Methylome Studies Research Articles

Perfluorooctanesulfonic acid (PFOS) induced cancer related DNA methylation alterations in human breast cells: A whole genome methylome study

DNA methylation plays a pivotal role in cancer. The ubiquitous contaminant perfluorooctanesulfonic acid (PFOS) has been epidemiologically associated with breast cancer, and can induce proliferation and malignant transformation of normal human breast epithelial cells (MCF-10A), but the information about its effect on DNA methylation is sparse. The aim of this study was to characterize the whole-genome methylome effects of PFOS in our breast cell model and compare the findings with previously demonstrated DNA methylation alterations in breast tumor tissues. The DNA methylation profile was assessed at single CpG resolution in MCF-10A cells treated with 1 μM PFOS for 72 h by using Enzymatic Methyl sequencing (EM-seq). We found 12,591 differentially methylated CpG-sites and 13,360 differentially methylated 100 bp tiles in the PFOS exposed breast cells. These differentially methylated regions (DMRs) overlapped with 2406 genes of which 494 were long non-coding RNA and 1841 protein coding genes. We identified 339 affected genes that have been shown to display altered DNA methylation in breast cancer tissue and several other genes related to cancer development. This includes hypermethylation of GACAT3, DELEC1, CASC2, LCIIAR, MUC16, SYNE1 and hypomethylation of TTN and KMT2C. DMRs were also found in estrogen receptor genes (ESR1, ESR2, ESRRG, ESRRB, GREB1) and estrogen responsive genes (GPER1, EEIG1, RERG). The gene ontology analysis revealed pathways related to cancer phenotypes such as cell adhesion and growth. These findings improve the understanding of PFOS's potential role in breast cancer and illustrate the value of whole-genome methylome analysis in uncovering mechanisms of chemical effects, identifying biomarker candidates, and strengthening epidemiological associations, potentially impacting risk assessment.

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.

Multi-omics portrait of ductal carcinoma in situ in young women.

Young patients with breast ductal carcinoma in situ (DCIS) often face a poorer prognosis. The genomic intricacies in young-onset DCIS, however, remain underexplored. To address this gap, we undertook a comprehensive study encompassing exome, transcriptome, and vmethylome analyses. Our investigation included 20 DCIS samples (including 15 young-onset DCIS) and paired samples of normal breast tissue and blood. Through RNA sequencing, we identified two distinct DCIS subgroups: "immune hot" and "immune cold". The "immune hot" subgroup was characterized by increased infiltration of lymphocytes and macrophages, elevated expression of PDCD1 and CTLA4, and reduced GATA3 expression. This group also exhibited active immunerelated transcriptional regulators. Mutational analysis revealed alterations in TP53 (38%), GATA3 (25%), and TTN (19%), with two cases showing mutations in APC, ERBB2, and SMARCC1. Common genomic alterations, irrespective of immune status, included gains in copy numbers at 1q, 8q, 17q, and 20q, and losses at 11q, 17p, and 22q. Signature analysis highlighted the predominance of signatures 2 and 1, with "immune cold" samples showing a significant presence of signature 8. Our methylome study on 13 DCIS samples identified 328 hyperdifferentially methylated regions (DMRs) and 521 hypo-DMRs, with "immune cold" cases generally showing lower levels of methylation. In summary, the molecular characteristics of young-onset DCIS share similarities with invasive breast cancer (IBC), potentially indicating a poor prognosis. Understanding these characteristics, especially the immune microenvironment of DCIS, could be pivotal in identifying new therapeutic targets and preventive strategies for breast cancer.

