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CUT&RUN Sequencing of EZH2 binding sites in HEK293T cells expressing a Dual-specificity Aptamer that specifically increases O-GlcNAcylation on beta-catenin

ABSTRACT: O-GlcNAc is a dynamic post-translational modification on thousands of intracellular proteins, it regulates protein functions and is involved in many metabolic diseases. Using a dual-specificity aptamer, we targeted the O-GlcNAc transferase (OGT) to endogenous β-catenin and specifically increased O-GlcNAcylation of β-catenin. We found that O-GlcNAc on β-catenin promoted its interaction with EZH2 regardless of the Wnt signaling status. To study how this interaction regulates the distribution of EZH2 on the genome, we performed Cleavage Under Targets and Release Using Nuclease (CUT&RUN) on HEK293T cells expressing individual (Ctrl.) or dual-specificity aptamers. Under Wnt - and Wnt + conditions, the dual-specificity aptamer increased the binding of EZH2 on 923 and 3473 genomic sites, respectively. These two sets of differential binding sites showed little overlap.

ORGANISM(S): Homo sapiens

PROVIDER: GSE190172 | GEO | 2022/11/30

REPOSITORIES: GEO

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CUT&RUN Sequencing of EZH2 binding sites in HEK293T cells expressing a Dual-specificity Aptamer that specifically increases O-GlcNAcylation on beta-catenin, when beta-catein was knocked-down

Project description:O-GlcNAc is a dynamic post-translational modification on thousands of intracellular proteins, it regulates protein functions and is involved in many metabolic diseases. Using a dual-specificity aptamer, we targeted the O-GlcNAc transferase (OGT) to endogenous β-catenin and specifically increased O-GlcNAcylation of β-catenin. We found that O-GlcNAc on β-catenin promoted its interaction with EZH2 regardless of the Wnt signaling status. To study how this interaction regulates the distribution of EZH2 on the genome, we performed Cleavage Under Targets and Release Using Nuclease (CUT&RUN) on HEK293T cells expressing individual (Ctrl.) or dual-specificity aptamers, under Wnt - and Wnt + conditions. This is a control experiment in beta-catenin knockdown cells.

2022-10-11 | GSE214775 | GEO

CUT&RUN Sequencing of EZH2 binding sites in HEK293T cells expressing a Dual-specificity Aptamer that specifically increases O-GlcNAcylation on beta-catenin when O-GlcNAc transferase was inhibited

2022-10-11 | GSE214774 | GEO

RNA Sequencing of HEK293T cells expressing a Dual-specificity Aptamer that specifically increases O-GlcNAcylation on beta-catenin

Project description:O-GlcNAc is a dynamic post-translational modification on thousands of intracellular proteins, it regulates protein functions and is involved in many metabolic diseases. Using a dual-specificity aptamer, we targeted the O-GlcNAc transferase (OGT) to endogenous β-catenin and specifically increased O-GlcNAcylation of β-catenin. To study how O-GlcNAcylation of β-catenin regulates the transcriptome, we performed RNA sequencing on HEK293T cells expressing individual (Ctrl.) or dual-specificity aptamers. We found that O-GlcNAcylation of β-catenin shifts the transcriptome in a Wnt-dependent manner: it repressed the expression of about 100 genes in the absence of Wnt, while it activated the expression of these genes when Wnt signaling was turned on.

2022-11-01 | GSE189378 | GEO

RNA Sequencing of HEK293T cells expressing a Dual-specificity Aptamer that specifically increases O-GlcNAcylation on beta-catenin when EZH2 was knocked-down

Project description:O-GlcNAc is a dynamic post-translational modification on thousands of intracellular proteins, it regulates protein functions and is involved in many metabolic diseases. Using a dual-specificity aptamer, we targeted the O-GlcNAc transferase (OGT) to endogenous β-catenin and specifically increased O-GlcNAcylation of β-catenin. To study how O-GlcNAcylation of β-catenin regulates the transcriptome, we performed RNA sequencing on HEK293T cells expressing individual (Ctrl.) or dual-specificity aptamers. This dataset is from the control experiment in which EZH2 was knocked-down with a shRNA in all samples.

2022-10-11 | GSE214777 | GEO

RNA Sequencing of HEK293T cells expressing a Dual-specificity Aptamer that specifically increases O-GlcNAcylation on beta-catenin when O-GlcNAc transferase was inhibited

Project description:O-GlcNAc is a dynamic post-translational modification on thousands of intracellular proteins, it regulates protein functions and is involved in many metabolic diseases. Using a dual-specificity aptamer, we targeted the O-GlcNAc transferase (OGT) to endogenous β-catenin and specifically increased O-GlcNAcylation of β-catenin. To study how O-GlcNAcylation of β-catenin regulates the transcriptome, we performed RNA sequencing on HEK293T cells expressing individual (Ctrl.) or dual-specificity aptamers. We found that O-GlcNAcylation of β-catenin shifts the transcriptome in a Wnt-dependent manner: it repressed the expression of about 100 genes in the absence of Wnt, while it activated the expression of these genes when Wnt signaling was turned on. This dataset is from the control experiment in which all samples were treated with the OGT inhibitor Ac5S.

