Identification of Pyruvate Dehydrogenase A1 (p-PDH A1 S293) and Pyruvate Kinase M2 (PKM2) target genes by ChIP Seq in HepG2 Cells Upon Insulin stimulation.
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ABSTRACT:
PROVIDER: PRJNA800819 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR17772240.fastq.gz | Fastqsanger.gz | |||
| SRR17772241.fastq.gz | Fastqsanger.gz | |||
| SRR17772242.fastq.gz | Fastqsanger.gz |
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Project description:Goal and Objectives: Here we first time report that p-PDH A1 (S293 in mouse but in Human is S264), and PKM2 can regulates the many genes upon Insulin in Human Heptocellular Carcinoma HepG2 cells Line. The goal of this study is to identify the target genes, of which the promoters are associated with p-PDHA1 and PKM2, which may regulate target genes' trascription. We performed Chromatin immunoprecipitation-sequencing (ChIP-Seq) experiments by using antibodies agaist p-PDHA1 and PKM2. Methods: HepG2 cells were cultured in DMEM media, supplemented with 10% FBS and 1% penicillin/Streptomycin antibiotics. In 100 mm (diameter) dishes, the cells were seeded (1×107 – 3×107 cells per dish) and then incubated for 24 h. The media was exchanged for DMEM without serum for 12 h and the cells were then treated with 100 nM insulin for 48 h. The cells were then washed with PBS. To crosslink proteins to DNA, we added formaldehyde drop-wise directly to PBS containing samples to a final concentration of 0.75% and incubated at room temperature for 1 h. The crosslinking was stopped by adding glycine to the samples to a final concentration of 125 mM, followed by incubation for 10 min at RT. The cells were then rinsed twice with ice cold PBS and were harvested. The cell pellets were suspended in ChIP lysis buffer with freshly added cocktail of protease/phosphatase inhibitors and incubated for 10 min on ice. Sonication of the sample was then performed with an OMNI Sonic Ruptor-400 sonicator (OMNI International, Kennesaw, GA, USA) on ice for 20 impulses of 20 sec each. This was repeated five times with a 30 sec interval for each. The sonicated sample was centrifuged at 13,000 rpm for 15 min and 200 μg of the protein–chromatin complex was used for each round of immunoprecipitation {Cap, 2020, 32046944}, performed with anti-p-PDH A1 (S293), and PKM2 antibodies overnight at 4°C. IgG antibody used as a control. The formed antibody–protein complex was captured by incubation with pre-blocked Pierce Protein A/G Beads (Pierce). After sequential washing the beads with wash buffer I, II, III and IV several times and DNA sequencing of ChIP was performed by eBiogen (Seoul, Korea). Results: From ChIP Seq data analysis, we found that the promoters of many genes are associated with both p-PDH and PKM2. p-PDHA1 and PKM2 alone or together may regulate transcription of many genes upon insulin. In our current manuscript, we sort out the common genes which may be regulated p-PDHA1/PKM2 complex common genes in hepatocellular carcinoma cell line in response to insulin stimulation. Conclusions: Our study represents the first detailed analysis of p-PDHA1/PKM2 complex regarding to transcriptional regulation through ChIP-seq. The optimized data analysis workflows reported here may provide a framework for comparative transcritional investigations of p-PDHA1 and PKM2. We conclude that ChIP-Seq data of p-PDHA1 and PKM2 would expedite genetic network analyses and permit the dissection of complex biologic functions as well as cancer biology studies.
2022-02-02 | GSE195504 | GEO
Project description:Pyruvate dehydrogenase (PDH) catalyses the irreversible decarboxylation of pyruvate to acetyl-CoA, which feeds the tricarboxylic acids cycle. We analysed how the lack of PDH affects Pseudomonas putida metabolism. Inactivating PDH generated a strain that can no longer use compounds whose assimilation converges into pyruvate, including sugars and several amino acids. Compounds generating acetyl-CoA supported growth. Interestingly, inactivation of PDH led to loss of the carbon catabolite repression (CCR) effect that inhibits the assimilation of non-preferred compounds when other preferred ones are also present. P. putida can degrade many aromatic compounds, most of which generate acetyl-CoA, and is a useful bacterium for biotransformation and bioremediation processes. However, the genes involved in these metabolic pathways are frequently inhibited by CCR when substrates such as glucose or mixtures of amino acids are also present. Our results show that the PDH-null strain could efficiently degrade aromatic compounds in the presence of other preferred substrates, a condition in which the wild type strain could not mineralise them. Since lack of PDH limits the assimilation of many sugars and amino acids, and relieved CCR, the PDH-null strain could be useful in biotransformation or bioremediation processes that imply growth with mixtures of preferred substrates and aromatic compounds.
