SET And RING-associated Domain Research Articles

Structural-Guided Identification of Small Molecule Inhibitor of UHRF1 Methyltransferase Activity.

Ubiquitin-like containing plant homeodomain Ring Finger 1 (UHRF1) protein is recognized as a cell-cycle-regulated multidomain protein. UHRF1 importantly manifests the maintenance of DNA methylation mediated by the interaction between its SRA (SET and RING associated) domain and DNA methyltransferase-1 (DNMT1)-like epigenetic modulators. However, overexpression of UHRF1 epigenetically responds to the aberrant global methylation and promotes tumorigenesis. To date, no potential molecular inhibitor has been studied against the SRA domain. Therefore, this study focused on identifying the active natural drug-like candidates against the SRA domain. A comprehensive set of in silico approaches including molecular docking, molecular dynamics (MD) simulation, and toxicity analysis was performed to identify potential candidates. A dataset of 709 natural compounds was screened through molecular docking where chicoric acid and nystose have been found showing higher binding affinities to the SRA domain. The MD simulations also showed the protein ligand interaction stability of and in silico toxicity analysis has also showed chicoric acid as a safe and nontoxic drug. In addition, chicoric acid possessed a longer interaction time and higher LD50 of 5000 mg/kg. Moreover, the global methylation level (%5 mC) has been assessed after chicoric acid treatment was in the colorectal cancer cell line (HCT116) at different doses. The result showed that 7.5 µM chicoric acid treatment reduced methylation levels significantly. Thus, the study found chicoric acid can become a possible epidrug-like inhibitor against the SRA domain of UHRF1 protein.

A Molecular Tool Targeting the Base-Flipping Activity of Human UHRF1.

During DNA replication, ubiquitin-like, containing PHD and RING fingers domains 1 (UHRF1) plays key roles in the inheritance of methylation patterns to daughter strands by recognizing through its SET and RING-associated domain (SRA) the methylated CpGs and recruiting DNA methyltransferase 1 (DNMT1). Herein, our goal is to identify UHRF1 inhibitors targeting the 5'-methylcytosine (5mC) binding pocket of the SRA domain to prevent the recognition and flipping of 5mC and determine the molecular and cellular consequences of this inhibition. 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. We also showed in cells that this hit impaired the UHRF1/DNMT1 interaction and decreased 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.

Novel Therapeutic Targeting of UHRF1 Restores 5'-Azacytidine Response in Resistant Acute Myeloid Leukemia

