UHRF1/DNMT1 tandem: mechanisms and inhibition P. Didier & Y. Mély

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.

In collaboration with the team of O. Mauffret (ENS Paris Saclay), we investigated by NMR a 12-mer duplex labeled by 3HCnt (4). Our data showed an excellent intercalation of 3HCnt in the DNA double helix. 3HCnt induces only localized perturbations that includes an extrusion from the double helix of the nucleobase opposite to 3HC. This confirms 3HCnt as a highly promising tool for nucleic acid labeling and sensing.

Searching for UHRF1 inhibitors

Since alteration of the DNA methylation pattern is involved in a variety of complex diseases, including cancers, DNMTs are recognized as major therapeutic targets. Several inhibitors of DNMTs have already been identified, but their lack of selectivity, poor stability and toxicity limit their use. As a result, demethylating agents acting by different mechanisms are actively searched. Due to its ability to favor DNMT1 binding onto the appropriate sites of newly replicating DNA and its accumulation in cancer cells, UHRF1 appears as a particularly attractive target to develop a new class of demethylating agents. In this context, our goal was to identify UHRF1 inhibitors targeting the 5'-methylcytosine (5mC) binding pocket of the SRA domain in order to prevent the recognition and flipping of 5mC and determine the molecular and cellular consequences of this inhibition (5). For this, we used a multidisciplinary strategy combining virtual screening and molecular modeling with biophysical assays in solution and cells. We identified an anthraquinone compound able to bind to the 5mC binding pocket and inhibit the base-flipping process in the low micromolar range. In cells, this hit impairs the UHRF1/DNMT1 interaction and decreases the overall methylation of DNA, highlighting the critical role of base flipping for DNMT1 recruitment and providing the first proof of concept of the druggability of the 5mC binding pocket. The selected anthraquinone appears thus as a key tool to investigate the role of UHRF1 in the inheritance of methylation patterns, as well as a starting point for hit-to-lead optimizations.

Natural anticancer compounds such as thymoquinone are able to down-regulate UHRF1 in Jurkat cells. We showed that thymoquinone induced degradation of UHRF1 that concomitantly underwent a rapid ubiquitination (6). This effect was not observed in cells expressing mutant UHRF1 RING domain, suggesting that UHRF1 commits an auto-ubiquitination through its RING domain in response to thymoquinone treatment. Exposure of cells to Z-DEVD, an inhibitor of caspase-3 markedly reduced the thymoquinone-induced down-regulation of UHRF1, while the proteosomal inhibitor MG132 had no such effect. Thymoquinone-induced UHRF1 auto-ubiquitination followed by its degradation appears as a key event in inducing apoptosis through a proteasome-independent mechanism.

In normal cells, the levels of both UHRF1 and DNMT1 and therefore, the DNA methylation level, are regulated by the Tat Interaction protein 60 (Tip60), which functions as an acetyl-transferase. Following Tip60-induced acetylation, the Ubiquitin-like domain of UHRF1 ubiquitinylates DNMT1 as well as UHRF1 itself, leading to their degradation. In cancer cells, Tip60 and UHRF1 are overexpressed, which affects the level of DNMT1 as well as the DNA methylation level in the cell. In this context, our aim was to investigate the interaction between UHRF1 and TIP60 in order to elucidate the dialogue between these two proteins (7). UHRF1, TIP60 and DNMT1 were found in the same epigenetic macro-molecular complex. Deletion of either the zinc fingers in the MYST domain or deletion of whole MYST domain from TIP60 significantly reduced its interaction with UHRF1. Confocal and FLIM microscopy showed that UHRF1 co-localized with TIP60 in the nucleus and confirmed that both proteins interacted together through the MYST domain of TIP60. Moreover, overexpression of TIP60 reduced the DNA methylation levels in HeLa cells along with downregulation of UHRF1 and DNMT1.

(1) Bronner, C.; Alhosin, M.; Hamiche, A.; Mousli, M. Coordinated Dialogue between UHRF1 and DNMT1 to Ensure Faithful Inheritance of Methylated DNA Patterns. Genes 2019, 10 (1), 65. doi.org/10.3390/genes10010065.

(2) Ashraf, W.; Ibrahim, A.; Alhosin, M.; Zaayter, L.; Ouararhni, K.; Papin, C.; Ahmad, T.; Hamiche, A.; Mély, Y.; Bronner, C.; Mousli, M. The Epigenetic Integrator UHRF1: On the Road to Become a Universal Biomarker for Cancer. Oncotarget 2017, 8 (31), 51946–51962. doi.org/10.18632/oncotarget.17393.

(3) Kilin, V.; Gavvala, K.; Barthes, N. P. F.; Michel, B. Y.; Shin, D.; Boudier, C.; Mauffret, O.; Yashchuk, V.; Mousli, M.; Ruff, M.; Granger, F.; Eiler, S.; Bronner, C.; Tor, Y.; Burger, A.; Mély, Y. Dynamics of Methylated Cytosine Flipping by UHRF1. J. Am. Chem. Soc. 2017, 139 (6), 2520–2528. doi.org/10.1021/jacs.7b00154.

(4) Zargarian, L.; Ben Imeddourene, A.; Gavvala, K.; Barthes, N. P. F.; Michel, B. Y.; Kenfack, C. A.; Morellet, N.; René, B.; Fossé, P.; Burger, A.; Mély, Y.; Mauffret, O. Structural and Dynamical Impact of a Universal Fluorescent Nucleoside Analogue Inserted Into a DNA Duplex. J. Phys. Chem. B 2017, 121 (50), 11249–11261. doi.org/10.1021/acs.jpcb.7b08825.

(5) Zaayter, L.; Mori, M.; Ahmad, T.; Ashraf, W.; Boudier, C.; Kilin, V.; Gavvala, K.; Richert, L.; Eiler, S.; Ruff, M.; Botta, M.; Bronner, C.; Mousli, M.; Mély, Y. A Molecular Tool Targeting the Base‐Flipping Activity of Human UHRF1. Chem. – Eur. J. 2019, 25 (58), 13363–13375. doi.org/10.1002/chem.201902605.

(6) Ibrahim, A.; Alhosin, M.; Papin, C.; Ouararhni, K.; Omran, Z.; Zamzami, M. A.; Al-Malki, A. L.; Choudhry, H.; Mély, Y.; Hamiche, A.; Mousli, M.; Bronner, C. Thymoquinone Challenges UHRF1 to Commit Auto-Ubiquitination: A Key Event for Apoptosis Induction in Cancer Cells. Oncotarget 2018, 9 (47), 28599–28611. doi.org/10.18632/oncotarget.25583.

(7) Ashraf, W.; Bronner, C.; Zaayter, L.; Ahmad, T.; Richert, L.; Alhosin, M.; Ibrahim, A.; Hamiche, A.; Mely, Y.; Mousli, M. Interaction of the Epigenetic Integrator UHRF1 with the MYST Domain of TIP60 inside the Cell. J. Exp. Clin. Cancer Res. 2017, 36 (1), 188. doi.org/10.1186/s13046-017-0659-1.