Maintenance Of DNA Methylation Research Articles

Zebularine induces enzymatic DNA-protein crosslinks in 45S rDNA heterochromatin of Arabidopsis nuclei.

Loss of genome stability leads to reduced fitness, fertility and a high mutation rate. Therefore, the genome is guarded by the pathways monitoring its integrity and neutralizing DNA lesions. To analyze the mechanism of DNA damage induction by cytidine analog zebularine, we performed a forward-directed suppressor genetic screen in the background of Arabidopsis thaliana zebularine-hypersensitive structural maintenance of chromosomes 6b (smc6b) mutant. We show that smc6b hypersensitivity was suppressed by the mutations in EQUILIBRATIVE NUCLEOSIDE TRANSPORTER 3 (ENT3), DNA METHYLTRANSFERASE 1 (MET1) and DECREASE IN DNA METHYLATION 1 (DDM1). Superior resistance of ent3 plants to zebularine indicated that ENT3 is likely necessary for the import of the drug to the cells. Identification of MET1 and DDM1 suggested that zebularine induces DNA damage by interference with the maintenance of CG DNA methylation. The same holds for structurally similar compounds 5-azacytidine and 2-deoxy-5-azacytidine. Based on our genetic and biochemical data, we propose that zebularine induces enzymatic DNA–protein crosslinks (DPCs) of MET1 and zebularine-containing DNA in Arabidopsis, which was confirmed by native chromatin immunoprecipitation experiments. Moreover, zebularine-induced DPCs accumulate preferentially in 45S rDNA chromocenters in a DDM1-dependent manner. These findings open a new avenue for studying genome stability and DPC repair in plants.

The phytochemical brazilin suppress DNMT1 expression by recruiting p53 to its promoter resulting in the epigenetic restoration of p21 in MCF7cells

BackgroundCancer is an outcome of uncontrolled cell division eventually associated with dysregulated epigenetic mechanisms, including DNA methylation. DNA methyltransferase 1 is ubiquitously expressed in the proliferating cells and is essential for the maintenance of DNA methylation. It causes the abnormal silencing of tumor suppressor genes in human cancer which is necessary for proliferation, cell cycle progression, and survival. DNMT1 is involved in tumorigenesis of several cancers, its upregulation potentially upscale the promoter level inactivation of transcription of a tumor inhibitory gene by introducing repressive methylation marks on the CpG islands. This epigenetic perturbation caused by DNMT is targeted for cancer therapeutics. PurposeTo demonstrate the proliferative inhibitory potential of brazilin in human breast cancer cell line (MCF-7) with concurrent mitigation of DNMT1 functional expression and to understand its effect on downstream targets like cell cycle inhibitor p21. Study Design/ MethodsThe impact of brazilin on the growth and proliferation of the MCF-7 cells was determined using the XTT assay. The global DNA 5-methyl cytosine methylation pattern was analyzed upon brazilin treatment. The gene and protein expression of DNMTs were determined with quantitative RTPCR and western blots respectively. The potential binding sites of transcription factors in the human DNMT1 promoter were predicted using the MatInspector tool on the Genomatix software. The chromatin immunoprecipitation (ChIP) assay was performed to demonstrate the transcription factors occupancy at the promoter. Methylation of promoter CpG islands was determined by the methylation-specific PCR (MSP) upon brazilin treatment. The molecular docking of the human DNMT1 with brazilin (ligand) was performed using the Schrödinger suite. ResultsThe heterotetracyclic compound brazilin, present in the wood of Caesalpinia sappan, inhibited the proliferation of the human breast cancer cell line (MCF-7) and reduced the DNMT1 expression with a decrease in global DNA methylation. Brazilin, by activating p38 MAPK and elevating p53 levels within the exposed cells. The elevated level of p53 enriched the occupancy at binding sites within 200 bp upstream to the transcription start site in the DNMT1 promoter, resulting in reduced DNMT1 gene expression. Furthermore, the brazilin restored the p21 levels in the exposed cells as the CpGs in the p21 promoter (-128 bp/+17 bp) were significantly demethylated as observed in the methylation-specific PCR (MSP). ConclusionHighly potential anti-proliferative molecule brazilin can modulate the DNMT1 functional expression and restore the cell cycle inhibitor p21expression. We propose that brazilin can be used in therapeutic interventions to restore the deregulated epigenetic mechanisms in cancer.

