Epigenetic and transcriptional signatures of ex situ conserved golden snub-nosed monkeys (Rhinopithecus roxellana)

Cytosine Methylation Patterns As Biomarkers in MDS

DNA methylation plays a central role in regulating many aspects of growth and development in mammals through regulating gene expression. The development of next generation sequencing technologies have paved the way for genome-wide, high resolution analysis of DNA methylation landscapes using methodology known as reduced representation bisulfite sequencing (RRBS). While RRBS has proven to be effective in understanding DNA methylation landscapes in humans, mice, and rats, to date, few studies have utilised this powerful method for investigating DNA methylation in agricultural animals. Here we describe the utilisation of RRBS to investigate DNA methylation in sheep Longissimus dorsi muscles. RRBS analysis of ∼1% of the genome from Longissimus dorsi muscles provided data of suitably high precision and accuracy for DNA methylation analysis, at all levels of resolution from genome-wide to individual nucleotides. Combining RRBS data with mRNAseq data allowed the sheep Longissimus dorsi muscle methylome to be compared with methylomes from other species. While some species differences were identified, many similarities were observed between DNA methylation patterns in sheep and other more commonly studied species. The RRBS data presented here highlights the complexity of epigenetic regulation of genes. However, the similarities observed across species are promising, in that knowledge gained from epigenetic studies in human and mice may be applied, with caution, to agricultural species. The ability to accurately measure DNA methylation in agricultural animals will contribute an additional layer of information to the genetic analyses currently being used to maximise production gains in these species.

DNA methylation in CpG dinucleotides is an important epigenetic mark in fish spermatozoa since it has been shown that some sperm methylome features are transmitted to the offspring. Reduced representation bisulfite sequencing (RRBS) is one genome-scale methods developed to assess DNA methylation at CpG sites. It allows the sequencing of a reduced fraction of the genome expected to be enriched in CpGs. The aim of this study is to characterize the extent of the CpG sites that can be identified in the RRBS-reduced sequenced fraction of rainbow trout spermatozoa, in order to evaluate the potential of RRBS for sperm DNA methylation studies. We observed that RRBS did provide a reduced amount of genomic data, the sum of the CpGs analyzed on 12 males spanning 9% of the total genomic CpGs. CpGs were only slightly enriched in the RRBS data (×1.7 times the sequenced nucleotides), the possible causes being linked to trout genome structure and sequenced fragments size. All genomic functional features were represented in our CpG dataset, with a noticeable enrichment in exons but, strikingly, not in promoters. The number of CpGs shared between biological replicates was low, but this proportion reached workable values from six biological replicates (46% of the analyzed cytosines) on. The choices that are to be made regarding fragment size selection and the options during bioinformatic data processing are discussed. In all, RRBS is a relevant first-approach method to scan the CpG DNA methylation status of spermatozoa along rainbow trout genome, although in a very reduced pattern among biological replicates.

