Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that the GAPDH control western blots shown in Fig. 5A on p. 9 for the experiments without MG132 treatment ('‑ MMGM132') were strikingly similar to the GAPDH control western blots shown in Fig. 5D, albeit with some horizontal and vertical resizing of the bands. In addition, an independent analysis of the data in this paper revealed that the flow cytometric plots for the TIP60‑transfected cells experiment in Fig. 8B on p. 12 were also strikingly similar to the plots shown to represent the TIP60‑eGFP Positive cells experiment in Fig. 8E. The authors have been contacted by the Editorial Office to offer an explanation for the apparent re‑use of the abovementioned data in this paper, and we are awaiting their response. Owing to the fact that the Editorial Office has been made aware of potential issues surrounding the scientific integrity of this paper, we are issuing an Expression of Concern to notify readers of this potential problem while the Editorial Office continues to investigate this matter further. [International Journal of Oncology 59: 89, 2021; DOI: 10.3892/ijo.2021.5269].

The histone variant macroH2A (mH2A), the most deviant variant, is about threefold larger than the conventional histone H2A and consists of a histone H2A-like domain fused to a large Non-Histone Region responsible for recruiting PARP-1 to chromatin. The available data suggest that the histone variant mH2A participates in the regulation of transcription, maintenance of heterochromatin, NAD+ metabolism, and double-strand DNA repair. Here, we describe a novel function of mH2A, namely its implication in DNA oxidative damage repair through PARP-1. The depletion of mH2A affected both repair and cell survival after the induction of oxidative lesions in DNA. PARP-1 formed a specific complex with mH2A nucleosomes in vivo. The mH2A nucleosome-associated PARP-1 is inactive. Upon oxidative damage, mH2A is ubiquitinated, PARP-1 is released from the mH2A nucleosomal complex, and is activated. The in vivo-induced ubiquitination of mH2A, in the absence of any oxidative damage, was sufficient for the release of PARP-1. However, no release of PARP-1 was observed upon treatment of the cells with either the DNA alkylating agent MMS or doxorubicin. Our data identify a novel pathway for the repair of DNA oxidative lesions, requiring the ubiquitination of mH2A for the release of PARP-1 from chromatin and its activation.

DNA methylation is one of the most important epigenetic mark involved in many physiologic cellular processes and pathologies. During mitosis, the transmission of DNA methylation patterns from a mother to the daughter cells is ensured through the action of the Ubiquitin-like, containing PHD and RING domains, 1/DNA methyltransferase 1 (UHRF1/DNMT1) tandem. UHRF1 is involved in the silencing of many tumor suppressor genes (TSGs) via mechanisms that remain largely to be deciphered. The present study investigated the role and the regulation of UHRF1 poly-ubiquitination induced by thymoquinone, a natural anti-cancer drug, known to enhance or re-activate the expression of TSGs. We found that the auto-ubiquitination of UHRF1, induced by TQ, is mediated by reactive oxygen species, and occurs following DNA damage. We demonstrated that the poly-ubiquitinated form of UHRF1 is K63-linked and can still silence the tumor suppressor gene p16INK4A/CDKN2A. We further showed that TQ-induced auto-ubiquitination is mediated via the activity of Tip60. Since this latter is known as a nuclear receptor co-factor, we investigated if the glucocorticoid receptor (GR) might be involved in the regulation of UHRF1 ubiquitination. Activation of the GR, with dexamethasone, did not influence auto-ubiquitination of UHRF1. However, we could observe that TQ induced a K48-linked poly-ubiquitination of GR, probably involved in the proteosomal degradation pathway. Mass-spectrometry analysis of FLAG-HA-tagged UHRF1 identified UHRF1 partners involved in DNA repair and showed that TQ increased their association with UHRF1, suggesting that poly-ubiquitination of UHRF1 is involved in the DNA repair process. We propose that poly-ubiquitination of UHRF1 serves as a scaffold to recruit the DNA repair machinery at DNA damage sites.

