Methylated Cytosines Mutate to Transcription Factor Binding Sites that Drive Tetrapod Evolution

Abstract

In mammals, the cytosine in CG dinucleotides is typically methylated producing 5-methylcytosine (5mC), a chemically less stable form of cytosine that can spontaneously deaminate to thymidine resulting in a T•G mismatched base pair. Unlike other eukaryotes that efficiently repair this mismatched base pair back to C•G, in mammals, 5mCG deamination is mutagenic, sometimes producing TG dinucleotides, explaining the depletion of CG dinucleotides in mammalian genomes. It was suggested that new TG dinucleotides generate genetic diversity that may be critical for evolutionary change. We tested this conjecture by examining the DNA sequence properties of regulatory sequences identified by DNase I hypersensitive sites (DHSs) in human and mouse genomes. We hypothesized that the new TG dinucleotides generate transcription factor binding sites (TFBS) that become tissue-specific DHSs (TS-DHSs). We find that 8-mers containing the CG dinucleotide are enriched in DHSs in both species. However, 8-mers containing a TG and no CG dinucleotide are preferentially enriched in TS-DHSs when compared with 8-mers with neither a TG nor a CG dinucleotide. The most enriched 8-mer with a TG and no CG dinucleotide in tissue-specific regulatory regions in both genomes is the AP-1 motif (TGAC/GTCAN), and we find evidence that TG dinucleotides in the AP-1 motif arose from CG dinucleotides. Additional TS-DHS-enriched TFBS containing the TG/CA dinucleotide are the E-Box motif (GCAGCTGC), the NF-1 motif (GGCA—TGCC), and the GR (glucocorticoid receptor) motif (G-ACA—TGT-C). Our results support the suggestion that cytosine methylation is mutagenic in tetrapods producing TG dinucleotides that create TFBS that drive evolution.

The Epstein-Barr Virus B-ZIP Protein Zta Recognizes Specific DNA Sequences Containing 5-Methylcytosine and 5-Hydroxymethylcytosine

“…We next examined three additional B-ZIP domains, the cJun|cFos heterodimer (data from 19 ), CREB1 (data from this study), and CEBPB (data from 20 ) binding to DNA 8-mers containing methylated CG dinucleotides (Figure 2B–D, S4B–D). For cJun|cFos heterodimers, the best bound 8-mers contain the TRE motif, as expected (Figure 2B, highlighted gray spots).…”

“…Zta was obtained as a GST construct cloned into a modified pDEST15 MAGIC vector (N-terminal GST) vector 18 . The DBD of cFOS was obtained as a construct cloned into a pT5 expression plasmid 19 . Proteins were expressed using the PURExpress In vitro protein synthesis kit (NEB) according to the manufacturer’s protocol in a 25μL reaction volume containing 180 ng of plasmid 20 .…”

5-Hydroxymethylcytosine in E-box motifs ACAT|GTG and ACAC|GTG increases DNA-binding of the B-HLH transcription factor TCF4

“…This results in a hemi-methylated or hemi-hydroxymethylated state. DNA double-stranding was performed as previously described 1, 27 .…”

“…The remaining 2% of the genome is CG dinucleotide rich regions, known as CG islands (CGIs) that are typically unmethylated and often have housekeeping gene regulatory functions 1 . Two recent observations in mammals have expanded the possibilities for sequence-specific DNA binding of transcription factors (TFs).…”

The Genomic Impact of DNA CpG Methylation on Gene Expression; Relationships in Prostate Cancer

“…Methylated cytosine is a dynamic modification not only because it is added and removed by enzymatic processes, but also because it can spontaneously deaminate and, therefore, mutate [15,16,17]. By contrast, spontaneous deamination of unmethylated cytosine is readily recognized by the DNA repair machinery and corrected.…”