Study question Determine the presence of DNA methylation modifying enzymes in sperm and assess whether the sperm DNA methylome changes after exposure to different culture media conditions. Summary answer We identified 2,151 sperm-borne proteins, including 144 novel ones. DNA methylation remained unchanged after 24-hour exposure to culture media supplemented with H2O2 or palmitate. What is known already Children conceived via assisted reproduction techniques (ART) have a higher prevalence of DNA methylation-related disorders. Sperm DNA methylation is crucial for embryo development and is influenced by environmental and lifestyle factors such as diet, weight loss, and exercise. ART procedures, including semen preparation and incubation in embryo culture media, may impact sperm DNA integrity. However, whether culture conditions alter the DNA methylome of mature spermatozoa during ART remains unknown. Study design, size, duration Sperm from four donors was analysed using targeted shotgun proteomics, with a focus on protein sizes corresponding to epigenetic modifying enzymes. Sperm samples collected from fifteen men were incubated for 24 hours with either standard media, media supplemented with H2O2 or media supplemented with palmitate. DNA methylation profiles of incubated sperm were assessed by Reduced Representation Bisulfite Sequencing (RRBS) and compared to that of sperm before incubation. Participants/materials, setting, methods Sperm samples were collected from fifteen healthy Caucasian men aged 20-40 years. Exclusion criteria included daily use of prescription medication and/or testicular disease. Motile sperm were isolated using the swim-up procedure. Protein analysis was performed using in-gel digestion and mass spectrometry. DNA methylation was assessed using RRBS and further validated by targeted bisulfite pyrosequencing for a subset of genomic regions. Main results and the role of chance Proteomic analysis identified 2,151 sperm-borne proteins, including 144 newly reported in mature spermatozoa. DNMT1 was confirmed, while DNMT3A and DNMT3B were not detected. Sperm motility declined after 24-hour incubation in control, H2O2 and palmitate media, with a more rapid decrease in the H2O2 condition. To assess genome-wide DNA methylation, reduced representation bisulfite sequencing (RRBS) was performed, yielding an average depth of 21.4 million aligned reads per sample. No significant differences in global or region-specific DNA methylation levels were observed across the three culture conditions compared to pre-incubation. These findings suggest that the sperm DNA methylome remains highly stable. Limitations, reasons for caution The RRBS analysis does not allow analysis of the full genome therefore, DNA methylation may be affected in other, not detected genomic regions. Additionally, only three culture media conditions were tested in this study, we cannot rule out that other media compositions could induce DNA methylation changes. Wider implications of the findings This study suggests the sperm DNA methylome is stable despite DNA methylation machinery presence. DNA methylation changes via lifestyle changes likely occur earlier in spermatogenesis, with ART media unlikely to alter patterns. However, selection for sperm with specific methylation signatures remains possible. Trial registration number Yes

S-adenosylmethionine Levels Regulate the Schwann Cell DNA Methylome

Measuring stress-induced DNA methylation in apomictic Dandelions

Reduced representation bisulfite sequencing (RRBS) has been used to profile DNA methylation patterns in mammalian genomes such as human, mouse and rat. The methylome of the zebrafish, an important animal model, has not yet been characterized at base-pair resolution using RRBS. Therefore, we evaluated the technique of RRBS in this model organism by generating four single-nucleotide resolution DNA methylomes of adult zebrafish brain. We performed several simulations to show the distribution of fragments and enrichment of CpGs in different in silico reduced representation genomes of zebrafish. Four RRBS brain libraries generated 98 million sequenced reads and had higher frequencies of multiple mapping than equivalent human RRBS libraries. The zebrafish methylome indicates there is higher global DNA methylation in the zebrafish genome compared with its equivalent human methylome. This observation was confirmed by RRBS of zebrafish liver. High coverage CpG dinucleotides are enriched in CpG island shores more than in the CpG island core. We found that 45% of the mapped CpGs reside in gene bodies, and 7% in gene promoters. This analysis provides a roadmap for generating reproducible base-pair level methylomes for zebrafish using RRBS and our results provide the first evidence that RRBS is a suitable technique for global methylation analysis in zebrafish.

Robust Discovery of Candidate DNA Methylation Cancer Drivers

Whole-genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS) are widely used for measuring DNA methylation levels on a genome-wide scale. Both methods have limitations: WGBS is expensive and prohibitive for most large-scale projects; RRBS only interrogates 6–12% of the CpGs in the human genome. Here, we introduce methylation-sensitive restriction enzyme bisulfite sequencing (MREBS) which has the reduced sequencing requirements of RRBS, but significantly expands the coverage of CpG sites in the genome. We built a multiple regression model that combines the two features of MREBS: the bisulfite conversion ratios of single cytosines (as in WGBS and RRBS) as well as the number of reads that cover each locus (as in MRE-seq). This combined approach allowed us to estimate differential methylation across 60% of the genome using read count data alone, and where counts were sufficiently high in both samples (about 1.5% of the genome), our estimates were significantly improved by the single CpG conversion information. We show that differential DNA methylation values based on MREBS data correlate well with those based on WGBS and RRBS. This newly developed technique combines the sequencing cost of RRBS and DNA methylation estimates on a portion of the genome similar to WGBS, making it ideal for large-scale projects of mammalian genomes.

