Abstract
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.
Highlights
Methylation of cytosine bases in DNA (5mC) represents a major epigenetic mark found in vertebrates genomes associated with transcriptional silencing1. 5mC is being introduced and maintained by DNA methyltransferases[2,3,4,5], and has been shown to play important roles in a number of biological processes including genomic imprinting, X-chromosome inactivation, cellular differentiation, and development[3, 6]
Discovery of the products of 5mC sequential oxidation mediated by ten-eleven translocation enzymes 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]
To determine the distribution of 5hmC in brain tissue sections, we performed co-detection of this epigenetic modification with a marker for postmitotic neurons, NeuN, employing commercial anti-5hmC antibody that interacts with this mark but not with other forms of modified cytosine[20, 25]
Summary
Methylation of cytosine bases in DNA (5mC) represents a major epigenetic mark found in vertebrates genomes associated with transcriptional silencing1. 5mC is being introduced and maintained by DNA methyltransferases[2,3,4,5], and has been shown to play important roles in a number of biological processes including genomic imprinting, X-chromosome inactivation, cellular differentiation, and development[3, 6]. 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]. Www.jove.com circumvents the challenges of detecting the very low amounts of 5fC and 5caC This technique can be used to co-detect the modified forms of cytosine with lineage specific markers, effectively complementing other approaches in elucidating the biological functions of these epigenetic marks. All the animal-involved procedures were performed in accordance with the University of Nottingham's ethical review board
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