Other Publications (1)
Articles by Lara C. Lewis in JoVE
Immunostaining for DNA Modifications: Computational Analysis of Confocal Images Ashley H. Ramsawhook*1, Lara C. Lewis*1, Maria Eleftheriou1, Abdulkadir Abakir1, Paulina Durczak1, Robert Markus2, Seema Rajani2, Nicholas R.F. Hannan1, Beth Coyle3, Alexey Ruzov1 1Division of Cancer and Stem Cells, School of Medicine, Centre for Biomolecular Sciences, University of Nottingham, 2School of Life Sciences Imaging (SLIM), School of Life Sciences, University of Nottingham, 3Children's Brain Tumour Research Centre, School of Medicine, QMC, University of Nottingham Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.
Other articles by Lara C. Lewis on PubMed
Dynamics of 5-carboxylcytosine During Hepatic Differentiation: Potential General Role for Active Demethylation by DNA Repair in Lineage Specification Epigenetics. Apr, 2017 | Pubmed ID: 28267381 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.