Abstract 3170: Epigenetic identification and functional characterization of a novel RING finger protein tumor suppressor in nasopharyngeal carcinoma and esophageal carcinoma

Abstract RING finger proteins play crucial roles in normal physiology and pathophysiology as important regulators of ubiquitin-dependent proteasomal protein degradation. Many RING finger proteins have also been implicated in tumorigenesis either as oncogenes or tumor suppressors. Through cancer CpG methylome studies, we identified RING finger protein 150 (RNF150) as a methylated target gene in nasopharyngeal carcinoma (NPC) and esophageal carcinoma (ESCC). RNF150 was found to be ubiquitously expressed in human normal tissues but frequently downregulated in NPC and ESCC cells due to its promoter methylation. Its downregulation is associated with poor survival in patients with head and neck cancer and esophageal cancer. We also found that RNF150 is a functional tumor suppressor by inhibiting tumor cell growth and inducing cell cycle arrest in NPC and ESCC cells. Furthermore, ectopic expression of RNF150 inhibited NPC and ESCC cell migration/invasion by suppressing epithelial-mesenchymal transition (EMT) through upregulation of epithelial marker E-cadherin and downregulation of mesenchymal markers (vimentin, N-cadherin, Snail, and Slug). RNF150 is located in the cytoplasm and represses several oncogenic signaling pathways including the Wnt/β-catenin and NF-kB signaling pathways. Liquid chromatography-mass spectrometry (LC-MS) was performed to elucidate RNF150 binding proteins. KEGG enrichment analysis showed that glycolysis/gluconeogenesis, pyruvate metabolism, central carbon metabolism in cancer, and AMPK signaling were the top 4 pathways with significant enrichment, suggesting potential targeting of metabolic molecules by RNF150. Thus, our results provide insights into a new ring finger protein member, RNF150, as a functional tumor suppressor for NPC and ESCC. Our study also suggests a potential epigenetic biomarker for the early dection of NPC and ESCC. Citation Format: Xiaoxue Chai, Feifei Luo, Shujie Bao, Liuzhen Meng, Anthony T. Chan, Brigette B. MA, Qian Tao, li LiLi. Epigenetic identification and functional characterization of a novel RING finger protein tumor suppressor in nasopharyngeal carcinoma and esophageal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3170.

Understanding the Genetic and Non-Genetic Interconnections in the Aetiology of Syndromic Congenital Heart Disease: An Updated Review: Part 2.

Approximately 30% of syndromic cases diagnosed with CHD, which lure us to further investigate the molecular and clinical challenges behind syndromic CHD (sCHD). The aetiology of sCHD in a majority of cases remains enigmatic due to involvement of multiple factors, namely genetic, epigenetic and environmental modifiable risk factors for the development of the disease. Here, we aim to update the role of genetic contributors including chromosomal abnormalities, copy number variations (CNVs) and single gene mutations in cardiac specific genes, maternal lifestyle conditions, environmental exposures and epigenetic modifiers in causing CHD in different genetic syndromes. The exact aetiology of sCHD is still unknown. With the advancement of next-generation technologies including WGS, WES, transcriptome, proteome and methylome study, numerous novel genes and pathways have been identified. Moreover, our recent knowledge regarding epigenetic and environmental regulation during cardiogenesis is still evolving and may solve some of the mystery behind complex sCHD. Here, we focus to understand how the complex combination of genetic, environmental and epigenetic factors interact to interfere with developmental pathways, culminating into cardiac and extracardiac defects in sCHD.

Dynamic roles of small RNAs and DNA methylation associated with heterosis in allotetraploid cotton (Gossypium hirsutum L.)

