5-hydroxymethylcytosine (5-hmC) is a modified form of cytosine recently found in mammalians and is believed, like 5-methylcytosine, to also play an important role in switching genes on and off. By utilizing a newly developed 5-hmC immunoassay, we determined the abundance of 5-hmC in human tissues and compared 5-hmC states in normal colorectal tissue and cancerous colorectal tissue. Significant differences of 5-hmC content in different tissues were observed. The percentage of 5-hmC measured is high in brain, liver, kidney and colorectal tissues (0.40–0.65%), while it is relatively low in lung (0.18%) and very low in heart, breast, and placenta (0.05-0.06%). Abundance of 5-hmC in the cancerous colorectal tissues was significantly reduced (0.02–0.06%) compared to that in normal colorectal tissues (0.46–0.57%). Our results showed for the first time that 5-hmC distribution is tissue dependent in human tissues and its abundance could be changed in the diseased states such as colorectal cancer. 1. Introduction DNA methylation is an epigenetic modification which is catalyzed by DNA cytosine-5-methyltransferases (DNMTs) and occurs at the 5-position (C5) of the cytosine ring, within CpG dinucleotides. DNA methylation is essential in regulating gene expression in nearly all biological processes including development, growth, and differentiation [1–3]. Alterations in DNA methylation have been demonstrated to cause the changes in gene expression. For example, hypermethylation leads to gene silencing or decreased gene expression while hypomethylation activates the genes or increases gene expression. Region-specific DNA methylation is mainly found in 5′-CpG-3′dinucleotides within the promoters or in the first exon of genes, which is an important pathway for the repression of gene transcription in diseased cells. Very recently, a modified nucleotide, 5-hydroxymethylcytosine (5-hmC) was detected to be abundant in mouse brain and embryonic stem cells [4–6]. 5-hmC is a hydroxylated and methylated form of cytosine and was first seen in bacteriophages in 1952 [7]. In mammals, it can be generated by a TET protein-mediated reaction [5]. The exact function of 5-hmC in epigenetics is still a mystery today. However, a line of evidence showed that conversion of 5-mC to 5-hmC greatly reduced affinity of MBD proteins to methylated DNA [8], and 5-hmC accounts for roughly 40 percent of the methylated cytosine in Purkinje cells and is also specifically localized in CpG regions [4]. Thus, 5-hmC might also play an important and different role in regulation of DNA methylation, chromatin
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