At first, UHRF1 is in a closed conformation: SRA combines with PHD. The diversity region (PBR) between SRA and RING binds to TTD competitively with the linker (but mainly PBR), thus preventing TTD from binding to H3k9me3. However, this state does not hinder the interaction along UBL, RING, and ubiquitin-binding enzyme (E2)-ubiquitin molecules (36).

UHRF1 undergoes a conformational change from a closed state to an open conformation in the following three scenarios: i) HAUSP binds to PBR of UHRF1 and hemi-methylated DNA (hmDNA; where only one of the two complementary strands is methylated); ii) SRA binds to hmDNA and iii) phosphorylation of S651 by phosphatidyl-5-phosphate (PI5P) leads to the release of the PBR's binding to TTD, allowing TTD to bind histone with the PHD domain and linker (37). Since the concentration of PI5P is different in the G1 and S phases, the localization of UHRF1 in chromatin in the cell cycle can be determined by analyzing the concentration of PI5P (38).

In the open state, TTD-PHD binds to H3K9me2/3, UBL and histone H3 ubiquitinated by RING, and then UHRF1 recruits DNMT1 (39) in a cell cycle-dependent manner (40–42) and relieves the autoinhibitory activity of DNMT1 (37). In addition, UBL and SRA also recruit DNMT1, releasing its catalytic domain. Moreover, H3K27me3 can affect DNA methylation by inhibiting UHRF1-mediated H3 ubiquitination in this process (43).

The binding of the replication foci targeting (RFT) domain of DNMT1 with two monoubiquitinated histones H3 disrupts the interaction between RFT and its C-terminal catalytic domain, resulting in the conformational change of DNMT1, allowing hmDNA to enter the catalytic center and undergo DNA methylation. It is also considered that DNMT1 is unlikely to dissociate and bind repeatedly from UHRF1 or the UHRF1 complex during methylation modification of multiple methylation sites of hmDNA. Otherwise, the time for UHRF1 to recruit DNMT1 will be too long. Therefore, Bronner et al (33) proposed a new possibility that a macromolecular complex with DNMT1 formed after recruitment. The complex slides along the newly synthesized DNA for DNA replication and modification, controlled by the semi-methylation state of DNA. If hmDNA is not encountered, the SRA domain may not interact with the RFT sequencing (RFTS) domain of DNMT1, as DNMT1 has no enzymatic activity and abnormal DNA methylation will not occur. At the same time, the two combination methods can be used as a double check or double lock to ensure the fidelity of methylation map transmission. However, there is no clear and recognized binding mode, which needs to be confirmed through the interaction between the two protein structures and in vitro experiments (33).

HAUSP removes the ubiquitin labeling from histone H3 in the synthesized intact methylated DNA region after DNA methylation modification. DNMT1 dissociates from ubiquitinated histone H3 and repeats the process when it encounters new hmDNA sites (44).

In the replication coupling stage of DNA methylation, the arginine binding cavity of the TTD domain of UHRF1 can recognize the guanidine group in the Arg121 side chain of DNA ligase 1 (LIG1), which helps the TTD domain to be recruited to the replication site through the Lys126 di/trimethylation (45–47) on LIG1. Subsequently, UHRF1 monoubiquitinates Lys15 and Lys24 of the PCNA-associated factor 15 (PAF15) (48,49). PAF15 with double monoubiquitin recruits DNMT1 into the new replication chain (50). Afterwards, PAF15ub2 may be deubiquitinated by HAUSP in two cases: i) One case is the dissociation of UHRF1 from chromatin after the semi methylated DNA is converted to fully methylated DNA. The second case is the binding of DNMT1 to semi methylated DNA, which induces conformational changes in USP7 or PAF15ub2, although the mechanism is not yet fully clear (51). The two modes of methylation complement each other and work together to maintain the stable inheritance of epigenetic information.

TTD Domain includes two subdomains: i) TTDn and ii) TTDc. The aromatic structures constructed by F152, Y188 and Y191 in TTDn residues recognize dimethylated and trimethylated lysine residues (H3K9me2/me3). During DNA methylation, TTD and PHD domains read histone code information and transmit it to the SRA domain, allowing the SRA domain to flip methylated cytosine out of the DNA double-strand to locate the CpG site that needs to be methylated. The combination of H3K9me2/me3 and LIG1 lysine 126 dimethylation (LIG1K126me2) with UHRF1 may not play a vital role in maintaining DNA methylation (52). However, a previous study demonstrated that the arginine binding cavity in the TTD domain is crucial for the interaction of LIG1. A specific inhibitor, 5-amino-2,4-dimethylpyridine, was developed to target the Arg binding cavity. This inhibitor binds with the Arg binding cavity and effectively inhibits the binding of LIG1 and UHRF1 (53).