2024-07-03 | GSE263208 | GEO
Project description:Pyruvate dehydrogenase (PDH) is the central enzyme connecting glycolysis and the tricarboxylic acid (TCA) cycle. The importance of PDH function in Th17 cells is unknown. Here, we show that PDH is essential for the generation of a unique glucose-derived citrate pool needed for Th17 cell proliferation, survival and effector function. In vivo, mice harboring a T cell-specific deletion of PDH were less susceptible to developing experimental autoimmune encephalomyelitis. Mechanistically, the absence of PDH in Th17 cells increased glutaminolysis, glycolysis, and lipid uptake in an mTOR-dependent manner. However, cellular citrate remained critically low in mutant Th17 cells, which interfered with oxidative phosphorylation (OXPHOS), lipid synthesis and histone acetylation crucial for the transcription of Th17 signature genes. Increasing cellular citrate in PDH-deficient Th17 cells restored their metabolism and function, identifying a metabolic feedback loop within central carbon metabolism that may offer possibilities for therapeutically targeting Th17 cell-driven autoimmunity.
2023-03-02 | GSE222878 | GEO
Project description:Pyruvate dehydrogenase (PDH) is the gatekeeper enzyme of the tricarboxylic acid (TCA) cycle. Here we show that the deglycase DJ-1 (encoded by PARK7, a key familial Parkinson's disease gene) is a pacemaker regulating PDH activity in CD4+ regulatory T cells (Treg cells). DJ-1 binds to PDHE1-β (PDHB), inhibiting phosphorylation of PDHE1-α (PDHA), thus promoting PDH activity and oxidative phosphorylation (OXPHOS). Park7 (Dj-1) deletion impairs Treg survival starting in young mice and reduces Treg homeostatic proliferation and cellularity only in aged mice. This leads to increased severity in aged mice during the remission of experimental autoimmune encephalomyelitis (EAE). Dj-1 deletion also compromises differentiation of inducible Treg cells especially in aged mice, and the impairment occurs via regulation of PDHB. These findings provide unforeseen insight into the complicated regulatory machinery of the PDH complex. As Treg homeostasis is dysregulated in many complex diseases, the DJ-1-PDHB axis represents a potential target to maintain or re-establish Treg homeostasis.
2022-04-19 | GSE115269 | GEO
Project description:Pyruvate dehydrogenase (PDH) is the central enzyme connecting glycolysis and the tricarboxylic acid (TCA) cycle. The importance of PDH function in Th17 cells is unknown. Here, we show that PDH is essential for the generation of a unique glucose-derived citrate pool needed for Th17 cell proliferation, survival and effector function. In vivo, mice harboring a T cell-specific deletion of PDH were less susceptible to developing experimental autoimmune encephalomyelitis. Mechanistically, the absence of PDH in Th17 cells increased glutaminolysis, glycolysis, and lipid uptake in an mTOR-dependent manner. However, cellular citrate remained critically low in mutant Th17 cells, which interfered with oxidative phosphorylation (OXPHOS), lipid synthesis and histone acetylation crucial for the transcription of Th17 signature genes. Increasing cellular citrate in PDH-deficient Th17 cells restored their metabolism and function, identifying a metabolic feedback loop within central carbon metabolism that may offer possibilities for therapeutically targeting Th17 cell-driven autoimmunity.
2023-03-02 | GSE222880 | GEO
Project description:Pyruvate dehydrogenase (PDH) is the central enzyme connecting glycolysis and the tricarboxylic acid (TCA) cycle. The importance of PDH function in Th17 cells is unknown. Here, we show that PDH is essential for the generation of a unique glucose-derived citrate pool needed for Th17 cell proliferation, survival and effector function. In vivo, mice harboring a T cell-specific deletion of PDH were less susceptible to developing experimental autoimmune encephalomyelitis. Mechanistically, the absence of PDH in Th17 cells increased glutaminolysis, glycolysis, and lipid uptake in an mTOR-dependent manner. However, cellular citrate remained critically low in mutant Th17 cells, which interfered with oxidative phosphorylation (OXPHOS), lipid synthesis and histone acetylation crucial for the transcription of Th17 signature genes. Increasing cellular citrate in PDH-deficient Th17 cells restored their metabolism and function, identifying a metabolic feedback loop within central carbon metabolism that may offer possibilities for therapeutically targeting Th17 cell-driven autoimmunity.
