The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain

Abstract

Despite the importance of epigenetic regulation in neurological disorders, little is known about neuronal chromatin. Cerebellar Purkinje neurons have large and euchromatic nuclei, whereas granule cell nuclei are small and have a more typical heterochromatin distribution. While comparing the abundance of 5-methylcytosine in Purkinje and granule cell nuclei, we detected the presence of an unusual DNA nucleotide. Using thin-layer chromatography, high-pressure liquid chromatography, and mass spectrometry, we identified the nucleotide as 5-hydroxymethyl-2'-deoxycytidine (hmdC). hmdC constitutes 0.6% of total nucleotides in Purkinje cells, 0.2% in granule cells, and is not present in cancer cell lines. hmdC is a constituent of nuclear DNA that is highly abundant in the brain, suggesting a role in epigenetic control of neuronal function.

Fig. 1

Quantification of mC and “x” abundance in Purkinje and granule neurons. A, 2D TLC separation of nucleoside monophosphates from genomic DNA in Purkinje and granule cells. B, reference map to the TLC spots (A is dAMP, etc) (14), with added “x” position. C, percentage shows the abundance of a nucleotide neighboring G. Error bars show SEM (n=11), p values were derived from Matt-Whitney statistics. Neither abundance of dTMP or dAMP were different between the samples (p=0.743 and p=0.793 respectively).

Fig. 2

2D TLC, HPLC and MS identification of hmC. A, 2D TLC analysis of synthetic DNA templates indicates that hmC comigrates with the “x” spot (Fig 1). B, HPLC chromatograms (A, 254 nm) of the nucleosides derived from synthetic and cerebellum DNA. The peaks were identified using MS. The black arrow points to the peak, which elutes at the same time as hmdC. C, MS of the fraction corresponding to the HPLC peak indicated above. Closed black arrows indicate the masses of 5-hydroxymethylcytosine and 5-hydroxymethyl-2′-deoxylcytidine sodium ions (structures are shown on the insets). Open arrows indicate the ions generated by 2′-deoxycytidine, which elutes in a large nearby peak and spills over into the analyzed fraction.