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Isabelle Hernandez Cantão Renato Borges Tesser Taiza Stumpp 《Biological procedures online》2017,19(1):9
Background
Primordial germ cells (PGC) are the precursors of the gametes. During pre-natal development, PGC undergo an epigenetic reprogramming when bulk DNA demethylation occurs and is followed by sex-specific de novo methylation. The de novo methylation and the maintenance of the methylation patterns depend on DNA methyltransferases (DNMTs). PGC reprogramming has been widely studied in mice but not in rats. We have previously shown that the rat might be an interesting model to study germ cell development. In face of the difficulties of getting enough PGC for molecular studies, the aim of this study was to propose an alternative method to study rat PGC DNA methylation. Rat embryos were collected at 14, 15 and 19 days post-coitus (dpc) for the analysis of 5mC, 5hmC, DNMT1, DNMT3a and DNMT3b expression or at 16dpc for treatment 5-Aza-CdR, a DNMT inhibitor, in vitro.Methods
Once collected, the gonads were placed in 24-well plates previously containing 45μm pore membrane and medium with or without 5-Aza-CdR. The culture was kept for five days and medium was changed daily. The gonads were either fixed or submitted to RNA extraction.Results
5mC and DNMTs labelling suggests that PGC are undergoing epigenetic reprogramming around 14/15dpc. The in vitro treatment of rat embryonic gonads with 1 μM of 5-Aza-CdR lead to a loss of 5mC labelling and to the activation of Pax6 expression in PGC, but not in somatic cells, suggesting that 5-Aza-CdR promoted a PGC-specific global DNA hypomethylation.Conclusions
This study suggests that the protocol used here can be a potential method to study the wide DNA demethylation that takes place during PGC reprogramming.4.
A streamlined method for analysing genome-wide DNA methylation patterns from low amounts of FFPE DNA
Jackie L. Ludgate James Wright Peter A. Stockwell Ian M. Morison Michael R. Eccles Aniruddha Chatterjee 《BMC medical genomics》2017,10(1):54
Background
Formalin fixed paraffin embedded (FFPE) tumor samples are a major source of DNA from patients in cancer research. However, FFPE is a challenging material to work with due to macromolecular fragmentation and nucleic acid crosslinking. FFPE tissue particularly possesses challenges for methylation analysis and for preparing sequencing-based libraries relying on bisulfite conversion. Successful bisulfite conversion is a key requirement for sequencing-based methylation analysis.Methods
Here we describe a complete and streamlined workflow for preparing next generation sequencing libraries for methylation analysis from FFPE tissues. This includes, counting cells from FFPE blocks and extracting DNA from FFPE slides, testing bisulfite conversion efficiency with a polymerase chain reaction (PCR) based test, preparing reduced representation bisulfite sequencing libraries and massively parallel sequencing.Results
The main features and advantages of this protocol are:
- An optimized method for extracting good quality DNA from FFPE tissues.
- An efficient bisulfite conversion and next generation sequencing library preparation protocol that uses 50 ng DNA from FFPE tissue.
- Incorporation of a PCR-based test to assess bisulfite conversion efficiency prior to sequencing.
Conclusions
We provide a complete workflow and an integrated protocol for performing DNA methylation analysis at the genome-scale and we believe this will facilitate clinical epigenetic research that involves the use of FFPE tissue.5.
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Background
An important feature in many genomic studies is quality control and normalization. This is particularly important when analyzing epigenetic data, where the process of obtaining measurements can be bias prone. The GAW20 data was from the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN), a study with multigeneration families, where DNA cytosine-phosphate-guanine (CpG) methylation was measured pre- and posttreatment with fenofibrate. We performed quality control assessment of the GAW20 DNA methylation data, including normalization, assessment of batch effects and detection of sample swaps.Results
We show that even after normalization, the GOLDN methylation data has systematic differences pre- and posttreatment. Through investigation of (a) CpGs sites containing a single nucleotide polymorphism, (b) the stability of breeding values for methylation across time points, and (c) autosomal gender-associated CpGs, 13 sample swaps were detected, 11 of which were posttreatment.Conclusions
This paper demonstrates several ways to perform quality control of methylation data in the absence of raw data files and highlights the importance of normalization and quality control of the GAW20 methylation data from the GOLDN study.7.