DNA methylation is an epigenetic mark that plays an essential role in regulating gene expression. CpG islands are DNA methylations regions in promoters known to regulate gene expression through transcriptional silencing of the corresponding gene. DNA methylation at CpG islands is crucial for gene expression and tissue-specific processes. At the current time, a limited number of studies have reported on gene expression associated with DNA methylation in diverse adult tissues at the genome-wide level. Expression levels are rarely affected by DNA methylation in normal adult tissues; however, statistical differences in gene expression level correlated with DNA methylation have recently been revealed. In this study, we examined 20 pairs of DNA methylomes and transcriptomes from RNA-seq and reduced representation bisulfite sequencing (RRBS) data using adult Ogye chicken tissues. A total of 3,133 CpG islands were identified from 20 tissue data in a single chicken sample which could affect downstream genes. Analyzing these CpG island and gene pairs, 121 significant units were statistically correlated. Among them, six genes (CLDN3, DECR2, EVA1B, NME4, NTSR1, and XPNPEP2) were highly significantly changed by altered DNA methylation. Finally, our data demonstrated how DNA methylation correlated to gene expression in normal adult tissues. Our source codes can be found at https://github.com/wjlim/correlation-between-rna-seq-and-RRBS.

Genomic DNA methylation is one of the most important epigenetic modifications in eukaryotes necessary for cellular differentiation and plays a crucial role in gene regulation of many oncogenes and tumor suppressor genes. Aberrant DNA methylation at the level of both individual gene promoters and on a genome-wide scale has been heavily implicated in cancer initiation and progression. Whole genome bisulfite sequencing (WGBS) is the gold standard method for studying alterations in DNA methylation at a single base pare resolution. However, the high cost of WGBS is still prohibitive for high-throughput observational studies on large sample cohorts. Moreover, the WGBS data analysis requires substantial IT resources and a complex bioinformatics analysis. Targeted bisulfite sequencing enables a cost-effective and focused analysis of genomic regions of interest where most of DNA methylation alterations occur, such as promoters, CpG islands and shores, and enhancer regions. Currently, there are 2 main approaches for targeted bisulfite sequencing of medium-to-large genomic regions, reduced representation bisulfite sequencing (RRBS) and hybridization-based enrichment. We have performed a comparison of 5 commercially available platforms for RRBS and in-solution hybridization-based enrichment panels. We analyzed a panel of two cancer cell lines (Hela and Coriell NA12878) and a normal blood reference gDNA sample, in duplicate on each platform using recommended sample inputs, and compared the results to WGBS data. Given that sample input requirements differ significantly between RRBS (∼100ng or less), and hybridization based methods (500ng - 1 μg), we have analyzed the normal blood reference gDNA sample at 500ng input in duplicate across all platforms. All samples were sequenced to a ∼30x depth on an Illumina HiSeq 2500 sequencer. We compared the genomic coverage, precision and off-target effects and overall concordance in methylome profiles for each method. In summary, here we provide a comprehensive comparison of the performance of 5 different commercially available methods for medium to large scale targeted bisulfite sequencing, facilitating information to researchers for selection of appropriate method depending on required application. Citation Format: Miljana Tanic, Simon Rodney, James Barrett, Hannah Parker, Andrew Feber, Stephan Beck. EpiCapture: Benchmarking commercially available targeted bisulfite-sequencing platforms to gold-standard whole genome bisulfite sequencing. [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 4530.

Integrated DNA methylation analysis of peripheral blood from asbestos exposed populations and patients with malignant mesothelioma reveals novel methylation driver genes of diagnostic and prognostic relevance