DNA hypermethylation plays a pivotal role in the pathogenesis of acute myeloid leukemia (AML). Most of the recurrent driver mutations and chromosomal translocations in AML involve genes encoding chromatin modifiers and DNA methylation relevant enzymes. Hypo-methylating drugs such as 5-Azacytidine (AZA) that target DNMTs prolong overall survival in AML patients. However, their long term treatments lead to emergence of acquired therapy resistance mostly through unknown mechanisms and hence there is an urgent need for alternate therapeutics to address AZA resistance in AML patients. Recently, it has been shown that AZA resistant leukemic cells are relatively quiescent with higher expression of many components of DNA methylation machinery that also includes UHRF1 (ubiquitin-like with PHD and ring finger domains 1). UHRF1 is a key epigenetic modulator that regulates DNA methylation and gene expression. It is a multi-domain nuclear protein with an SRA (SET-and-RING-associated) domain to recognize hemi-methylated DNA immediately after replication. It plays a crucial role in the maintenance of DNA methylation by recruiting DNMT1 to replication sites and facilitates methylation on newly synthesized DNA strand. UHRF1 is frequently overexpressed in multiple human neoplasms including AML and in the absence of UHRF1, hematopoietic stem cells undergo erythroid-biased differentiation at the expense of self-renewal capacity. Despite UHRF1 being key a therapeutic target against AML, specific, and cell-permeable inhibitors of UHRF1 have not been identified yet.In this study, we hypothesized that targeting UHRF1 using novel small molecule inhibitor will interfere with DNMT1-dependent DNA methylation at newly synthesized DNA strand, which may further synergize with antiproliferative effect of classical DNMT inhibitors in AML cells. In this study, we used in silico strategy to discover novel putative UHRF1 inhibitors by screening NCI compound database. For in vitro validation, we have first purified the SRA domain of UHRF1 followed by analysis of total DNA methylation levels using 5'-methyl cytosine (5mC) dot blot in the presence of each inhibitor. After a series of stringent in vitro and cell based assays we have identified lead compound 20 (C20) as a potent UHRF1 inhibitor which suppresses DNA methylation without affecting DNMTs in leukemic cells. Specificity of C20 against SRA domain was further established by isothermal titration calorimetry (ITC). We next found that C20 treatment significantly decreased UHRF1 and DNMT1 foci formation in the nucleus of mouse embryonic fibroblast and stem cells. Based on the its critical role in DNA methylation and enhanced expression in resistant cells, we assumed that AZA resistance in AML may be mediated by UHRF1 and C20 might restore AZA sensitivity by attenuating enhanced UHRF1 activity. To validate this, we pretreated AZA resistant leukemic cells (HL60R) with suboptimal dose of C20 followed by AZA treatment. Interestingly, we found a synergistic increase in antiproliferative effect by flow cytometry and colony formation assay. By analyzing the surface expression of myeloid differentiation markers, we found that C20 treatment promotes differentiation and decreases quiescent leukemic cell population. In conclusion, we report a novel UHRF1 inhibitor as a sensitizer of resistant AML cells towards AZA treatment potentially by promoting differentiation, suggesting a novel combination approach for future clinical evaluations. DisclosuresNo relevant conflicts of interest to declare.

Targeting the SET and RING-associated (SRA) domain of ubiquitin-like, PHD and ring finger-containing 1 (UHRF1) for anti-cancer drug development.

Ubiquitin-like containing PHD Ring Finger 1 (UHRF1) is a multi-domain protein with a methyl-DNA binding SRA (SET and RING-associated) domain, required for maintenance DNA methylation mediated by DNMT1. Primarily expressed in proliferating cells, UHRF1 is a cell-cycle regulated protein that is required for S phase entry. Furthermore, UHRF1 participates in transcriptional gene regulation by connecting DNA methylation to histone modifications. Upregulation of UHRF1 may serve as a biomarker for a variety of cancers; including breast, gastric, prostate, lung and colorectal carcinoma. To this end, overexpression of UHRF1 promotes cancer metastasis by triggering aberrant patterns of DNA methylation, and subsequently, silencing tumor suppressor genes. Various small molecule effectors of UHRF1 have been reported in the literature, although the mechanism of action may not be fully characterized. Small molecules that potentially bind to the SRA domain may affect the ability of UHRF1 to bind hemimethylated DNA; thereby reducing aberrant DNA methylation. Therefore, in a subset of cancers, small molecule UHRF1 inhibitors may restore normal gene expression and serve as useful anti-cancer therapeutics.

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

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.

Site-Selective Monitoring of the Interaction of the SRA Domain of UHRF1 with Target DNA Sequences Labeled with 2-Aminopurine.

UHRF1 plays a central role in the maintenance and transmission of epigenetic modifications by recruiting DNMT1 to hemimethylated CpG sites via its SET and RING-associated (SRA) domain, ensuring error-free duplication of methylation profiles. To characterize SRA-induced changes in the conformation and dynamics of a target 12 bp DNA duplex as a function of the methylation status, we labeled duplexes by the environment-sensitive probe 2-aminopurine (2-Ap) at various positions near or far from the central CpG recognition site containing either a nonmodified cytosine (NM duplex), a methylated cytosine (HM duplex), or methylated cytosines on both strands (BM duplex). Steady-state and time-resolved fluorescence indicated that binding of SRA induced modest conformational and dynamical changes in NM, HM, and BM duplexes, with only slight destabilization of base pairs, restriction of global duplex flexibility, and diminution of local nucleobase mobility. Moreover, significant restriction of the local motion of residues flanking the methylcytosine in the HM duplex suggested that these residues are more rigidly bound to SRA, in line with a slightly higher affinity of the HM duplex as compared to that of the NM or BM duplex. Our results are consistent with a "reader" role, in which the SRA domain scans DNA sequences for hemimethylated CpG sites without perturbation of the structure of contacted nucleotides.