BackgroundHeterosis is a complex phenomenon wherein the hybrids outperform their parents. Understanding the underlying molecular mechanism by which hybridization leads to higher yields in allopolyploid cotton is critical for effective breeding programs. Here, we integrated DNA methylation, transcriptomes, and small RNA profiles to comprehend the genetic and molecular basis of heterosis in allopolyploid cotton at three developmental stages.ResultsTranscriptome analysis revealed that numerous DEGs responsive to phytohormones (auxin and salicylic acid) were drastically altered in F1 hybrid compared to the parental lines. DEGs involved in energy metabolism and plant growth were upregulated, whereas DEGs related to basal defense were downregulated. Differences in homoeologous gene expression in F1 hybrid were greatly reduced after hybridization, suggesting that higher levels of parental expression have a vital role in heterosis. Small RNAome and methylome studies showed that the degree of DNA methylation in hybrid is higher when compared to the parents. A substantial number of allele-specific expression genes were found to be strongly regulated by CG allele-specific methylation levels. The hybrid exhibited higher 24-nt-small RNA (siRNA) expression levels than the parents. The regions in the genome with increased levels of 24-nt-siRNA were chiefly related to genes and their flanking regulatory regions, demonstrating a possible effect of these molecules on gene expression. The transposable elements correlated with siRNA clusters in the F1 hybrid had higher methylation levels but lower expression levels, which suggest that these non-additively expressed siRNA clusters, reduced the activity of transposable elements through DNA methylation in the hybrid.ConclusionsThese multi-omics data provide insights into how changes in epigenetic mechanisms and gene expression patterns can lead to heterosis in allopolyploid cotton. This makes heterosis a viable tool in cotton breeding.

SAT426 Long-term Nanomolar Bisphenol A (BPA) Exposure of MCF7 Cells Induces DNA Methylation Changes

Abstract Disclosure: S.L. Dela Cruz: None. M.C. Velarde: None. P.D. Bagamasbad: None. Breast cancer (BCa) has the highest worldwide prevalence and mortality rate among cancers. For ER-positive luminal BCa, hormone deprivation therapy using drugs that inhibit the estrogen signaling axis is commonly employed. However, activation of ER may persist due to the presence of endocrine disrupting chemicals (EDCs) that are structurally similar to endogenous estrogens. Bisphenol A (BPA) is a known EDC found in consumer products and has been associated with increased BCa risk in various animal models. Mechanistically, BPA weakly binds and activates ER resulting in transcriptional changes. In addition, studies show that BPA potentially reprograms the epigenome through changes in DNA methylation. While methylome studies on BPA-exposed cancer models have been conducted, to the best of our knowledge, this has not been done on luminal BCa cells chronically exposed to environmentally relevant doses of BPA. The use of a chronic, nanomolar-dose treatment paradigm is significant as it more accurately reflects non-occupational environmental exposure. In this study, we investigated the epigenetic effects of chronic (8 weeks) nanomolar doses (10 nM) of BPA on the ER-positive BCa cell line MCF7. We probed single gene-level DNA methylation through Bisulfite Sequencing PCR (BSP) and genome-wide methylation through Reduced Representation Bisulfite PCR (RRBS). In silico analysis of publicly available data identified potential differentially methylated regions (DMRs) upon chronic BPA treatment. BSP showed minimal changes in methylation levels of CpGs within the target DMRs, while gene expression analysis via RT-qPCR did not find significant changes in mRNA levels of genes encompassing the target regions. Methylome-wide profiling of chronically treated cells identified DMRs that fall under gene promoters (DMPs) and gene coding regions (DMGs). Pathway analysis of DMPs indicated an association with death receptor signaling, with an overall predicted effect in inhibiting apoptosis. Network analysis revealed genes encompassing DMPs that interact with the BCa markers ESR1/2, BRCA1, and PGR. Based on the pathway and network analyses, we selected 8 genes for gene expression analysis and found that the expression of all 8 genes are unchanged following chronic BPA treatment. Taken together, our results show that chronic BPA exposure of MCF7 can induce subtle changes in DNA methylation. Although initial validation did not reveal changes in gene expression associated with differential methylation, we speculate that the incremental DNA methylation changes could accumulate over time resulting in further ER signaling dysregulation, exacerbating the BCa phenotype. Presentation: Saturday, June 17, 2023

Evaluation of circulating tumor DNA by electropherogram analysis and methylome profiling in high-risk neuroblastomas.

