Epigenetic modifications play a vital role in the preservation of genome integrity and in the regulation of gene expression. DNA methylation, one of the key mechanisms of epigenetic control, impacts growth, development, stress response and adaptability of all organisms, including plants. The detection of DNA methylation marks is crucial for understanding the mechanisms underlying these processes and for developing strategies to improve productivity and stress resistance of crop plants. There are different methods for detecting plant DNA methylation, such as bisulfite sequencing, methylation-sensitive amplified polymorphism, genome-wide DNA methylation analysis, methylated DNA immunoprecipitation sequencing, reduced representation bisulfite sequencing, MS and immuno-based techniques. These profiling approaches vary in many aspects, including DNA input, resolution, genomic region coverage, and bioinformatics analysis. Selecting an appropriate methylation screening approach requires an understanding of all these techniques. This review provides an overview of DNA methylation profiling methods in crop plants, along with comparisons of the efficacy of these techniques between model and crop plants. The strengths and limitations of each methodological approach are outlined, and the importance of considering both technical and biological factors are highlighted. Additionally, methods for modulating DNA methylation in model and crop species are presented. Overall, this review will assist scientists in making informed decisions when selecting an appropriate DNA methylation profiling method.

• Klíčová slova
• DNA methylation modulation, DNA methylation profiling, bisulfite sequencing, crop epigenome, immunological techniques, mass spectrometry, next-generation sequencing,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Chordoma is a rare malignant tumor with notochordal differentiation, usually affecting the axial skeleton of young patients. We report a case of a high-grade epithelioid tumor involving the synovium and soft tissues of the knee in a 74-year-old male patient. The preliminary biopsy was inconclusive, but a diagnosis of metastatic clear-cell carcinoma of unknown origin was suggested. However, imaging studies did not reveal any primary lesions. The resection specimen consisted of nests and sheets of oval to polygonal cells with discernible cell borders, clear or lightly amphophilic cytoplasm, and round to oval nuclei with occasional well-visible eosinophilic nucleoli. Rare atypical mitoses, necrotic areas, and bizarre nuclei were noted. The biopsy and resection specimens underwent a wide molecular genetic analysis which included methylation profiling. The DKFZ sarcoma classifier assigned the methylation class chordoma (dedifferentiated) with a calibrated score of 0.96, and additionally, a loss of SMARCB1 locus was noted in the copy number variation plot. To verify these findings, T-brachyury and SMARCB1 immunostaining was performed afterward, showing diffuse nuclear positivity and complete loss in the tumor cells, respectively. To assess the prevalence of T-brachyury immunopositivity among SWI/SNF-deficient tumors and to evaluate its specificity for poorly differentiated chordoma, we analyzed a series of 23 SMARCB1- or SMARCA4-deficient tumors, all of which were negative. After incorporating all the available data, including the absence of any morphological features of conventional chordoma, the case was diagnosed as poorly differentiated chordoma. As illustrated herein, the utilization of methylation profiling in the diagnostic process of some carefully selected unclassifiable soft tissue neoplasms may lead to an increased detection rate of such extremely rare soft tissue tumors and enable their better characterization.

• Klíčová slova
• Brachyury, Extra-axial chordoma, INI1, Knee, Methylation, Poorly differentiated chordoma, SMARCB1,
• MeSH
• buněčná diferenciace MeSH
• chordom * patologie   genetika   diagnóza   metabolismus   MeSH
• fetální proteiny * genetika   metabolismus   MeSH
• gen SMARCB1 * genetika   MeSH
• lidé MeSH
• metylace DNA * MeSH
• nádorové biomarkery * analýza   genetika   MeSH
• nádory měkkých tkání patologie   diagnóza   genetika   metabolismus   MeSH
• proteiny T-boxu * genetika   metabolismus   MeSH
• senioři MeSH
• transkripční faktory genetika   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• Názvy látek
• Brachyury protein MeSH Prohlížeč
• fetální proteiny * MeSH
• gen SMARCB1 * MeSH
• nádorové biomarkery * MeSH
• proteiny T-boxu * MeSH
• SMARCB1 protein, human MeSH Prohlížeč
• transkripční faktory MeSH

Nonalcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease in children and adolescents, increasing the risk of its progression toward nonalcoholic steatohepatitis (NASH), cirrhosis, and cancer. There is an urgent need for noninvasive early diagnostic and prognostic tools such as epigenetic marks (epimarks), which would replace liver biopsy in the future. We used plasma samples from 67 children with biopsy-proven NAFLD, and as controls we used samples from 20 children negative for steatosis by ultrasound. All patients were genotyped for patatin-like phospholipase domain containing 3 (PNPLA3), transmembrane 6 superfamily member 2 (TM6SF2), membrane bound O-acyltransferase domain containing 7 (MBOAT7), and klotho-β (KLB) gene variants, and data on anthropometric and biochemical parameters were collected. Furthermore, plasma cell-free DNA (cfDNA) methylation was quantified using a commercially available kit, and ImageStream(X) was used for the detection of free circulating histone complexes and variants. We found a significant enrichment of the levels of histone macroH2A1.2 in the plasma of children with NAFLD compared to controls, and a strong correlation between cfDNA methylation levels and NASH. Receiver operating characteristic curve analysis demonstrated that combination of cfDNA methylation, PNPLA3 rs738409 variant, coupled with either high-density lipoprotein cholesterol or alanine aminotransferase levels can strongly predict the progression of pediatric NAFLD to NASH with area under the curve >0.87. Conclusion: Our pilot study combined epimarks and genetic and metabolic markers for a robust risk assessment of NAFLD development and progression in children, offering a promising noninvasive tool for the consistent diagnosis and prognosis of pediatric NAFLD. Further studies are necessary to identify their pathogenic origin and function.

• MeSH
• dítě MeSH
• histony genetika   MeSH
• lidé MeSH
• lipasa genetika   MeSH
• membránové proteiny genetika   MeSH
• metylace DNA genetika   MeSH
• mladiství MeSH
• nealkoholová steatóza jater * diagnóza   MeSH
• pilotní projekty MeSH
• volné cirkulující nukleové kyseliny * metabolismus   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• mladiství MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• histony MeSH
• lipasa MeSH
• membránové proteiny MeSH
• volné cirkulující nukleové kyseliny * MeSH

The risk of exposure to nanoparticles (NPs) has rapidly increased during the last decade due to the vast use of nanomaterials (NMs) in many areas of human life. Despite this fact, human biomonitoring studies focused on the effect of NP exposure on DNA alterations are still rare. Furthermore, there are virtually no epigenetic data available. In this study, we investigated global and gene-specific DNA methylation profiles in a group of 20 long-term (mean 14.5 years) exposed, nanocomposite, research workers and in 20 controls. Both groups were sampled twice/day (pre-shift and post-shift) in September 2018. We applied Infinium Methylation Assay, using the Infinium MethylationEPIC BeadChips with more than 850,000 CpG loci, for identification of the DNA methylation pattern in the studied groups. Aerosol exposure monitoring, including two nanosized fractions, was also performed as proof of acute NP exposure. The obtained array data showed significant differences in methylation between the exposed and control groups related to long-term exposure, specifically 341 CpG loci were hypomethylated and 364 hypermethylated. The most significant CpG differences were mainly detected in genes involved in lipid metabolism, the immune system, lung functions, signaling pathways, cancer development and xenobiotic detoxification. In contrast, short-term acute NP exposure was not accompanied by DNA methylation changes. In summary, long-term (years) exposure to NP is associated with DNA epigenetic alterations.

• Klíčová slova
• 850K microarray, CpG sites, DNA methylation, epigenetic adaptation, human, nanoparticles, occupational exposure,
• MeSH
• dospělí MeSH
• epigeneze genetická MeSH
• genom lidský MeSH
• lidé středního věku MeSH
• lidé MeSH
• metylace DNA účinky léků   MeSH
• mladý dospělý MeSH
• nanočástice škodlivé účinky   MeSH
• nanokompozity škodlivé účinky   MeSH
• pracovní expozice * MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladý dospělý MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH

Epigenetic modifications established during gametogenesis regulate transcription and other nuclear processes in gametes, but also have influences in the zygote, embryo and postnatal life. This is best understood for DNA methylation which, established at discrete regions of the oocyte and sperm genomes, governs genomic imprinting. In this review, we describe how imprinting has informed our understanding of de novo DNA methylation mechanisms, highlight how recent genome-wide profiling studies have provided unprecedented insights into establishment of the sperm and oocyte methylomes and consider the fate and function of gametic methylation and other epigenetic modifications after fertilization.