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Lei Luo Zhiqiu Yao Jing Ye Yuan Tian Chen Yang Xiaoxiao Gao Min Song Ya Liu Yunhai Zhang Yunsheng Li Xiaorong Zhang Fugui Fang 《Reproductive biology and endocrinology : RB&E》2017,15(1):81
Background
There are many variables affecting the onset of puberty in animals, including genetic, nutritional, and environmental factors. Recent studies suggest that epigenetic regulation, especially DNA methylation, plays a majorrole in the regulation of puberty. However, there have been no reports on DNA methylation of the pubertal genome.Methods
We investigated DNA methylation in the female rat hypothalamus at prepuberty and puberty using reduced representation bisulfite sequencing technology. The identified genes and signaling pathways exhibiting changes to DNA methylation in pubertal rats were determined by Gene Ontogeny and Kyoto Encyclopedia of Genes and Genomes analysis.Results
The distribution of the three types of methylated C bases in promoter and CpG island (CGI) regions in the hypothalamus was as follows: 87.79% CG, 3.05% CHG, 9.16% CHH for promoters, and 88.35% CG, 3.21% CHG, 88.35% CHH for CGI in prepubertal rats; and 90.78% CG, 2.13% CHG, 7.09% CHH for promoters, and 88.59% CG, 88.59% CHG, 8.35% CHH for CGI in pubertal animals. CG showed the highest percentage of methylation, and was the highest methylation state in CGI. Compared to prepubertal hyoyhalamus samples, we identified ten genes with altered methylation in promoter regions in the pubertal hypothalamus samples, and 43 genes with altered methylation in the CGI. Changes in DNA methylation were found in gonadotropin-releasing hormone signaling pathways, and the oocyte meiosis pathway.Conclusion
Our results demonstrate changes in DNA methylation occur in female rats from prepuberty to puberty suggestng DNA methylation may play a crucial role in the regulation of puberty onset. This study provides essential information for future studies on the role of epigenetics in the regulation of puberty.10.
Kai-Le Li Lei Zhang Xiao-Mei Yang Qiang Fang Xue-Fang Yin Hui-Min Wei Ting Zhou Ya-Bin Li Xue-Lin Chen Fan Tang Yong-Hao Li Jian-Feng Chang Wei Li Feng Sun 《BMC developmental biology》2018,18(1):20
Background
Histone modifications are critical in regulating neuronal processes. However, the impacts of individual histone modifications on learning and memory are elusive. Here, we investigated the contributions of histone H3 lysine modifications to learning and memory in Drosophila by using histone lysine-to-alanine mutants.Results
Behavioural analysis indicated that compared to the H3WT group, mutants overexpressing H3K23A displayed impaired courtship learning. Chromatin immunoprecipitation analysis of H3K23A mutants showed that H3K23 acetylation (H3K23ac) levels were decreased on learning-related genes. Knockdown of CREB-binding protein (CBP) decreased H3K23ac levels, attenuated the expression of learning-related genes, led to a courtship learning defect and altered development of the mushroom bodies. A decline in courtship learning ability was observed in both larvae and adult treatments with ICG-001. Furthermore, treatment of Drosophila overexpressing mutated H3K23A with a CBP inhibitor did not aggravate the learning defect.Conclusions
H3K23ac, catalysed by the acetyltransferases dCBP, contributes to Drosophila learning, likely by controlling the expression of specific genes. This is a novel epigenetic regulatory mechanism underlying neuronal behaviours.11.
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Background
The rise in popularity and accessibility of DNA methylation data to evaluate epigenetic associations with disease has led to numerous methodological questions. As part of GAW20, our working group of 8 research groups focused on gene searching methods.Results
Although the methods were varied, we identified 3 main themes within our group. First, many groups tackled the question of how best to use pedigree information in downstream analyses, finding that (a) the use of kinship matrices is common practice, (b) ascertainment corrections may be necessary, and (c) pedigree information may be useful for identifying parent-of-origin effects. Second, many groups also considered multimarker versus single-marker tests. Multimarker tests had modestly improved power versus single-marker methods on simulated data, and on real data identified additional associations that were not identified with single-marker methods, including identification of a gene with a strong biological interpretation. Finally, some of the groups explored methods to combine single-nucleotide polymorphism (SNP) and DNA methylation into a single association analysis.Conclusions
A causal inference method showed promise at discovering new mechanisms of SNP activity; gene-based methods of summarizing SNP and DNA methylation data also showed promise. Even though numerous questions still remain in the analysis of DNA methylation data, our discussions at GAW20 suggest some emerging best practices.13.
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Background
Methylation analysis of cell-free DNA is a encouraging tool for tumor diagnosis, monitoring and prognosis. Sensitivity of methylation analysis is a very important matter due to the tiny amounts of cell-free DNA available in plasma. Most current methods of DNA methylation analysis are based on the difference of bisulfite-mediated deamination of cytosine between cytosine and 5-methylcytosine. However, the recovery of bisulfite-converted DNA based on current methods is very poor for the methylation analysis of cell-free DNA.Results
We optimized a rapid method for the crucial steps of bisulfite conversion with high recovery of cell-free DNA. A rapid deamination step and alkaline desulfonation was combined with the purification of DNA on a silica column. The conversion efficiency and recovery of bisulfite-treated DNA was investigated by the droplet digital PCR. The optimization of the reaction results in complete cytosine conversion in 30 min at 70 °C and about 65% of recovery of bisulfite-treated cell-free DNA, which is higher than current methods.Conclusions
The method allows high recovery from low levels of bisulfite-treated cell-free DNA, enhancing the analysis sensitivity of methylation detection from cell-free DNA.15.
