In silico APC/C substrate discovery reveals cell-cycle-dependent degradation of UHRF1 and other chromatin regulators
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PROVIDER: PRJNA573856 | ENA |
REPOSITORIES: ENA
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Project description:E3 ubiquitin ligases are the final regulatory enzymes in the ubiquitin cascade, responsible for choosing substrates for ubiquitylation. Despite their central role in governing ubiquitin pathways, technical challenges have precluded development of simple and robust methods to systematically map their substrates. The Anaphase Promoting Complex/Cyclosome (APC/C) is an E3 ligase and master regulator of cell cycle progression and genome maintenance. Here, we develop a sensitive and specific computational strategy to predict APC/C substrates, leveraging orthogonal features among known targets. Interestingly, chromatin regulatory proteins are enriched among putative substrates and we validate several here. We reveal detailed ubiquitylation mechanisms of a key reader and writer of histone modifications, UHRF1, and show that its degradation regulates cell cycle and D methylation maintenance. Our results uncover mechanisms underlying the coordination between cell cycle and the chromatin landscape, and suggest an extensive, post-translational crosstalk with far-reaching consequences in normal cell physiology and disease.
2020-09-23 | GSE137913 | GEO
Project description:The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase and critical regulator of cell cycle progression. Despite its vital role, it has remained challenging to globally map APC/C substrates. By combining orthogonal features of known substrates, we predicted APC/C substrates in silico. This analysis identified many known substrates and suggested numerous candidates. Unexpectedly, chromatin regulatory proteins are enriched among putative substrates, and we show experimentally that several chromatin proteins bind APC/C, oscillate during the cell cycle, and are degraded following APC/C activation, consistent with being direct APC/C substrates. Additional analysis revealed detailed mechanisms of ubiquitylation for UHRF1, a key chromatin regulator involved in histone ubiquitylation and DNA methylation maintenance. Disrupting UHRF1 degradation at mitotic exit accelerates G1-phase cell cycle progression and perturbs global DNA methylation patterning in the genome. We conclude that APC/C coordinates crosstalk between cell cycle and chromatin regulatory proteins. This has potential consequences in normal cell physiology, where the chromatin environment changes depending on proliferative state, as well as in disease.
| S-EPMC7758050 | biostudies-literature
Project description:UHRF1 (Ubiquitin-like, containing PHD and RING finger domains, 1) recruits DNMT1 to hemimethylated DNA during replication, is essential for maintaining DNA methylation patterns during cell division and is suggested to direct additional repressive epigenetic marks. Uhrf1 mutation in zebrafish results in multiple embryonic defects including failed hepatic outgrowth, but the epigenetic basis of these phenotypes is not known. We find that DNA methylation is the only epigenetic mark that is depleted in uhrf1 mutants and make the surprising finding that despite the reduced organ size in uhrf1 mutants, genes regulating DNA replication and S-phase progression were highly upregulated. Further, there is a striking increase in BrdU incorporation in uhrf1 mutant cells, and they retained BrdU labeling over several days, indicating they are arrested in S-phase. Moreover, some of the label retaining nuclei co-localized with TUNEL positive nuclei, suggesting that arrested cells are responsible for apoptosis. Importantly, dnmt1 mutation phenocopies the S-phase arrest and hepatic outgrowth defects in uhrf1 mutants and Dnmt1 knock-down enhances the uhrf1 hepatic phenotype. Together, these data indicate that DNA hypomethylation is sufficient to generate the uhrf1 mutant phenotype by promoting an S-phase arrest. We thus propose that cell cycle arrest is a mechanism to restrict propagation of epigenetically deranged cells during embryogenesis. Genome-wide expression profiling was performed on 2 uhrf1 mutant and 2 wildtype zebrafish larvae (120 hours post fertilization) by using Zebrafish Genome Array (Affymetrix) according to manufacturer's instruction.