Arabidopsis VIM Proteins Regulate Epigenetic Silencing by Modulating DNA Methylation and Histone Modification in Cooperation with MET1

Methylcytosine-binding proteins containing SRA (SET- and RING-Associated) domain are required for the establishment and/or maintenance of DNA methylation in both plants and animals. We previously proposed that Arabidopsis VIM/ORTH proteins with an SRA domain maintain DNA methylation and epigenetic gene silencing in heterochromatic regions. However, their endogenous targets of epigenetic gene silencing have not been analyzed globally and the mechanisms by which VIM proteins coordinate DNA methylation and epigenetic silencing are largely unknown. In this study, a genome-wide transcript profiling analysis revealed 544 derepressed genes in a vim1/2/3 triple mutant, including 133 known genes. VIM1 bound to promoter and transcribed regions of the up-regulated genes in vim1/2/3 and VIM deficiency caused severe DNA hypomethylation in all sequence contexts at direct VIM1 targets. We found a drastic loss of H3K9me2 at heterochromatic chromocenters in vim1/2/3 nuclei. Furthermore, aberrant changes in transcriptionally active and repressive histone modifications were observed at VIM1 targets in vim1/2/3. VIM1-binding capacity to target genes was significantly reduced in the met1 background, indicating that VIM1 primarily recognizes CG methylation deposited by MET1. Overall, our data indicate that VIM proteins regulate genome-wide epigenetic gene silencing through coordinated modulation of DNA methylation and histone modification status in collaboration with MET1.

The UHRF1 Protein Stimulates the Activity and Specificity of the Maintenance DNA Methyltransferase DNMT1 by an Allosteric Mechanism

The ubiquitin-like, containing PHD and RING finger domains protein 1 (UHRF1) is essential for maintenance DNA methylation by DNA methyltransferase 1 (DNMT1). UHRF1 has been shown to recruit DNMT1 to replicated DNA by the ability of its SET and RING-associated (SRA) domain to bind to hemimethylated DNA. Here, we demonstrate that UHRF1 also increases the activity of DNMT1 by almost 5-fold. This stimulation is mediated by a direct interaction of both proteins through the SRA domain of UHRF1 and the replication focus targeting sequence domain of DNMT1, and it does not require DNA binding by the SRA domain. Disruption of the interaction between DNMT1 and UHRF1 by replacement of key residues in the replication focus targeting sequence domain led to a strong reduction of DNMT1 stimulation. Additionally, the interaction with UHRF1 increased the specificity of DNMT1 for methylation of hemimethylated CpG sites. These findings show that apart from the targeting of DNMT1 to the replicated DNA UHRF1 increases the activity and specificity of DNMT1, thus exerting a multifaceted influence on the maintenance of DNA methylation.

The multi-domain protein Np95 connects DNA methylation and histone modification

DNA methylation and histone modifications play a central role in the epigenetic regulation of gene expression and cell differentiation. Recently, Np95 (also known as UHRF1 or ICBP90) has been found to interact with Dnmt1 and to bind hemimethylated DNA, indicating together with genetic studies a central role in the maintenance of DNA methylation. Using in vitro binding assays we observed a weak preference of Np95 and its SRA (SET- and Ring-associated) domain for hemimethylated CpG sites. However, the binding kinetics of Np95 in living cells was not affected by the complete loss of genomic methylation. Investigating further links with heterochromatin, we could show that Np95 preferentially binds histone H3 N-terminal tails with trimethylated (H3K9me3) but not acetylated lysine 9 via a tandem Tudor domain. This domain contains three highly conserved aromatic amino acids that form an aromatic cage similar to the one binding H3K9me3 in the chromodomain of HP1ß. Mutations targeting the aromatic cage of the Np95 tandem Tudor domain (Y188A and Y191A) abolished specific H3 histone tail binding. These multiple interactions of the multi-domain protein Np95 with hemimethylated DNA and repressive histone marks as well as with DNA and histone methyltransferases integrate the two major epigenetic silencing pathways.

