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.

Enhanced immunological detection of epigenetic modifications of DNA in healthy and cancerous cells by fluorescence microscopy

“…The application of acid can vary according to a range of conditions, including concentration and incubation. The 2-4 N HCl has been used for the unmasking of methylation (Zhong et al, 2017;Zhou et al, 2011) and for hydroxymethylation (Lewis et al, 2017;Zhong et al, 2017) at 37 C (Alioui et al, 2012) or at room temperature (Jafarpour et al, 2017). Some studies have included the use of trypsin following an acid treatment for the detection of methylation (Heras, Smits, De Schauwer, & Van Soom, 2017;Li & O'Neill, 2012), and hydroxymethylation (Heras et al, 2017).…”

“…The standard agent used for antigen retrieval is hydrochloric acid, which denatures the proteins around DNA. For cytosine modifications, an acid treatment has been commonly used for antigen retrieval (Lewis et al, ; Zhong et al, ; Zhou et al, ), but few studies have also included heat‐induced retrieval with buffers (Baker, Zhang, Jin, & Jass, ). To the best of knowledge, there is no study including treatment(s) with proteolytic enzymes to discard proteins from methylated (modified) DNA.…”

The Dynamic DNA Demethylation during Postnatal Neuronal Development and Neural Stem Cell Differentiation

“…During neuronal development, 5hmC not only displayed cell- and tissue-specific features but also displayed dynamic features in the genome [ 7 , 18 , 19 ]. 5hmC, 5fC, and 5caC were shown to be enriched in distinct genomic regions including promoters and enhancers [ 7 , 31 – 33 ] and to display dynamic alteration during lineage specification [ 34 , 35 ]. Thus, it is rational to speculate that the dynamic and specific distribution of active DNA demethylation could contribute to the proper gene expression during neuronal development.…”

Epigenetic Regulations in Neural Stem Cells and Neurological Diseases

“…Interestingly, for individual tissues, the levels of 5hmC, 5fC, and 5caC were not significantly related; for example, although 5hmC is more abundant in mouse brains than in ESCs, the levels of 5fC and 5caC are less abundant [ 45 ]. 5fC and 5caC can be further removed by the base excision repair (BER) pathway and thymine-DNA glycosylase [ 60 ]. This pattern suggests that the different steps of demethylation cycle are different in different tissues [ 61 ].…”