 and 732,348,182 clean reads were generated from the TLI and SI groups, respectively. TLI-cultured embryos had similar genome-wide methylation patterns to SI-cultured embryos. There were no significant differences in the methylation and transcription levels of transposable elements and imprinted control regions. Although a total of 198 differentially methylated genes (DMGs) were identified, only five DMGs had significantly different transcription levels between the two groups. CONCLUSIONS: This is the first study to compare the DNA methylation profiles of embryos cultured in TLI and SI and will provide a foundation for evaluating the safety of TLI application in assisted reproductive technologies. However, further study with a larger cohort of samples was needed for the data validation.

The Change in Whole-Genome Methylation and Transcriptome Profile under Autophagy Defect and Nitrogen Starvation.

Through whole-genome bisulfite sequencing and RNA-seq, we determined the potential impact of autophagy in regulating DNA methylation in Arabidopsis, providing a solid foundation for further understanding the molecular mechanism of autophagy and how plants cope with nitrogen deficiency. A total of 335 notable differentially expressed genes (DEGs) were discovered in wild-type Arabidopsis (Col-0-N) and an autophagic mutant cultivated under nitrogen starvation (atg5-1-N). Among these, 142 DEGs were associated with hypomethylated regions (hypo-DMRs) and were upregulated. This suggests a correlation between DNA demethylation and the ability of Arabidopsis to cope with nitrogen deficiency. Examination of the hypo-DMR-linked upregulated DEGs indicated that the expression of MYB101, an ABA pathway regulator, may be regulated by DNA demethylation and the recruitment of transcription factors (TFs; ERF57, ERF105, ERF48, and ERF111), which may contribute to the growth arrest induced by abscisic acid (ABA). Additionally, we found that DNA methylation might impact the biosynthesis of salicylic acid (SA). The promoter region of ATGH3.12 (PBS3), a key enzyme in SA synthesis, was hypomethylated, combined with overexpression of PBS3 and its potential TF AT3G46070, suggesting that autophagy defects may lead to SA-activated senescence, depending on DNA demethylation. These findings suggest that DNA hypomethylation may impact the mechanism by which Arabidopsis autophagy mutants (atg5-1) respond to nitrogen deficiency, specifically in relation to ABA and SA regulation. Our evaluation of hormone levels verified that these two hormones are significantly enriched under nitrogen deficiency in atg5-1-N compared to Col-0-N.

Study on detection of CNVs using human whole genome bisulfite sequencing data.

It has been reported that the aberrant DNA methylation may result in copy number variations (CNVs), and the CNVs may alter the levels of DNA methylation. Whole genome bisulfite sequencing (WGBS) is able to generate the sequencing data of DNAs, and shows the potential ability to detect CNVs. However, the evaluations and performances on the detections of CNVs using WGBS data is still unclear. In this study, five software with different strategies for CNV detections, e.g., BreakDancer, cn.mops, CNVnator, DELLY and Pindel, were selected to explore and benchmark the performances of CNV detections with WGBS data. Based on the real (2.62 billion reads) and simulated (12.35 billion reads) WGBS data of humans, we calculated the number, precision, recall, relative ability, memory usage, and running time of CNV detections by 150 times, and tried to figure out the optimal strategy for CNV detections with WGBS data. Based on the real WGBS data, Pindel detected the most deletions and duplications, CNVnator detected the deletions with the highest precision, cn.mops detected the duplications with the highest precision, Pindel detected the deletions with the highest recall, and cn.mops detected the duplications with the highest recall. Based on the simulated WGBS data, BreakDancer detected the most deletions, and cn.mops detected the most duplications. The CNVnator showed the highest precision and recall for both deletions and duplications. In real and simulated WGBS data, the ability of CNVnator to detect CNVs was likely to overtake that in the whole genome sequencing data. Additionally, DELLY and BreakDancer displayed the lowest peak of memory usage and the lest CPU runtime, while CNVnator expressed the highest peak of memory usage and the most CPU runtime. Taken together, CNVnator and cn.mops showed the excellent performances of CNV detections with WGBS data. These results suggested that it was feasible to detect CNVs using WGBS data, and provided the useful information to further investigate both CNVs and DNA methylation using WGBS data alone.