Dynamics of Methylated Cytosine Flipping by UHRF1

Abstract

DNA methylation patterns, which are critical for gene expression, are replicated by DNA methyltransferase 1 (DNMT1) and ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) proteins. This replication is initiated by the recognition of hemimethylated CpG sites and further flipping of methylated cytosines (mC) by the Set and Ring Associated (SRA) domain of UHRF1. Although crystallography has shed light on the mechanism of mC flipping by SRA, tools are required to monitor in real time how SRA reads DNA and flips the modified nucleobase. To accomplish this aim, we have utilized two distinct fluorescent nucleobase surrogates, 2-thienyl-3-hydroxychromone nucleoside (3HCnt) and thienoguanosine (thG), incorporated at different positions into hemimethylated (HM) and nonmethylated (NM) DNA duplexes. Large fluorescence changes were associated with mC flipping in HM duplexes, showing the outstanding sensitivity of both nucleobase surrogates to the small structural changes accompanying base flipping. Importantly, the nucleobase surrogates marginally affected the structure of the duplex and its affinity for SRA at positions where they were responsive to base flipping, illustrating their promise as nonperturbing probes for monitoring such events. Stopped-flow studies using these two distinct tools revealed the fast kinetics of SRA binding and sliding to NM duplexes, consistent with its reader role. In contrast, the kinetics of mC flipping was found to be much slower in HM duplexes, substantially increasing the lifetime of CpG-bound UHRF1, and thus the probability of recruiting DNMT1 to faithfully duplicate the DNA methylation profile. The fluorescence-based approach using these two different fluorescent nucleoside surrogates advances the mechanistic understanding of the UHRF1/DNMT1 tandem and the development of assays for the identification of base flipping inhibitors.

A Molecular Tool Targeting the Base‐Flipping Activity of Human UHRF1

“…To experimentally evidence that UM63 targets the 5mC binding pocket on SRA, we replaced the wild‐type SRA with a SRA‐G448D mutant where the glycine 448 residue is replaced by a more bulky aspartic acid to block the binding pocket and prevent base flipping . As expected, only marginal binding was observed with this mutant (Figure B), confirming that the 5mC binding pocket of SRA is the target of UM63, in line with molecular modeling predictions.…”

“…As expected, only marginal binding was observed with this mutant (Figure B), confirming that the 5mC binding pocket of SRA is the target of UM63, in line with molecular modeling predictions. To further explore the base‐flipping inhibition by UM63, we investigated by the stopped‐flow technique how UM63 alters the kinetics of the SRA‐induced 5mC flipping in the th G‐labeled DNA . In line with our previous study, the kinetic trace of the th G‐labeled DNA in the presence of SRA showed a slow component with a rate constant of approximately 6.5 s −1 attributed to the 5mC base‐flipping process (Figure , red curve).…”

“…This assay is based on the use of a HM DNA labeled by thienoguanosine ( th G) (Figure A), an isomorphic guanosine derivative that has been shown to perfectly replace the guanine residue next to the methylated cytosine in the CpG motif . Addition of SRA to this labeled DNA is accompanied by a 4‐fold increase in the fluorescence intensity, as a result of the SRA‐induced flipping of the 5mC residue (Figures B and C). Among the 26 compounds, UM63 was the most promising hit candidate, as it induced a concentration‐dependent decrease in the fluorescence intensity, suggesting that it could inhibit the SRA‐induced base flipping with an IC 50 value of (4.4±0.5) μ m (Figure B).…”

La première étape de la transmission d’un caractère épigénétique suivie par fluorescence

“…Dans ce contexte, l'objectif de notre travail publié dans la revue J Am Chem Soc, a été de suivre et de caractériser la dynamique du basculement des cytosines méthylées sous l'effet du domaine SRA de la protéine UHRF1 qui reconnaît l'ADN hémi-méthylé [6]. Pour atteindre cet objectif, nous avons utilisé deux analogues fluorescents de nucléosides très innovants, la 2-thiényl-3-hydroxychromone (3HCnt) capable de substituer toutes les nucléobases de l'ADN [7] et la thiénoguanosine (thG) qui remplace parfaitement la guanine [8,9].…”

“…Par ailleurs, nous avons véri-fié que (1) les analogues thG et 3HCnt n'affectent pas ou peu la stabilité du double brin d'ADN dans lequel ils sont insérés, et (2) qu'aux positions où ces sondes sont sensibles au basculement de base induit par SRA, ce domaine conserve une affinité préférentielle pour les séquences hémi-méthylées par rapport aux séquences non méthylées. Ces sondes ont permis également de déter-miner par une technique de « stoppedflow 1 » que la liaison du domaine SRA avec l'ADN est rapide et donc compatible avec l'activité de lecture de l'UHRF1 pour trouver les sites à méthyler sur le brin d'ADN néo-synthétisé [6,11]. En revanche, l'interaction de SRA avec la cytosine méthylée et le bascu-…”