Liquid biopsy has emerged as a promising, non-invasive diagnostic approach in oncology because the analysis of circulating tumor DNA (ctDNA) reflects the precise status of the disease at diagnosis, progression, and response to treatment. DNA methylation profiling is also a potential solution for sensitive and specific detection of many cancers. The combination of both approaches, DNA methylation analysis from ctDNA, provides an extremely useful and minimally invasive tool with high relevance in patients with childhood cancer. Neuroblastoma is an extracranial solid tumor most common in children and responsible for up to 15% of cancer-related deaths. This high death rate has prompted the scientific community to search for new therapeutic targets. DNA methylation also offers a new source for identifying these molecules. However, the limited blood sample size which can be obtained from children with cancer and the fact that ctDNA content may occasionally be diluted by non-tumor cell-free DNA (cfDNA) complicate optimal quantities of material for high-throughput sequencing studies. In this article, we present an improved method for ctDNA methylome studies of blood-derived plasma from high-risk neuroblastoma patients. We assessed the electropherogram profiles of ctDNA-containing samples suitable for methylome studies, using 10 ng of plasma-derived ctDNA from 126 samples of 86 high-risk neuroblastoma patients, and evaluated several bioinformatic approaches to analyze DNA methylation sequencing data. We demonstrated that enzymatic methyl-sequencing (EM-seq) outperformed bisulfite conversion-based method, based on the lower proportion of PCR duplicates and the higher percentage of unique mapping reads, mean coverage, and genome coverage. The analysis of the electropherogram profiles revealed the presence of nucleosomal multimers, and occasionally high molecular weight DNA. We established that 10% content of the mono-nucleosomal peak is sufficient ctDNA for successful detection of copy number variations and methylation profiles. Quantification of mono-nucleosomal peak also showed that samples at diagnosis contained a higher amount of ctDNA than relapse samples. Our results refine the use of electropherogram profiles to optimize sample selection for subsequent high-throughput analysis and support the use of liquid biopsy followed by enzymatic conversion of unmethylated cysteines to assess the methylomes of neuroblastoma patients.

Biomarqueurs des carcinomes pulmonaires à petites cellules en 2022

AbstractConversely of non-small cell lung cancer, the small cell lung cancer (SCLC) made little profit since last twenty years from discoveries of molecules based notably of genomic alteration, in order to develop new therapeutic targets. Recent data, based on the expression of dominant transcriptional signatures, then on methylome studies, permitted classification of SCLCs into four biologically distinct subtypes, namely, SCLC-A, SCLC-N, SCLC-P, and SCLC-I. This molecular characterization led to new classification of these cancers. The different subtypes can be identified on variable immunohistochemical profiles thanks to the use of anti-ASCL1, NEUROD1, POU2F3 and YAP1 antibodies. These histo-molecular subtypes are associated with a predictive potential for emerging and promising therapies, including PARP inhibitors, immune checkpoint inhibitors, and DLL3 therapies. In this context, an immunohistochemistry analysis combining the four antibodies cited above, plus anti-DLL3 and anti-SLFN11 antibodies might be soon necessary in clinical practice. The new findings offer a glimmer of hope for patients and open room for increasing the quality of life as well as the over all survival of SCLC patients in the near future.

Haplotype-resolved DNA methylome of African cassava genome.

Haplotype-resolved DNA methylome of African cassava genome.

A Transcriptome and Methylome Study Comparing Tissues of Early and Late Onset Colorectal Carcinoma.

There is an increase in the incidence of early onset colorectal carcinoma (EOCRC). To better understand if there is any difference in molecular pathogenesis of EOCRC and late onset colorectal carcinoma (LOCRC), we compared the clinical, histological, transcriptome, and methylome profile of paired CRC and healthy colonic tissue from 67 EOCRC and 98 LOCRC patients. The frequency of stage 3 CRC, lymph node involvement, lymphovascular invasion, and perineural invasion was higher in the EOCRC group. Many of the cancer related pathways were differentially expressed in CRC tissue in both EOCRC and LOCRC patients. However, the magnitude of differential expression for some groups of genes, such as DNA damage repair genes and replication stress genes, were significantly less pronounced in the EOCRC group, suggesting less efficient DNA damage repair to be associated with EOCRC. A more marked methylation of "growth factor receptor" genes in LOCRC correlated with a more pronounced down-regulation of those genes in that group. From a therapeutic point of view, more over-expression of fatty acid synthase (FASN) among the LOCRC patients may suggest a better response of FASN targeted therapy in that group. The age of onset of CRC did not appear to modify the response of cis-platin or certain immune checkpoint inhibitors. We found some differences in the molecular pathogenesis in EOCRC and LOCRC that may have some biological and therapeutic significance.

