In silico structural analysis of sequences containing 5-hydroxymethylcytosine reveals its potential as binding regulator for development, aging and cancer-related transcription factors

The presence of 5-hydroxymethyl cytosine in DNA has been previously associated with aging. Using in silico analysis of normal liver samples we presently observed that in 5-hydroxymethyl cytosine sequences, DNA methylation is dependent on the co-presence of G-quadruplexes and palindromes. This association exhibits discrete patterns depending on G-quadruplex and palindrome densities. DNase-Seq data show that 5-hydroxymethyl cytosine sequences are common among liver nucleosomes (<i>p</i>&lt;2.2x10⁻¹⁶) and 3-fold more frequent than nucleosome sequences. Nucleosomes lacking palindromes and potential G-quadruplexes are rare in vivo (1%) and nucleosome occupancy potential decreases with increasing G4s. Palindrome distribution is similar to that previously reported in nucleosomes. In low and mixed complexity sequences 5-hydroxymethyl cytosine is frequently located next to three elements: G-quadruplexes or imperfect G-quadruplexes with CpGs, or unstable hairpin loops (TCCCAY₆TGGGA) mostly located in antisense strands, or finally A-/T-rich segments near these motifs. The high frequencies and selective distribution of pentamer sequences (including TCCCA, TGGGA) probably indicates the positive contribution of 5-hydroxymethyl cytosine to stabilize the formation of structures unstable in the absence this cytosine modification. Common motifs identified in all total 5-hydroxymethyl cytosine-containing sequences exhibit high homology to recognition sites of several transcription factor families: homeobox, factors involved in growth, mortality/aging, cancer, neuronal function, vision and reproduction. We conclude that cytosine hydroxymethylation could play a role in the recognition of sequences with G-quadruplexes/palindromes by forming epigenetically regulated DNA “springs” and governing expansions or compressions recognized by different transcription factors or stabilizing nucleosomes. The balance of these epigenetic elements is lost in hepatocellular carcinoma.