Detection of modified forms of cytosine using sensitive immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts

5-Carboxylcytosine is localized to euchromatic regions in the nuclei of follicular cells in axolotl ovary

5-Methylcytosine (5-mC) is an epigenetic modification associated with gene repression. Recent studies demonstrated that 5-mC can be enzymatically oxidised into 5-hydroxymethylcytosine and further into 5-formylcytosine (5-fC) and 5-carboxylcytsine (5-caC). 5-caC has been found in embryonic stem cells and in mouse pre-implantation embryos but no detectable levels of this modification have been reported for somatic tissues to date. Whereas it has been suggested that 5-caC can serve as an intermediate in the process of active demethylation, the function of this form of modified cytosine remains obscure. Here we show that 5-caC is immunochemically detectable in somatic cells of axolotl ovary. We demonstrate that both 5-hmC and 5-caC are localized to the euchromatin in the nuclei of axolotl follicular cells with similar patterns of spatial distribution. Our results suggest that 5-carboxylcytosine may play a distinct functional role in certain biological contexts

Molecular mechanisms governing the stem cell's fate in brain cancer: factors of stemness and quiescence

Cellular quiescence is a reversible, non-cycling state controlled by epigenetic, transcriptional and niche-associated molecular factors. Quiescence is a condition where molecular signaling pathways maintain the poised cell-cycle state whilst enabling rapid cell cycle re-entry. To achieve therapeutic breakthroughs in oncology it is crucial to decipher these molecular mechanisms employed by the cancerous milieu to control, maintain and gear stem cells towards re-activation. Cancer stem-like cells (CSCs) have been extensively studied in most malignancies, including glioma. Here, the aberrant niche activities skew the quiescence/activation equilibrium, leading to rapid tumor relapse after surgery and/or chemotherapy. Unraveling quiescence mechanisms promises to afford prevention of (often multiple) relapses, a key problem in current glioma treatment. This review article covers the current knowledge regarding normal and aberrant cellular quiescence control whilst also exploring how different molecular mechanisms and properties of the neighboring cells can influence the molecular processes behind glioma stem cell quiescence.

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 enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans

Dynamics of 5-carboxylcytosine during hepatic differentiation: potential general role for active demethylation by DNA repair in lineage specification

Patterns of DNA methylation (5-methylcytosine, 5mC) are rearranged during differentiation contributing to the regulation of cell type-specific gene expression. TET proteins oxidise 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Both 5fC and 5caC can be recognised 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 operative in this system, however it is still unknown if TDG/BER-dependent demethylation is utilised during other types of cellular differentiation. Here we analyse dynamics of the global levels of 5hmC and 5caC during differentiation of human pluripotent stem cells (hPSCs) towards 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 Alpha 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 commencement of their expression. Our data indicate that transient 5caC accumulation is a common feature of two 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

Molecular Mechanisms Governing the Stem Cell’s Fate in Brain Cancer: Factors of Stemness and Quiescence

Cellular quiescence is a reversible, non-cycling state controlled by epigenetic, transcriptional and niche-associated molecular factors. Quiescence is a condition where molecular signaling pathways maintain the poised cell-cycle state whilst enabling rapid cell cycle re-entry. To achieve therapeutic breakthroughs in oncology it is crucial to decipher these molecular mechanisms employed by the cancerous milieu to control, maintain and gear stem cells towards re-activation. Cancer stem-like cells (CSCs) have been extensively studied in most malignancies, including glioma. Here, the aberrant niche activities skew the quiescence/activation equilibrium, leading to rapid tumor relapse after surgery and/or chemotherapy. Unraveling quiescence mechanisms promises to afford prevention of (often multiple) relapses, a key problem in current glioma treatment. This review article covers the current knowledge regarding normal and aberrant cellular quiescence control whilst also exploring how different molecular mechanisms and properties of the neighboring cells can influence the molecular processes behind glioma stem cell quiescence

5-hydroxymethyl-cytosine enrichment of non-committed cells is not a universal feature of vertebrate development

5-hydroxymethyl-cytosine (5-hmc) is a cytosine modification that is relatively abundant in mammalian pre-implantation embryos and embryonic stem cells (Esc) derived from mammalian blastocysts. Recent observations imply that both 5-hmc and Tet1/2/3 proteins, catalyzing the conversion of 5-methyl-cytosine to 5-hmc, may play an important role in self renewal and differentiation of Escs. here we assessed the distribution of 5-hmc in zebrafish and chick embryos and found that, unlike in mammals, 5-hmc is immunochemically undetectable in these systems before the onset of organogenesis. In addition, Tet1/2/3 transcripts are either low or undetectable at corresponding stages of zebrafish development. however, 5-hmc is enriched in later zebrafish and chick embryos and exhibits tissue-specific distribution in adult zebrafish. Our findings show that 5-hmc enrichment of non-committed cells is not a universal feature of vertebrate development and give insights both into evolution of embryonic pluripotency and the potential role of 5-hmc in its regulation