Mechanistic insights into the recognition of 5-methylcytosine oxidation derivatives by the SUVH5 SRA domain

Abstract

5-Methylcytosine (5 mC) is associated with epigenetic gene silencing in mammals and plants. 5 mC is consecutively oxidized to 5-hydroxymethylcytosine (5 hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by ten-eleven translocation enzymes. We performed binding and structural studies to investigate the molecular basis of the recognition of the 5 mC oxidation derivatives in the context of a CG sequence by the SET- and RING-associated domain (SRA) of the SUVH5 protein (SUVH5 SRA). Using calorimetric measurements, we demonstrate that the SRA domain binds to the hydroxymethylated CG (5hmCG) DNA duplex in a similar manner to methylated CG (5mCG). Interestingly, the SUVH5 SRA domain exhibits weaker affinity towards carboxylated CG (5caCG) and formylated CG (5fCG). We report the 2.6 Å resolution crystal structure of the SUVH5 SRA domain in a complex with fully hydroxymethyl-CG and demonstrate a dual flip-out mechanism, whereby the symmetrical 5hmCs are simultaneously extruded from the partner strands of the DNA duplex and are positioned within the binding pockets of individual SRA domains. The hydroxyl group of 5hmC establishes both intra- and intermolecular interactions in the binding pocket. Collectively, we show that SUVH5 SRA recognizes 5hmC in a similar manner to 5 mC, but exhibits weaker affinity towards 5 hmC oxidation derivatives.

UHRF1-SRA recognizes symmetric non-CG methylated DNA through dual-flip out of 5-methyl cytosines

“…NKR finger in UHRF1 SRA-fully-mCHG DNA (present study) is also unstructured suggesting that this region is highly flexible or dynamic in nature. The dynamic behavior of NKR finger may allow it to adapt different conformations to recognize cytosine methylation or hydroxy-methylation through dual-flip out mechanism (Rajakumara et al 2011(Rajakumara et al , 2016Zhou et al 2014).Otherwise, NKR fingers of the two SRA molecules involved in dual-flip out recognition may have steric clashes (Rajakumara et al 2011(Rajakumara et al , 2016Zhou et al 2014) ( Supplementary Fig. 1).…”

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

“…The UHRF family member UHRF2, which features a highly similar domain architecture to UHRF1 [17,18], is a reader with increased affinity for hmC in neuronal progenitor cells [19]. Other examples include SUVH5, which binds both mC and hmC with similar strength [20], while POL II, WT1 and TET3 specifically recognize caC [21][22][23]. It is currently unclear how frequent certain CpG modification patterns occur in vivo.…”

“…In contrast to mC and hmC, the structural effects of fC and caC variants on UHRF1-DNA binding are still not well elucidated. Investigations of several SRA domains by Rajakumara et al suggest a reduced affinity of UHRF1 towards hemi-hmC, -fC and-caC containing DNA [20]. Crystal structures of POL II and TDG, which exhibit specific activity towards caC, show that the caC carboxyl group participates in specific hydrogen bond networks, which are crucial for binding key recognition residues in the protein [21,34].…”

Biochemical and dynamic basis for combinatorial recognition of H3R2K9me2 by dual domains of UHRF1

“…1) [16,17], and combinatorial recognition is required for the epigenetic inheritance of DNA methylation in vivo [18,19]. The SRA domain, of the plant SUVH histone methyltransferase, recognizes 5-methylcytosine (5mC) and 5-hydroxymethyl cytosine (5hmC) in different sequence contexts and methylation statuses [20,21]. Recognition of hemi-mCG by the UHRF1 SRA leads to allosteric activation of the UHRF1 to recognize the H3R2 and H3K9me2 marks by its TTD-PHD cassette [2,3,19,[22][23][24][25][26].…”