Distinct H3K9me3 and DNA methylation modifications during mouse spermatogenesis

Ectopic NSD2 Remodels H3K36me2 and DNA Methylation to Promote Oncogenic Gene Expression in Multiple Myeloma

Pericentromeric DNA, consisting of high-copy-number tandem repeats and transposable elements, is normally silenced through DNA methylation and histone modifications to maintain chromosomal integrity and stability. Although histone deacetylase 6 (HDA6) has been known to participate in pericentromeric silencing, the mechanism is still yet unclear. Here, using whole genome bisulfite sequencing (WGBS) and chromatin immunoprecipitation-sequencing (ChIP-Seq), we mapped the genome-wide patterns of differential DNA methylation and histone H3 lysine 18 acetylation (H3K18ac) in wild-type and hda6 mutant strains. Results show pericentromeric CHG hypomethylation in hda6 mutants was mediated by DNA demethylases, not by DNA methyltransferases as previously thought. DNA demethylases can recognize H3K18ac mark and then be recruited to the chromatin. Using biochemical assays, we found that HDA6 could function as an ‘eraser’ enzyme for H3K18ac mark to prevent DNA demethylation. Oxford Nanopore Technology Direct RNA Sequencing (ONT DRS) also revealed that hda6 mutants with H3K18ac accumulation and CHG hypomethylation were shown to have transcriptionally active pericentromeric DNA.