DNA Methylome and Transcriptome Study of Triterpenoid CDDO in TPA-Mediated Skin Carcinogenesis Model.

Overexposure to ultraviolet radiation and environmental carcinogens drive skin cancer development through redox imbalance and gene mutation. Antioxidants such as triterpenoids have exhibited anti-oxidative and anti-inflammatory potentials to alleviate skin carcinogenesis. This study investigated the methylome and transcriptome altered by tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) or TPA with 2-cyano 2,3-dioxoolean-1,9-dien-28-oic acid (CDDO). The results show that CDDO blocks TPA-induced transformation dose dependently. Several differential expressed genes (DEGs) involved in skin cell transformation, while counteracted by CDDO, were revealed by differential expression analysis including Lyl1, Lad1, and Dennd2d. In CpG methylomic profiles, the differentially methylated regions (DMRs) in the promoter region altered by TPA while showing the opposite methylation status in the CDDO treatment group were identified. The correlation between DNA methylation and RNA expression has been established and DMRs showing inverse correlation were further studied as potential therapeutic targets. From the CpG methylome and transcriptome results, CDDO significantly restored gene expression of NAD(P)H:quinone oxidoreductase 1 (Nqo1) inhibited by TPA by decreasing their promoter CpG methylation. Ingenuity Pathways Analysis (IPA) shows that CDDO neutralized the effect of TPA through modulating cell cycles, cell migration, and inflammatory and immune response regulatory pathways. Notably, Tumor Necrosis Factor Receptor 2 (TNFR2) signaling was significantly downregulated by CDDO potentially contributing to prevention of TPA-induced cell transformation. Overall, incorporating the transcriptome, CpG methylome, and signaling pathway network, we reveal potential therapeutic targets and pathways by which CDDO could reverse TPA-induced carcinogenesis. The results could be useful for future human study and targets development for skin cancer.

At age 9, the methylome of assisted reproductive technology children that underwent embryo culture in different media is not significantly different on a genome-wide scale.

Can we detect DNA methylation differences between ART children that underwent embryo culture in different media? We identified no significant differences in site-specific or regional DNA methylation between the different culture medium groups. Embryo culture in G3 or K-SICM medium leads to differences in embryonic, neonatal and childhood outcomes, including growth and weight. The methylome may mediate this association as the period of in vitro culture of ART treatments coincides with epigenetic reprogramming. This study was conducted as a follow-up to a previous culture medium comparison study in which couples were pseudo-randomized to embryo culture in G3 or K-SICM medium. Of the resultant singletons, 120 (n = 65 G3, n = 55 K-SICM), were recruited at age 9. The ART children provided a saliva sample from which the methylome was analysed using the Infinium MethylationEPIC array. After quality and context filtering, 106 (n = 57 G3, n = 49 K-SICM) samples and 659 708 sites were retained for the analyses. Differential methylation analyses were conducted using mixed effects linear models corrected for age, sex, sample plate and cell composition. These were applied to all cytosine-guanine dinucleotide (CpG) sites, various genomic regions (genes, promoters, CpG Islands (CGIs)) and as a targeted analysis of imprinted genes and birth weight-associated CpG sites. Differential variance was assessed using the improved epigenetic variable outliers for risk prediction analysis (iEVORA) algorithm and methylation outliers were identified using a previously defined threshold (upper or lower quartile plus or minus three times the interquartile range, respectively). After correcting for multiple testing, we did not identify any significantly differentially methylated CpG sites, genes, promoters or CGIs between G3 and K-SICM children despite a lenient corrected P-value threshold of 0.1. Targeted analyses of (sites within) imprinted genes and birth weight-associated sites also did not identify any significant differences. The number of DNA methylation outliers per sample was comparable between the culture medium groups. iEVORA identified 101 differentially variable CpG sites of which 94 were more variable in the G3 group. Gene Expression Omnibus (GEO) GSE196432. To detect significant methylation differences with a magnitude of <10% between the groups many more participants would be necessary; however, the clinical relevance of such small differences is unclear. The results of this study are reassuring, suggesting that if there is an effect of the culture medium on DNA methylation (and methylation-mediated diseases risk), it does not differ between the two media investigated here. The findings concur with other methylome studies of ART neonates and children that underwent embryo culture in different media, which also found no significant methylome differences. Study funded by March of Dimes (6-FY13-153), EVA (Erfelijkheid Voortplanting & Aanleg) specialty programme (grant no. KP111513) of Maastricht University Medical Centre (MUMC+) and the Horizon 2020 innovation (ERIN) (grant no. EU952516) of the European Commission. The authors do not report any conflicts of interest relevant to this study. Dutch Trial register-NL4083.

