Regulatory Mechanism Of AMPK-mediated Histone O-GlcNAc

Objective1. To investigate mechanism of the influence of metformin on the proliferation of cancer cells.2. To investigate the role of AMPK in metformin-mediated tumor-suppressing effects.3. To study the mechanism of AMPK-mediated histone modifications, H2B K120ub and H2B S112GlcNAc for instance; to investigate the molecular mechanism of AMPK and OGT interaction and its biological effect to histone O-GlcNAc, and how AMPK regulates OGT recruitment to chromatin.4. To explore the molecular mechanism of OGT-mediated AMPK O-GlcNAc and the impact of AMPK O-GlcNAc on the activation of AMPK.Methods1. T47D cell proliferation curve was determined by cell counting after metformin treatment with different dose or different time course. Cell cycle was analyzed by PI staining with flow cytometer. The activation of AMPK and Histone H2B monoubiquitination was valued by histone extraction and western blot. Histone H2B monoubiquitination downstream gene expression level was determined by qRT-PCR.To test whether the influence of metformin on T47D cells is dependent on AMPK.2. Treat cancer cell and AMPK-/-or AMPK+/+MEFs with AMPK activator AICAR and inhibitor compound C, and then detect the protein level of H2B K120ub and H2B S112GlcNAc by western-blot.3. To make out the molecular mechanism of the interaction of AMPK and OGT, and find out the actual phosphorylation site of OGT by AMPK with MS (mass spectra), CO-IP, in-vitro kinase assay and autoradiography, employ WB, q-RT-PCR to determine whether AMPK-mediated regulation of H2B K120ub and H2B S112GlcNAc is rely on the phosphorylation of OGT by AMPK.4. Adopt In vitro OGT assay, Chromatin extraction, ChlP-qPCR, O oil red staining to evaluate AMPK regulates OGT recruitment to chromatin but not OGT activity and OGT can O-GlcNAcylate AMPK a and regulate its activity.Results1. Metformin inhibit H2B K120ub is dependent on AMPK, AMPK can suppress the expression of H2B S112O-Glc and H2B K120ub, and the transcription of downstream gene of H2B GlcNAc.2. AMPK can interact with OGT and phosphorylate OGT at T444.3. AMPK regulates OGT recruitment to chromatin but not OGT activity.4. AMPK functions in histone epigenetic modification and downstream gene transcription through OGT T444phosphorylation.5. OGT can O-GlcNAcylate AMPK and regulate its activity.Conclusion1. The restraintion on cancer cells of metformin may benefit from AMPK phosphorylates OGT and regulates OGT recruitment to chromatin, suppress the expression of H2B S112O-Glc and the transcription of downstream gene of H2B GlcNAc.2. AMPK can interact with OGT and phosphorylate OGT at T444, and this site is conserved in many species, the phosphorylation of OGT by AMPK regulates OGT recruitment to chromatin but not OGT activity. 3. OGT can O-GlcNAcylate AMPKa, regulate its activity and the activation of downstream proteins of AMPK.