HIRIP3 is a mammalian protein homologous to the yeast H2A.Z deposition chaperone Chz1. However, the structural basis underlying Chz’s binding preference for H2A.Z over H2A, as well as the mechanism through which Chz1 modulates histone deposition or replacement, remains enigmatic. In this study, we aimed to characterize the function of HIRIP3 and to identify its interacting partners in HeLa cells. Our findings reveal that HIRIP3 is specifically associated in vivo with H2A–H2B dimers and CK2 kinase. While bacterially expressed HIRIP3 exhibited a similar binding affinity towards H2A and H2A.Z, the associated CK2 kinase showed a notable preference for H2A phosphorylation at serine 1. The recombinant HIRIP3 physically interacted with the H2A αC helix through an extended CHZ domain and played a crucial role in depositing the canonical core histones onto naked DNA. Our results demonstrate that mammalian HIRIP3 acts as an H2A histone chaperone, assisting in its selective phosphorylation by Ck2 kinase at serine 1 and facilitating its deposition onto chromatin.

Background Inherited defects in the base-excision repair gene MBD4 predispose individuals to adenomatous polyposis and colorectal cancer, which is characterized by an accumulation of C > T transitions resulting from spontaneous deamination of 5’-methylcytosine. Methods Here, we have investigated the potential role of MBD4 in regulating DNA methylation levels using genome-wide transcriptome and methylome analyses. Additionally, we have elucidated its function through a series of in vitro experiments. Results Here we show that the protein MBD4 is required for DNA methylation maintenance and G/T mismatch repair. Transcriptome and methylome analyses reveal a genome-wide hypomethylation of promoters, gene bodies and repetitive elements in the absence of MBD4 in vivo. Methylation mark loss is accompanied by a broad transcriptional derepression phenotype affecting promoters and retroelements with low methylated CpG density. MBD4 in vivo forms a complex with the mismatch repair proteins (MMR), which exhibits high bi-functional glycosylase/AP-lyase endonuclease specific activity towards methylated DNA substrates containing a G/T mismatch. Experiments using recombinant proteins reveal that the association of MBD4 with the MMR protein MLH1 is required for this activity. Conclusions Our data identify MBD4 as an enzyme specifically designed to repair deaminated 5-methylcytosines and underscores its critical role in safeguarding against methylation damage. Furthermore, it illustrates how MBD4 functions in normal and pathological conditions.

Tat interactive protein, 60 kDa (TIP60) is an important partner of ubiquitin-like, containing PHD and RING finger domains 1 (UHRF1), ensuring various cellular processes through its acetyltransferase activity. TIP60 is believed to play a tumor suppressive role, partly explained by its downregulated expression in a number of cancers. The aim of the present study was to investigate the role and mechanisms of action of TIP60 in the regulation of UHRF1 expression. The results revealed that TIP60 overexpression downregulated the UHRF1 and DNA methyltransferase 1 (DNMT1) expression levels. TIP60 interfered with USP7-UHRF1 association and induced the degradation of UHRF1 in an auto-ubiquitination-dependent manner. Moreover, TIP60 activated the p73-mediated apoptotic pathway. Taken together, the data of the present study suggest that the tumor suppressor role of TIP60 is mediated by its regulation to UHRF1.

The Rett syndrome protein MeCP2 was described as a methyl-CpG-binding protein, but its exact function remains unknown. Here we show that mouse MeCP2 is a microsatellite binding protein that specifically recognizes hydroxymethylated CA repeats. Depletion of MeCP2 alters chromatin organization of CA repeats and lamina-associated domains and results in nucleosome accumulation on CA repeats and genome-wide transcriptional dysregulation. The structure of MeCP2 in complex with a hydroxymethylated CA repeat reveals a characteristic DNA shape, with considerably modified geometry at the 5-hydroxymethylcytosine, which is recognized specifically by Arg133, a key residue whose mutation causes Rett syndrome. Our work identifies MeCP2 as a microsatellite DNA binding protein that targets the 5hmC-modified CA-rich strand and maintains genome regions nucleosome-free, suggesting a role for MeCP2 dysfunction in Rett syndrome.