A mathematical model which examines age-related stochastic fluctuations in DNA maintenance methylation.

Due to its complexity and its ubiquitous nature the ageing process remains an enduring biological puzzle. Many molecular mechanisms and biochemical process have become synonymous with ageing. However, recent findings have pinpointed epigenetics as having a key role in ageing and healthspan. In particular age related changes to DNA methylation offer the possibility of monitoring the trajectory of biological ageing and could even be used to predict the onset of diseases such as cancer, Alzheimer's disease and cardiovascular disease. At the molecular level emerging evidence strongly suggests the regulatory processes which govern DNA methylation are subject to intracellular stochasticity. It is challenging to fully understand the impact of stochasticity on DNA methylation levels at the molecular level experimentally. An ideal solution is to use mathematical models to capture the essence of the stochasticity and its outcomes. In this paper we present a novel stochastic model which accounts for specific methylation levels within a gene promoter. Uncertainty of the eventual site-specific methylation levels for different values of methylation age, depending on the initial methylation levels were analysed. Our model predicts the observed bistable levels in CpG islands. In addition, simulations with various levels of noise indicate that uncertainty predominantly spreads through the hypermethylated region of stability, especially for large values of input noise. A key outcome of the model is that CpG islands with high to intermediate methylation levels tend to be more susceptible to dramatic DNA methylation changes due to increasing methylation age.

Structure-based screening combined with computational and biochemical analyses identified the inhibitor targeting the binding of DNA Ligase 1 to UHRF1

The accumulation of epigenetic alterations is one of the major causes of tumorigenesis. Aberrant DNA methylation patterns cause genome instability and silencing of tumor suppressor genes in various types of tumors. Therefore, drugs that target DNA methylation-regulating factors have great potential for cancer therapy. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) is an essential factor for DNA methylation maintenance. UHRF1 is overexpressed in various cancer cells and down-regulation of UHRF1 in these cells reactivates the expression of tumor suppressor genes, thus UHRF1 is a promising target for cancer therapy. We have previously shown that interaction between the tandem Tudor domain (TTD) of UHRF1 and DNA ligase 1 (LIG1) di/trimethylated on Lys126 plays a key role in the recruitment of UHRF1 to replication sites and replication-coupled DNA methylation maintenance. An arginine binding cavity (Arg-binding cavity) of the TTD is essential for LIG1 interaction, thus the development of inhibitors that target the Arg-binding cavity could potentially repress UHRF1 function in cancer cells. To develop such an inhibitor, we performed in silico screening using not only static but also dynamic metrics based on all-atom molecular dynamics simulations, resulting in efficient identification of 5-amino-2,4-dimethylpyridine (5A-DMP) as a novel TTD-binding compound. Crystal structure of the TTD in complex with 5A-DMP revealed that the compound stably bound to the Arg-binding cavity of the TTD. Furthermore, 5A-DMP inhibits the full-length UHRF1:LIG1 interaction in Xenopus egg extracts. Our study uncovers a UHRF1 inhibitor which can be the basis of future experiments for cancer therapy.

Evidences of the PI5P Increasing the Expression of HBG and Gamma Globin Concentrations

