Systematic analysis of the binding behaviour of UHRF1 towards different methyl- and carboxylcytosine modification patterns at CpG dyads

Abstract

The multi-domain protein UHRF1 is essential for DNA methylation maintenance and binds DNA via a base-flipping mechanism with a preference for hemi-methylated CpG sites. We investigated its binding to hemi-and symmetrically modified DNA containing either 5methylcytosine (mC), 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), or 5-carboxylcytosine (caC). Our experimental results indicate that UHRF1 binds symmetrically carboxylated and hybrid methylated/carboxylated CpG dyads in addition to its previously reported substrates. Complementary molecular dynamics simulations provide a possible mechanistic explanation of how the protein could differentiate between modification patterns. First, we observe different local binding modes in the nucleotide binding pocket as well as the protein's NKR finger. Second, both DNA modification sites are coupled through key residues within the NKR finger, suggesting a communication pathway affecting protein-DNA binding for carboxylcytosine modifications. Our results suggest a possible additional function of the hemi-methylation reader UHRF1 through binding of carboxylated CpG sites. This opens the possibility of new biological roles of UHRF1 beyond DNA methylation maintenance and of oxidised methylcytosine derivates in epigenetic regulation.

Structural dynamics of double-stranded DNA with epigenome modification

“…During preparation of this manuscript, it was reported that UHRF1 binds to fully carboxyl-methylated dsDNA (5caC/5caC) with high affinity ( 48 ). Therefore, we additionally prepared this type of dsDNA and analysed its structural dynamics by the CLEANEX-PM and imino 1 H R 1ρ relaxation dispersion experiments.…”

Unraveling the functional role of DNA methylation using targeted DNA demethylation by steric blockage of DNA methyltransferase with CRISPR/dCas9

“…1), but the results leave us with unanswered questions on whether DNA demethylation of the basal promoter was causal for this activation. First, TET enzymes are not enzymatically demethylases but monooxygenases which oxidize 5-methylcytosine to 5hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine, which have demonstrated stability [43,44], demonstrated differential protein interactors [21][22][23][24][25][26], and demonstrated structural effects on DNA [27], suggesting that each derivative may be a unique epigenetic mark in its own right. We show here that dCas9-TET causes hydroxymethylation of the Il33-002 promoter that is maintained in culture (Supp.…”

“…More recently, the TET dioxygenases -which oxidize the methyl moiety in cytosine and can lead to passive loss of methylation by either inhibiting methylation during replication or through repair of the oxidized methylcytosine and its replacement with an unmethylated cytosine -were targeted to specific sites using a fusion of TET dioxygenase domains to catalytically inactive CRISPR/Cas9 (deltaCas9 or dCas9) [17][18][19][20]. However, this method still introduces several confounding factors that preclude causational inferences, such as the fact that oxidized methylcytosines are new epigenetic modifications that are not unmethylated cytosines [21][22][23][24][25][26][27] and the fact that TET has methylation-independent transcriptional activation activity [28,29]. We propose and optimize instead an enzyme-free CRISPR/dCas9-based system for targeted methylation editing which we show to be able to achieve selective methylation in vitro and passive demethylation in cells through steric interference with DNA methyltransferase activity.…”

Staying true to yourself: mechanisms of DNA methylation maintenance in mammals

“…However, despite this seemingly critical function of the TTD, in mouse ES cells ( 111 , 112 ), and in human cancer lines ( 113 ), a mutant version of UHRF1 with an inactivated TTD can almost fully substitute for the wild-type protein to ensure steady-state DNA methylation levels. The last structural domain of UHRF1 is the SRA (SET and RING finger-associated), which binds hemi-methylated CpGs and also, with less affinity, fully-methylated or even unmethylated CpGs ( 53 , 114 ); this activity is essential for DNA methylation maintenance in cells ( 111 , 113 ).…”