2015-02-09 | E-GEOD-55339 | biostudies-arrayexpress
Project description:We used microarrays to explore the expression profile from cells expressing wild type and UHRF1 S674A mutant. HeLa cells expressing UHRF1 WT and S674A mutant showed similar gene expression pattern without significant affecting the transcription of DNA repari genes. UHRF1 was depleted in HeLa cells by shRNA treatment. Total RNA was purified and used to determine the global gene transcription profiles by microarray assays. The UHRF1-related genes expression profiles were compared among control cells, UHRF1-depleted cells, UHRF1 WT reconstituting cells and UHRF1 S674A mutant reconstituting cells.
2016-01-05 | E-GEOD-73751 | biostudies-arrayexpress
Project description:Deletion of Uhrf1 resulted in stage 1-specific defects during iNKT cell development. To investigate the molecular mechanism, we sorted WT and Uhrf1-KO stage 1 iNKT cells and performed RNA-seq. By comparing gene expression profile, we found metabolic defects in Uhrf1-KO stage 1 iNKT cells. The expression of CD71 (Tfrc), two subunits of CD98 (Slc3a2 and Slc7a5) and Glut3 (Slc2a3) was reduced in stage 1 iNKT cells. Besides, the downstream pathways of AKT-mTOR axis were significantly reduced. Collectively, our results suggest that Uhrf1 is required for iNKT cell development by regulating the Akt-mTOR signaling pathway. We first sorted WT and Uhrf1-KO stage 1 iNKT cells, extracted the mRNA and performed RNA-seq. We then analyzed the differentially expressed genes and performed KEGG pathway analysis. We used RT-PCR to verify the expression of the key nutrient related genes (Tfrc, Slc3a2, Slc7a5 and Slc2a3) and used flow cytometry to test the protein level of metabolic related molecules. Besides, we also analyzed the expression of genes of mTOR downstream pathways to demonstrate that Uhrf1 mediated AKt-mTOR axis regulates iNKT cell development.
2016-03-10 | E-GEOD-77941 | biostudies-arrayexpress
Project description:Background-The endothelial protein C receptor (EPCR) plays an important role within the protein C (PC) pathway in regulating coagulation and inflammation. Recently, we described a novel mechanism of EPCR release from the surface of primary physiological cells. Induced by exogenous activated protein C (APC), EPCR is released in microparticulate form. Its bound APC retains proteolytic anticoagulant activity and we now hypothesise that this microparticulate EPCR-APC complex can also cleave endothelial protease activated receptor 1 (PAR1) to modulate inflammation and cytoprotection. Methods & Results-The gene profiling effect on human endothelial cells by 40nM APC, in free or microparticulate form, was assessed. Transcript profile results showed upregulation of anti-apoptotic and inflammatory genes by APC in either form, which were confirmed by RT-PCR. These translated into increased GM-CSF and interleukin 8 secretion and cytoprotection against staurosporine-induced apoptosis. PC or PAR1 antagonism reversed these results to demonstrate that induced effects by microparticles were APC specific and PAR1-dependent. Further analysis of human plasma from septic patients, by confocal microscopy and ELISA, showed evidence of circulating microparticle-associated EPCR during recombinant APC treatment. Functional coagulation and cellular studies demonstrate that these express APC-specific effects on anticoagulation with PAR1-mediated gene induction and anti-apoptotic function. Conclusions-APC induction of microparticle-associated EPCR release can occur in vivo. These microparticles could potentially disseminate the function of the EPCR-APC complex to PAR1 on different cells and vascular sites. Keywords: free apc versus mp-apc MP were isolated from huvec after estimulation with apc; the apc content of the mp was estimated by chromogenic substrate and a standard curve. 40nM apc on mp was used for estimulation of huvec for 4 hours for study of gene expression. APC relevance was studied by blocking experiments with antibody and par1 involvement was studied by including a par1 antagonist. All experiments were done in triplicate. A basline control was obtained by looking at the profile of non-estimulated cells under the same conditions.