Efforts have been made to identify modulators of increasing fetal hemoglobin (HbF - α2ɣ2) that may act as promising targets for hemoglobinopathies, such as in sickle cell disease and beta thalassemia. The inositide PI5P is a second messenger from the inositol pathway that comprehends in the main phosphorylation target of the enzyme phosphatidylinositol-4-phosphate-5-kinase-II-alpha (PIPKIIα), found in high concentrations in the erythrocyte. Some evidences have postulated a relation between this enzyme and the expression of the hemoglobin (Hb) genes (1,2). More recently, PI5P was found to stabilize UHRF1 protein (3), which has been reported as a possible repressor of HBG (from ɣ globin gene), in the switch from ɣ to β in the transition HbF- HbA (4). To evaluate the effects of the inositide PI5P (substrate of PIPKIIα) in globins, a preliminary time course experiment was performed. KU812 cells (ATCC CRL-2099), which express all three globin genes (HBA, HBB and HBG), were treated with 1μM of PI5P (Thermo Fischer Scientific, USA) in triplicate (4, 8 and 12 hours of treatment) - time 0h used as control. Gene expression and protein concentrations were determined by qPCR - SYBR Green Master Mix amplification and detection kit (Thermo Fischer Scientific, USA, in equipment StepOnePlus Real Time PCR System, Applied Biosystems - USA) using 2DDCT method with the ACTB and GAPDH genes as internal controls - and immunoblotting. Primers were design according to Zacariotto et al (2). No additional phosphate was imputed into the cell culture and the ATP consumption was monitored by ATP Determination Kit (Thermo Fisher Scientific, EUA). PI5P treatment resulted in significant increasement of HBG RNA transcripts (p= 0.001; 8 hours / treatment) and decrease of PIP4K2A (from PIPKIIα enzyme) (p=0.02; 12 hours / treatment), with no other significant changes into globin genes (HBA and HBB). At the protein level, there was a prominent increase in ɣ globin concentrations according to time course. No other consistent change in the concentrations of PIPKIIα or globin chains were observed. Due to protein similarities between the subfamilies of phosphatidylinositol-phosphate kinases (PIPKins) (5), the gene expression of these enzymes was also monitored by qPCR. There was a significant reduction of PIP5K1A (from PIPKIα enzyme) (p=0.01 - 4h; p=0.01 - 8h, and p=0.009 - 12h) as well as PIP4K2B (from PIPKIIβ enzyme) (p=0.03 - 4 hours/ treatment). No significant changes were observed in the other PIPKins (PIPKIβ, PIPKIɣ, PIPKIIɣ and PIPKIII). Considering that a significant consumption of intracellular ATP was observed in time course (p=0.006) it is possible to infer that the high concentrations of PI5P have shifted the inositol pathway resulting in the downregulation of its own PIPKin genes and culminating into the upregulation of HBG, reflected into the protein level. The mechanisms involving the inositide as second messenger, the role of PIPKins or other genes into the modulation of hemoglobin genes, particularly in HBG, should be further investigated. However, despite preliminary, these results reinforce the involvement of the PIPKins and its molecular targets (mainly PIPKIα and PI5P) in globin gene regulation and could represent a promising target for future therapeutic target for hemoglobinopathies. Financial Support: Fapesp, CNPq, CAPES, Faepex-Unicamp.1.Wenning MR, Mello MP, Andrade TG, et al. PIP4KIIA and beta-globin: transcripts differentially expressed in reticulocytes and associated with high levels of Hb H in two siblings with Hb H disease. Eur J Haematol. 2009; 83: 490-3.2.Zaccariotto TR, Lanaro C, Albuquerque DM, Santos MN, Bezerra MA, Cunha FG, et al. Expression profiles of phosphatidylinositol phosphate kinase genes during normal human in vitro erythropoiesis. Genet Mol Res. 2012; 11: 3861-8.3.Gelato KA, Tauber M, Ong MS, Winter S, et al. Accessibility of different histone H3-binding domains of UHRF1 is allosterically regulated by phosphatidylinositol 5-phosphate. Mol Cell. 2014;54(6):905-19.4.Ruopeng Feng, Phillip A Doerfler, Yu Yao XT, et al. The DNA Methylation Maintenance Protein UHRF1 Regulates Fetal Globin Expression Independent of H BG Promoter DNA Methylation. Blood. 2018;132:410.5.Heck JN, Mellman DL, Ling K, et al. A conspicuous connection: structure defines function for the phosphatidylinositol-phosphate kinase family. Crit Rev Biochem Mol Biol. 2007; 42: 15-39. DisclosuresCosta: Novartis: Consultancy.

HIGH-FAT DIET INDUCED OBESITY ALTERS Dnmt1 AND Dnmt3a LEVELS AND GLOBAL DNA METHYLATION IN MOUSE OVARY AND TESTES

DNA methylation is one of the epigenetic mechanisms that play crucial roles in oogenesis, spermatogenesis, and embryo development. DNA methylation mechanisms, de novo and maintenance methylation, are catalyzed by DNA methyltransferases (DNMTs) enzymes. Dnmt1, responsible for the maintenance of global DNA methylation and Dnmt3a for the de novo methylation. Obesity, which is considered as one of the factors for decreased fertility, alters DNA methylation in umbilical cord, peripheral blood or fetal tissues and inherited to offspring.

