Molecular mechanisms of the UHRF1/DNMT1 tandem in DNA methylation
Gene expression is under the control of epigenetic mechanisms that do not modify the DNA sequence and are faithfully inherited during cell division. The genomic and epigenomic sequences, together, thus determine the expression of genes and provide a form of cell memory for the maintenance of cell functions and phenotype. DNA methylation plays a key role in setting the epigenome of a cell that has to be inherited during cell proliferation. This requires that the daughter cells faithfully inherit the DNA methylation pattern of the mother strand, through postreplicative methylation of the newly synthesized DNA strand by DNA methyltransferases (DNMTs) and notably DNMT1, which is mainly responsible for the maintenance of the cell methylation profile. This enzyme is recruited to replication foci during the S-phase of the cell cycle by a proliferating cell nuclear antigen and UHRF1, a histone-binding protein able to sense methylcytosines (mC) and to bind preferentially to hemimethylated DNA (1). DNA methylation occurs almost exclusively in the context of CpG dinucleotides that are concentrated in regions called CpG islands found in gene promoter regions, transposons and repeated elements. Alteration of the DNA methylation pattern is involved in a variety of complex diseases, including cancer, neurodegenerative disorders and genetic and metabolic diseases (2). Using a multi-disciplinary approach combining innovative fluorescent nucleobase analogues and state-of-the-art fluorescence techniques, our objective is to unravel the molecular mechanisms of the UHRF1/DNMT1 tandem in DNA methylation.
Base flipping mechanism of UHRF1
Our first objective was to monitor in real time how the set and ring domain (SRA) of UHRF1 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 (3). 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.