Christian Bronner

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 1.7u2009Å crystal structure of the apo SRA domain of human UHRF1 and a 2.2u2009Å 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.

Recognition of Multivalent Histone States Associated with Heterochromatin by UHRF1 Protein

Histone modifications and DNA methylation represent two layers of heritable epigenetic information that regulate eukaryotic chromatin structure and gene activity. UHRF1 is a unique factor that bridges these two layers; it is required for maintenance DNA methylation at hemimethylated CpG sites, which are specifically recognized through its SRA domain and also interacts with histone H3 trimethylated on lysine 9 (H3K9me3) in an unspecified manner. Here we show that UHRF1 contains a tandem Tudor domain (TTD) that recognizes H3 tail peptides with the heterochromatin-associated modification state of trimethylated lysine 9 and unmodified lysine 4 (H3K4me0/K9me3). Solution NMR and crystallographic data reveal the TTD simultaneously recognizes H3K9me3 through a conserved aromatic cage in the first Tudor subdomain and unmodified H3K4 within a groove between the tandem subdomains. The subdomains undergo a conformational adjustment upon peptide binding, distinct from previously reported mechanisms for dual histone mark recognition. Mutant UHRF1 protein deficient for H3K4me0/K9me3 binding shows altered localization to heterochromatic chromocenters and fails to reduce expression of a target gene, p16INK4A, when overexpressed. Our results demonstrate a novel recognition mechanism for the combinatorial readout of histone modification states associated with gene silencing and add to the growing evidence for coordination of, and cross-talk between, the modification states of H3K4 and H3K9 in regulation of gene expression.

Induction of apoptosis by thymoquinone in lymphoblastic leukemia Jurkat cells is mediated by a p73-dependent pathway which targets the epigenetic integrator UHRF1

The salvage anti-tumoral pathway which implicates the p53-related p73 gene is not yet fully characterized. We therefore attempted to identify the up- and down-stream events involved in the activation of the p73-dependent pro-apoptotic pathway, by focusing on the anti-apoptotic and epigenetic integrator UHRF1 which is essential for cell cycle progression. For this purpose, we analyzed the effects of a known anti-neoplastic drug, thymoquinone (TQ), on the p53-deficient acute lymphoblastic leukemia (ALL) Jurkat cell line. Our results showed that TQ inhibits the proliferation of Jurkat cells and induces G1 cell cycle arrest in a dose-dependent manner. Moreover, TQ treatment triggers programmed cell death, production of reactive oxygen species (ROS) and alteration of the mitochondrial membrane potential (DeltaPsim). TQ-induced apoptosis, confirmed by the presence of hypodiploid G0/G1 cells, is associated with a rapid and sharp re-expression of p73 and dose-dependent changes of the levels of caspase-3 cleaved subunits. These modifications are accompanied by a dramatic down-regulation of UHRF1 and two of its main partners, namely DNMT1 and HDAC1, which are all involved in the epigenetic code regulation. Knockdown of p73 expression restores UHRF1 expression, reactivates cell cycle progression and inhibits TQ-induced apoptosis. Altogether our results showed that TQ mediates its growth inhibitory effects on ALL p53-mutated cells via the activation of a p73-dependent mitochondrial and cell cycle checkpoint signaling pathway which subsequently targets UHRF1.

Down-regulation of UHRF1, associated with re-expression of tumor suppressor genes, is a common feature of natural compounds exhibiting anti-cancer properties

Over-expressed in numerous cancers, Ubiquitin-like containing PHD Ring Finger 1 (UHRF1, also known as ICBP90 or Np95) is characterized by a SRA domain (Set and Ring Associated) which is found only in the UHRF family. UHRF1 constitutes a complex with histone deacetylase 1 (HDAC1) and DNA methyltransferase 1 (DNMT1) via its SRA domain and represses the expression of several tumour suppressor genes (TSGs) including p16INK4A, hMLH1, BRCA1 and RB1. Conversely, UHRF1 is regulated by other TSGs such as p53 and p73. UHRF1 is hypothetically involved in a macro-molecular protein complex called ECREM for Epigenetic Code Replication Machinery. This complex would be able to duplicate the epigenetic code by acting at the DNA replication fork and by activating the right enzymatic activity at the right moment. There are increasing evidence that UHRF1 is the conductor of this replication process by ensuring the crosstalk between DNA methylation and histone modifications via the SRA and Tandem Tudor Domains, respectively. This cross-talk allows cancer cells to maintain the repression of TSGs during cell proliferation. Several studies showed that down-regulation of UHRF1 expression in cancer cells by natural pharmacological active compounds, favors enhanced expression or re-expression of TSGs, suppresses cell growth and induces apoptosis. This suggests that hindering UHRF1 to exert its role in the duplication of the methylation patterns (DNA + histones) is responsible for inducing apoptosis. In this review, we present UHRF1 expression as a target of several natural products and we discuss their underlying molecular mechanisms and benefits for chemoprevention and chemotherapy.