PurposeTransposable elements (TEs) or “jumping genes” are genetic sequences that make up ~50% of the mammalian genome. The TE LINE‐1 (L1) has the ability to move within the genome via a “copy and paste” mechanism termed retrotransposition, which ultimately results in amplification of L1. Retrotransposition has shown to be advantageous in certain context, namely by introducing regulatory elements as well as adding genetic diversity both between and within species. These occurrences, although evolutionarily advantageous, have the ability to cause damage through increased mutagenesis. Indeed, L1 insertions have been the subject of intense investigation with respect to cancer causing mutations, genome instability, and specific cases of Duchenne's muscular dystrophy. Recent research has revealed that L1 gene expression and DNA content is increased in aged tissue and is attenuated with caloric restriction. These findings have been attributed to epigenetic modifications at L1 repeats such as DNA methylation and histone modifications. The above observations suggest that L1 gene regulation is affected by environmental factors. With the current study, we sought to investigate if long‐term voluntary wheel running alters skeletal muscle L1 activity.MethodsRats selectively bred for high voluntary wheel running (EX) and sedentary age‐ and strain‐matched controls (SED) were compared for L1 mRNA expression, L1 DNA content, L1 5′‐UTR DNA methylation, heterochromatic L1 content via nuclease digestion, and gene expression of endogenous inhibitors of L1 activity. EX animals were housed with running wheels for 22 weeks, and the gastrocnemius was harvested from both groups at 27 weeks of age. Due to the rich evolutionary history of L1, multiple subfamilies exist within the L1 gene family. For this reason, a qPCR primer set was designed for the most active form of L1 along with primers that encompass non‐functional L1 elements, termed L1.3 and L1.tot, respectively.ResultsNeither L1.3 (p=0.52) or L1.tot (p=0.51) gDNA was different between groups. Both L1.3 (p=0.01) and L1.tot (p<0.01) mRNA expression showed a significant decrease in EX rats compared to SED rats. Methylated DNA at the L1 promoter was significantly higher in EX rats for both L1.3 (p=0.02) and L1.tot (p<0.01) when compared to SED rats. Gene expression for purported L1 inhibitors was not different between groups for TREX1 (p=0.55) or PIWIL2 (p=0.88). L1.3 within heterochromatin was not different in EX versus SED rats (p=0.22), while L1.tot was significantly greater in EX versus SED rats (p=0.03). Interestingly, there was an inverse correlation with citrate synthase activity and L1 gene expression for the active L1.3 primer set (r = −0.52, p=0.01).ConclusionsThe data herein provide evidence that skeletal muscle L1 activity can be regulated by voluntary wheel running in rodents. Given that there are now multiple studies suggesting exercise affects skeletal muscle L1 activity, one can speculate that exercise may serve a larger role than expected in genome regulation.Support or Funding InformationM.A.R. is funded by the William Townsend Porter Predoctoral Fellowship from the American Physiological Society.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ewing sarcoma is an excellent model for studying the role of epigenetic deregulation and tumor heterogeneity, given its low mutation rates and the well-defined oncogenic driver. We have recently shown that the fusion oncogene EWS-FLI1 induces widespread epigenetic rewiring in proximal and distal enhancers (Tomazou et al. Cell Reports 2015). In the current study, we validate the clinical relevance of our results in a large cohort of primary tumors, and we explore the prevalence, characteristics, and clinical impact of epigenetic tumor heterogeneity in Ewing sarcoma. We used reduced representation bisulfite sequencing (RRBS) to generate genome-wide profiles of DNA methylation in 141 Ewing sarcoma primary tumors, 17 Ewing sarcoma cell lines, and 32 primary mesenchymal stem cell (MSC) samples. Deep sequencing resulted in DNA methylation measurements for an average of 3.5 million unique CpGs per sample with excellent data quality (>98% bisulfite conversion efficiency). In addition, for three primary tumors we generated comprehensive reference epigenome maps using whole genome bisulfite sequencing (WGBS) and ChIP-seq for seven histone marks (H3K4me3, H3K4me1, H3K27me3, H3K27ac, H3K56ac, H3K36me3, and H3K9me3). We show that DNA methylation data can be used to infer enhancer activity differences among tumors, allowing us to exploit our large primary tumor dataset to systematically compare the regulation of EWS-FLI1 correlated and anticorrelated enhancers. We also identified Ewing-specific DNA methylation patterns. For example, Ewing sarcoma samples consistently show higher DNA methylation than MSCs at AP-1 binding sites, but lower DNA methylation at EWS-FLI1 binding sites. To explore epigenetic heterogeneity within individual tumors, we developed a bioinformatic algorithm that quantifies DNA methylation disorder. Using individual reads containing multiple DNA methylation measurements from single cells, we assign scores at single-nucleotide resolution. This method uses a probabilistic model to account for overall methylation rate and expected disorder levels. By evaluating the likelihood of the data in a model that assumes that the DNA methylation status of a CpG is independent of the methylation status of a nearby CpG, we identify extremely heterogeneous as well as highly epigenetically conserved genomic elements. These different region types show distinct patterns of enrichment for regulatory modes and transcription factor binding. We also compared the observed DNA methylation disorder in 141 Ewing tumors to those observed in 17 Ewing sarcoma cell lines, 32 primary MSC samples, and several hundred additional tumor and normal samples that are unrelated to Ewing sarcoma. This analysis identified Ewing-specific patterns of DNA methylation heterogeneity and stratifies patients based on epigenetic heterogeneity. Our dataset constitutes the largest available resource of genome-scale DNA methylation maps in a solid pediatric tumor. It strongly confirms the relevance of enhancer reprogramming and tumor heterogeneity in Ewing sarcoma, and it constitutes a starting point to develop DNA methylation biomarkers for prognosis and patient stratification. This study is supported by the Austrian National Bank (OeNB project #15714) and the Kapsch group (https://www.kapsch.net/). This abstract is also presented as Poster A24. Citation Format: Nathan C. Sheffield, Franck Tirode, Sandrine Grossetete-Lalami, Paul Datlinger, Andreas Schönegger, Johanna Hadler, Diana Walder, Ingeborg M. Ambros, Ana Teresa Amaral, Enrique de Álava, Katharina Schallmoser, Dirk Strunk, Beate Rinner, Bernadette Liegl-Atzwanger, Berthold Huppertz, Andreas Leithner, Uta Dirksen, Peter Ambros, Olivier Delattre, Heinrich Kovar, Christoph Bock, Eleni M. Tomazou. DNA methylation mapping and computational modeling in a large Ewing sarcoma cohort identifies principles of tumor heterogeneity and their impact on clinical phenotypes. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr PR13.

The influence of increasing concentrations of heavy metals solutions (Pb(NO3)2, CuSO4, ZnSO4) on the modification of content of proline in the plants Cannabis sativa L was studied. It is supposed that accumulation of amino acid in the plants has an adaptive character concerning stress factors. The dependence of modification of the level of free proline from concentrations of salt in the medium of cultivation was exposed.