Disclosure: A. Hattori: None. A. Seki: None. Y. Naiki: None. A. Nakamura: None. T. Michigami: None. Y. Katoh-Fukui: None. T. Ogata: None. M. Kagami: None. M. Fukami: None.Introduction: Male-specific growth genes, alongside gonadal sex steroids, have been proposed as contributors to sex differences in height; however, no such gene has been identified. SHOX, a key growth gene located in the pseudoautosomal region on the short arm (PAR1) of the sex chromosomes, influences height in a dose-dependent manner. Conventionally, genes on PAR1, including SHOX, were thought to escape X-chromosome inactivation (XCI), thus being expressed equally in men and women. In 2017, Tukiainen et al. reported that most genes on PAR1 are expressed in a male-dominant manner. Nevertheless, sex differences in SHOX expression remain largely unexplored due to its highly tissue-specific expression. Objective: To explore the role of SHOX in sex differences in height. Methods: We compared male and female cartilage and/or chondrocyte samples using the following methods: (i) microarray analysis for X chromosome gene expression (n = 16), (ii) RT-qPCR for SHOX expression (n = 36), (iii) reduced representation bisulfite sequencing (RRBS) for X chromosome DNA methylation (n = 4), and (iv) pyrosequencing for DNA methylation of SHOX-flanking regions (n = 22). Furthermore, we established fibroblast clones derived from a boy and a girl with polydactyly, which enabled us to compare DNA methylation patterns around SHOX among the active and inactive X chromosomes and the Y chromosome using long-read sequencing. Results: Microarray analysis showed male-dominant expression of PAR1 genes, including SHOX. RT-qPCR confirmed male-dominant SHOX expression. RRBS revealed that the X chromosome exhibited female-dominant DNA methylation, which was pronounced in the X-specific region and mild in PAR1. RRBS also showed that SHOX-flanking regions exhibited sexually dimorphic DNA methylation. Specifically, male-dominant DNA methylation was observed in a region upstream of SHOX and a region spanning intron 5-exon 6a, whereas female-dominant DNA methylation was detected in intron 2. These findings were confirmed by pyrosequencing. In silico analysis suggested that the region with female-dominant DNA methylation in intron 2 functions as an enhancer of SHOX. Long-read sequencing of fibroblast clones revealed hypomethylation of intron 2 in the active X and Y chromosomes and hypermethylation in the inactive X chromosome. Discussion: Male-dominant SHOX expression in cartilage tissue indicates a role of SHOX in sex differences in height. Sexually dimorphic DNA methylation around SHOX implies that XCI represses SHOX expression in women. In particular, allele-specific DNA methylation in intron 2 (hypomethylation of the active X and hypermethylation of the inactive X) suggests that this region functions as an enhancer involved in the repression of SHOX expression due to XCI. Conclusion: Male-dominant SHOX expression, driven by the spreading of XCI to SHOX, likely contributes to sex differences in height.Presentation: Saturday, July 12, 2025

Proliferation-Dependent Alterations of the DNA Methylation Landscape Underlie Hematopoietic Stem Cell Aging

Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes. Emerging evidence suggests that aberrant DNA methylation may play a critical role in the pathogenesis of diabetic complications. Yet, its involvement in DPN is not fully characterized. In this study, we performed a large-scale genome-wide methylation profiling of sural nerve samples obtained from subjects with type 2 diabetes mellitus (T2DM) using Reduced Representation Bisulfite Sequencing (RRBS). A total of 78 samples were sequenced and mapped to the human reference genome. Differential analysis was performed on two groups, identified by our previous transcriptomic analysis of the same samples, which showed significantly different HbA1c levels: Group 1 (=8.5%±1.6%) and Group 2 (<7.0%±1.4%). Using MethylKit R package, with an adjusted p-value < 0.01 and 15% methylation change as significance cutoffs, we identified 2,066 differentially methylated CpG sites and 440 regions, corresponding to 1,519 and 429 unique differentially methylated genes (DMGs). These genes were highly enriched in biological processes related to nervous system development, neuron fate specification and neuron differentiation. KEGG pathway enrichment analysis demonstrated that genes involved in cancer, ECM-receptor interaction and axon guidance pathways were significantly differentially methylated between the two groups, highlighting the importance of DNA methylation in DPN pathogenesis. In summary, we demonstrate that type 2 diabetic patients with peripheral neuropathy and high HbA1c show distinct variations in sural nerve methylome, suggesting that DNA methylation and DPN are associated and that these associations are at least in part HbA1c-dependent. Our results provide new insights into the role of HbA1c in epigenetic variation, and identify candidate genes relevant to the pathogenesis of DPN in human sural nerves of subjects with T2DM. Disclosure K. Guo: None. S. Elzinga: None. S. Eid: None. C. Figueroa-Romero: None. B.A. McGregor: None. G. de Anda-Jáuregui: None. C. Pacut: None. E.L. Feldman: None. J. Hur: None.