KNOCKDOWN OF Dnmt1 AND Dnmt3a ENZYMES DISRUPTS PREIMPLANTATION EMBRYO DEVELOPMENT BY AFFECTING GLOBAL DNA METHYLATION

DNA methylation is one of the epigenetic mechanisms that play critical roles in preimplantation embryo development executed by DNA methyltransferase (Dnmt) enzymes. Dnmt1, responsible for the maintenance of global DNA methylation and Dnmt3a for the de novo methylation, are gradually erased from zygote to blastocyst stage by a demethylation process, and then reestablished by de novo methylation around blastocyst stage. This important differentiation mechanism indicates pivotal roles of global DNA methylation during the course of preimplantation embryo development. So, a fundamental question which also served as the aim of the current study was to address the spatio-temporal expression of Dnmt1 and Dnmt3a and thus the establishment of DNA methylation could affect the embryo development. We set up four groups as control, Dnmt1 siRNA, Dnmt3a siRNA, and non-targeted siRNA. In vivo developed Balb/c mouse zygotes were collected, divided into groups and then cultured for 96 h to reach blastocyst stage. Zygotes were transfected with 50 nM Dnmt1 and Dnmt3a-spesific small interfering RNA (siRNA) duplexes vs non-targeting control siRNA duplexes for 96 h using DharmaFECT as a transfection reagent. Nontargeting siRNA duplex served as the negative control. Knockdown of Dnmt genes using siRNA was confirmed by analyzing the proteins using immunofluorescence and Western blot techniques after 96 h in culture. Following knockdown of Dnmt1 and Dnmt3a, we analyzed the developmental competence of embryos. We also evaluated global DNA methylation levels by 5 methylcytosine staining in blastocyst stage. We found that both Dnmt genes were successfully knocked-down (approximately 80%) in 2-4 cell embryos. In Dnmt1 siRNA group, embryo arrest rates were 29.5% in 2-cell, 15.7 in %4-cell, and 9.8% in morula stages. Only 16.7% embryos reached to blastocyst stage in this group. In Dnmt3a siRNA group, embryos arrested in 26.2% 2-cell, 21.9% 4-cell, 9.2% morula and 29.5% embryos reached to blastocyst stage. Degenerated embryo rates were 28.2% in Dnmt1 siRNA, and 13.1% in Dnmt3a siRNA group. Interestingly, we noted that global DNA methylation level significantly decreased in Dnmt1 knockdown group while increased in Dnmt3a knockdown group (P<0.001). These results suggested that temporal Dnmt enzymes could contribute to the process of early embryonic development and thus play vital roles in the mouse embryos. Therefore it seems evident that these enzymes should also be elucidated in human to check whether they play any major role in the human preimplantation embryo development.

Increased de novo DNA Methylation Enzymes in Sperm of Individuals with Varicocele.

Objective Chronic genital heat-stress associated with varicocele leads to DNA hypo-methylation of spermatozoa. The objective of this study was comparing level of DNA methyl-transferases (DNMTs) in sperm of men suffering varicocele with fertile individuals. Materials and Methods In this case-control study, semen samples were obtained from 35 infertile men with varicocele (grade II or III) and 26 fertile men. Sperm parameters were assessed according to World Health Organization (WHO) protocol. DNMTs enzymes level were assessed by flow cytometer and fluorescence microscope. mRNAs expression of these DNMTs were also assessed by real-time reverse transcription polymerase chain reaction (RT-PCR). Results DNMT1 and DNMT3A proteins were mainly localized in equatorial and mid-piece regions of sperm head, respectively, while DNMT3B protein appeared to be localized mainly in equatorial and anterior regions of sperm head. In contrast to DNMT1, expression and percentage of DNMT3A and DNMT3B at RNA and protein levels were significantly higher in the varicocele group compared to the fertile group (P<0.05). In addition, significant correlations were found between sperm concentration and motility as well as DNMT1 and DNMT3B proteins levels in the infertile individuals with varicocele (P<0.05). Additionally, significant correlations were observed between abnormal sperm morphology with DNMTs proteins in the infertile individuals with varicocele. ConclusionUnlike DNMT1, which is involved in maintenance of DNA methylation at both RNA and protein levels, expression of de novo methylation enzymes (DNMT3A and DNMT3B) at both levels were increased in the varicocele group compared to the fertile group. Based on literature, this increase might be due to the dual roles played by DNMT3A and DNMT3B, as methyl-transferases in normal condition as well as dehydroxymethylases in stress condition, like varicocele. Although, this hypothesis needs further validation.