The identification of early markers of dementia is important for higher-risk populations such as those with type 2 diabetes (T2D). Retrotransposons, including long interspersed nuclear element 1 (LINE-1) and Alu, comprise ~40% of the human genome. Although dysregulation of these retrotransposons can induce aberrant gene regulation and genomic instability, their role in the development of pre-symptomatic dementia (PSD) among T2D patients is unknown. Here, we examined locus-specific changes in LINE-1 and Alu methylation in PSD and the potential to offset these changes via supplementation with folate and vitamin B12. We interrogated DNA methylation patterns corresponding to 22,352 probes for LINE-1 and Alu elements using publicly-available Illumina Infinium 450K methylation datasets from i) an 18-month prospective study in 28 T2D patients (GSE62003) and ii) an intervention study in which 44 individuals were supplemented with folic acid (400 μg/day) and vitamin B12 (500 μg/day) over two years (GSE74548). We identified 714 differentially methylated positions (DMP) mapping to retrotransposons in T2D patients who developed PSD in comparison to those who did not (PFDR < 0.05), comprised of 2.4% (228 probes) of all LINE-1 probes and 3.8% (486 probes) of all Alu probes. These loci were enriched in genes with functions related to Alzheimer's disease and cognitive decline, including GNB5, GNG7 and PKN3 (p < 0.05). In older individuals supplemented with folate/vitamin B12, 85 (11.9%) PSD retrotransposon loci showed significant changes in methylation (p < 0.05): participants with the MTHFR CC genotype predominantly showed hypermethylation at these loci, while hypomethylation was observed more frequently in those with the TT genotype. In T2D patients, LINE-1 and Alu elements are differentially methylated in PSD in a locus-specific manner and may offer clinical utility in monitoring risk of dementia. Further work is required to examine the potential for dietary supplementation in lowering the risk of PSD.

Methylation Levels of LINE-1 As a Useful Marker for Venous Invasion in Both FFPE and Frozen Tumor Tissues of Gastric Cancer.

Few studies have reported the existence of imprinted genes in cattle compared to the human and mouse. Genomic imprinting is expressed in monoallelic form and it depends on a single parent-specific form of the allele. Comparative analysis of mammals other than the human is a valuable tool for explaining the genomic basis of imprinted genes. In this study, we investigated 34 common imprinted genes in the human and mouse as well as 35 known non-imprinted genes in the human. We found short interspersed nuclear elements (SINEs), long interspersed nuclear elements (LINEs), and long terminal repeats (LTRs) in imprinted (human and mouse) and control (cattle) genes. Pair-wise comparisons for the three species were conducted using SINEs, LINEs, and LTRs. We also calculated 95% confidence intervals of frequencies of repetitive sequences for the three species. As a result, most genes had a similar interval between species. We found 11 genes with conserved SINEs, LINEs, and LTRs in the human, mouse, and cattle. In conclusion, eleven genes (CALCR, Grb10, HTR2A, KCNK9, Kcnq1, MEST, OSBPL5, PPP1R9A, Sgce, SLC22A18, and UBE3A) were identified as candidate imprinted genes in cattle.

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

Positive association of low-level environmental phthalate exposure with sperm motility was mediated by DNA methylation: A pilot study

Any treatment that affects seed germination and seedling development is of paramount importance from an agricultural point of view since they are critical prerequisites for successful crop production. In present study, we have examined the after-effect of ultrasonication (at 30 kHz, 70 W for 5min) of winter wheat (Triticum aestivum L. cv. SE15) seeds on the early seedling growth and development, and accompanying changes in the DNA methylation and transcriptomic pattern in 7-day-old seedlings. We used mRNA-sequencing and whole genome bisulfite sequencing (WGBS) to identify significantly differentially expressed genes (DEGs), significantly differently methylated regions (DMRs) and genes (DMGs). Ultrasonication of seeds did not alter the germination rate but increased both the length and weight of roots and shoots of 7-day-old seedlings significantly by 23%-68% and 16%-28%, respectively. Analyzing the expression intensity of 107,891 genes, significantly differentially expressed sequences related mainly to starch biosynthesis, IAA biosynthesis, photosynthesis and TCA cycle pathways. The same pathways were also affected by DNA-methylation changes. DNA hypomethylation occurred in the global methylation profile after ultrasound treatment altering the accessibility of some genes for transcription. Transcriptomic changes suggested alterations in the crosstalk between IAA and sucrose signaling, enhancement of growth processes, and increased activity of nuclear transcription factor stimulating the transcription of genes having CCAAT motif in the promoter. In the present first whole genome level study, we have identified seed ultrasonication as a priming technique that can act as a hypomethylating agent and thereby is able to modify the mRNA transcription allowing enhanced seedling growth.

Effects of DNA Methylation on Progression to Interstitial Fibrosis and Tubular Atrophy in Renal Allograft Biopsies: A Multi-Omics Approach.