2023-03-02 | GSE222881 | GEO
Project description:Pyruvate kinase (PK) catalyzes the conversion of phosphoenolpyruvate to pyruvate during glycolysis. The PK isoform PKM2 has additional roles in regulation of gene transcription and protein phosphorylation. PKM2 controls macrophage metabolic remodeling in inflammation, but its role in T cell biology is poorly understood. These results show that TEPP-46, an activator of PKM2, reduces CD4+ T cell activation, proliferation, and cytokine production by inhibiting essential signaling pathways and preventing glycolysis.
2019-11-26 | GSE139378 | GEO
Project description:In eukaryotes, pyruvate, a key metabolite produced by glycolysis, is converted by a mitochondrial pyruvate dehydrogenase complex (PDH) to acetyl-coenzyme A, which is fed into the tricarboxylic acid cycle. Two additional enzyme complexes catalyze similar oxidative decarboxylation reactions albeit using different substrates, the branched-chain ketoacid dehydrogenase (BCKDH) and the 2-oxoglutarate dehydrogenase (OGDH). In diplonemids, one of the most abundant and diverse groups of oceanic protists, comparative transcriptome analyses indicated that their PDH complex is compositionally unique, with the conventional E1 subunit replaced by an aceE protein of prokaryotic origin. Here we demonstrate in the model diplonemid Paradiplonema papillatum that the pyruvate dehydrogenase activity is comparable to that in other euglenozoan protists. By protein mass spectrometry we revealed that the aceE protein is twice as abundant as the E1 subunits of OGDH and enriched in the mitochondrion to the same level as the BCKDH E1 subunits, corroborating the functional relevance of the proposed aceE subunit of the PDH complex. Importantly, the acquisition of the archaeal aceE by the diplonemid ancestor led not only to the complete loss of the eukaryotic-type E1 of the PDH complex, but also its dedicated E2 and E3 subunits, still present in other euglenozoans. Hence, to reconstitute the diplonemid PDH, the aceE protein needs to partner with E2 and E3 subunits of BCKDH and/or OGDH. The diplonemid example illustrates that the acquisition and successful integration of a foreign E1p can profoundly reorganize the entire PDH complex.
2023-07-28 | PXD035104 | Pride
Project description:The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl-CoA is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of a pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required the simultaneous expression of E. faecalis genes encoding its E1α, E1β, E2 and E3 subunits, as well as genes involved in lipoylation of E2 and addition of lipoate to growth media. A strain lacking ACS, that expressed these E. faecalis genes, grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs+ reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial micro-organisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways. For both strains - mutant strain IMY104 and reference strain CEN.PK113-7D' three independent chemostat cultures were performed. Each of the chemosta was sampled for transcriptome analysis. Samples were processed as described below.
2014-11-04 | E-GEOD-59814 | biostudies-arrayexpress
Project description:T cells rely on different metabolic pathways to differentiate into effector or memory cells, and metabolic intervention is a promising strategy to optimize T cell function for immunotherapy. Pyruvate dehydrogenase (PDH) is a nexus between glycolytic and mitochondrial metabolism, regulating pyruvate conversion either to lactate or acetyl-CoA. Here, we retrovirally transduced pyruvate dehydrogenase kinase 1 (PDK1) or pyruvate dehydrogenase phosphatase 1 (PDP1), which control PDH activity, into CD8+ T cells to test effects on T cell function. Although PDK1 and PDP1 were expected to influence PDH in opposing directions, by several criteria they induced similar changes relative to control T cells. Seahorse metabolic flux assays showed both groups exhibited increased glycolysis and oxidative phosphorylation. Both groups had improved primary and memory recall responses following infection with murine gammaherpesvirus-68 (MHV-68). However, metabolomics using labeled fuels indicated differential usage of key fuels by metabolic pathways. Importantly, after B cell lymphoma challenge the engineered CD8+ T cells produced smaller effector populations in the tumor, resulting in larger tumors and poorer survival compared with control cells. Overall, this study indicates that PDK1 and PDP1 both enhance metabolic capacity, but the context of the antigenic challenge significantly influences the consequences for T cell function.
2024-10-16 | GSE279041 | GEO