Pathway conversion enables a double-lock mechanism to maintain DNA methylation and genome stability

The CMT2 and RNA-directed DNA methylation (RdDM) pathways have been proposed to separately maintain CHH methylation in specific regions of the Arabidopsis thaliana genome. Here, we show that dysfunction of the chromatin remodeler DDM1 causes hundreds of genomic regions to switch from CMT2 dependency to RdDM dependency in DNA methylation. These converted loci are enriched at the edge regions of long transposable elements (TEs). Furthermore, we found that dysfunction in both DDM1 and RdDM causes strong reactivation of TEs and a burst of TE transposition in the first generation of mutant plants, indicating that the DDM1 and RdDM pathways together are critical to maintaining TE repression and protecting genomic stability. Our findings reveal the existence of a pathway conversion-based backup mechanism to guarantee the maintenance of DNA methylation and genome integrity.

Pre-implantation alcohol exposure induces lasting sex-specific DNA methylation programming errors in the developing forebrain

BackgroundPrenatal alcohol exposure is recognized for altering DNA methylation profiles of brain cells during development, and to be part of the molecular basis underpinning Fetal Alcohol Spectrum Disorder (FASD) etiology. However, we have negligible information on the effects of alcohol exposure during pre-implantation, the early embryonic window marked with dynamic DNA methylation reprogramming, and on how this may rewire the brain developmental program.ResultsUsing a pre-clinical in vivo mouse model, we show that a binge-like alcohol exposure during pre-implantation at the 8-cell stage leads to surge in morphological brain defects and adverse developmental outcomes during fetal life. Genome-wide DNA methylation analyses of fetal forebrains uncovered sex-specific alterations, including partial loss of DNA methylation maintenance at imprinting control regions, and abnormal de novo DNA methylation profiles in various biological pathways (e.g., neural/brain development).ConclusionThese findings support that alcohol-induced DNA methylation programming deviations during pre-implantation could contribute to the manifestation of neurodevelopmental phenotypes associated with FASD.

Helical and β-Turn Conformations in the Peptide Recognition Regions of the VIM1 PHD Finger Abrogate H3K4 Peptide Recognition.

The PHD finger-containing VARIANT IN METHYLATION/ORTHRUS (VIM/ORTH) family of proteins in Arabidopsis consists of functional homologues of mammalian UHRF1 and is required for the maintenance of DNA methylation. Comparison of the sequence with those of other PHD fingers implied that VIM1 and VIM3 PHD could recognize lysine 4 of histone H3 (H3K4) through interactions mediated by a conserved aspartic acid. However, our calorimetric and modified histone peptide array binding studies suggested that neither H3K4 nor other histone marks are recognized by VIM1 and VIM3 PHD fingers. Here, we report a 2.6 Å resolution crystal structure of the VIM1 PHD finger and demonstrate significant structural changes in the putative H3 recognition segments in contrast to canonical H3K4 binding PHD fingers. These changes include (i) the H3A1 binding region, (ii) strand β1 that forms an intermolecular β-sheet with the H3 peptide, and (iii) an aspartate-containing motif involved in salt bridge interaction with H3K4, which together appear to abrogate recognition of H3K4 by the VIM1 PHD finger. To understand the significance of the altered structural features in the VIM1 PHD that might prevent histone H3 recognition, we modeled a chimeric VIM1 PHD (chmVIM1 PHD) by grafting the peptide binding structural features of the BHC80 PHD onto the VIM1 PHD. Molecular dynamics simulation and metadynamics analyses revealed that the chmVIM1 PHD-H3 complex is stable and also showed a network of intermolecular interactions similar to those of the BHC80 PHD-H3 complex. Collectively, this study reveals that subtle structural changes in the peptide binding region of the VIM1 PHD abrogate histone H3 recognition.