In addition, there is a number of studies targeting the tight binding between TTD and H3K9me2 or H3K9me3, screening and optimizing the small molecule antagonist, NV01, that disrupts this binding. This has significant reference value for studying drugs that target the structural domain (54). In addition, a recent study indicated that TTD prefers binding to histone H3 tails containing K4me1 in the context of H3K9me2/3. Moreover, the H3K4me1-K9me2/3 specific binding of UHRF1-TTD to enhancers and promoters of transcription factor binding sites downregulates these genes (55).

As one of the most common families of chromatin reader domains, the PHD finger domain has a shallow acidic groove used to identify the N-terminal of the ligand. Recognition of the K4 methylation state of the H3N terminal tail is a relatively stable and common recognition pattern in the PHD family (56–58). TTD/PHD tandem module can stimulate H3K9 methyltransferase (H3K9MT) and methylate adjacent H3K9 of adjacent nucleosomes. During this process, UHRF1-related inhibitory complexes (including DNMT1, H3K9MT, PCNA and HDAC1) may play a synergistic role (59,60), while the phosphorylation of H3 threonine 3, symmetric or asymmetric demethylation of H3R2, and acetylation of H3A1N terminal may disrupt this binding (61).

In addition, the combination of H3K9me3 with TTD-PHD also leads to a conformational change of TTD-PHD. However, this conformational change does not affect its ubiquitination activity or binding affinity with semi-methylated DNA. The potential function of this conformational change requires further studies (62). Moreover, the combination of PHD and H3 could affect the combination of TTD and H3. When the PHD domain mutates (mainly D334) or the N-terminal of H3 is modified, the PHD separates from the H3 tail, thereby disrupting or weakening the binding of TTD and methylated H3K9. Conversely, TTD mutation or histone modification (such as H3K4me3) does not affect the interaction between PHD and the unmodified H3 N-terminal (62–65). This indicates that PHD is the critical domain to identify H3, which is also consistent with the aforementioned situation of TTD (66).

Previous studies have shown that the C-terminal region of Stella (also known as Dppa3/PGC7) competitively inhibits the binding of UHRF1 to H3K9me3 by binding to the PHD domain of UHRF1, thereby damaging the DNA methylation function of UHRF1. In addition, Stella can also inhibit DNA methylation by antagonizing UHRF1 activity and isolating UHRF1 from the nucleus. Disrupting Stella's interaction with UHRF1 may be a new potential direction for developing UHRF1-targeted drugs (56,67).

SRA domain is the unique domain of UHRF1 and UHRF2 (32). Therefore, a compound targeting the SRA domain will be a specific inhibitor for UHFR1, such as chicoric acid (75), uracil derivative NSC232003 (71) and epigallocatechin-3-gallate (EGCG). EGCG can downregulate UHRF1 and DNMT1, reverse the methylation of tumor suppressor p16INK4a and induce cell cycle arrest and apoptosis in Jurkat cells (24). The anthraquinone compound UM63 (an inhibitor of this process) performs the role of 5mC in DNA methylation. UM63 can merge with a 5mC binding pocket to inhibit the base reversal process and reduce the overall DNA methylation by damaging the UHRF1/DNMT1 interaction (76).

Based on the UM63 structure, some studies have identified new inhibitors of UHRF1-SRA, such as AMSA2 and MPB7, using multidisciplinary methods. Similar to UM63, they can inhibit SRA-mediated base flipping at low concentrations but do not intercalate into DNA. These inhibitors prevent the involvement of UHRF1 and DNMT1 in DNA methylation. In addition, because they prioritize affecting cells with high levels of UHRF1, they will reduce damage to normal cells (77). Another inhibitor targeting the SRA domain, UF146, has been shown to effectively eradicate leukemia-initiating cells, confirming the potential of UHRF1 inhibitors in cancer treatment (78). However, due to UF146 being a pan-assay interference compound (79), its specificity is not high, and it may have unpredictable reactions with numerous biological targets, resulting in false positive results (80). Through molecular docking, molecular dynamics simulation and toxicity analysis, other inhibitors targeting the SRA domain can also be screened, such as chicoric acid. However, the specific therapeutic effects and indications require extensive experiments for further verification and screening (75).