• Klíčová slova
• DNA methylation, embryo, imprinting, oogenesis, spermatogenesis, transgenerational inheritance,
• MeSH
• genomový imprinting * MeSH
• histonový kód MeSH
• lidé MeSH
• metylace DNA * MeSH
• zárodečné buňky metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

BACKGROUND: Gametogenesis in mammals entails profound re-patterning of the epigenome. In the female germline, DNA methylation is acquired late in oogenesis from an essentially unmethylated baseline and is established largely as a consequence of transcription events. Molecular and functional studies have shown that imprinted genes become methylated at different times during oocyte growth; however, little is known about the kinetics of methylation gain genome wide and the reasons for asynchrony in methylation at imprinted loci. RESULTS: Given the predominant role of transcription, we sought to investigate whether transcription timing is rate limiting for de novo methylation and determines the asynchrony of methylation events. Therefore, we generated genome-wide methylation and transcriptome maps of size-selected, growing oocytes to capture the onset and progression of methylation. We find that most sequence elements, including most classes of transposable elements, acquire methylation at similar rates overall. However, methylation of CpG islands (CGIs) is delayed compared with the genome average and there are reproducible differences amongst CGIs in onset of methylation. Although more highly transcribed genes acquire methylation earlier, the major transitions in the oocyte transcriptome occur well before the de novo methylation phase, indicating that transcription is generally not rate limiting in conferring permissiveness to DNA methylation. Instead, CGI methylation timing negatively correlates with enrichment for histone 3 lysine 4 (H3K4) methylation and dependence on the H3K4 demethylases KDM1A and KDM1B, implicating chromatin remodelling as a major determinant of methylation timing. We also identified differential enrichment of transcription factor binding motifs in CGIs acquiring methylation early or late in oocyte growth. By combining these parameters into multiple regression models, we were able to account for about a fifth of the variation in methylation timing of CGIs. Finally, we show that establishment of non-CpG methylation, which is prevalent in fully grown oocytes, and methylation over non-transcribed regions, are later events in oogenesis. CONCLUSIONS: These results do not support a major role for transcriptional transitions in the time of onset of DNA methylation in the oocyte, but suggest a model in which sequences least dependent on chromatin remodelling are the earliest to become permissive for methylation.

• Klíčová slova
• DNA methylation, Histone modifications, Imprinting, Oocytes, Transcription,
• MeSH
• chromatin genetika   MeSH
• CpG ostrůvky genetika   MeSH
• DNA-(cytosin-5-)methyltransferasa genetika   MeSH
• genetická transkripce * MeSH
• genomový imprinting genetika   MeSH
• histony genetika   MeSH
• metylace DNA genetika   MeSH
• myši MeSH
• oocyty růst a vývoj   metabolismus   MeSH
• oogeneze genetika   MeSH
• restrukturace chromatinu MeSH
• transkriptom genetika   MeSH
• zárodečné buňky MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromatin MeSH
• DNA-(cytosin-5-)methyltransferasa MeSH
• histony MeSH

BACKGROUND: In order to gain insight into the contribution of DNA methylation to disease progression of chronic lymphocytic leukemia (CLL), using 450K Illumina arrays, we determined the DNA methylation profiles in paired pre-treatment/relapse samples from 34 CLL patients treated with chemoimmunotherapy, mostly (n = 31) with the fludarabine-cyclophosphamide-rituximab (FCR) regimen. RESULTS: The extent of identified changes in CLL cells versus memory B cells from healthy donors was termed "epigenetic burden" (EB) whereas the number of changes between the pre-treatment versus the relapse sample was termed "relapse changes" (RC). Significant (p < 0.05) associations were identified between (i) high EB and short time-to-first-treatment (TTFT); and, (ii) few RCs and short time-to-relapse. Both the EB and the RC clustered in specific genomic regions and chromatin states, including regulatory regions containing binding sites of transcription factors implicated in B cell and CLL biology. CONCLUSIONS: Overall, we show that DNA methylation in CLL follows different dynamics in response to chemoimmunotherapy. These epigenetic alterations were linked with specific clinical and biological features.

• Klíčová slova
• CLL, Chemoimmunotherapy, DNA methylation, Microarray analysis, Relapse,
• MeSH
• chronická lymfatická leukemie farmakoterapie   genetika   MeSH
• cyklofosfamid farmakologie   terapeutické užití   MeSH
• dospělí MeSH
• epigeneze genetická účinky léků   MeSH
• genové regulační sítě účinky léků   MeSH
• imunoterapie MeSH
• lidé středního věku MeSH
• lidé MeSH
• longitudinální studie MeSH
• metylace DNA účinky léků   MeSH
• progrese nemoci MeSH
• rituximab farmakologie   terapeutické užití   MeSH
• sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
• senioři MeSH
• vidarabin analogy a deriváty   farmakologie   terapeutické užití   MeSH
• výsledek terapie MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cyklofosfamid MeSH
• fludarabine MeSH Prohlížeč
• rituximab MeSH
• vidarabin MeSH

Examination of changes in the methylation profile of DNA in cancer is currently used to determine the diagnosis or prognostic and predictive biomarkers. It complements histological or molecular biological examinations. At the same time, it helps to identify new diagnostic groups and subgroups. Currently, this diagnosis is most common in brain tumors, where it has become a routine examination. The established methylation profile may help even where the diagnosis or subgroup classification of the disease cannot be determined in any other way, as is the case with medulloblastoma.