Recognition of hemi-methylated DNA by the SRA protein UHRF1 by a base-flipping mechanism

DNA methylation of CpG dinucleotides is an important epigenetic modification of mammalian genomes and is essential for the regulation of chromatin structure, of gene expression and of genome stability. Differences in DNA methylation patterns underlie a wide range of biological processes, such as genomic imprinting, inactivation of the X chromosome, embryogenesis, and carcinogenesis. Inheritance of the epigenetic methylation pattern is mediated by the enzyme DNA methyltransferase 1 (Dnmt1), which methylates newly synthesized CpG sequences during DNA replication, depending on the methylation status of the template strands. The protein UHRF1 (also known as Np95 and ICBP90) recognizes hemi-methylation sites via a SET and RING-associated (SRA) domain and directs Dnmt1 to these sites. Here we report the crystal structures of the SRA domain in free and hemi-methylated DNA-bound states. The SRA domain folds into a globular structure with a basic concave surface formed by highly conserved residues. Binding of DNA to the concave surface causes a loop and an amino-terminal tail of the SRA domain to fold into DNA interfaces at the major and minor grooves of the methylation site. In contrast to fully methylated CpG sites recognized by the methyl-CpG-binding domain, the methylcytosine base at the hemi-methylated site is flipped out of the DNA helix in the SRA-DNA complex and fits tightly into a protein pocket on the concave surface. The complex structure suggests that the successive flip out of the pre-existing methylated cytosine and the target cytosine to be methylated is associated with the coordinated transfer of the hemi-methylated CpG site from UHRF1 to Dnmt1.

Structural basis for recognition of hemi-methylated DNA by the SRA domain of human UHRF1

Epigenetic inheritance in mammals is characterized by high-fidelity replication of CpG methylation patterns during development. UHRF1 (also known as ICBP90 in humans and Np95 in mouse) is an E3 ligase important for the maintenance of global and local DNA methylation in vivo. The preferential affinity of UHRF1 for hemi-methylated DNA over symmetrically methylated DNA by means of its SET and RING-associated (SRA) domain and its association with the maintenance DNA methyltransferase 1 (DNMT1) suggests a role in replication of the epigenetic code. Here we report the 1.7 A crystal structure of the apo SRA domain of human UHRF1 and a 2.2 A structure of its complex with hemi-methylated DNA, revealing a previously unknown reading mechanism for methylated CpG sites (mCpG). The SRA-DNA complex has several notable structural features including a binding pocket that accommodates the 5-methylcytosine that is flipped out of the duplex DNA. Two specialized loops reach through the resulting gap in the DNA from both the major and the minor grooves to read the other three bases of the CpG duplex. The major groove loop confers both specificity for the CpG dinucleotide and discrimination against methylation of deoxycytidine of the complementary strand. The structure, along with mutagenesis data, suggests how UHRF1 acts as a key factor for DNMT1 maintenance methylation through recognition of a fundamental unit of epigenetic inheritance, mCpG.

The SRA domain of UHRF1 flips 5-methylcytosine out of the DNA helix

Maintenance methylation of hemimethylated CpG dinucleotides at DNA replication forks is the key to faithful mitotic inheritance of genomic methylation patterns. UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is required for maintenance methylation by interacting with DNA nucleotide methyltransferase 1 (DNMT1), the maintenance methyltransferase, and with hemimethylated CpG, the substrate for DNMT1 (refs 1 and 2). Here we present the crystal structure of the SET and RING-associated (SRA) domain of mouse UHRF1 in complex with DNA containing a hemimethylated CpG site. The DNA is contacted in both the major and minor grooves by two loops that penetrate into the middle of the DNA helix. The 5-methylcytosine has flipped completely out of the DNA helix and is positioned in a binding pocket with planar stacking contacts, Watson-Crick polar hydrogen bonds and van der Waals interactions specific for 5-methylcytosine. Hence, UHRF1 contains a previously unknown DNA-binding module and is the first example of a non-enzymatic, sequence-specific DNA-binding protein domain to use the base flipping mechanism to interact with DNA.