The splicing regulator SLU7 is required to preserve DNMT1 protein stability and DNA methylation.

Gene expression is finely and dynamically controlled through the tightly coordinated and interconnected activity of epigenetic modulators, transcription and splicing factors and post-translational modifiers. We have recently identified the splicing factor SLU7 as essential for maintaining liver cell identity and genome integrity and for securing cell division both trough transcriptional and splicing mechanisms. Now we uncover a new function of SLU7 controlling gene expression at the epigenetic level. We show that SLU7 is required to secure DNMT1 protein stability and a correct DNA methylation. We demonstrate that SLU7 is part in the chromatome of the protein complex implicated in DNA methylation maintenance interacting with and controlling the integrity of DNMT1, its adaptor protein UHRF1 and the histone methyl-transferase G9a at the chromatin level. Mechanistically, we found that SLU7 assures DNMT1 stability preventing its acetylation and degradation by facilitating its interaction with HDAC1 and the desubiquitinase USP7. Importantly, we demonstrate that this DNMT1 dependency on SLU7 occurs in a large panel of proliferating cell lines of different origins and in in vivo models of liver proliferation. Overall, our results uncover a novel and non-redundant role of SLU7 in DNA methylation and present SLU7 as a holistic regulator of gene expression.

Arabidopsis MORC proteins function in the efficient establishment of RNA directed DNA methylation

The Microrchidia (MORC) family of ATPases are required for transposable element (TE) silencing and heterochromatin condensation in plants and animals, and C. elegans MORC-1 has been shown to topologically entrap and condense DNA. In Arabidopsis thaliana, mutation of MORCs has been shown to reactivate silent methylated genes and transposons and to decondense heterochromatic chromocenters, despite only minor changes in the maintenance of DNA methylation. Here we provide the first evidence localizing Arabidopsis MORC proteins to specific regions of chromatin and find that MORC4 and MORC7 are closely co-localized with sites of RNA-directed DNA methylation (RdDM). We further show that MORC7, when tethered to DNA by an artificial zinc finger, can facilitate the establishment of RdDM. Finally, we show that MORCs are required for the efficient RdDM mediated establishment of DNA methylation and silencing of a newly integrated FWA transgene, even though morc mutations have no effect on the maintenance of preexisting methylation at the endogenous FWA gene. We propose that MORCs function as a molecular tether in RdDM complexes to reinforce RdDM activity for methylation establishment. These findings have implications for MORC protein function in a variety of other eukaryotic organisms.

UHRF1 regulates alternative splicing by interacting with splicing factors and U snRNAs in a H3R2me involved manner.

The well-established functions of UHRF1 converge to DNA biological processes, as exemplified by DNA methylation maintenance and DNA damage repair during cell cycles. However, the potential effect of UHRF1 on RNA metabolism is largely unexplored. Here, we revealed that UHRF1 serves as a novel alternative RNA splicing regulator. The protein interactome of UHRF1 identified various splicing factors. Among them, SF3B3 could interact with UHRF1 directly and participate in UHRF1-regulated alternative splicing events. Furthermore, we interrogated the RNA interactome of UHRF1, and surprisingly, we identified U snRNAs, the canonical spliceosome components, in the purified UHRF1 complex. Unexpectedly, we found H3R2 methylation status determines the binding preference of U snRNAs, especially U2 snRNAs. The involvement of U snRNAs in UHRF1-containing complex and their binding preference to specific chromatin configuration imply a finely orchestrated mechanism at play. Our results provided the resources and pinpointed the molecular basis of UHRF1-mediated alternative RNA splicing, which will help us better our understanding of the physiological and pathological roles of UHRF1 in disease development.

Roles of IDM3 and SDJ1/2/3 in Establishment and/or Maintenance of DNA Methylation in Arabidopsis.