2022-11-01 | GSE189622 | GEO

RNA-sequencing and ChIP-sequencing in murine mesenchymal stem cells treated with Ezh2 inhibitor GSK126 or β-catenin siRNA

Project description:During mesenchymal stem cell (MSC) differentiation, both Wnt signaling and the development of a rigid cytoskeleton promote commitment to the osteoblastic over adipogenic lineage. β-catenin is thought to play a critical role in Wnt effects. We show that β-catenin was additive with cytoskeletal signals to prevent adipogenesis, and β-catenin knockdown promoted adipogenesis even when the actin cytoskeleton was depolymerized. β-catenin also prevented osteoblast commitment in a cytoskeletal-independent manner, with β-catenin knockdown enhancing lineage commitment. Chip-seq showed that β-catenin associated with the promoter of EZH2, a key component of the PRC2 complex that governs genome methylation. Knocking down β-catenin lowered EZH2 levels and H3K27me3 at osteogenic loci. Further, when EZH2 was inhibited, β-catenin ’s anti-differentiation effects were lost. These results indicate that regulating EZH2 activity is key to β-catenin effects on MSC to preserve MSC multipotentiality.

2019-10-17 | GSE138980 | GEO

Leeuwen2007 - Elucidating the interactions between the adhesive and transcriptional functions of beta-catenin in normal and cancerous cells

Project description:Elucidating the interactions between the adhesive and transcriptional functions of beta-catenin in normal and cancerous cells. van Leeuwen IM1, Byrne HM, Jensen OE, King JR. Author information 1 Centre for Mathematical Medicine and Biology, Division of Applied Mathematics, School of Mathematical Sciences, University of Nottingham, Nottingham, NG7 2RD, UK. pmzivl@exmail.nottingham.ac.uk Abstract Wnt signalling is involved in a wide range of physiological and pathological processes. The presence of an extracellular Wnt stimulus induces cytoplasmic stabilisation and nuclear translocation of beta-catenin, a protein that also plays an essential role in cadherin-mediated adhesion. Two main hypotheses have been proposed concerning the balance between beta-catenin's adhesive and transcriptional functions: either beta-catenin's fate is determined by competition between its binding partners, or Wnt induces folding of beta-catenin into a conformation allocated preferentially to transcription. The experimental data supporting each hypotheses remain inconclusive. In this paper we present a new mathematical model of the Wnt pathway that incorporates beta-catenin's dual function. We use this model to carry out a series of in silico experiments and compare the behaviour of systems governed by each hypothesis. Our analytical results and model simulations provide further insight into the current understanding of Wnt signalling and, in particular, reveal differences in the response of the two modes of interaction between adhesion and signalling in certain in silico settings. We also exploit our model to investigate the impact of the mutations most commonly observed in human colorectal cancer. Simulations show that the amount of functional APC required to maintain a normal phenotype increases with increasing strength of the Wnt signal, a result which illustrates that the environment can substantially influence both tumour initiation and phenotype.

2020-01-09 | MODEL2001090001 | BioModels

CUT&RUN experiment at four time points in HEK293T and hESC cells, assessing the time-resolved response of β-catenin genomic occupancy upon Wnt signaling activation

Project description:Wnt/β-catenin signaling is a highly organized biochemical cascade that triggers a gene expression program in the signal-receiving cell. The Wnt/β-catenin-driven transcriptional response is involved in virtually all cellular processes during development, homeostasis, and its deregulation causes human disease. However, outstanding questions remain unanswered. A first question concerns cell-specificity: how this response is integrated into lineage-specific choices is still unknown. A second question concerns time: it is not known whether β-catenin associates with its targets simultaneously or in a time-dependent fashion. For instance, while TCF/LEF and other components of the Wnt transcriptional complex are constitutively associated with the chromatin, it is β-catenin arrival, upon Wnt induction, that launches target genes transcription. Therefore, discovering the dynamics of the genome-wide β-catenin binding pattern is required to unambiguously define the direct targets of Wnt signaling To address these questions, we realized a time-resolved atlas of β-catenin genome-wide occupancy in two human cell types, human embryonic kidney cells 293T (HEK293T) and human embryonic stem cells (hESCs). To this end, we treated HEK293T and hESCs with the GSK3 inhibitor/Wnt activator CHIR99021 (10 mM) for 3 days, and assessed β-catenin binding via CUT&RUN-LoV-U (Zambanini et al., 2022) 90 minutes, 4 hours, 24 hours and 3 days after the onset of the stimulation. This approach allowed us to establish that β-catenin repositions to different genomic loci along stimulation time, showing that a definition of Wnt target genes must take into account the time-dimension. Moreover, β-catenin physical targets are largely cell-type specific, as only a subset of them is present across the examined contexts.

2022-09-30 | E-MTAB-12077 | biostudies-arrayexpress

ACE-Scan of the cocaine DNA aptamer on 10^12 Gen1 DNA microarray

Project description:The experiments were carried out to map the ligand binding landscape of various DNA and RNA duplexed aptamer families. Duplexed Aptamer (DA) constructs were engineered from (i) natural and synthetic DNA and RNA aptamers and (i) synthetic oligonucleotide aptamer-complementary elements synthesized on custom DNA microarrays. The aptamers tested consist of the ATP DNA aptamer, the ATP RNA aptamer, the cocaine DNA aptamer, the human alpha-thrombin DNA aptamer, and the natural add riboswitch aptamer from the pathogenic bacteria Vibrio vulnificus. Each duplexed aptamer family consists of 1000's of synthetic constructs, each formed by hybridizing the aptamer with an aptamer-complementary element (ACE) - here, ACEs consisted of various DNA oligonucleotides synthesized as a custom DNA microarray.

2017-12-20 | E-MTAB-6377 | biostudies-arrayexpress

ACE-Scan of the ATP DNA aptamer on various density Gen2 DNA microarrays

2017-12-20 | E-MTAB-6375 | biostudies-arrayexpress

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