Integrated methylome and phenome study of the circulating proteome reveals markers pertinent to brain health

Characterising associations between the methylome, proteome and phenome may provide insight into biological pathways governing brain health. Here, we report an integrated DNA methylation and phenotypic study of the circulating proteome in relation to brain health. Methylome-wide association studies of 4058 plasma proteins are performed (N = 774), identifying 2928 CpG-protein associations after adjustment for multiple testing. These are independent of known genetic protein quantitative trait loci (pQTLs) and common lifestyle effects. Phenome-wide association studies of each protein are then performed in relation to 15 neurological traits (N = 1,065), identifying 405 associations between the levels of 191 proteins and cognitive scores, brain imaging measures or APOE e4 status. We uncover 35 previously unreported DNA methylation signatures for 17 protein markers of brain health. The epigenetic and proteomic markers we identify are pertinent to understanding and stratifying brain health.

Chromosome-level assembly and annotation of the blue catfish Ictalurus furcatus, an aquaculture species for hybrid catfish reproduction, epigenetics, and heterosis studies.

BackgroundThe blue catfish is of great value in aquaculture and recreational fisheries. The F1 hybrids of female channel catfish (Ictalurus punctatus) × male blue catfish (Ictalurusfurcatus) have been the primary driver of US catfish production in recent years because of superior growth, survival, and carcass yield. The channel–blue hybrid also provides an excellent model to investigate molecular mechanisms of environment-dependent heterosis. However, transcriptome and methylome studies suffered from low alignment rates to the channel catfish genome due to divergence, and the genome resources for blue catfish are not publicly available.ResultsThe blue catfish genome assembly is 841.86 Mbp in length with excellent continuity (8.6 Mbp contig N50, 28.2 Mbp scaffold N50) and completeness (98.6% Eukaryota and 97.0% Actinopterygii BUSCO). A total of 30,971 protein-coding genes were predicted, of which 21,781 were supported by RNA sequencing evidence. Phylogenomic analyses revealed that it diverged from channel catfish approximately 9 million years ago with 15.7 million fixed nucleotide differences. The within-species single-nucleotide polymorphism (SNP) density is 0.32% between the most aquaculturally important blue catfish strains (D&B and Rio Grande). Gene family analysis discovered significant expansion of immune-related families in the blue catfish lineage, which may contribute to disease resistance in blue catfish.ConclusionsWe reported the first high-quality, chromosome-level assembly of the blue catfish genome, which provides the necessary genomic tool kit for transcriptome and methylome analysis, SNP discovery and marker-assisted selection, gene editing and genome engineering, and reproductive enhancement of the blue catfish and hybrid catfish.

Integrated Methylome and Transcriptome Analysis Provides Insights into the DNA Methylation Underlying the Mechanism of Cytoplasmic Male Sterility in Kenaf (Hibiscus cannabinus L.).