Abstract Background: The epigenetic regulator UHRF1 (Ubiquitin-like, containing PHD and RING finger domains 1) plays an essential role in faithful transmission of DNA methylation during replication. It has tumorogenesis potential and is overexpressed in cancers. TIP60 (Tat interactive protein, 60 kDa) is an important partner of UHRF1, ensuring various cellular processes through its acetyltransferase activity. TIP60 is believed to exert a tumor suppressive role partly explained by its down-regulation in many cancers. Both proteins participate in various cellular functions such as chromatin remodeling, cell cycle, DNA damage repair and regulation of protein stability.Methods: Immunoprecipitation and confocal microscopy techniques were performed to study the ubiquitination of UHRF1 and USP-UHRF1 association. Fluorescence lifetime imaging microscopy (FLIM) technique was performed to analyze the interaction between UHRF1 and ubiquitin inside the nucleus. Western blotting was used to assess the effect of TIP60 overexpression on p73, pro- and anti-apoptotic proteins. TIP60-mediated apoptosis in HeLa cells was investigated by flow cytometry. Results were statistically analyzed using Graphpad prism.Results: Herein, our goal was to investigate the role and mechanism of TIP60 in the regulation of UHRF1 expression. Our results showed that TIP60 overexpression down-regulated UHRF1 and DNMT1 (DNA methyltransferase 1) expressions. TIP60 interfered with USP7-UHRF1 association and induced degradation of UHRF1 in an auto-ubiquitination dependent pathway. Moreover, TIP60 activated the p73-mediated apoptotic pathway.Conclusion: Taken together, our data suggest that the tumor suppressor role of TIP60 is mediated by its regulation to UHRF1.

Epigenetic modifications and nucleosome positioning play an important role in modulating gene expression. However, how the patterns of epigenetic modifications and nucleosome positioning are established around promoters is not well understood. Here, we have addressed these questions in a series of genome-wide experiments coupled to a novel bioinformatic analysis approach. Our data reveal a clear correlation between CpG density, promoter activity and accumulation of active or repressive histone marks. CGI boundaries define the chromatin promoter regions that will be epigenetically modified. CpG-rich promoters are targeted by histone modifications and histone variants, while CpG-poor promoters are regulated by DNA methylation. CGIs boundaries, but not transcriptional activity, are essential determinants of H2A.Z positioning in vicinity of the promoters, suggesting that the presence of H2A.Z is not related to transcriptional control. Accordingly, H2A.Z depletion has no impact on gene expression of arrested mouse embryonic fibroblasts. Therefore, the underlying DNA sequence, the promoter CpG density and, to a lesser extent, transcriptional activity, are key factors implicated in promoter chromatin architecture.

Natural polyamines such as putrescine, spermidine, and spermine are crucial in the cell proliferation and maintenance in all the eukaryotes. However, the requirement of polyamines in tumor cells is stepped up to maintain tumorigenicity. Many synthetic polyamine analogues have been designed recently to target the polyamine metabolism in tumors to induce apoptosis. N4-Erucoyl spermidine (designed as N4-Eru), a novel acylspermidine derivative, has been shown to exert selective inhibitory effects on both hematological and solid tumors, but its mechanisms of action are unknown. In this study, RNA sequencing was performed to investigate the anticancer mechanisms of N4-Eru-treated T-cell acute lymphoblastic leukemia (ALL) cell line (Jurkat cells), and gene expression was examined through different tools. We could show that many key oncogenes including NDRG1, CACNA1G, TGFBR2, NOTCH1,2,3, UHRF1, DNMT1,3, HDAC1,3, KDM3A, KDM4B, KDM4C, FOS, and SATB1 were downregulated, whereas several tumor suppressor genes such as CDKN2AIPNL, KISS1, DDIT3, TP53I13, PPARG, FOXP1 were upregulated. Data obtained through RNA-Seq further showed that N4-Eru inhibited the NOTCH/Wnt/JAK-STAT axis. This study also indicated that N4-Eru-induced apoptosis could involve several key signaling pathways in cancer. Altogether, our results suggest that N4-Eru is a promising drug to treat ALL.

