GSE195504
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.
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. Overall design: We prepared three samples. The first sample as control was used for that imunopricipited with only IgG antibody without insulin treatment. The second sample was immunopricipited with p-PDH A1 antibody and the last sample was immunopreicipited with PKM2 antibody with insulin stimulation. All samples are prepared human hepatocellular carcinoma HepG2 Cells line.