UBL domain is also known as the N-terminal of a novel Np95/icbp90-like ring finger protein, which is the target of single- and multi-ubiquitination (81). Some studies have proved that UHRF1 cannot play its role in DNA methylation modification without the UBL domain (47,82,83). This highlights the two crucial functions of UBL in DNA methylation inheritance (47): i) Cooperating with the RING domain to ubiquitinate histone H3, recruiting the ubiquitin-binding enzyme E2 to form a stable E2/ubiquitin ligase E3 /chromatin complex. Subsequently, ubiquitin is transferred from E2 to histone H3 (this binding is universal, meaning that other proteins containing the UBL domain can also occur); ii) UBL recruits DNMT1 into chromatin through a hydrophobic patch and enhances DNMT activity by binding to DNMT1-621 (amino acids: 621–1,616) (47,82,83).

RING Domain has ubiquitin ligase activity, including ubiquitination of UHRF1 itself. UHRF1 protects itself from ubiquitination by interacting with HAUSP (69). The natural compound thymoquinone (TQ) eliminates the protective effect on UHRF1 by reducing the expression of HAUSP, leading to the ubiquitination of UHRF1 under the action of ubiquitin ligase, reactivating TSG, inhibiting cell proliferation, promoting cell cycle arrest and inducing apoptosis (84). TQ can selectively induce the degradation of UHRF1 in cancer cells without affecting its expression level in normal cells (85). In addition to TQ, a recent study has observed that diosgenin (DSG) induces the dissociation of UHRF1 and HAUSP protein complexes by directly binding to UHRF1, inhibiting the protective effect of HAUSP to UHRF1, thereby reducing the expression of UHRF1 and increasing the expression of TSGs. However, the specific binding sites between DSG and UHRF1 are not clear, and the effect of DSG needs to be achieved at high drug concentrations (86). Therefore, further research is needed.

As a ubiquitin ligase, RING ubiquitinates the target genes of DNMT1, which are histone H3Lys23 and Lys18 (87,88). The ubiquitin ligase activity of the RING domain is crucial for the growth of tumor cells, and inhibitors targeting this activity may be a method to produce anticancer drugs (16). In addition to tumor cells, a previous study indicated that the RING domain can interact with the human immunodeficiency virus type 1 (HIV-1) protein Tat, which is involved in virus replication, thus promoting the ubiquitination degradation of Tat, inhibiting HIV-1 transcription and maintaining HIV-1 latency (89).

In the methylation modification involving UHRF1, the linker replaces the H3 tail in the TTD peptide binding tank so that the H3 tail connects PHD at the N-terminal and the TTD domain at K9me3. The two arginine and one lysine of the linker residue (R295-R296-K297) are essential to stabilize this TTD-PHD conformation (64,65). Additionally, phosphorylation of S298 in linker can change the interaction between UHRF1 and H3, potentially serving as a functional switch for UHRF1 and participating in a variety of regulatory pathways, including DNA methylation maintenance, transcriptional inhibition and cell cycle progression (17). In vitro studies have confirmed that PIM1, an essential regulatory factor of aging, can regulate the function of UHRF1 through Ser311 phosphorylation. This regulation inhibits the binding of TTD-PHD to H3K9me3, thereby affecting the activation of DNMT1 and triggering DNA hypomethylation (90).

PBR Domain has five kinds of functions: i) It binds to TTD in the closed state of UHRF1, inhibiting its interaction with H3K9me3; ii) it promotes the recognition and binding of SRA and hmDNA; iii) it enhances the interaction between RFTS of DNMT1 and SRA (36); iv) it interacts with the UBL1 and UBL2 domains of HAUSP, keeping UHRF1 in the open state and facilitating the binding of UHRF1 to H3K9me3 (65,91,92); and v) it binds to PI5P, opening the closed conformation of UHRF1 and increasing the affinity of H3K9me3 for TTD (38). All domains of UHRF2 and UHRF1 have a very high sequence similarity, except for pRb (93). This may be why UHRF2 cannot replace UHRF1 to maintain DNA methylation (94).