Previous studies had demonstrated that in Arabidopsis, IDM3 is involved in ROS1-mediated DNA demethylation pathway, and SUVH-SDJ complex functions as a DNA methylation reader complex for enhancing gene transcription, which presumably recruits ROS1 to the promoters of target genes for DNA demethylation. Here, our analyses, however, showed that the IDM3 and SDJ1/2/3, the components of the SUVH-SDJ complex, are implicated in establishing and/or maintaining DNA methylation as well through DDR (DRD1-DMS3-RDM1) complex. idm3-3 or sdj1/2/3 mutations led to genome-wide DNA hypomethylation, and both mutants shared a large number of common hypo-DMRs (Differentially Methylated Regions) with rdm1-4 and dms3-4, suggesting that IDM3 and SDJ1/2/3 help establish and/or maintain DNA methylation, mediated by RdDM pathway, at a subset of genomic regions largely through DDR complex. IDM3 is able to strongly interact with RDM1 and DMS3, but weakly with SDJ1 and SDJ3; SDJ1 and SDJ3 is capable of interacting separately with RDM1 and DMS3. Furthermore, comparisons of DNA methylation features in idm3-3 and sdj1/2/3 indicated that idm3-3 and sdj1/2/3 mutations make differential impacts on DNA methylation levels and patterns on a genome-wide scale, indicating that they are targeted to quite distinct genomic regions for aiding in DNA methylation. Further analyses on ChIP-seq data demonstrated that RDM1, DMS3 and NRPE1 are enriched in IDM3- and SDJ1/2/3-targted regions. Altogether, our results provide clear demonstration that IDM3 and SDJ1/2/3 play a part in establishing and/or maintaining DNA methylation of a group of genomic regions, through the DDR complex.

RNA-directed DNA methylation prevents rapid and heritable reversal of transposon silencing under heat stress in Zea mays.

In large complex plant genomes, RNA-directed DNA methylation (RdDM) ensures that epigenetic silencing is maintained at the boundary between genes and flanking transposable elements. In maize, RdDM is dependent on Mediator of Paramutation1 (Mop1), a gene encoding a putative RNA dependent RNA polymerase. Here we show that although RdDM is essential for the maintenance of DNA methylation of a silenced MuDR transposon in maize, a loss of that methylation does not result in a restoration of activity. Instead, heritable maintenance of silencing is maintained by histone modifications. At one terminal inverted repeat (TIR) of this element, heritable silencing is mediated via histone H3 lysine 9 dimethylation (H3K9me2), and histone H3 lysine 27 dimethylation (H3K27me2), even in the absence of DNA methylation. At the second TIR, heritable silencing is mediated by histone H3 lysine 27 trimethylation (H3K27me3), a mark normally associated with somatically inherited gene silencing. We find that a brief exposure of high temperature in a mop1 mutant rapidly reverses both of these modifications in conjunction with a loss of transcriptional silencing. These reversals are heritable, even in mop1 wild-type progeny in which methylation is restored at both TIRs. These observations suggest that DNA methylation is neither necessary to maintain silencing, nor is it sufficient to initiate silencing once has been reversed. However, given that heritable reactivation only occurs in a mop1 mutant background, these observations suggest that DNA methylation is required to buffer the effects of environmental stress on transposable elements.

Molecular pathogenesis of ICF syndrome

Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is characterized by frequent appearance of multiradial chromosomes, which are distinctive chromosome fusions that occur at hypomethylated pericentromeric regions comprising repetitive sequences, in activated lymphocytes. The syndrome is caused by mutations in DNMT3B, ZBTB24, CDCA7, or HELLS. Dr. Motoko Unoki, Medical Institute of Bioregulation, Kyushu University, Japan, was involved in the team to identify the CDCA7 and HELLS genes as causative genes of ICF syndrome. Recent studies including Unoki's studies suggested that de novo DNA methylation is likely defective in patients with ICF syndrome harboring mutations in DNMT3B, whereas accumulating evidence suggests that replication-uncoupled maintenance DNA methylation of late-replicating regions is impaired in patients with ICF syndrome harboring mutations in ZBTB24, CDCA7, or HELLS. ZBTB24 is a transcriptional activator of CDCA7, and CDCA7 and HELLS compose a chromatin remodeling complex and are involved in the maintenance DNA methylation through an interaction with UHRF1 in a feed-forward manner. The latest study by Unoki possibly provided the missing link between DNA hypomethylation and the formation of the abnormal chromosomes; it could occur via aberrant transcription from the hypomethylated regions, followed by pathological R-loop formation. The homologous-recombination dominant condition caused by a defect in non-homologous end joining observed in several types of ICF syndrome could facilitate the formation of multiradial chromosomes.