DNA methylation is an essential epigenetic modification involved in numerous biological processes. Here, we present a cell-based system pLTR-Luc2P-EGFP for evaluation of DNA methylation in mammalian cells. In this system, the expression of reporter gene luciferase2P (Luc2P)-EGFP is under the control of HIV-1 promoter 5' long terminal repeat (LTR), which contains multiple CpG sites. Once these sites are methylated, the expression of Luc2P-EGFP is turned off, which may be visualized under fluorescence microscopy, with quantification performed in luciferase activity assay. As a proof of principle, pLTR-Luc2P-EGFP was methylated in vitro, and transfected into 293T cells, where the reduction of Luc2P-EGFP expression was confirmed. Premixed reporter DNA samples with the methylation levels varying from 0 to 100% were used for quantitative measurements of DNA methylation. The resulting standard curves indicated the accuracy of luciferase activity exceeding that of the Western blotting against EGFP. The Bland–Altman analysis showed that data from luciferase activity assay were in good agreement with the actual DNA methylation levels. In summary, we have established a reporter system coupled with reliable detection technique capable of efficient quantifying the changes in methylation in mammalian cells. This system may be utilized as a high throughput screening tool for identifying molecules that modulate DNA methylation.

Low concentrations of folate, other B vitamins, and methionine are associated with colorectal cancer risk, possibly by changing DNA methylation patterns. Here, we examine whether plasma concentrations of B vitamins and methionine are associated with methylation of long interspersed nuclear element-1 (LINE-1) among those at high risk of colorectal cancer, i.e. patients with at least one histologically confirmed colorectal adenoma (CRA) in their life. We used LINE-1 bisulfite pyrosequencing to measure global DNA methylation levels in leukocytes of 281 CRA patients. Multivariable linear regression was used to assess associations between plasma B vitamin concentrations and LINE-1 methylation levels. Plasma folate was inversely associated with LINE-1 methylation in CRA patients, while plasma methionine was positively associated with LINE-1 methylation. This study does not provide evidence that in CRA patients, plasma folate concentrations are positively related to LINE-1 methylation in leukocytes but does suggest a direct association between plasma methionine and LINE-1 methylation in leukocytes.

Multiple myeloma is a malignancy of terminally differentiated, antibody secreting B cells known as plasma cells. Normal plasma cell differentiation and cell fate are coupled to epigenetic and transcriptional reprogramming, including a proliferation-dependent global loss of DNA methylation. However, relatively little is known about the epigenetic changes that underlie myelomagenesis and how these contribute to disease etiology. To this end, we have analyzed the DNA methylome of 119 myeloma specimens from the CoMMpass study (NCT01454297) by whole genome bisulfite sequencing (WGBS) and more than 90% of these same specimens were also characterized for structural variants (long-insert whole genome sequencing) and gene expression (RNA-seq). Unsupervised hierarchical clustering grouped together specimens with t(4;14) translocations that upregulate the H3K36 dimethyltransferase NSD2 (also known as MMSET and WHSC1), which likely impacts the DNA methylation state through epigenetic cross-talk. These data also revealed a dramatic genome-wide hypomethylation where myeloma samples had a median global CpG methylation level of 41% as compared to 71% and 89% in normal plasma cells and B cells, respectively. Demethylation of the myeloma methylome occurred in large megabase domains encompassing genes that were devoid of gene expression. Conversely, DNA methylation remained mostly unchanged in the bodies of genes that were highly expressed. Although the majority of these hypomethylated domains were common across myelomas, many regions of variable methylation exist and these differences corresponded with proximal gene expression differences. These variably methylated regions were compared to PFS and OS and this identified 6,314 CpG loci where the level of DNA methylation was prognostic of outcome (P≤0.00001). These loci were clustered into discrete regions and in the majority of cases (79%), reduced DNA methylation at these loci corresponded with poor outcome. For example, several loci in the gene bodies of PRKCE, MGMT, FHIT, WWOX were prognostic of poor survival. Interestingly, myeloma t(14;16) translocations disrupt the tumor suppressor WWOX and induce the oncogene MAF. These data suggest that WWOX expression may also be lost by epigenetic mechanisms. Finally, we analyzed the DNA methylome of primary and relapsed samples for 22 patients, including multiple relapsed samples for 2 patients. These data identified genome-wide DNA methylation remodeling with a median of 1.9 million differential methylated CpGs between the newly diagnosed and relapsed specimens. These relapse differentially methylated loci coincided at the same regions in several patients and significantly overlapped loci where the DNA methylation level was prognostic of outcome. Integrative genetic, epigenetic, and transcriptional analyses for 120 myeloma samples will be presented. Citation Format: Benjamin G. Barwick, Doris R. Powell, Daniel Penaherrera, Sheri Skerget, Jonathan J. Keats, Daniel Auclair, Sagar Lonial, Lawrence H. Boise, Paula M. Vertino. Whole genome DNA methylation analysis of multiple myeloma identifies pervasive hypomethylation and biomarkers of survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 839.