Down-regulation of UHRF1 (Ubiquitin-like containing PHD and Ring Finger 1) in Jurkat cells, induced by natural anticancer compounds such as thymoquinone, allows re-expression of tumor suppressor genes such as p73 and p16INK4A . In order to decipher the mechanisms of UHRF1 down-regulation, we investigated the kinetic of expression of HAUSP (herpes virus-associated ubiquitin-specific protease), UHRF1, cleaved caspase-3 and p73 in Jurkat cells treated with thymoquinone. We found that thymoquinone induced degradation of UHRF1, correlated with a sharp decrease in HAUSP and an increase in cleaved caspase-3 and p73. UHRF1 concomitantly underwent a rapid ubiquitination in response to thymoquinone and this effect was not observed in the 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 proteosomal inhibitor MG132 had no such effect. The present findings indicate that thymoquinone induces in cancer cells a fast UHRF1 auto-ubiquitination through its RING domain associated with HAUSP down-regulation. They further suggest that thymoquinone-induced UHRF1 auto-ubiquitination followed by its degradation is a key event in inducing apoptosis through a proteasome-independent mechanism.

Background: The nuclear epigenetic integrator UHRF1 is known to play a key role with DNMT1 in maintaining the DNA methylation patterns during cell division. Among UHRF1 partners, TIP60 takes part in epigenetic regulations through its acetyltransferase activity. Both proteins are involved in multiple cellular functions such as chromatin remodeling, DNA damage repair and regulation of stability and activity of other proteins. The aim of this work was to investigate the interaction between UHRF1 and TIP60 in order to elucidate the dialogue between these two proteins. Methods: Biochemical (immunoprecipitation and pull-down assays) and microscopic (confocal and fluorescence lifetime imaging microscopy; FLIM) techniques were used to analyze the interaction between TIP60 and UHRF1 in vitro and in vivo. Global methylation levels were assessed by using a specific kit. The results were statistically analyzed using Graphpad prism and Origin. Results: Our study shows that UHRF1, TIP60 and DNMT1 were found in the same epigenetic macro-molecular complex. In vitro pull-down assay showed that deletion of either the zinc finger in 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. Conclusion: Our data demonstrate for the first time that TIP60 through its MYST domain directly interacts with UHRF1 which might be of high interest for the development of novel oncogenic inhibitors targeting this interaction.

Cancer is one of the deadliest diseases in the world causing record number of mortalities in both developed and undeveloped countries. Despite a lot of advances and breakthroughs in the field of oncology still, it is very hard to diagnose and treat the cancers at early stages. Here in this review we analyze the potential of Ubiquitin-like containing PHD and Ring Finger domain 1 (UHRF1) as a universal biomarker for cancers. UHRF1 is an important epigenetic regulator maintaining DNA methylation and histone code in the cell. It is highly expressed in a variety of cancers and is a well-known oncogene that can disrupt the epigenetic code and override the senescence machinery. Many studies have validated UHRF1 as a powerful diagnostic and prognostic tool to differentially diagnose cancer, predict the therapeutic response and assess the risk of tumor progression and recurrence. Highly sensitive, non-invasive and cost effective approaches are therefore needed to assess the level of UHRF1 in patients, which can be deployed in diagnostic laboratories to detect cancer and monitor disease progression.

DNA methylation is an essential epigenetic modification, present in both unique DNA sequences and repetitive elements, but its exact function in repetitive elements remains obscure. Here, we describe the genome-wide comparative analysis of the 5mC, 5hmC, 5fC, and 5caC profiles of repetitive elements in mouse embryonic fibroblasts and mouse embryonic stem cells. We provide evidence for distinct and highly specific DNA methylation/oxidation patterns of the repetitive elements in both cell types, which mainly affect CA repeats and evolutionarily conserved mouse-specific transposable elements including IAP-LTRs, SINEs B1m/B2m, and L1Md-LINEs. DNA methylation controls the expression of these retroelements, which are clustered at specific locations in the mouse genome. We show that TDG is implicated in the regulation of their unique DNA methylation/oxidation signatures and their dynamics. Our data suggest the existence of a novel epigenetic code for the most recently acquired evolutionarily conserved repeats that could play a major role in cell differentiation.

