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

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. 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE189622 | GEO | 2022/11/01

REPOSITORIES: GEO

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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

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

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 which all samples were treated with the OGT inhibitor Ac5S.

2022-10-11 | GSE214774 | 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 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

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, 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.

2022-11-30 | GSE190172 | 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

Effect of O-GlcNAc transferase deficiency on intestinal epithelial cells in mice

Project description:O-GlcNAcylation is the modification of serine and threonine residues with beta-N-acetylglucosamine (O-GlcNAc) on intracellular proteins. To investigate the role of protein O-GlcNAcylation on intestinal homeostasis, we generated intestinal epithelial cell (IEC)-specific O-GlcNAc transferase (OGT) knockout in mice. The KO mice developed spontanous intestinal inflammation. To determine the underlying molecular mechanisms, we performed RNA sequencing of ileum and colon epithelial cells of wildtype and IEC-OGT KO mice.

2017-06-27 | GSE100473 | GEO

TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

Project description:TET proteins convert 5-methylcytosine to 5-hydroxymethylcytosine, an emerging dynamic epigenetic state of DNA that can influence transcription. Evidence has linked TET1 function to epigenetic repression complexes, yet mechanistic information, especially for the TET2 and TET3 proteins, remains limited. Here, we show a direct interaction of TET2 and TET3 with O-GlcNAc transferase (OGT). OGT does not appear to influence hmC activity, rather TET2 and TET3 promote OGT activity. TET2/3-OGT co-localize on chromatin at active promoters enriched for H3K4me3 and reduction of either TET2/3 or OGT activity results in a direct decrease in H3K4me3 and concomitant decreased transcription. Further, we show that Host Cell Factor 1 (HCF1), a component of the H3K4 methyltransferase SET1/COMPASS complex, is a specific GlcNAcylation target of TET2/3-OGT, and modification of HCF1 is important for the integrity of SET1/COMPASS. Additionally, we find both TET proteins and OGT activity promote binding of the SET1/COMPASS H3K4 methyltransferase, SETD1A, to chromatin. Finally, studies in Tet2 knockout mouse bone marrow tissue extend and support the data as decreases are observed of global GlcNAcylation and also of H3K4me3, notably at several key regulators of haematopoiesis. Together, our results unveil a step-wise model, involving TET-OGT interactions, promotion of GlcNAcylation, and influence on H3K4me3 via SET1/COMPASS, highlighting a novel means by which TETs may induce transcriptional activation. ChIP-Seq experiments were performed on Illumina HiScanSQ sequencer in wild-type HEK293T cells for H3K4me3 histone marks, O-GlcNAc and HCF1, for HT-TET2, HT-TET3 and HT-OGT in HEK293T cells overexpressing those three fusion proteins and in TET2 Kd HEK293T cells for H3K4me3 histone marks. ChIP-Seqs were also performed in mouse bone marrow tissues for H3K4me3 histone marks, O-GlcNAc, endogenous Tet2 and in Tet2 Ko bone marrow tissues for H3K4me3 histone marks.

2013-02-07 | E-GEOD-36620 | biostudies-arrayexpress

OGT, H2B S112GlcNAc, TET2 ChIP-seq results

Project description:TET2 directly interacts with OGT, which is important for the chromatin association of OGT in vivo. Although this specific interaction does not regulate the enzymatic activity of TET2, it facilitates OGT-dependent histone O-GlcNAcylation. Moreover, OGT associates with TET2 at transcription starting sites (TSS). Down-regulation of TET2 reduces the amount of H2B S112 GlcNAc marks in vivo, which are associated with gene transcription regulation. We found that OGT interacts with TET2 tightly. Using ChIP-seq with specific antibodies, we tested the co-localization of TET2 and OGT in genome level.

2012-12-11 | E-GEOD-41720 | biostudies-arrayexpress

O-glcnAc reprograms cellular energetics

Project description:Dysfunctional mitochondria and generation of reactive oxygen species (ROS) promote chronic diseases, which have spurred interest in the molecular mechanisms underlying these conditions. Previously, we have demonstrated that disruption of post-translational modification of proteins with β-linked N-acetylglucosamine (O- glcnAcylation) via overexpression of the O-glcnAc–regulating enzymes O- glcnAc transferase (OGT) or O- glcnAcase (OGA) impairs mitochondrial function. Here, we report that sustained alterations in O- glcnAcylation either by pharmacological or genetic manipulation also alters metabolic function. Sustained O-glcnAc elevation in SH-SY5Y neuroblastoma cells increased OGA expression and reduced cellular respiration and ROS generation. Cells with elevated O-glcnAc levels had elongated mitochondria and increased mitochondrial membrane potential, and RNA-Seq in SH-SY5Y cells indicated transcriptome reprogramming and down regulation of the NRF2-mediated antioxidant response. Sustained O-glcnAcylation in mice brain and liver validated the metabolic phenotypes observed in the cells, and OGT knockdown in the liver elevated ROS levels, impaired respiration, and increased the NRF2 antioxidant response. Moreover, elevated O-glcnAc levels promoted weight loss and lowered respiration in mice and skewed the mice toward carbohydrate-dependent metabolism as determined by indirect calorimetry. In summary, sustained elevation in O-glcnAcylation coupled with increased OGA expression reprograms energy metabolism, a finding that has potential implications for the etiology, development, and management of metabolic diseases.

2017-07-20 | GSE101642 | GEO

Dot1L recruits OGT target chromatin to regulate histones O-GlcNAcylation

Project description:OGT (O-GlcNAc transferase) is the distinctive enzyme responsible for catalyzing O-GlcNAc to the serine or threonine residues of thousands of cytoplasm and nuclear proteins that are involved in DNA damage, RNA splicing, and transcription preinitiation and initiation complex assembly. However, the molecular mechanism by OGT regulating gene transcription remains elusive. Using proximity labeling based mass spectrometry, we searched for functional partners of OGT and found that Dot1L, the conserved and unique histone methyltransferase mediated histone H3 lys79 methylation required for gene transcription, DNA damage repair, cell proliferation, and embryo development, interacts with OGT. Although this specific interaction does not regulate the enzymatic activity of Dot1L, it facilitates OGT-dependent histones O-GlcNAcylation. Moreover, OGT associates with Dot1L at transcription start sites, and depleting Dot1L decreased OGT associated with chromatin globally. Notably, downregulation of Dot1L reduces the levels of histone H2B S112 O-GlcNAcylation and histone H2B K120 ubiquitination in vivo, which are associated with gene transcription regulation. Taken together, these results reveal a Dot1L-dependent O-GlcNAcylation of chromatin.

2022-04-06 | GSE200059 | GEO

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