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

Arai, Yoshikazu; Fukukawa, Hisho; Atozi, Takanori; Matsumoto, Shoma; Hanazono, Yutaka; Nagashima, Hiroshi; Ohgane, Jun

doi:10.1371/journal.pone.0146498

Cited by 7 publications

(7 citation statements)

References 26 publications

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“…Since each read in a WGBS library is the clonal derivative of a DNA molecule originating from a single cell, cell-specific information is inherent in WGBS data 27 , as it is in clonal BS-seq. The use of read-level bisulfite sequencing data to infer cell type- and allele-specific methylation patterns is well established in the literature 28,29 , though until now it has not yet been deployed on a genome-wide scale.…”

Section: Resultsmentioning

confidence: 99%

“…To begin to understand downstream, cell type-specific effects, we implemented a read-level analysis of our WGBS data, analogous to previously described approaches using ultra-deep BS-seq to identify minor cell populations in pituitary tissue 29 . This led us to conclude that TGF-β-associated SMAD binding motifs enriched in hypermethylated DMRs are a signature of secondary effects in non-AgRP cell types, and that specific hypomethylated read clusters in the promoter region of Bmp7 originate from AgRP neurons.…”

Section: Discussionmentioning

confidence: 99%

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DNA methylation in AgRP neurons regulates voluntary exercise behavior in mice

et al. 2019

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DNA methylation regulates cell type-specific gene expression. Here, in a transgenic mouse model, we show that deletion of the gene encoding DNA methyltransferase Dnmt3a in hypothalamic AgRP neurons causes a sedentary phenotype characterized by reduced voluntary exercise and increased adiposity. Whole-genome bisulfite sequencing (WGBS) and transcriptional profiling in neuronal nuclei from the arcuate nucleus of the hypothalamus (ARH) reveal differentially methylated genomic regions and reduced expression of AgRP neuron-associated genes in knockout mice. We use read-level analysis of WGBS data to infer putative ARH neural cell types affected by the knockout, and to localize promoter hypomethylation and increased expression of the growth factor Bmp7 to AgRP neurons, suggesting a role for aberrant TGF-β signaling in the development of this phenotype. Together, these data demonstrate that DNA methylation in AgRP neurons is required for their normal epigenetic development and neuron-specific gene expression profiles, and regulates voluntary exercise behavior.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

DNA methylation in AgRP neurons regulates voluntary exercise behavior in mice

et al. 2019

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“…This strongly suggested that DNA methylation is at least in part related to transcriptional repression of FBN1 . To further examine the correlation between DNA methylation and FBN1 expression, the previously proposed Hypo-allele concept [ 27 ] was applied to FBN1 . CpG island shores are involved in the tissue/cell-type-dependent gene expression [ 14 , 15 ].…”

Section: Resultsmentioning

confidence: 99%

“…DNA methylation analysis by sodium bisulfite polymerase chain reaction (PCR) followed by sequencing of cloned PCR fragments enabled us to identify DNA methylation patterns of each sequenced fragment that can be recognized as one allele/cell in a tissue. Based on this concept, we previously focused on transcriptionally active DNA hypomethylated alleles (Hypo-alleles), which can be recognized by the high ratio of unmethylated CpGs in each sequenced fragment (allele), and indicated that Hypo-allele ratio of a non-imprinted and autosomal gene can be used to estimate the proportion of the cell type expressing the corresponding gene in the tissue [ 27 ]. Therefore, the Hypo-allele concept can be applied to the detection of transcriptional regulation in each allele involved in certain tissues or cell populations.…”

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confidence: 99%

Establishment of DNA methylation patterns of the <i>Fibrillin1</i> (<i>FBN1</i>) gene in porcine embryos and tissues

Arai

Umeyama

Takeuchi

et al. 2017

Journal of Reproduction and Development

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DNA methylation in transcriptional regulatory regions is crucial for gene expression. The DNA methylation status of the edges of CpG islands, called CpG island shore, is involved in tissue/cell-type-specific gene expression. Haploinsufficiency diseases are caused by inheritance of one mutated null allele and are classified as autosomal dominant. However, in the same pedigree, phenotypic variances are observed despite the inheritance of the identical mutated null allele, including Fibrillin1 (FBN1), which is responsible for development of the haploinsufficient Marfan disease. In this study, we examined the relationship between gene expression and DNA methylation patterns of the FBN1 CpG island shore focusing on transcriptionally active hypomethylated alleles (Hypo-alleles). No difference in the DNA methylation level of FBN1 CpG island shore was observed in porcine fetal fibroblast (PFF) and the liver, whereas FBN1 expression was higher in PFF than in the liver. However, Hypo-allele ratio of the FBN1 CpG island shore in PFF was higher than that in the liver, indicating that Hypo-allele ratio of the FBN1 CpG island shore likely correlated with FBN1 expression level. In addition, oocyte-derived DNA hypermethylation in preimplantation embryos was erased until the blastocyst stage, and re-methylation of the FBN1 CpG island shore was observed with prolonged in vitro culture of blastocysts. These results suggest that the establishment of the DNA methylation pattern within the FBN1 CpG island shore occurs after the blastocyst stage, likely during organogenesis. In conclusion, Hypo-allele ratios of the FBN1 CpG island shore correlated with FBN1 expression levels in porcine tissues.

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“…Another method, epiG [12], does utilize methylation patterns in WGBS reads, but identifies only one dominant epi-haplotype in each sample, essentially disregarding cell type heterogeneity. Other attempts at exploring readlevel information in WGBS data sets have focused on minority regions such as those exhibiting "bipolar methylation" [13] or containing "hypo-methylated alleles" [14], rather than assessing the full breadth and depth of potential cell type-specific signals.…”

Section: Introductionmentioning

confidence: 99%

Identification of cell type-specific methylation signals in bulk whole genome bisulfite sequencing data

et al. 2020

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Background: The traditional approach to studying the epigenetic mechanism CpG methylation in tissue samples is to identify regions of concordant differential methylation spanning multiple CpG sites (differentially methylated regions). Variation limited to single or small numbers of CpGs has been assumed to reflect stochastic processes. To test this, we developed software, Cluster-Based analysis of CpG methylation (CluBCpG), and explored variation in read-level CpG methylation patterns in whole genome bisulfite sequencing data. Results: Analysis of both human and mouse whole genome bisulfite sequencing datasets reveals read-level signatures associated with cell type and cell type-specific biological processes. These signatures, which are mostly orthogonal to classical differentially methylated regions, are enriched at cell type-specific enhancers and allow estimation of proportional cell composition in synthetic mixtures and improved prediction of gene expression. In tandem, we developed a machine learning algorithm, Precise Read-Level Imputation of Methylation (PReLIM), to increase coverage of existing whole genome bisulfite sequencing datasets by imputing CpG methylation states on individual sequencing reads. PReLIM both improves CluBCpG coverage and performance and enables identification of novel differentially methylated regions, which we independently validate. Conclusions: Our data indicate that, rather than stochastic variation, read-level CpG methylation patterns in tissue whole genome bisulfite sequencing libraries reflect cell type. Accordingly, these new computational tools should lead to an improved understanding of epigenetic regulation by DNA methylation.

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

Cited by 7 publications

References 26 publications

DNA methylation in AgRP neurons regulates voluntary exercise behavior in mice

DNA methylation in AgRP neurons regulates voluntary exercise behavior in mice

Establishment of DNA methylation patterns of the <i>Fibrillin1</i> (<i>FBN1</i>) gene in porcine embryos and tissues

Identification of cell type-specific methylation signals in bulk whole genome bisulfite sequencing data

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