title: Dynamics of 5-carboxylcytosine during hepatic differentiation: Potential general role for active demethylation by DNA repair in lineage specification.
abstract: Patterns of DNA methylation (5-methylcytosine, 5mC) are rearranged during differentiation contributing to the regulation of cell type-specific gene expression. TET proteins oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be recognized and excised from DNA by thymine-DNA glycosylase (TDG) followed by the subsequent incorporation of unmodified cytosine into the abasic site via the base excision repair (BER) pathway. We previously demonstrated that 5caC accumulates during lineage specification of neural stem cells (NSCs) suggesting that such active demethylation pathway is operational in this system; however, it is still unknown if TDG/BER-dependent demethylation is used during other types of cellular differentiation. Here we analyze dynamics of the global levels of 5hmC and 5caC during differentiation of human pluripotent stem cells toward hepatic endoderm. We show that, similar to differentiating NSCs, 5caC transiently accumulates during hepatic differentiation. The levels of 5caC increase during specification of foregut, peak at the stage of hepatic endoderm commitment, and drop in differentiating cells concurrently with the onset of expression of α fetoprotein, a marker of committed hepatic progenitors. Moreover, we show that 5caC accumulates at promoter regions of several genes expressed during hepatic specification at differentiation stages corresponding to the beginning of their expression. Our data indicate that transient 5caC accumulation is a common feature of 2 different types (neural/glial and endoderm/hepatic) of cellular differentiation. This suggests that oxidation of 5mC may represent a general mechanism of rearrangement of 5mC profiles during lineage specification of somatic cells in mammals.
authors.affiliation: a Division of Cancer and Stem Cells , School of Medicine, Centre for Biomolecular Sciences, University of Nottingham , Nottingham , UK.
authors.forename: Lara C
authors.initials: LC
authors.lastname: Lewis
authors.affiliation: a Division of Cancer and Stem Cells , School of Medicine, Centre for Biomolecular Sciences, University of Nottingham , Nottingham , UK.|b National Institute for Health Research (NIHR) Nottingham Digestive Diseases Biomedical Research Unit, Nottingham University Hospitals NHS Trust and University of Nottingham , Nottingham , UK.
authors.forename: Peggy Cho Kiu
authors.initials: PC
authors.lastname: Lo
authors.affiliation: c New England Biolabs, Inc. , Ipswich , USA.
authors.forename: Jeremy M
authors.initials: JM
authors.lastname: Foster
authors.affiliation: c New England Biolabs, Inc. , Ipswich , USA.
authors.forename: Nan
authors.initials: N
authors.lastname: Dai
authors.affiliation: c New England Biolabs, Inc. , Ipswich , USA.
authors.forename: Ivan R
authors.initials: IR
authors.lastname: Corrêa
authors.affiliation: a Division of Cancer and Stem Cells , School of Medicine, Centre for Biomolecular Sciences, University of Nottingham , Nottingham , UK.
authors.forename: Paulina M
authors.initials: PM
authors.lastname: Durczak
authors.affiliation: a Division of Cancer and Stem Cells , School of Medicine, Centre for Biomolecular Sciences, University of Nottingham , Nottingham , UK.
authors.forename: Gary
authors.initials: G
authors.lastname: Duncan
authors.affiliation: a Division of Cancer and Stem Cells , School of Medicine, Centre for Biomolecular Sciences, University of Nottingham , Nottingham , UK.
authors.forename: Ashley
authors.initials: A
authors.lastname: Ramsawhook
authors.affiliation: b National Institute for Health Research (NIHR) Nottingham Digestive Diseases Biomedical Research Unit, Nottingham University Hospitals NHS Trust and University of Nottingham , Nottingham , UK.
authors.forename: Guruprasad Padur
authors.initials: GP
authors.lastname: Aithal
authors.affiliation: a Division of Cancer and Stem Cells , School of Medicine, Centre for Biomolecular Sciences, University of Nottingham , Nottingham , UK.
authors.forename: Chris
authors.initials: C
authors.lastname: Denning
authors.affiliation: a Division of Cancer and Stem Cells , School of Medicine, Centre for Biomolecular Sciences, University of Nottingham , Nottingham , UK.|b National Institute for Health Research (NIHR) Nottingham Digestive Diseases Biomedical Research Unit, Nottingham University Hospitals NHS Trust and University of Nottingham , Nottingham , UK.
authors.forename: Nicholas R F
authors.initials: NR
authors.lastname: Hannan
authors.affiliation: a Division of Cancer and Stem Cells , School of Medicine, Centre for Biomolecular Sciences, University of Nottingham , Nottingham , UK.
authors.forename: Alexey
authors.initials: A
authors.lastname: Ruzov
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D019070:Cell Lineage; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D019175:DNA Methylation; D004260:DNA Repair; D006801:Humans; D008099:Liver / Q000166:cytology*
chemical_list: C560974:5-carboxylcytosine; D003596:Cytosine
keywords: 5-carboxylcytosine; 5-hydroxymethylcytosine; DNA methylation; TET1/2/3 proteins; definitive endoderm specification; hepatic differentiation; hepatocytes; human pluripotent stem cells; immunohistochemistry