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5‐Hydroxymethylcytosine (5hmC) is produced from 5‐methylcytosine (5mC) by Ten‐eleven translocation (TET) dioxygenases. The epigenetic modification 5hmC has crucial roles in both cellular development and differentiation. The 5hmC level is particularly high in the brain. While 5mC is generally associated with gene silencing/reduced expression, 5hmC is a more permissive epigenetic mark. To understand its physiological function, an easy and accurate quantification method is required. Here, we have developed a novel LC‐MS/MS‐based approach to quantify both genomic 5mC and 5hmC contents. The method is based on the liberation of nucleobases by formic acid. Applying this method, we characterized the levels of DNA methylation and hydroxymethylation in mouse brain and liver, primary hepatocytes, and various cell lines. Using this approach, we confirm that the treatment of different cell lines with the DNA methyltransferase inhibitor 5‐aza‐2′‐deoxycytidine leads to a decrease in 5mC content. This decrease was accompanied by an increase in 5hmC levels in cell lines of hematopoietic origin. Finally, we showed that ascorbate elevates the levels of 5hmC and augments the effect of 5‐aza‐2′‐deoxycytidine without significantly influencing 5mC levels.

The methylation status of CpG dinucleotides located in or near regulatory elements affects gene expression. The CpG-rich sequence located outside the 5' promoter region of the human Tyrosine Hydroxylase (TH) gene appears to influence the functional effect of the adjacent intronic HUMTH01 microsatellite. In order to identify new regulatory elements in this region acting on gene expression, the methylation profile of the TH CpG island was investigated using the bisulfite sequencing method. The overall methylation level of this region is correlated to TH-expressing and non-expressing status in cell lines and DNA demethylation treatment with 5-azacytidine increased TH expression. Moreover, in a homogeneous background of methylated CpGs, a single CpG in the first exon of the gene is constantly either unmethylated or methylated in, respectively, TH-expressing or non-expressing cell lines, tissues and single cells. Further analysis ascertained that this CpG is contained in a sequence characterized by putative binding sites for the AP2, Sp1 and KAISO factors. Characterization of this sequence shows that these factors specifically bind their respective sites. Finally, the binding of KAISO, a transcriptional repressor, is conditioned by the methylation of this sequence, which may, thus, participate in the regulation of TH gene expression according to its methylation pattern.

Predicting the functional consequence of a given amino acid replacement, that is, the difference between the wild type protein and its nonsynonymous single nucleotide polymorph variants (nsSNPs) is a great challenge. This is especially true in the case of large membrane proteins, like ABC transporters. Kelly et al. has published such an ambitious study in a recent issue of Protein Science focusing on nsSNP in structurally conserved segments within the nucleotide binding domains (NBDs) of ABC transporters supposed to be involved in interdomain communication.1 With the aid of structural rationalization the authors predicted the impact of 40 nsSNPs found in seven clinically important human ABC transporters using a bivalent scoring: disease or neutral. We have performed a deep search of the available literature to find published data on the 40 nsSNPs to delineate them with the predictions. We have incorporated only data from original peer-reviewed publications into our dataset, and our hits were checked in various gene-specific databases too [BioBase and ABCC6 Database]. Based on the available information, we categorized the published data on a given nsSNP as (i) “disease-associated” when the publication clearly demonstrates that the variant segregates with the disease but is absent from at least 200 alleles of unrelated, nonaffected individuals; or (ii) in vitro experimental evidence when the specific variant was heterologously expressed and its (transport) function and/or folding-stability was investigated (Table I). We considered functional differences only if the level of expression of the given nsSNP variant was comparable to the wild type expressed in the same system. Our results, in comparison with the predictions of Kelly et al. are summarized in Table I. The left five columns of the table is taken from the publication of Kelly et al. On the right we list our findings and give the related primary reference. In several cases more than one independent studies were found showing the same result. To keep the list of references short, we indicated only one reference, if more publications were found we added +n (where n is the number of additional independent publications). Table I Predicted and Published Phenotype of nsSNPs in Human ABC Transporters We found the published data on 19 of the 40 ABC nsSNPs. Of these, 16 nsSNPs had been tested in in vitro experiments and in seven examples the given nsSNP resulted in impaired function and/or folding. In one case the published nsSNP generates a new splice site (exon skipping), what affects the mRNA maturation therefore the function or folding of the protein with the given nsSNP can not be concluded (ABCB11, E1186K). One of the nsSNPs was characterized solely by its association to a genetic disease (ABCC6, A1291T associated to pseudoxanthoma elasticum). One additional nsSNP was found as homozygous nsSNP in several healthy individuals therefore it was categorized as neutral, on the basis of this human genetic data (ABCC6, R1268Q). There are two nsSNPs with both published genetic association and in vitro experimental characterization in our data set (ABCB11 V444A associated to intrahepatic cholestasis of pregnancy; ABCG2, Q141K associated to gout). Our conclusions on the nsSNPs are listed in column “Published phenotype” of Table I.; for the sake of simplicity we kept the bivalent terminology of Kelly et al.: disease or neutral. Comparing the predicted phenotype of the Kelly's paper with the phenotype extracted from the published data reveals misprediction in roughly half of the cases (10/19) thus demonstrating the inherent difficulties of rationalizing and predicting the functional impact of snSNPs.