Ubiquitin-like containing PHD and Ring finger 1 (UHRF1) contributes to silencing of tumor suppressor genes by recruiting DNA methyltransferase 1 (DNMT1) to their hemi-methylated promoters. Conversely, demethylation of these promoters has been ascribed to the natural anti-cancer drug, epigallocatechin-3-gallate (EGCG). The aim of the present study was to investigate whether the UHRF1/DNMT1 pair is an important target of EGCG action. Here, we show that EGCG down-regulates UHRF1 and DNMT1 expression in Jurkat cells, with subsequent up-regulation of p73 and p16(INK4A) genes. The down-regulation of UHRF1 is dependent upon the generation of reactive oxygen species by EGCG. Up-regulation of p16(INK4A) is strongly correlated with decreased promoter binding by UHRF1. UHRF1 over-expression counteracted EGCG-induced G1-arrested cells, apoptosis, and up-regulation of p16(INK4A) and p73. Mutants of the Set and Ring Associated (SRA) domain of UHRF1 were unable to down-regulate p16(INK4A) and p73, either in the presence or absence of EGCG. Our results show that down-regulation of UHRF1 is upstream to many cellular events, including G1 cell arrest, up-regulation of tumor suppressor genes and apoptosis.

The microtubule-targeting agents derived from natural products, such as vinca-alkaloids and taxanes are an important family of efficient anti-cancer drugs with therapeutic benefits in both haematological and solid tumors. These drugs interfere with the assembly of microtubules of α/β tubulin heterodimers without altering their expression level. The aim of the present study was to investigate the effect of thymoquinone (TQ), a natural product present in black cumin seed oil known to exhibit putative anti-cancer activities, on α/β tubulin expression in human astrocytoma cells (cell line U87, solid tumor model) and in Jurkat cells (T lymphoblastic leukaemia cells). TQ induced a concentration- and time-dependent degradation of α/β tubulin in both cancer cell types. This degradation was associated with the up-regulation of the tumor suppressor p73 with subsequent induction of apoptosis. Interestingly, TQ had no effect on α/β tubulin protein expression in normal human fibroblast cells, which were used as a non-cancerous cell model. These data indicate that TQ exerts a selective effect towards α/β tubulin in cancer cells. In conclusion, the present findings indicate that TQ is a novel anti-microtubule drug which targets the level of α/β tubulin proteins in cancer cells. Furthermore, they highlight the interest of developing anti-cancer therapies that target directly tubulin rather than microtubules dynamics.

Background: UHRF1 (Ubiquitin-like containing PHD Ring Finger), an anti-apoptotic protein essential for cell proliferation, is over-expressed in several types of cancer. UHRF1 participates in a huge macro-molecular complex including DNMT1 (DNA methyltransferase 1), Tip60 (a histone acetyltransferase), HAUSP (a ubiquitin specific protease) and HDAC1 (a histone deacetylase). It has been shown that HAUSP protects, in vitro, UHRF1 from auto-ubiquitination and regulates its stability in vivo. We previously showed that thymoquinone (TQ), an anti-cancer drug, induced UHRF1 down-regulation by a p73and caspase 3dependent process. The goal of the present study was to determine more precisely the pathway involved in the degradation of UHRF1 by TQ. Material and Methods: Jurkat cells (T lymphoblastic leukaemia cells) and human astrocytoma cells (cell line U87) were used as cancer cell models. Western blot experiments were performed to detect UHRF1, HAUSP and p73 in both cell lines. Results: We have observed that TQ induced a dose-dependent downregulation of UHRF1 and HAUSP accompanied with p73 up-regulation. Interestingly, kinetic study revealed the presence of higher bands of UHRF1 expression as assessed by western blotting on both cell lines. Co-immunoprecipitation experiments allowed us to demonstrate, in Jurkat cells, that these bands were due to ubiquitination of UHRF1. These data show that the degradation of UHRF1, challenged by TQ, is due to its ubiquitination through an as yet unknown mechanism but which appears dependent upon HAUSP down-regulation. Conclusion: In conclusion, we propose that UHRF1 ubiquitination is a key event in the TQ-induced apoptosis in cancer cells. This ubiquitination might result from the auto-ubiquitination activity of UHRF1, following HAUSP down-regulation.