Chronic intake of red wine polyphenols by young rats prevents aging-induced endothelial dysfunction and decline in physical performance: Role of NADPH oxidase

Aging is associated with oxidative stress-mediated endothelial dysfunction and decline in physical performance, which promote cardiovascular diseases. This study examined whether chronic intake of red wine polyphenols (RWPs), a rich source of natural antioxidants, prevents aging-related impairment of vascular function and physical exercise capacity. Vascular reactivity from 12, 20 and 40 week-old rats was assessed in organ chambers. Rats received from week 16 to 40 either solvent, RWPs or the antioxidant and NADPH oxidase inhibitor, apocynin. Aging was associated with blunted endothelium-dependent relaxations, oxidative stress (dihydroethidine staining), and an upregulation of eNOS, arginase I, NADPH oxidase p22phox and nox1 subunits, and AT1 and AT2 receptors (assessed by immunohistochemistry) in the mesenteric artery. RWPs and apocynin improved the endothelial dysfunction, normalized oxidative stress and the expression of the different proteins. RWPs also improved aging-related decline in physical exercise. Thus, intake of RWPs protects against aging-induced endothelial dysfunction and decline in physical performance. These effects likely involve the ability of RWPs to normalize oxidative stress and the expression of proteins involved in the formation of NO and the angiotensin II pathway.

Anti-proliferative effect of curcumin on melanoma cells is mediated by PDE1A inhibition that regulates the epigenetic integrator UHRF1

SCOPEnCurcumin inhibits proliferation of many cancer cells. Cyclic nucleotide phosphodiesterases (PDEs), by hydrolyzing intracellular cyclic adenosine-3,5-monophosphate (cAMP) and/or cyclic guanosine-3,5-monophosphate (cGMP), play a pivotal role in signalling pathways involved in cell proliferation. Therefore, this study investigated PDE1-5 participations in the anti-proliferative properties of curcumin in B16F10 murine melanoma cells.nnnMETHODS AND RESULTSnWe report that curcumin inhibits PDE1-5 activities (IC(50) ≅10(-5) u2009M), indicating that curcumin acts as a non-selective PDE inhibitor. In melanoma cells, PDE4 and PDE1 represent the major cAMP-PDEs and cGMP-PDEs activities, respectively. Curcumin treatment decreased PDE1 and PDE4 activities and dose dependently increased intracellular cGMP levels, whereas cAMP levels were unchanged. Curcumin inhibited cell proliferation and cell cycle progression by accumulating cells in the S- and G2/M-phases with enhanced expressions of cyclin-dependent kinase inhibitors. In contrast, expressions of PDE1A, cyclin A and the epigenetic integrator ubiquitin-like containing PHD and Ring Finger domains 1 (UHRF1) and DNA methyltransferase 1 (DNMT1) were decreased by curcumin. Interestingly, PDE1A overexpression increased UHRF1 and DNMT1 expressions and rescued the B16F10 cells from curcumin anti-proliferative effects. Nimodipine, a PDE1 inhibitor, mimicked the curcumin effects.nnnCONCLUSIONnCurcumin exerts its anti-cancer property by targeting PDE1 that inhibits melanoma cell proliferation via UHRF1, DNMT1, cyclin A, p21 and p27 regulations. This suggests that natural PDE1 inhibitors present in food might be effective in preventing cancer.

Red wine polyphenols cause growth inhibition and apoptosis in acute lymphoblastic leukaemia cells by inducing a redox-sensitive up-regulation of p73 and down-regulation of UHRF1

Several epidemiological studies suggest that a diet rich in fruits and vegetables, which contain high levels of polyphenols, is associated with a reduced risk of cancer. The aim of the present study was to determine whether a red wine polyphenolic extract (RWPs, a rich source of polyphenols; 2.9g/L) affects the proliferation of human lymphoblastic leukaemia cells (Jurkat cells) and, if so, to determine the underlying mechanism. Cell proliferation and viability were determined by the MTS and trypan blue exclusion assays, respectively. Cell cycle analysis, apoptosis activity and oxidative stress levels were assessed by flow cytometry, and the expression of p73, UHRF1 and active caspase-3 by Western blot analysis. RWPs inhibited the proliferation of Jurkat cells and induced G0/G1 cell cycle arrest in a concentration-dependent manner. Moreover, RWPs triggered apoptosis, which is associated with an increased expression level of the pro-apoptotic protein p73 and the active caspase-3. RWPs induced apoptosis confirmed by DNA fragmentation analysis, and this effect was associated with down-regulation of the antiapoptotic protein UHRF1. Furthermore RWPs significantly increased the formation of reactive oxygen species (ROS). Intracellular scavengers of superoxide anions (MnTMPyP, MnTBAP, PEG-SOD) prevented the RWPs-induced formation of ROS and apoptosis, while native extracellular superoxide dismutase (SOD) was without effect. In addition, the effect of RWPs on the expression levels of p73, active caspase-3 and UHRF1 was also prevented by MnTMPyP. Thus, these findings indicate that RWPs induce apoptosis in Jurkat cells by a redox-sensitive mechanism involving the intracellular formation of superoxide anions and consequently the up-regulation of p73 and down-regulation of UHRF1.

UHRF1 recruits the histone acetyltransferase Tip60 and controls its expression and activity

Tat-interactive protein, 60kDa (Tip60) is a histone acetyltransferase with specificity toward lysine 5 of histone H2A (H2AK5) and plays multiple roles in chromatin remodeling processes. Co-immunoprecipitation experiments performed on Jurkat cells, showed that Tip60 is present in the same macro-molecular complex as UHRF1 (Ubiquitin-like containing PHD and RING domain 1), DNMT1 (DNA methyltransferase 1), and HDAC1 (histone deacetylase 1). Furthermore, immunocytochemistry experiments confirmed that Tip60 co-localizes with the UHRF1/DNMT1 complex. Although down-regulation of UHRF1 by RNA interference enhanced Tip60 expression, a significant decrease of the level of acetylated H2AK5 was observed. Consistently, we have observed that down-regulation of Tip60 and DNMT1 by RNA interference, dramatically reduced the levels of acetylated H2AK5. Altogether, these results suggest that Tip60 is a novel partner of the epigenetic integration platform interplayed by UHRF1, DNMT1 and HDAC1 involved in the epigenetic code replication.

Losartan Prevents Portal Hypertension-Induced, Redox-Mediated Endothelial Dysfunction in the Mesenteric Artery in Rats

BACKGROUND & AIMSnAdvanced stages of portal hypertension are characterized by generalized vasodilatation and a hyperdynamic syndrome that leads to complications such as hepatopulmonary syndrome. We assessed the endothelial function--particularly the formation of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF)--in rats following common bile duct ligation (CBDL) to determine the underlying mechanisms of these processes.nnnMETHODSnReactivity of mesenteric artery rings from male Wistar rats was determined in organ chambers. The expression levels of connexins (Cx) (Cx37, Cx40, Cx43), intermediate and small conductance Ca(2+)-activated K(+) channels (IK(Ca), SK(Ca)), endothelial NO synthase (eNOS), NADPH oxidase subunits, and nitrotyrosines were assessed by immunohistochemistry in mesenteric and pulmonary arteries. The vascular formation of reactive oxygen species (ROS) was evaluated using dihydroethidine. Control rats or those that had undergone CBDL were given either the NADPH oxidase inhibitor apocynin or the angiotensin II receptor type 1 antagonist losartan.nnnRESULTSnDecreased EDHF-mediated relaxations to acetylcholine and red wine polyphenols were observed in CBDL rats, compared with controls, whereas the level of NO-mediated relaxation was similar. Impaired EDHF-mediated relaxations were associated with reduced vascular expression of Cx37, Cx40, Cx43, IK(Ca) and SK(Ca); increased expression of eNOS and NADPH oxidase subunits; and increased vascular formation of ROS and peroxynitrites. These effects were prevented by exposure to apocynin or losartan.nnnCONCLUSIONSnCBDL is associated with reduced EDHF-mediated relaxations in the mesenteric artery, whereas NO-mediated relaxations persisted. These findings indicate that impaired EDHF-mediated relaxation involves an excessive vascular oxidative stress, most likely following activation of angiotensin II type 1 receptors.

Red wine polyphenols improve an established aging-related endothelial dysfunction in the mesenteric artery of middle-aged rats: role of oxidative stress.

Aging is associated with blunted endothelium-dependent relaxations and vascular oxidative stress. Our previous study has indicated that daily intake of red wine polyphenols (RWPs) by young rats retards aging-related endothelial dysfunction in middle-aged rats. The aim of the present study is to determine whether intake of RWPs also improves an established endothelial dysfunction in middle-aged rats and, if so, to determine the underlying mechanism. Middle-aged rats (51 weeks) received either solvent (3% ethanol), RWPs extract (100mg/kg/day) or the antioxidant and NADPH oxidase inhibitor apocynin (100mg/kg/day) in the drinking water for 4 weeks. Vascular reactivity of mesenteric artery rings from control young (12 weeks) and middle-aged rats was assessed in organ chambers. The expression level of endothelial NO synthase (eNOS), arginase I, angiotensin II receptors (AT1R and AT2R), NADPH oxidase subunits and nitrotyrosines was assessed by immunohistochemistry, and the vascular formation of reactive oxygen species (ROS) by dihydroethidine. Aging is associated with blunted endothelium-dependent relaxations, an excessive vascular formation of ROS and peroxynitrites, and an up-regulation of eNOS, arginase I, NADPH oxidase subunits (nox-1, p22phox), and AT1R and AT2R expression. RWPs and apocynin treatments improved endothelial dysfunction, normalized oxidative stress and the expression of the different proteins in the mesenteric artery of middle-aged rats. The present findings indicate that aging is associated with blunted endothelium-dependent relaxations involving an increased oxidative stress, and that these responses are improved by the intake of RWPs or apocynin for 4weeks most likely by normalizing the expression of eNOS, arginase I, NADPH oxidase and angiotensin receptors.