Three SRA-Domain Methylcytosine-Binding Proteins Cooperate to Maintain Global CpG Methylation and Epigenetic Silencing in Arabidopsis

Methylcytosine-binding proteins decipher the epigenetic information encoded by DNA methylation and provide a link between DNA methylation, modification of chromatin structure, and gene silencing. VARIANT IN METHYLATION 1 (VIM1) encodes an SRA (SET- and RING-associated) domain methylcytosine-binding protein in Arabidopsis thaliana, and loss of VIM1 function causes centromere DNA hypomethylation and centromeric heterochromatin decondensation in interphase. In the Arabidopsis genome, there are five VIM genes that share very high sequence similarity and encode proteins containing a PHD domain, two RING domains, and an SRA domain. To gain further insight into the function and potential redundancy among the VIM proteins, we investigated strains combining different vim mutations and transgenic vim knock-down lines that down-regulate multiple VIM family genes. The vim1 vim3 double mutant and the transgenic vim knock-down lines showed decreased DNA methylation primarily at CpG sites in genic regions, as well as repeated sequences in heterochromatic regions. In addition, transcriptional silencing was released in these plants at most heterochromatin regions examined. Interestingly, the vim1 vim3 mutant and vim knock-down lines gained ectopic CpHpH methylation in the 5S rRNA genes against a background of CpG hypomethylation. The vim1 vim2 vim3 triple mutant displayed abnormal morphological phenotypes including late flowering, which is associated with DNA hypomethylation of the 5′ region of FWA and release of FWA gene silencing. Our findings demonstrate that VIM1, VIM2, and VIM3 have overlapping functions in maintenance of global CpG methylation and epigenetic transcriptional silencing.

ICBP90, a Novel Methyl K9 H3 Binding Protein Linking Protein Ubiquitination with Heterochromatin Formation

Methylation of histone H3 on lysine 9 is critical for diverse biological processes including transcriptional repression, heterochromatin formation, and X inactivation. The biological effects of histone methylation are thought to be mediated by effector proteins that recognize and bind to specific patterns of methylation. Using an unbiased in vitro biochemical approach, we have identified ICBP90, a transcription and cell cycle regulator, as a novel methyl K9 H3-specific binding protein. ICBP90 and its murine homologue Np95 are enriched in pericentric heterochromatin of interphase nuclei, and this localization is dependent on H3K9 methylation. Specific binding of ICBP90 to methyl K9 H3 depends on two functional domains, a PHD (plant homeodomain) finger that defines the binding specificity and an SRA (SET- and RING-associated) domain that promotes binding activity. Furthermore, we present evidence that ICBP90 is required for proper heterochromatin formation in mammalian cells.

The interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 is involved in the regulation of VEGF gene expression

Inverted CCAAT box-binding protein of 90 kDa (ICBP90) is over-expressed in several types of cancer, including breast, prostate and lung cancers. In search for proteins that interact with the set and ring-associated (SRA) domain of ICBP90, we used the two-hybrid system and screened a placental cDNA library. Several clones coding for a new domain of DNMT1 were found. The interaction, between the ICBP90 SRA domain and the DNMT1 domain, has been confirmed with purified proteins by glutathione-S-transferase pull-down experiments. We checked whether ICBP90 and DNMT1 are present in the same macro-molecular complexes in Jurkat cells and immortalized human vascular smooth muscle cells (HVTs-SM1). Co-immunoprecipitation experiments showed that ICBP90 and DNMT1 are present in the same molecular complex, which was further confirmed by co-localization experiments as assessed by immunocytochemistry. Downregulation of ICBP90 and DNMT1 decreased VEGF gene expression, a major pro-angiogenic factor, whereas those of p16(INK4A) gene and RB1 gene were significantly enhanced. Together, these results indicate that DNMT1 and ICBP90 are involved in VEGF gene expression, possibly via an interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 and an upregulation of p16(INK4A). They further suggest a new role of ICBP90 in the relationship between histone ubiquitination and DNA methylation in the context of tumoral angiogenesis and tumour suppressor genes silencing.

