immunostaining-for-dna-modifications-computational-analysis-confocal

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

generation-hipsc-derived-intestinal-organoids-for-developmental

Generation of hiPSC-Derived Intestinal Organoids for Developmental and Disease Modelling Applications

hiPSC-derived intestinal organoids are epithelial structures that self-assemble from differentiated cells into complex 3D structures, representative of ...

University of Nottingham

University of Nottingham Biodiscovery Institute

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.

Paulina Durczak

Division of Cancer and Stem Cells,
School of Medicine,
Centre for Biomolecular Sciences,
Division of Cancer and Stem Cells, School of Medicine, Centre for Biomolecular Sciences

University of Nottingham

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

Generation of hiPSC-Derived Intestinal Organoids for Developmental and Disease Modelling Applications

Paulina Durczak

Division of Cancer and Stem Cells,School of Medicine,Centre for Biomolecular Sciences,Division of Cancer and Stem Cells, School of Medicine, Centre for Biomolecular Sciences

University of Nottingham

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

Generation of hiPSC-Derived Intestinal Organoids for Developmental and Disease Modelling Applications

Division of Cancer and Stem Cells,
School of Medicine,
Centre for Biomolecular Sciences,
Division of Cancer and Stem Cells, School of Medicine, Centre for Biomolecular Sciences