Deciphering Epigenetic Cytosine Modifications by Direct Molecular Recognition

Abstract

Epigenetic modification at the 5-position of cytosine is a key regulatory element of mammalian gene expression with important roles in genome stability, development, and disease. The repertoire of cytosine modifications has long been confined to only 5-methylcytosine (mC) but has recently been expanded by the discovery of 5-hydroxymethyl-, 5-formyl-, and 5-carboxylcytosine. These are key intermediates of active mC demethylation but may additionally represent new epigenetic marks with distinct biological roles. This leap in chemical complexity of epigenetic cytosine modifications has not only created a pressing need for analytical approaches that enable unraveling of their functions, it has also created new challenges for such analyses with respect to sensitivity and selectivity. The crucial step of any such approach that defines its analytic potential is the strategy used for the actual differentiation of the cytosine 5-modifications from one another, and this selectivity can in principle be provided either by chemoselective conversions or by selective, molecular recognition events. While the former strategy has been particularly successful for accurate genomic profiling of cytosine modifications in vitro, the latter strategy provides interesting perspectives for simplified profiling of natural, untreated DNA, as well as for emerging applications such as single cell analysis and the monitoring of cytosine modification in vivo. We here review analytical techniques for the deciphering of epigenetic cytosine modifications with an emphasis on approaches that are based on the direct molecular recognition of these modifications in DNA.

Whole genome DNA methylation: beyond genes silencing

“…Furthermore, it is worth mentioning that enzymatic modification of cytosine is a complex dynamic involving DNMT1, DNMT3A and DNMT3B methyltrasferases, which methylates cytosines (5mC), and the TET family of cytosine oxygenase enzymes, which oxidizes 5mC to 5-hydroxymethylcytosine (5hmC), subsequently to 5-formylcytosine (5fC) and finally to 5-carboxycytosine (5caC) [50, 51]. These oxidized derivatives might also hinder TF binding.…”

Recognition of Oxidized 5‐Methylcytosine Derivatives in DNA by Natural and Engineered Protein Scaffolds

“…In contrast to proteins without or with limited sequence selectivity, a complementary potential lies in proteins that provide programmable sequence selectivity. These promise an increased resolution (defined by their interaction range with DNA, i. e. ∼20–30 nt), and a direct enrichment of only the target sequence of interest (for the cost of requiring a dedicated protein for each targeted sequence) . This requires scaffolds that bind DNA via the major groove, the DNA face typically recognized by sequence‐specific DNA binding proteins.…”

“…These promise an increased resolution (defined by their interaction range with DNA, i. e.~20-30 nt), and a direct enrichment of only the target sequence of interest (for the cost of requiring a dedicated protein for each targeted sequence). [104] This requires scaffolds that bind DNA via the major groove, the DNA face typically recognized by sequence-specific DNA binding proteins. The first scaffolds used for the evolutionary design of DNA binding domains with programmable sequence selectivity have been Cys 2 His 2 ZFP [105][106][107][108] (for reviews, see [109,110] ) Each zinc finger repeat thereby recognizes three nucleobases in dsDNA and multiple repeats with designed triplet selectivities can be concatenated to arrays for the recognition of longer DNA sequences.…”

The expanding scope and impact of epigenetic cytosine modifications

“…Exogenous proteins such as restriction endonucleases provide a powerful tool to probe and manipulate ox-mC bases in vitro and in vivo [41]. For example, AbaSI, initially found to restrict T-even phage, selectively cleaves DNA containing hmC or glucosyl-hmC; one sequencing method harnessed this enzyme to localize hmC in mammalian genomes [42].…”