title: Ultra-Deep Bisulfite Sequencing to Detect Specific DNA Methylation Patterns of Minor Cell Types in Heterogeneous Cell Populations: An Example of the Pituitary Tissue.
abstract: 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.
authors.affiliation: Laboratory of Genomic function Engineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Japan.
authors.forename: Yoshikazu
authors.initials: Y
authors.lastname: Arai
authors.affiliation: Laboratory of Genomic function Engineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Japan.
authors.forename: Hisho
authors.initials: H
authors.lastname: Fukukawa
authors.affiliation: Laboratory of Genomic function Engineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Japan.
authors.forename: Takanori
authors.initials: T
authors.lastname: Atozi
authors.affiliation: Laboratory of Genomic function Engineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Japan.
authors.forename: Shoma
authors.initials: S
authors.lastname: Matsumoto
authors.affiliation: Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University, Tochigi, Japan.|CREST, Japan Science and Technology Agency, Tokyo, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Hanazono
authors.affiliation: Laboratory of Developmental Engineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Japan.|Meiji University International Institute for Bio-Resource Research (MUIIBR), Kawasaki, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nagashima
authors.affiliation: Laboratory of Genomic function Engineering, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Ohgane
mesh_terms: D000483:Alleles; D000818:Animals; D018899:CpG Islands / Q000235:genetics; D019175:DNA Methylation / Q000235:genetics / Q000502:physiology*; D059014:High-Throughput Nucleotide Sequencing; D010902:Pituitary Gland / Q000378:metabolism*; D013552:Swine