Cytoplasmic male sterility (CMS) is widely exploited in hybrid seed production. Kenaf is an important fiber crop with high heterosis. The molecular mechanism of kenaf CMS remains unclear, particularly in terms of DNA methylation. Here, using the anthers of a kenaf CMS line (P3A) and its maintainer line (P3B), comparative physiological, DNA methylation, and transcriptome analyses were performed. The results showed that P3A had considerably lower levels of IAA, ABA, photosynthetic products and ATP contents than P3B. DNA methylome analysis revealed 650 differentially methylated genes (DMGs) with 313 up- and 337 down methylated, and transcriptome analysis revealed 1788 differentially expressed genes (DEGs) with 558 up- and 1230 downregulated genes in P3A compared with P3B. Moreover, 45 genes were characterized as both DEGs and DMGs, including AUX, CYP, BGL3B, SUS6, AGL30 and MYB21. Many DEGs may be regulated by related DMGs based on methylome and transcriptome studies. These DEGs were involved in carbon metabolism, plant hormone signal transduction, the TCA cycle and the MAPK signaling pathway and were shown to be important for CMS in kenaf. These results provide new insights into the epigenetic mechanism of CMS in kenaf and other crops.

Benchmarking DNA methylation analysis of 14 alignment algorithms for whole genome bisulfite sequencing in mammals

Whole genome bisulfite sequencing (WGBS) is an essential technique for methylome studies. Although a series of tools have been developed to overcome the mapping challenges caused by bisulfite treatment, the latest available tools have not been evaluated on the performance of reads mapping as well as on biological insights in multiple mammals. Herein, based on the real and simulated WGBS data of 14.77 billion reads, we undertook 936 mappings to benchmark and evaluate 14 wildly utilized alignment algorithms from reads mapping to biological interpretation in humans, cattle and pigs: Bwa-meth, BSBolt, BSMAP, Walt, Abismal, Batmeth2, Hisat_3n, Hisat_3n_repeat, Bismark-bwt2-e2e, Bismark-his2, BSSeeker2-bwt, BSSeeker2-soap2, BSSeeker2-bwt2-e2e and BSSeeker2-bwt2-local. Specifically, Bwa-meth, BSBolt, BSMAP, Bismark-bwt2-e2e and Walt exhibited higher uniquely mapped reads, mapped precision, recall and F1 score than other nine alignment algorithms, and the influences of distinct alignment algorithms on the methylomes varied considerably at the numbers and methylation levels of CpG sites, the calling of differentially methylated CpGs (DMCs) and regions (DMRs). Moreover, we reported that BSMAP showed the highest accuracy at the detection of CpG coordinates and methylation levels, the calling of DMCs, DMRs, DMR-related genes and signaling pathways. These results suggested that careful selection of algorithms to profile the genome-wide DNA methylation is required, and our works provided investigators with useful information on the choice of alignment algorithms to effectively improve the DNA methylation detection accuracy in mammals.

Extraction and high-throughput sequencing of oak heartwood DNA: Assessing the feasibility of genome-wide DNA methylation profiling.

Tree ring features are affected by environmental factors and therefore are the basis for dendrochronological studies to reconstruct past environmental conditions. Oak wood often provides the data for these studies because of the durability of oak heartwood and hence the availability of samples spanning long time periods of the distant past. Wood formation is regulated in part by epigenetic mechanisms such as DNA methylation. Studies of the methylation state of DNA preserved in oak heartwood thus could identify epigenetic tree ring features informing on past environmental conditions. In this study, we aimed to establish protocols for the extraction of DNA, the high-throughput sequencing of whole-genome DNA libraries (WGS) and the profiling of DNA methylation by whole-genome bisulfite sequencing (WGBS) for oak (Quercus robur) heartwood drill cores taken from the trunks of living standing trees spanning the AD 1776-2014 time period. Heartwood contains little DNA, and large amounts of phenolic compounds known to hinder the preparation of high-throughput sequencing libraries. Whole-genome and DNA methylome library preparation and sequencing consistently failed for oak heartwood samples more than 100 and 50 years of age, respectively. DNA fragmentation increased with sample age and was exacerbated by the additional bisulfite treatment step during methylome library preparation. Relative coverage of the non-repetitive portion of the oak genome was sparse. These results suggest that quantitative methylome studies of oak hardwood will likely be limited to relatively recent samples and will require a high sequencing depth to achieve sufficient genome coverage.