Fernndez-Rhodes Lindsay Howard Annie Green Tao Ran Young Kristin L. Graff Mariaelisa Aiello Allison E. North Kari E. Justice Anne E. 《BMC genetics》2018,19(1):69-14
Background
Transgenerational epigenetic inheritance has been posited as a possible contributor to the observed heritability of metabolic syndrome (MetS). Yet the extent to which estimates of epigenetic inheritance for DNA methylation sites are inflated by environmental and genetic covariance within families is still unclear. We applied current methods to quantify the environmental and genetic contributors to the observed heritability and familial correlations of four previously associated MetS methylation sites at three genes (CPT1A, SOCS3 and ABCG1) using real data made available through the GAW20.Results
Our findings support the role of both shared environment and genetic variation in explaining the heritability of MetS and the four MetS cytosine-phosphate-guanine (CpG) sites, although the resulting heritability estimates were indistinguishable from one another. Familial correlations by type of relative pair generally followed our expectation based on relatedness, but in the case of sister and parent pairs we observed nonsignificant trends toward greater correlation than expected, as would be consistent with the role of shared environmental factors in the inflation of our estimated correlations.Conclusions
Our work provides an interesting and flexible statistical framework for testing models of epigenetic inheritance in the context of human family studies. Future work should endeavor to replicate our findings and advance these methods to more robustly describe epigenetic inheritance patterns in human populations.16.
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Background
Integrative analysis on multi-omics data has gained much attention recently. To investigate the interactive effect of gene expression and DNA methylation on cancer, we propose a directed random walk-based approach on an integrated gene-gene graph that is guided by pathway information.Methods
Our approach first extracts a single pathway profile matrix out of the gene expression and DNA methylation data by performing the random walk over the integrated graph. We then apply a denoising autoencoder to the pathway profile to further identify important pathway features and genes. The extracted features are validated in the survival prediction task for breast cancer patients.Results
The results show that the proposed method substantially improves the survival prediction performance compared to that of other pathway-based prediction methods, revealing that the combined effect of gene expression and methylation data is well reflected in the integrated gene-gene graph combined with pathway information. Furthermore, we show that our joint analysis on the methylation features and gene expression profile identifies cancer-specific pathways with genes related to breast cancer.Conclusions
In this study, we proposed a DRW-based method on an integrated gene-gene graph with expression and methylation profiles in order to utilize the interactions between them. The results showed that the constructed integrated gene-gene graph can successfully reflect the combined effect of methylation features on gene expression profiles. We also found that the selected features by DA can effectively extract topologically important pathways and genes specifically related to breast cancer.18.
Cristina Maria Pinto de Paula Fausto Souza Sobrinho Vânia Helena Techio 《Plant cell reports》2016,35(6):1359-1369
Key message
Assessment of chromosomal distribution of modified histones and 5-methylcytosine shown that there are diversification of chromosomal types among species of Brachiaria and its interspecific hybrids.Abstract
Histone post-translational modifications and DNA methylation are epigenetic processes that are involved in structural and functional organization of the genome. This study compared the chromosomal distribution of modified histones and 5-methylcytosine (5-mCyt) in species and interspecific hybrids of Brachiaria with different ploidy levels and reproduction modes. The relation between H3K9me2 and 5-mCyt was observed in the nucleolus organizer region, centromeric central domain and pericentromeric region. H3K4me2 was detected in euchromatic domains, mainly in the terminal chromosomal regions. Comparison of chromosomal distribution among species and hybrids showed greater variation of chromosomal types for the H3K9me2 in B. decumbens (tetraploid and apomictic species) and the 963 hybrid, while, for the H3K4me2, the variation was higher in B. brizantha and B. decumbens (tetraploid and apomictic species) and 963 hybrid. The chromosome distribution of 5-mCyt was similar between B. brizantha and B. decumbens, which differ from the distribution observed in B. ruziziensis (diploid and sexual species). Significant alterations in DNA methylation were observed in the artificially tetraploidized B. ruziziensis and in the interspecific hybrids, possibly as result of hybridization and polyploidization processes. The monitoring of histone modifications and DNA methylation allowed categorizing nuclear and chromosomal distribution of these epigenetic marks, thus contributing to the knowledge of composition and structure of the genome/epigenome of Brachiaria species and hybrids. These data can be useful for speciation and genome evolution studies in genus Brachiaria, and represent important markers to explore relationships between genomes.19.
Jian-Rong Guo Lei Yin Yong-Quan Chen Xiao-Ju Jin Xun Zhou Na-Na Zhu Xiao-Qian Liu Han-Wei Wei Li-Shuang Duan 《Cell communication and signaling : CCS》2018,16(1):84