• Klíčová slova
• Methylation, SNP array, brain tumors, classification,
• MeSH
• lidé MeSH
• metylace DNA MeSH
• nádorové biomarkery genetika   metabolismus   MeSH
• nádory mozečku * genetika   MeSH
• nádory mozku * diagnóza   genetika   MeSH
• prognóza MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• nádorové biomarkery MeSH

BACKGROUND: Reproductive biology methods rely on in vitro follicle cultures from mature follicles obtained by hormonal stimulation for generating metaphase II oocytes to be fertilised and developed into a healthy embryo. Such techniques are used routinely in both rodent and human species. DNA methylation is a dynamic process that plays a role in epigenetic regulation of gametogenesis and development. In mammalian oocytes, DNA methylation establishment regulates gene expression in the embryos. This regulation is particularly important for a class of genes, imprinted genes, whose expression patterns are crucial for the next generation. The aim of this work was to establish an in vitro culture system for immature mouse oocytes that will allow manipulation of specific factors for a deeper analysis of regulatory mechanisms for establishing transcription regulation-associated methylation patterns. RESULTS: An in vitro culture system was developed from immature mouse oocytes that were grown to germinal vesicles (GV) under two different conditions: normoxia (20% oxygen, 20% O2) and hypoxia (5% oxygen, 5% O2). The cultured oocytes were sorted based on their sizes. Reduced representative bisulphite sequencing (RRBS) and RNA-seq libraries were generated from cultured and compared to in vivo-grown oocytes. In the in vitro cultured oocytes, global and CpG-island (CGI) methylation increased gradually along with oocyte growth, and methylation of the imprinted genes was similar to in vivo-grown oocytes. Transcriptomes of the oocytes grown in normoxia revealed chromatin reorganisation and enriched expression of female reproductive genes, whereas in the 5% O2 condition, transcripts were biased towards cellular stress responses. To further confirm the results, we developed a functional assay based on our model for characterising oocyte methylation using drugs that reduce methylation and transcription. When histone methylation and transcription processes were reduced, DNA methylation at CGIs from gene bodies of grown oocytes presented a lower methylation profile. CONCLUSIONS: Our observations reveal changes in DNA methylation and transcripts between oocytes cultured in vitro with different oxygen concentrations and in vivo-grown murine oocytes. Oocytes grown under 20% O2 had a higher correlation with in vivo oocytes for DNA methylation and transcription demonstrating that higher oxygen concentration is beneficial for the oocyte maturation in ex vivo culture condition. Our results shed light on epigenetic mechanisms for the development of oocytes from an immature to GV oocyte in an in vitro culture model.

• Klíčová slova
• 5% oxygen, DNA methylation, In vitro culture, Mouse, Normoxia, Oocyte, Transcription,
• MeSH
• IVM techniky metody   MeSH
• kyslík metabolismus   MeSH
• metylace DNA * MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• oocyty růst a vývoj   MeSH
• transkriptom * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyslík MeSH

The potential clinical applications of human induced pluripotent stem cells (hiPSCs) are limited by genetic and epigenetic variations among hiPSC lines and the question of their equivalency with human embryonic stem cells (hESCs). We used MethylScreen technology to determine the DNA methylation profile of pluripotency and differentiation markers in hiPSC lines from different source cell types compared to hESCs and hiPSC source cells. After derivation, hiPSC lines compromised a heterogeneous population characterized by variable levels of aberrant DNA methylation. These aberrations were induced during somatic cell reprogramming and their levels were associated with the type of hiPSC source cells. hiPSC population heterogeneity was reduced during prolonged culture and hiPSCs acquired an hESC-like methylation profile. In contrast, the expression of differentiation marker genes in hiPSC lines remained distinguishable from that in hESCs. Taken together, in vitro culture facilitates hiPSC acquisition of hESC epigenetic characteristics. However, differences remain between both pluripotent stem cell types, which must be considered before their use in downstream applications.

• MeSH
• buněčná diferenciace genetika   MeSH
• buněčné linie MeSH
• fibroblasty cytologie   metabolismus   MeSH
• indukované pluripotentní kmenové buňky cytologie   metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• lidské embryonální kmenové buňky cytologie   metabolismus   MeSH
• metylace DNA * MeSH
• přeprogramování buněk genetika   MeSH
• shluková analýza MeSH
• stanovení celkové genové exprese MeSH
• vývojová regulace genové exprese MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH