Transient Accumulation of 5-Carboxylcytosine Indicates Involvement of Active Demethylation in Lineage Specification of Neural Stem Cells

Abstract

5-Methylcytosine (5mC) is an epigenetic modification involved in regulation of gene activity during differentiation. Tet dioxygenases oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be excised from DNA by thymine-DNA glycosylase (TDG) followed by regeneration of unmodified cytosine via the base excision repair pathway. Despite evidence that this mechanism is operative in embryonic stem cells, the role of TDG-dependent demethylation in differentiation and development is currently unclear. Here, we demonstrate that widespread oxidation of 5hmC to 5caC occurs in postimplantation mouse embryos. We show that 5fC and 5caC are transiently accumulated during lineage specification of neural stem cells (NSCs) in culture and in vivo. Moreover, 5caC is enriched at the cell-type-specific promoters during differentiation of NSCs, and TDG knockdown leads to increased 5fC/5caC levels in differentiating NSCs. Our data suggest that active demethylation contributes to epigenetic reprogramming determining lineage specification in embryonic brain.

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

“…NOTE: Sections processed without primary antibodies can serve as suitable negative controls for the staining procedure. The 12.5 days post coitum murine embryonic brain sections enriched in 5caC, 5fC and 5hmC 20 can be used as positive control.…”

“…Most of the biochemical and antibody based approaches do not provide any spatial information regarding the distribution of the modified forms of 5mC in different tissue and cell-types. In contrast, immunochemistry based techniques can provide a rapid tool for assessing the spatial distribution and nuclear localization of 5mC 28 and its oxidized derivatives 20 . That is said, the reported very low abundance of 5fC (20 in every 10 6 cytosine) and 5caC (3 in every 10 6 cytosine) in the mouse genome 18 represents a significant challenge for standard immunochemistry.…”

“…Discovery of the products of 5mC sequential oxidation mediated by ten-eleven translocation enzymes (tet1/2/3) such as 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) in eukaryotic DNA 16,17,18,19 prompted speculations whether they may serve as intermediates of DNA demethylation processes or act as stable epigenetic marks in their own right 13 . While the demonstration that the component of base excision repair, thymine-DNA glycosylase (TDG) can bind and remove both 5fC and 5caC from DNA 19,20 suggests a role for the modified 5mC derivatives in an active DNA demethylation. Recent evidence showing that 5fC/5caC can modulate the rate of RNA II processivity points to potential involvement of these marks in transcriptional regulation 29 .…”

“…Recent evidence showing that 5fC/5caC can modulate the rate of RNA II processivity points to potential involvement of these marks in transcriptional regulation 29 . Due to this potential biological importance of oxidized forms of 5mC, a range of biochemical and antibody based techniques have been employed for studying their genomic distribution and global content 16,[19][20][21][22][23][24] .…”

Detection of 5-carboxylcytosine in Mammalian Cells DNA

“…Cytosine (Cyt) modification in DNA is an important epigenetic marker that plays regulatory roles in a variety of cellular processes, such as embryogenesis, carcinogenesis and gene depression [1][2][3][4]. Mammalian DNA cytosine modification is a dynamic process ( Figure 1).…”