2010-06-24 | E-GEOD-5660 | biostudies-arrayexpress
Project description:In silico discovery of the blood cell interactome
| PRJNA817538 | ENA
Project description:The multi-domain protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) recruits DNMT1 for DNA methylation maintenance during DNA replication. Here, we show that MOF (Males absent On the First) is an acetyltransferase of UHRF1 to acetylate UHRF1 at Lys670 in the pre-RING linker region whereas HDAC1 is a deacetylase of UHRF1 at the same site. The MOF/HDAC1-mediated acetylation in UHRF1 is cell-cycle regulated and peaks at G1/S phase, in line with the function of UHRF1 in recruiting DNMT1 to maintain DNA methylation. In addition, UHRF1 acetylation significantly enhances its E3 ligase activity and elimination of UHRF1 acetylation at these sites attenuates UHRF1-mediated H3 ubiquitination, which in turn impairs the DNMT1 recruitment and DNA methylation. Taken together, these findings not only identify MOF as a new acetyltransferase for UHRF1 but also reveal a novel mechanism underlying the regulation of DNA methylation maintenance through MOF-mediated UHRF1 acetylation.
2024-03-06 | PXD040426 | Pride
Project description:Histone methylation occurs on both lysine and arginine residues and its dynamic regulation plays a critical role in chromatin biology. Here we identify the UHRF1 PHD domain (PHDUHRF1), an important regulator of DNA CpG methylation, as an unanticipated histone H3 unmodified arginine 2 (H3R2)-recognition modality. This conclusion is based on binding studies and co-crystal structures of the PHDUHRF1 bound to histone H3 peptides, where the guanidinium group of unmodified R2 forms an extensive intermolecular hydrogen bond network, with methylation of H3R2, but not H3K4 or H3K9, disrupting complex formation. We have identified direct target genes of UHRF1 from microarray and ChIP studies. Importantly, we show that UHRF1’s ability to repress its direct target gene expression is dependent on PHDUHRF1 binding to unmodified H3R2, thereby demonstrating the functional importance of this recognition event and supporting the potential for crosstalk between histone arginine methylation and UHRF1 function. UHRF1 protein was depleted in HCT116 cells by shRNA treatment. Total RNA was purified and used to determine the global gene transcription profiles by microarray assays. The UHRF1-regulated genes were identified by comparing the gene expression profiles of control and UHRF1-depleted HCT116 cells.
2011-07-07 | E-GEOD-30478 | biostudies-arrayexpress
Project description:The Anaphase Promoting Complex/Cyclosome (APC/C) is a mega-dalton ubiquitin ligase that initiates mitotic exit by targeting substrates for degradation. The APC/C is activated by Cdc20, which acts as a substrate receptor, and is inhibited by the mitotic checkpoint complex (MCC), which delays mitotic exit when the spindle assembly checkpoint (SAC) is activated. We previously identified apcin, a small molecule ligand of Cdc20, as an inhibitor of APC/CCdc20. Surprisingly, we found that apcin paradoxically accelerates substrate degradation and promotes mitotic exit in cells with high SAC activity. Biochemical studies indicate that apcin cooperates with p31comet to relieve MCC-dependent inhibition of APC/C. Apcin’s behavior as an antagonist of Cdc20 can thus result in either net inhibition of APC/C, delaying mitotic exit when SAC activity is slow, or net activation of APC/C, promoting mitotic exit when SAC activity is high. Genetic experiments suggest that the dual behaviors of apcin arise from targeting a common binding site in Cdc20 that is required for both substrate ubiquitination as well as efficient APC/C inhibition by MCC. We therefore establish a new mechanism through which a small molecule, by targeting a single site on a dynamic protein interface, can lead to opposing biological effects depending on the regulatory context.
2020-01-07 | PXD013786 | Pride