DNA methylation analysis of HCT116 after knockdown and rescue of UHRF1
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ABSTRACT:
PROVIDER: PRJNA560094 | ENA |
REPOSITORIES: ENA
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Project description:In HCT116 colorectal cancer cells, UHRF1 was knocked down by shRNA (puromycin) while simultaneously transduced with wildtype or mutant UHRF1 (blasticidin) or NDI1 (- control) followed by dual antibiotic selection. DNA was analyzed 11 days after viral transduction.
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Project description:DNA methylation analysis of HCT116 after knockdown of LIG1, UHRF1, or luciferase (control)
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Project description:In HCT116 colorectal cancer cells, UHRF1, LIG1, or luciferase was knocked down by shRNA followed by selection with puromycin for 2 days. DNA was analyzed 12 days after viral transduction.
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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:DNA methylation is a heritable chromatin modification essential to mammalian development that functions with histone post-translational modifications to regulate chromatin structure and gene expression programs. The epigenetic inheritance of DNA methylation requires the combined actions of DNMT1 and UHRF1, a histone- and DNA-binding RING E3 ubiquitin ligase that facilitates DNMT1 recruitment to sites of newly replicated DNA through the ubiquitylation of histone H3. UHRF1 binds DNA with modest selectivity towards hemi-methylated CpG dinucleotides (HeDNA); however, the contribution of HeDNA sensing to UHRF1 function remains elusive. Here, we reveal that the interaction of UHRF1 with HeDNA is required for DNA methylation inheritance but is dispensable for chromatin interaction, which is governed by reciprocal positive cooperativity between the UHRF1 histone- and DNA-binding domains. We further show that HeDNA functions as an allosteric regulator of UHRF1 ubiquitin ligase activity, directing ubiquitylation towards multiple lysines on the H3 tail adjacent to the UHRF1 histone-binding site. Collectively, our studies define a highly orchestrated epigenetic control mechanism involving modifications both to histones and DNA that facilitate UHRF1 chromatin targeting, H3 ubiquitylation, and DNA methylation inheritance.
2016-08-25 | PXD003983 | Pride
Project description:UHRF1 is a key regulator of DNA methylation maintenance. In this study, we investigated whether acetylation of UHRF1 affects its hemimethylated DNA binding affinity and alters genome-wide DNA methylation pattern. We show that cells with mutation in K490 of UHRF1 have distinct methylation profile versus wildtype UHRF1 expressing cells.
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Project description:HIV-1 latent reservoirs constitute a major barrier to viral eradication. The various potencies of latency-reversing agents reflect the multiplicity of the silencing mechanisms underlying latency, and the dynamic and heterogeneous nature of the HIV-1 latent reservoirs. Here, we investigated the mechanisms of the DNA methylation inhibitor 5-aza-2’-deoxycytidine (5-AzadC) potency in HIV-1 latency reversal. We report that this potency was directly linked to 5-AzadC-induced demethylation of the HIV-1 promoter, which occurred at non-random CpG dinucleotides. Specifically, the methylation status of one of those CpGs allowed the recruitment of the epigenetic integrator UHRF1 to the HIV-1 promoter. 5-AzadC-induced viral reactivation led to a decreased in vivo recruitment of UHRF1. RNA interference-mediated knockdown of UHRF1 demonstrated its role in DNA methylation-mediated silencing of the latent HIV-1 promoter. Pharmacological downregulation of UHRF1 in ex vivo HIV+ patient cell cultures resulted in potent reactivation of latent HIV-1, further highlighting that UHRF1 could constitute a new molecular target to devise innovative HIV-1 curative strategies.
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Project description:DNA methylation is an essential epigenetic mark in mammals. It controls gene expression and genome stability. Global DNA methylation pattern is abnormal in cancers. Ubiquitin like with PHD and RING finger domains 1 (UHRF1) is a key epigenetic regulator that recruits and activates DNA methyltransferase 1 (DNMT1), the methylation maintenance enzyme. UHRF1 is a proven oncogene and its overexpression transforms cells in vitro and causes cancer in animal models. Therefore, UHRF1 provides a unique entry point into the links between epigenetics and cancer. However, it is still not fully clear how UHRF1 works in cancer cells. To understand UHRF1 functions in cancer, we employed experimental strategy to use an advanced chemical/genetic system, the auxin-inducible degron (AID) technology, whereby the degron-fused protein can be totally and rapidly degraded upon the addition of a small molecule, auxin. We chose the human CRC cell line HCT116 as our model and successfully generated UHRF1-AID and DNMT1-AID. Through this study, we made the significant discovery that UHRF1 not only regulates DNMT1, but also influences the activities of de novo methyltransferases DNMT3A and DNMT3B, as well as the active demethylase TET2.
2024-02-12 | GSE236026 | GEO
Project description:DNA methylation is an essential epigenetic mark in mammals. It controls gene expression and genome stability. Global DNA methylation pattern is abnormal in cancers. Ubiquitin like with PHD and RING finger domains 1 (UHRF1) is a key epigenetic regulator that recruits and activates DNA methyltransferase 1 (DNMT1), the methylation maintenance enzyme. UHRF1 is a proven oncogene and its overexpression transforms cells in vitro and causes cancer in animal models. Therefore, UHRF1 provides a unique entry point into the links between epigenetics and cancer. However, it is still not fully clear how UHRF1 works in cancer cells. To understand UHRF1 functions in cancer, we employed experimental strategy to use an advanced chemical/genetic system, the auxin-inducible degron (AID) technology, whereby the degron-fused protein can be totally and rapidly degraded upon the addition of a small molecule, auxin. We chose the human CRC cell line HCT116 as our model and successfully generated UHRF1-AID and DNMT1-AID. Through this study, we made the significant discovery that UHRF1 not only regulates DNMT1, but also influences the activities of de novo methyltransferases DNMT3A and DNMT3B, as well as the active demethylase TET2.
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Project description:UHRF1 (ubiquitin-like with PHD and ring finger domains 1) is an epigenetic regulator that is involved in the regulation of DNA and histone methylation and many other cellular events. The UHRF1 is frequently found to be overexpressed in various human cancers, and its overexpression has been associated with pro-tumorigenic effects such as inhibition of apoptosis and high metastatic potential. However, the molecular mechanisms underlying these pro-tumorigenic effects of UHRF1 overexpression in cancers still remain unclear. Retinoblastoma (Rb) is an intraocular tumor which arises from developing retina by biallelic inactivation of Rb1 gene. In this study, we uncovered that the UHRF1 is highly expressed in retinoblastoma, and genomes of retinoblastoma have differential DNA methylation patterns compared to those of normal retina, characterized by global hypomethylation and promoter hypermethylation at key tumor suppressor genes. Given the well-documented functions of UHRF1 in the regulation of DNA methylation, we hypothesized that the overexpressed UHRF1 may contribute to the aberrant DNA methylation in retinoblastoma genomes. To test our hypothesis, we profiled the genome-wide methylation patterns in normal retina and Y79 retinoblastoma cell line by MeDIP-seq, and then investigated how the methylation patterns in Y79 are affected by down-modulation of UHRF1. For identification of differentially methylated regions between the control and UHRF1 knockdown Y79 cells, we analysed three independent sets of sequencing data to unambiguously determine the effects of UHRF1 down-modulation on the methylome of Y79 retinoblastoma cells.
2017-03-30 | GSE92712 | GEO