scholarly journals Whole-genome methylation analysis of testicular germ cells from cryptozoospermic men points to recurrent and functionally relevant DNA methylation changes

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Sara Di Persio ◽  
Elsa Leitão ◽  
Marius Wöste ◽  
Tobias Tekath ◽  
Jann-Frederik Cremers ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Germ Cells ◽  
Regulatory Elements ◽  
Obstructive Azoospermia ◽  
Whole Genome ◽  
Genome Methylation ◽  
Genome Level ◽  
Aberrant Dna Methylation ◽  
Almost All ◽  
Distal Regulatory Elements

Abstract Background Several studies have reported an association between male infertility and aberrant sperm DNA methylation patterns, in particular in imprinted genes. In a recent investigation based on whole methylome and deep bisulfite sequencing, we have not found any evidence for such an association, but have demonstrated that somatic DNA contamination and genetic variation confound methylation studies in sperm of severely oligozoospermic men. To find out whether testicular germ cells (TGCs) of such patients might carry aberrant DNA methylation, we compared the TGC methylomes of four men with cryptozoospermia (CZ) and four men with obstructive azoospermia, who had normal spermatogenesis and served as controls (CTR). Results There was no difference in DNA methylation at the whole genome level or at imprinted regions between CZ and CTR samples. However, using stringent filters to identify group-specific methylation differences, we detected 271 differentially methylated regions (DMRs), 238 of which were hypermethylated in CZ (binominal test, p < 2.2 × 10–16). The DMRs were enriched for distal regulatory elements (p = 1.0 × 10–6) and associated with 132 genes, 61 of which are differentially expressed at various stages of spermatogenesis. Almost all of the 67 DMRs associated with the 61 genes (94%) are hypermethylated in CZ (63/67, p = 1.107 × 10–14). As judged by single-cell RNA sequencing, 13 DMR-associated genes, which are mainly expressed during meiosis and spermiogenesis, show a significantly different pattern of expression in CZ patients. In four of these genes, the promoter is hypermethylated in CZ men, which correlates with a lower expression level in these patients. In the other nine genes, eight of which downregulated in CZ, germ cell-specific enhancers may be affected. Conclusions We found that impaired spermatogenesis is associated with DNA methylation changes in testicular germ cells at functionally relevant regions of the genome. We hypothesize that the described DNA methylation changes may reflect or contribute to premature abortion of spermatogenesis and therefore not appear in the mature, motile sperm.

P–529 Whole-genome methylation analysis of testicular germ cells from cryptozoospermic men points to recurrent and functionally relevant DNA methylation changes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
S D Persio ◽  
E Leitão ◽  
M Wöste ◽  
T Tekath ◽  
J F Cremers ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Germ Cell ◽  
Germ Cells ◽  
Small Sample ◽  
Global Dna Methylation ◽  
Obstructive Azoospermia ◽  
Whole Genome ◽  
Ploidy Analysis ◽  
Normal Spermatogenesis

Abstract Study question Do DNA methylation changes occur in testicular germ cells (TGCs) from patients with impaired spermatogenesis? Summary answer TGCs from men with cryptozoospermia exhibit altered DNA methylation levels at several genomic regions, many of which are associated with genes involved in spermatogenesis. What is known already In the last 15 years, several studies have described DNA methylation changes in sperm of infertile men. More recently, using whole genome bisulfite sequencing (WGBS) we were able to refute these findings by demonstrating that somatic DNA contamination and genetic variation confound methylation studies in swim-up purified sperm of severely oligozoospermic men. However, it remains unknown whether altered DNA methylation plays a role during the development of the germ cells in the testes of these patients. Study design, size, duration For identifying DNA methylation differences associated with impaired spermatogenesis, we compared the TGC methylomes of men with cryptozoospermia (CZ) and men with obstructive azoospermia (n = 4 each), who had normal spermatogenesis and served as controls (CTR). Study participants were selected among an age-matched cohort of 24 CTR and 10 CZ. The selection was based on similar composition of the TGC suspension evaluated by ploidy analysis and absence of somatic DNA. Participants/materials, setting, methods TGCs were isolated from biopsies after short-term cell culture. Presence of somatic DNA was evaluated by analyzing the DNA methylation levels of H19, MEST, DDX4 and XIST. WGBS was performed at ∼14× coverage. Bioinformatic tools were used to compare global DNA methylation levels, identify differentially methylated regions (DMRs) and functionally annotate the DMRs. Single-cell RNA sequencing (scRNA-seq) was used to associate the DNA methylation changes to gene expression. Main results and the role of chance We could not identify any difference in the global DNA methylation level or at imprinted regions between CZ and CTR samples. However, using stringent filters to identify group-specific methylation differences, we detected 271 DMRs, 238 of which were hypermethylated in CZ (binominal test, p &lt; 2.2 × 10–16). The DMRs are associated with 132 genes, 61 of which are known to be differentially expressed at various stages of spermatogenesis according to scRNA-seq studies. Almost all of the DMRs associated with the 61 genes are hypermethylated in CZ (63/67, p = 1.107 × 10–14). As assessed by scRNA-seq, 13 DMR-associated genes, which were mainly expressed during meiosis and spermiogenesis, show a significantly different pattern of expression in CZ patients. In four of these genes, the promoter was hypermethylated in CZ men, which correlates with a lower expression level in these patients. In the other nine genes, most of which downregulated in CZ, germ cell-specific enhancers may be affected. Limitations, reasons for caution The small sample size constitutes a limitation of this study. Furthermore, even though the cellular composition of samples was similar by ploidy analysis, we cannot rule out that the observed DNA methylation changes might be due to differences in the relative proportion of different germ cell types. Wider implications of the findings: Impaired spermatogenesis is associated with DNA methylation changes in testicular germ cells at functionally relevant regions of the genome, which points to an important role of DNA methylation in normal spermatogenesis. The DNA methylation changes may contribute to premature abortion of spermatogenesis and therefore not appear in mature sperm. Trial registration number N/A

DNA Methylation Profiling Predicts Clinical Outcomes and Reveals Unique Insights Into the Molecular Complexity of Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 707-707
Author(s):  
Maria E Figueroa ◽  
Sanne Lugthart ◽  
Yushan Li ◽  
Claudia Erpelinck-Verschueren ◽  
Xutao Deng ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bone Marrow ◽  
Risk Factor ◽  
Clinical Outcomes ◽  
Independent Risk Factor ◽  
Regulatory Elements ◽  
Hazard Ratio ◽  
Gene Methylation ◽  
Significant Difference ◽  
Aberrant Dna Methylation

Abstract Abstract 707 Epigenetic deregulation of genes through aberrant DNA methylation has been widely reported in cancer. We hypothesized that in AML this aberrant DNA methylation does not occur randomly, but rather occurs in specific and distinct patterns. Therefore, large-scale genome-wide analysis of the DNA methylome could help explain and define the complexity underlying leukemia biology and reveal the existence of epigenetically defined variants of AML. Using the HELP microarray assay, which measures DNA methylation at 50,000 CpG sites annotated to ∼14,000 promoters, we obtained DNA methylation profiles for 344 AML patients seen at Erasmus University Medical Center. Median follow-up based on survivors was 18.2 months (7-215); median age: 48 years (15-77). Unsupervised analysis (hierarchical clustering, correlation distance with Ward's clustering method) demonstrated that based on their methylation profiles AML patients distributed into 16 cohorts. 11 of these groups were also defined by the presence of specific molecular lesions: inv(16) [cluster 1], t(8;21) [cluster 3], t(15;17) [cluster 6], CEBPA-mutant [clusters 4 and 9], CEBPA-silenced [cluster 10] NPM1-mutant [clusters 12, 13, 14 and 16] and 11q23 abnormalities [cluster 11]. Enrichment for cases harboring a specific molecular lesion within a given cluster was determined using Fisher's exact test (p<0.01). Additionally, 5 new AML subtypes were defined based on epigenetic profiling alone and had no other clinical or molecular feature in common. Kaplan-Meier survival analysis revealed a significant difference in overall survival (OS) between these novel AML subtypes: 2-year OS±SE; 58.8%±8.4% and 45.2%±8.9% for clusters 5 and 7, respectively, vs. 23.6%±5.7%, 26.4%±9.2% and 33.3%±13.6%, for clusters 2, 8 and 15, respectively (log rank test, p=0.04). After adjustment for age, cytogenetic risk, NPM1 and FLT3-ITD status in a multivariate Cox proportional hazards regression model including all the clusters with ≥ 10 patients, 4 of these 5 novel clusters presented a statistically significant increased hazard ratio compared to the favorable risk inv(16) cluster. In contrast, the clinical outcomes of patients in cluster 5 were not significantly different from favorable risk patients with inv(16). In order to identify the genes affected by aberrant DNA methylation for each cluster, we performed a supervised analysis comparing each of the 16 clusters to normal CD34+ bone marrow progenitors (n=8) using ANOVA followed by Dunnet post hoc test, and selected genes with adjusted p values <0.05 and a methylation change >30%. The DNA methylation signatures of each cluster featured involvement of distinct gene networks and DNA regulatory elements, and displayed distinct degrees of hyper or hypomethylation with respect to normal CD34+ bone marrow cells. Of note, in spite of the variation in methylation across the 16 clusters, we identified a set of 45 genes that were almost universally aberrantly methylated (in >70% cases and present in at least 10/16 cluster signatures). This common epigenetic signature included the tumor suppressor PDZD2, the nuclear import proteins IPO8 and TNPO3, PIAS2, a regulator of MAP kinase signaling, CDK8, and CSDA, a regulator of CSF2. Gene expression profiling of the same patients indicated that at least 50% of these genes were also aberrantly silenced compared to normal CD34+ cells. Finally, we randomly divided the 344-patient cohort into a training group of 200 patients, a test group (n=95) and an independent validation group (n=49), and using the Supervised Principal Components algorithm identified a 15-gene methylation classifier that was predictive of OS (p<0.009) and event free survival (p<0.013). Furthermore, after adjustment for age, cytogenetic risk, NPM1, FLT3 and CEBPA status in a multivariate analysis, this classifier remained an independent risk factor for OS (Hazard ratio 1.29, 95% CI: 1.11-1.49; p<0.001). In summary, we have i) demonstrated that unique and distinct DNA methylation patterns characterize distinct forms of AML; ii) identified novel, epigenetically defined subgroups of AML with distinct clinical behavior; iii) revealed the presence of a consistently aberrantly methylated signature across AML subtypes, with confirmed silencing of the genes involved; and iv) report a 15-gene methylation classifier predictive of OS, and confirmed as an independent risk factor when adjusted for known AML covariates. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Aberrant DNA methylation at imprinted genes in testicular sperm retrieved from men with obstructive azoospermia and undergoing vasectomy reversal

Reproduction ◽  
2011 ◽  
Vol 141 (6) ◽  
pp. 749-757 ◽  
Author(s):  
Agata Minor ◽  
Victor Chow ◽  
Sai Ma
Keyword(s):  
Dna Methylation ◽  
Single Cells ◽  
Imprinted Genes ◽  
Obstructive Azoospermia ◽  
Testicular Sperm ◽  
Male Factor ◽  
Bisulphite Sequencing ◽  
The Status ◽  
Vasectomy Reversal ◽  
Aberrant Dna Methylation

Male factor infertility has been associated with abnormal DNA methylation at imprinted genes. Little information is available on the status of imprinting in the sperm of men with azoospermia, including the association between aberrant imprinting and obstructive azoospermia (OA) or non-OA (NOA). Analysis of DNA methylation at imprinted genes in the sperm of men undergoing vasectomy reversal would aid determination of whether aberrant imprinting is associated with obstruction. Testicular sperm was retrieved from testicular biopsies obtained from men with azoospermia (N=18), including OA (N=10), NOA (N=5), and unknown pathology (N=3), and from men undergoing vasectomy reversal (N=17). Sperm was also obtained from proven fertile men (N=9). DNA methylation was investigated at multiple CpG sites within the differentially methylated regions (DMRs) of three imprinted genes,H19,IG-GTL2andMEST, using bisulphite sequencing. Unique clones representative of single cells were analyzed. We found a significant decrease in DNA methylation at theH19DMR in testicular sperm of azoospermic men compared with proven fertile men. The decrease was also significant between OA and proven fertile men, and between men undergoing vasectomy reversal and proven fertile men, suggesting that aberrant DNA methylation may be associated with obstruction. Changes in DNA methylation atIG-GTL2andMESTDMRs among groups were not significant. Our data suggest that imprinting abnormalities may be associated with obstruction and may occur in response to changes in testicular environment and not only spermatogenesis failure, as previously reported. Methylation at theH19DMR was particularly prone to modification in testicular sperm.

DNA methylation profiling in cancer

2010 ◽  
Vol 12 ◽  
Author(s):  
Yutaka Kondo ◽  
Jean-Pierre J. Issa
Keyword(s):  
Dna Methylation ◽  
Clinical Diagnostics ◽  
Epigenetic Therapy ◽  
Tumour Suppressor Genes ◽  
Genome Wide ◽  
Dna Methylation Profiling ◽  
Aberrant Dna Methylation ◽  
Almost All ◽  
Multiple Tumour ◽  
Methylation Profiling

Aberrant DNA methylation in the genome is found in almost all types of cancer and contributes to malignant transformation by silencing multiple tumour-suppressor genes, sometimes simultaneously. Therefore, deciphering the signature of DNA methylation in each tumour is required to better understand tumour behaviour and might be of benefit for clinical diagnostics and therapy. Recent technologies for high-throughput genome-wide DNA methylation analyses are promising and potent tools for epigenetic profiling. Since epigenetic therapy is now in clinical use or trials for several types of cancers, efficient epigenetic profiling is required. In this review, the current key technologies available to assess genome-wide DNA methylation are introduced and the implications of DNA methylation profiling in human cancers are discussed.

scholarly journals Toward a More Robust Assessment of Intraspecies Diversity, Using Fewer Genetic Markers▿

2006 ◽  
Vol 72 (11) ◽  
pp. 7286-7293 ◽  
Author(s):  
Konstantinos T. Konstantinidis ◽  
Alban Ramette ◽  
James M. Tiedje
Keyword(s):  
Phylogenetic Reconstruction ◽  
Whole Genome ◽  
General Applicability ◽  
Elapsed Time ◽  
Improve Accuracy ◽  
Intraspecies Diversity ◽  
Genome Level ◽  
Almost All ◽  
Core Genes ◽  
Selection Of

ABSTRACT Phylogenetic sequence analysis of single or multiple genes has dominated the study and census of the genetic diversity among closely related bacteria. It remains unclear, however, how the results based on a few genes in the genome correlate with whole-genome-based relatedness and what genes (if any) best reflect whole-genome-level relatedness and hence should be preferentially used to economize on cost and to improve accuracy. We show here that phylogenies of closely related organisms based on the average nucleotide identity (ANI) of their shared genes correspond accurately to phylogenies based on state-of-the-art analysis of their whole-genome sequences. We use ANI to evaluate the phylogenetic robustness of every gene in the genome and show that almost all core genes, regardless of their functions and positions in the genome, offer robust phylogenetic reconstruction among strains that show 80 to 95% ANI (16S rRNA identity, >98.5%). Lack of elapsed time and, to a lesser extent, horizontal transfer and recombination make the selection of genes more critical for applications that target the intraspecies level, i.e., strains that show >95% ANI according to current standards. A much more accurate phylogeny for the Escherichia coli group was obtained based on just three best-performing genes according to our analysis compared to the concatenated alignment of eight genes that are commonly employed for phylogenetic purposes in this group. Our results are reproducible within the Salmonella, Burkholderia, and Shewanella groups and therefore are expected to have general applicability for microevolution studies, including metagenomic surveys.

scholarly journals Evolutionary Consequences of DNA Methylation on the GC Content in Vertebrate Genomes

2015 ◽  
Vol 5 (3) ◽  
pp. 441-447 ◽  
Author(s):  
Carina F Mugal ◽  
Peter F Arndt ◽  
Lena Holm ◽  
Hans Ellegren
Keyword(s):  
Dna Methylation ◽  
Gene Conversion ◽  
Local Equilibrium ◽  
Gc Content ◽  
Strong Impact ◽  
Whole Genome ◽  
Cpg Dinucleotides ◽  
Genome Methylation ◽  
Biased Gene Conversion ◽  
The Impact

Abstract The genomes of many vertebrates show a characteristic variation in GC content. To explain its origin and evolution, mainly three mechanisms have been proposed: selection for GC content, mutation bias, and GC-biased gene conversion. At present, the mechanism of GC-biased gene conversion, i.e., short-scale, unidirectional exchanges between homologous chromosomes in the neighborhood of recombination-initiating double-strand breaks in favor for GC nucleotides, is the most widely accepted hypothesis. We here suggest that DNA methylation also plays an important role in the evolution of GC content in vertebrate genomes. To test this hypothesis, we investigated one mammalian (human) and one avian (chicken) genome. We used bisulfite sequencing to generate a whole-genome methylation map of chicken sperm and made use of a publicly available whole-genome methylation map of human sperm. Inclusion of these methylation maps into a model of GC content evolution provided significant support for the impact of DNA methylation on the local equilibrium GC content. Moreover, two different estimates of equilibrium GC content, one that neglects and one that incorporates the impact of DNA methylation and the concomitant CpG hypermutability, give estimates that differ by approximately 15% in both genomes, arguing for a strong impact of DNA methylation on the evolution of GC content. Thus, our results put forward that previous estimates of equilibrium GC content, which neglect the hypermutability of CpG dinucleotides, need to be reevaluated.

scholarly journals Kaiso Regulates DNA Methylation Homeostasis

2021 ◽  
Vol 22 (14) ◽  
pp. 7587
Author(s):  
Darya Kaplun ◽  
Alexey Starshin ◽  
Fedor Sharko ◽  
Kristina Ganova ◽  
Galina Filonova ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Renal Cancer ◽  
Histone Modifications ◽  
De Novo ◽  
Cancer Cell Line ◽  
Dna Methyltransferases ◽  
Embryonic Fibroblasts ◽  
Genome Methylation ◽  
Renal Cancer Cell ◽  
Genome Level

Gain and loss of DNA methylation in cells is a dynamic process that tends to achieve an equilibrium. Many factors are involved in maintaining the balance between DNA methylation and demethylation. Previously, it was shown that methyl-DNA protein Kaiso may attract NCoR, SMRT repressive complexes affecting histone modifications. On the other hand, the deficiency of Kaiso resulted in reduced methylation of ICR in H19/Igf2 locus and Oct4 promoter in mouse embryonic fibroblasts. However, nothing is known about how Kaiso influences DNA methylation at the genome level. Here we show that deficiency of Kaiso led to whole-genome hypermethylation, using Kaiso deficient human renal cancer cell line obtained via CRISPR/CAS9 genome editing. However, Kaiso serves to protect genic regions, enhancers, and regions with a low level of histone modifications from demethylation. We detected hypomethylation of binding sites for Oct4 and Nanog in Kaiso deficient cells. Kaiso immunoprecipitated with de novo DNA methyltransferases DNMT3a/3b, but not with maintenance methyltransferase DNMT1. Thus, Kaiso may attract methyltransferases to surrounding regions and modulate genome methylation in renal cancer cells apart from being methyl DNA binding protein.

scholarly journals Mapping chromatin accessibility and active regulatory elements reveals new pathological mechanisms in human gliomas

2019 ◽  
Author(s):  
Karolina Stępniak ◽  
Magdalena A. Machnicka ◽  
Jakub Mieczkowski ◽  
Anna Macioszek ◽  
Bartosz Wojtaś ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Histone Modifications ◽  
Chromatin Structure ◽  
Malignant Gliomas ◽  
Regulatory Elements ◽  
Chromatin Accessibility ◽  
Cancer Development ◽  
Whole Genome ◽  
Human Gliomas

SummaryChromatin structure and accessibility, and combinatorial binding of transcription factors to regulatory elements in genomic DNA control transcription. Genetic variations in genes encoding histones, epigenetics-related enzymes or modifiers affect chromatin structure/dynamics and result in alterations in gene expression contributing to cancer development or progression. Gliomas are brain tumors frequently associated with epigenetics-related gene deregulation. We performed whole-genome mapping of chromatin accessibility, histone modifications, DNA methylation patterns and transcriptome analysis simultaneously in multiple tumor samples to unravel novel epigenetic dysfunctions driving gliomagenesis. Based on the results of the integrative analysis of the acquired profiles, we created an atlas of active enhancers and promoters in benign and malignant gliomas. We explored these elements and intersected with Hi-C data to uncover molecular mechanisms instructing gene expression in gliomas.SignificanceEpigenetics-driven deregulation of gene expression accompanies cancer development, but its comprehensive characterization in cancer patients is fragmentary. We performed whole-genome profiling of gene expression, open chromatin, histone modifications and DNA-methylation profiles in the same samples from benign and malignant gliomas. Our study provides a first comprehensive atlas of active regulatory elements in gliomas, which allowed identification of the functional enhancers and promoters in patient samples. This comprehensive approach revealed epigenetic patterns influencing gene expression in benign gliomas and a new pathogenic mechanism involving FOXM1-driven network in glioblastomas. This atlas provides a common set of elements for cross-comparisons of existing and new datasets, prompting novel discoveries and better understanding of gliomagenesis.HighlightsWe provide an atlas of cis-regulatory elements active in human gliomasEnhancer-promoter contacts operating in gliomas are revealedDiverse enhancer activation is pronounced in malignant gliomasChromatin loop activates FOXM1-ANXA2R pathological network in glioblastomas.

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