Targeted DNA Methylation Analysis by High Throughput Sequencing in Porcine Peri-attachment Embryos

Morrill, Benson H.; Cox, Lindsay; Ward, Anika; Heywood, Sierra; Prather, Randall S.; Isom, S. Clay

doi:10.1262/jrd.2012-144

Cited by 7 publications

(5 citation statements)

References 31 publications

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“…NGS generates thousands of read sequences for each sample, unlike traditional bisulfite sequencing with only 10–20 sequences. The greater the read number, the higher the statistical power of analysis for detecting even subtle differences in methylation between samples [33]. ChIP data are represented as the mean ± SD of 6 different treatments (n=6) and analyzed by two-tailed paired t-test with significance level P < 0.05 as above.…”

Section: Methodsmentioning

confidence: 99%

Nephrotoxicity of epigenetic inhibitors used for the treatment of cancer

Scholpa

Kolli

Moore

et al. 2016

Chemico-Biological Interactions

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This study determined the anti-neoplastic activity and nephrotoxicity of epigenetic inhibitors in vitro. The therapeutic efficacy of epigenetic inhibitors was determined in human prostate cancer cells (PC-3 and LNCaP) using the DNA methyltransferase inhibitor 5-azacytidine (5-Aza) and the histone deacetylase inhibitor trichostatin A (TSA). Cells were also treated with carbamazepine (CBZ), an anti-convulsant with histone deacetylase inhibitor-like properties. 5-Aza, TSA or CBZ alone did not decrease MTT staining in PC-3 or LNCaP cells after 48 hr. In contrast, docetaxel, a frontline chemotherapeutic induced concentration-dependent decreases in MTT staining. Pretreatment with 5-Aza or TSA increased docetaxel-induced cytotoxicity in LNCaP cells, but not PC-3 cells. TSA pretreatment also increased cisplatin-induced toxicity in LNCaP cells. Carfilzomib (CFZ), a protease inhibitor approved for the treatment of multiple myeloma had minimal effect on LNCaP cell viability, but reduced MTT staining 50% in PC-3 cells compared to control, and pretreatment with 5-Aza further enhanced toxicity. Treatment of normal rat kidney (NRK) and human embryonic kidney 293 (HEK293) cells with the same concentrations of epigenetic inhibitors used in prostate cancer cells significantly decreased MTT staining in all cell lines after 48 hr. Interestingly, we found that the toxicity of epigenetic inhibitors to kidney cells was dependent on both the compound and the stage of cell growth. The effect of 5-Aza and TSA on DNA methyltransferase and histone deacetylase activity, respectively, was confirmed by assessing the methylation and acetylation of the CDK inhibitor p21. Collectively, these data show that combinatorial treatment with epigenetic inhibitors alters the efficacy of chemotherapeutics in cancer cells in a compound- and cell-specific manner; however, this treatment also has the potential to induce nephrotoxic cell injury.

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Section: Methodsmentioning

confidence: 99%

Nephrotoxicity of epigenetic inhibitors used for the treatment of cancer

Scholpa

Kolli

Moore

et al. 2016

Chemico-Biological Interactions

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“…All the samples were pooled, purified and then sequenced using the 600 cycle v3 kits. An average of about 10,000 reads were obtained per sample, hence increasing the statistical power of analysis 40 as compared to the Sanger sequencing. The data from Bismark’s text file outputs were compiled together and the percent DNA methylation changes were displayed using heat-maps ( Figure 3B ).…”

Section: Resultsmentioning

confidence: 99%

Targeted Gene Bisulfite Sequencing Identifies Differential Methylation in p21

Kolli¹,

Glenn²,

Brown³

et al. 2018

Preprint

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35Next-generation sequencing (NGS) methods are widely available to assess 36 methylation of whole-genomes, reduced representation of genomes, and target capture 37 of many loci, but simple, flexible, and low-cost methods are needed to leverage NGS for 38 sequencing single-locus amplicons from large numbers of samples. We (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

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“…This is likely because each cell has two alleles of each gene, and alleles of minor cell types have stochastically lower chances of being sequenced than major cell types when only a few tens of clones are selected for bisulfite sequencing analysis. In addition, a previous study demonstrated the difficulty in detecting relatively subtle differences in methylation levels for small numbers of clones [ 26 ]. To our knowledge, the present study is the first to focus on hypomethylation of each sequenced read (transcriptionally active allele) obtained by ultra-deep bisulfite sequencing of cell type-restricted genes, which can reliably detect hypomethylation of minor cell types by sequencing hundreds or thousands of bisulfite PCR fragments of cell type-restricted genes.…”

Section: Discussionmentioning

confidence: 99%

Ultra-Deep Bisulfite Sequencing to Detect Specific DNA Methylation Patterns of Minor Cell Types in Heterogeneous Cell Populations: An Example of the Pituitary Tissue

et al. 2016

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DNA methylation is an epigenetic modification important for cell fate determination and cell type-specific gene expression. Transcriptional regulatory regions of the mammalian genome contain a large number of tissue/cell type-dependent differentially methylated regions (T-DMRs) with DNA methylation patterns crucial for transcription of the corresponding genes. In general, tissues consist of multiple cell types in various proportions, making it difficult to detect T-DMRs of minor cell types in tissues. The present study attempts to detect T-DMRs of minor cell types in tissues by ultra-deep bisulfite sequencing of cell type-restricted genes and to assume proportions of minor cell types based on DNA methylation patterns of sequenced reads. For this purpose, we focused on transcriptionally active hypomethylated alleles (Hypo-alleles), which can be recognized by the high ratio of unmethylated CpGs in each sequenced read (allele). The pituitary gland contains multiple cell types including five hormone-expressing cell types and stem/progenitor cells, each of which is a minor cell type in the pituitary tissue. By ultra-deep sequencing of more than 100 reads for detection of Hypo-alleles in pituitary cell type-specific genes, we identified T-DMRs specific to hormone-expressing cells and stem/progenitor cells and used them to estimate the proportions of each cell type based on the Hypo-allele ratio in pituitary tissue. Therefore, introduction of the novel Hypo-allele concept enabled us to detect T-DMRs of minor cell types with estimation of their proportions in the tissue by ultra-deep bisulfite sequencing.

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Targeted DNA Methylation Analysis by High Throughput Sequencing in Porcine Peri-attachment Embryos

Cited by 7 publications

References 31 publications

Nephrotoxicity of epigenetic inhibitors used for the treatment of cancer

Nephrotoxicity of epigenetic inhibitors used for the treatment of cancer

Targeted Gene Bisulfite Sequencing Identifies Differential Methylation in p21

Ultra-Deep Bisulfite Sequencing to Detect Specific DNA Methylation Patterns of Minor Cell Types in Heterogeneous Cell Populations: An Example of the Pituitary Tissue

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