Plant SET‐ and RING‐associated domain proteins in heterochromatinization

The heterochromatin of many eukaryotes is marked by both histone H3 lysine 9 (H3K9) methylation and DNA cytosine methylation. Several studies have revealed links between these two epigenetic markers. The molecular mechanisms involved in establishment of these links, however, remain largely unknown. In plants, H3K9 methylation is primarily carried out by a highly conserved family of proteins that contain SET and SRA (SET- and RING-associated) domains. Here, we show that the SRA-SET domain H3K9 methyltransferase NtSET1, as well as LIKE HETEROCHROMATIN PROTEIN1, binds heterochromatin DNA repeats. In the yeast two-hybrid assay, NtSET1 binds the DNA methylcytosine-binding protein VARIANT IN METHYLATION1 (VIM1), which contains conserved PHD, SRA and RING domains. This binding requires either the N-terminus of NtSET1 containing the SRA domain or the C-terminus of NtSET1 containing the SET domain and the PHD domain of VIM1. Consistent with a role in the establishment/maintenance of chromatin structure during cell division, VIM1 transcripts are abundant in actively dividing cells and the VIM1 protein is localized in the nucleus. While null vim1 mutant plants show a normal growth phenotype, transgenic Arabidopsis plants over-expressing VIM1 show inhibition in root growth and delay in flowering. We propose that SRA-SET domain H3K9 methyltransferases associate with the PHD-SRA-RING domain protein VIM1, mutually reinforcing H3K9 and DNA methylation in heterochromatinization.

UHRF1 Plays a Role in Maintaining DNA Methylation in Mammalian Cells

Epigenetic inheritance in mammals relies in part on robust propagation of DNA methylation patterns throughout development. We show that the protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1), also known as NP95 in mouse and ICBP90 in human, is required for maintaining DNA methylation. UHRF1 colocalizes with the maintenance DNA methyltransferase protein DNMT1 throughout S phase. UHRF1 appears to tether DNMT1 to chromatin through its direct interaction with DNMT1. Furthermore UHRF1 contains a methyl DNA binding domain, the SRA (SET and RING associated) domain, that shows strong preferential binding to hemimethylated CG sites, the physiological substrate for DNMT1. These data suggest that UHRF1 may help recruit DNMT1 to hemimethylated DNA to facilitate faithful maintenance of DNA methylation.

VIM1, a methylcytosine-binding protein required for centromeric heterochromatinization

Epigenetic regulation in eukaryotes is executed by a complex set of signaling interactions among small RNA species and chromatin marks, including histone modification and DNA methylation. We identified vim1 (VARIANT IN METHYLATION 1), an Arabidopsis mutation causing cytosine hypomethylation and decondensation of centromeres in interphase. VIM1 is a member of a small gene family, encoding proteins containing PHD, RING, and SRA (SET- and RING-associated) domains, which are found together in mammalian proteins implicated in regulation of chromatin modification, transcription, and the cell cycle. VIM1 is an unconventional methylcytosine-binding protein that interacts in vitro with 5mCpG- and 5mCpHpG-modified DNA (via its SRA domain), as well as recombinant histones (H2B, H3, H4, and HTR12) in plant extracts. VIM1 associates with methylated genomic loci in vivo and is enriched in chromocenters. Our findings suggest that VIM1 acts at the DNA methylation-histone interface to maintain centromeric heterochromatin.