Mapping epigenetic divergence in the massive radiation of Lake Malawi cichlid fishes

Epigenetic variation modulates gene expression and can be heritable. However, knowledge of the contribution of epigenetic divergence to adaptive diversification in nature remains limited. The massive evolutionary radiation of Lake Malawi cichlid fishes displaying extensive phenotypic diversity despite extremely low sequence divergence is an excellent system to study the epigenomic contribution to adaptation. Here, we present a comparative genome-wide methylome and transcriptome study, focussing on liver and muscle tissues in phenotypically divergent cichlid species. In both tissues we find substantial methylome divergence among species. Differentially methylated regions (DMR), enriched in evolutionary young transposons, are associated with transcription changes of ecologically-relevant genes related to energy expenditure and lipid metabolism, pointing to a link between dietary ecology and methylome divergence. Unexpectedly, half of all species-specific DMRs are shared across tissues and are enriched in developmental genes, likely reflecting distinct epigenetic developmental programmes. Our study reveals substantial methylome divergence in closely-related cichlid fishes and represents a resource to study the role of epigenetics in species diversification.

Severe Virilization in a Girl With the Homozygous G200SNicotinamide Nucleotide Transhydrogenase Mutation Is Surprisingly Caused by Rare Bilateral Para-Overian Adrenal Rest Tumors

Background: Patients with NNT (Nicotinamide Nucleotide Transhydrogenase) gene mutations, a rare cause of glucocorticoid and mineralocorticoid deficiency require hormone replacement therapy. Adrenal Rest Tumor (ART) in females is very rare (<20 cases world-wide) and was reported only in noncompliant patients with congenital adrenal hyperplasia having extremely elevated ACTH levels. This study characterizes the clinical characteristics, the pathophysiology and the molecular ontogeny of a unique ART in a female with adrenal failure due to the G200S mutation in NNT. Clinical presentation and Method: A 15-year-old girl, with homozygous G200S NNT-mutation that caused adrenal insufficiency reappeared to follow-up after several years with severe virilization and elevated serum testosterone (28.3 nmol/l) and ACTH (> 1500 pmol/l) levels. Pelvic MRI and Ultrasound demonstrated one sided para-ovarian round tumor with pathological vascularization. Laparoscopic exploration revealed bilateral para-ovarian mesosalpinx masses involving the serosa of the Fallopian tube (3 and 1 cm in diameter); the testosterone level normalized within one day after removal of those masses (0.2 nmol/l). Results: Histopathology demonstrated a pattern of adrenal rest tissue with strong intracellular positive staining for adrenal markers such as SF-1, calretinin, MART1, inhibin and the pituitary corticotroph marker-ACTH. The staining for ovarian characteristic markers such as PAX 8 was negative. Studying mRNA extracted from the tissue by RT-PCR revealed the presence of CYP17A1, CYP21A2 and MC2R (ACTH receptor) cDNA confirming typical adrenocortical transcriptional pattern in the tissue. cDNA of POMC was not detected suggesting that in spite of dense ACTH staining the tissue is not classically originated from pituitary corticotrophs. Methylome studies to further characterise the tissue are underway. Conclusion: This study exemplifies severe virilization that resulted from a unique and rare type of ART in ovarian related tissue that was caused by incompliance to treatment in a patient with NNT gene mutation. Given the ubiquitous expression of NNT and its reported pathophysiology as free radicals scavenger in all adrenocortical layers, it is surprising to have high ACTH induced severe virilization in spite of severe NNT dysfunction and adrenal insufficiency. This study may indicate timely testosterone screening in females with NNT mutation and when increased they should probably be laparoscopically surveyed for ART even when not detected by imaging. How NNT mutation damages mineralo and glucocorticoid secreting cells while androgen secreting cells are rescued is a theme for further studies.