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.Histone modifications and DNA methylation represent two layers of heritable epigenetic information that regulate chromatin structure and gene activity in eukaryotic organisms. Methylated DNA sequences are generally associated with long term transcriptional silencing through the recruitment of repressor complexes, including methyl-binding proteins, histone deacetylases, and chromatin remodeling machinery (1, 2). Likewise, specific histone methylation states can recruit multivalent adaptor proteins, which lead to chromatin condensation, further inhibiting gene expression. Accumulating evidence shows that these two methylation systems act cooperatively to establish the epigenetic state of the cell (3-5); however, the mechanisms of this cooperation remain vague.During replication, CpG methylation patterns are maintained in mammals by the DNA methyltransferase 1 with hemimethylated CpG dinucleotides serving as a substrate. This enzyme is aided by UHRF1 (ubiquitin-like, PHD and RING finger containing 1, also known as ICBP90 in humans and NP95 in mouse), which interacts with DNA methyltransferase 1 and specifically recognizes hemimethylated CpG dinucleotides through its SET-and RING-associated domain (SRA) 4 (6, 7). 4 The abbreviations used are: SRA, SET-and RING-associated domain; UHRF1, ubiquitin-like, PHD and RING finger containing 1; mUHRF1, murine UHRF1; TTD, tandem tudor domain; TTD N , N-terminal tudor subdomain; TTD C , C-terminal tudor subdomain; NMR, nuclear magnetic resonance; RDC, residual dipolar coupli...

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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-Stransferase pull-down experiments. We checked whether ICBP90 and DNMT1 are present in the same macromolecular complexes in Jurkat cells and immortalized human vascular smooth muscle cells (HVTs-SM1). Coimmunoprecipitation 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.

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 (m). 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.