| Abstract [eng] |
5-methylcytosine (5mC) is a DNA modification, important for eukaryotic gene regulation. TET proteins produce oxidized forms of 5mC (oxi-mCs) – 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). To understand the functions of oxi-mCs, accurate and cost-efficient genomic profiling methods are needed. In this work we investigated the 5caC decarboxylation by DNA methyltransferases and combined this reaction with the economical nucleotide-resolution TOP-seq method to develop a sensitive and specific 5caC profiling method caCLEAR (5caC clearance). caCLEAR analysis showed differences in the genomic distribution of 5caC between two pluripotency states of mouse embryonic stem cells, grown in serum-2i and serum conditions. Furthermore, we found an asymmetric distribution of 5caC in the antisense strand of active protein-coding genes of these cells. We also studied the basidiomycete fungi Laccaria bicolor and Coprinopsis cinerea, which have many homologues of TET genes, and for the first time, identified oxi-mCs in L. bicolor DNA. In these fungi we mapped genomic 5hmC and 5fC with the high-resolution 5hmC-specific hmTOP-seq method and developed its novel modification for 5fC profiling, foTOP-seq. A detailed assessment of 5hmC, 5fC and 5mC influence on gene expression showed differences between these organisms. For the first time, we detected oxi-mCs in RNA of both fungi and created a method facilitating the detection of 5caC in RNA by mass spectrometry which allowed us to analyze the abundance of 5caC in different RNA fractions. |