High throughput screening identifies SOX2 as a super pioneer factor that inhibits DNA methylation maintenance at its binding sites

Binding of mammalian transcription factors (TFs) to regulatory regions is hindered by chromatin compaction and DNA methylation of their binding sites. Nevertheless, pioneer transcription factors (PFs), a distinct class of TFs, have the ability to access nucleosomal DNA, leading to nucleosome remodelling and enhanced chromatin accessibility. Whether PFs can bind to methylated sites and induce DNA demethylation is largely unknown. Using a highly parallelized approach to investigate PF ability to bind methylated DNA and induce DNA demethylation, we show that the interdependence between DNA methylation and TF binding is more complex than previously thought, even within a select group of TFs displaying pioneering activity; while some PFs do not affect the methylation status of their binding sites, we identified PFs that can protect DNA from methylation and others that can induce DNA demethylation at methylated binding sites. We call the latter super pioneer transcription factors (SPFs), as they are seemingly able to overcome several types of repressive epigenetic marks. Finally, while most SPFs induce TET-dependent active DNA demethylation, SOX2 binding leads to passive demethylation, an activity enhanced by the co-binding of OCT4. This finding suggests that SPFs could interfere with epigenetic memory during DNA replication.

Maintenance DNA methylation in pre-meiotic germ cells regulates meiotic prophase by facilitating homologous chromosome pairing.

Heterochromatin-related epigenetic mechanisms, such as DNA methylation, facilitate pairing of homologous chromosomes during the meiotic prophase of mammalian spermatogenesis. In pro-spermatogonia, de novo DNA methylation plays a key role in completing meiotic prophase and initiating meiotic division. However, the role of maintenance DNA methylation in the regulation of meiosis, especially in the adult, is not well understood. Here, we reveal that NP95 (also known as UHRF1) and DNMT1 - two essential proteins for maintenance DNA methylation - are co-expressed in spermatogonia and are necessary for meiosis in male germ cells. We find that Np95- or Dnmt1-deficient spermatocytes exhibit spermatogenic defects characterized by synaptic failure during meiotic prophase. In addition, assembly of pericentric heterochromatin clusters in early meiotic prophase, a phenomenon that is required for subsequent pairing of homologous chromosomes, is disrupted in both mutants. Based on these observations, we propose that DNA methylation, established in pre-meiotic spermatogonia, regulates synapsis of homologous chromosomes and, in turn, quality control of male germ cells. Maintenance DNA methylation, therefore, plays a role in ensuring faithful transmission of both genetic and epigenetic information to offspring.

Chromatin remodeling in replication-uncoupled maintenance DNA methylation and chromosome stability: Insights from ICF syndrome studies.

Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is characterized by frequent appearance of multiradial chromosomes, which are distinctive chromosome fusions that occur at hypomethylated pericentromeric regions comprising repetitive sequences, in activated lymphocytes. The syndrome is caused by mutations in DNMT3B, ZBTB24, CDCA7, or HELLS. De novo DNA methylation is likely defective in patients with ICF syndrome harboring mutations in DNMT3B, whereas accumulating evidence suggests that replication‐uncoupled maintenance DNA methylation of late‐replicating regions is impaired in patients with ICF syndrome harboring mutations in ZBTB24, CDCA7, or HELLS. ZBTB24 is a transcriptional activator of CDCA7, and CDCA7 and HELLS compose a chromatin remodeling complex and are involved in the maintenance DNA methylation through an interaction with UHRF1 in a feed‐forward manner. Furthermore, our recent studies possibly provided the missing link between DNA hypomethylation and the formation of the abnormal chromosomes; it could occur via aberrant transcription from the hypomethylated regions, followed by pathological R‐loop formation. The homologous‐recombination dominant condition caused by a defect in nonhomologous end joining observed in several types of ICF syndrome could facilitate the formation of multiradial chromosomes. Here, the latest knowledge regarding maintenance DNA methylation and chromosome stability provided by those studies is reviewed.