Fig 1: Sen1 recruits Glc7 to dephosphorylate H3T11 at autophagy-related genes.a IP-MS analysis of proteins co-purified with Glc7-FLAG. The endogenously expressed Glc7-FLAG was immunoprecipitated from yeast cells with anti-FLAG agarose beads. The identified unique peptides and sequence coverage were listed. b The endogenously expressed Sen1 interacted with Glc7 as determined by Co-IP assay. Sen1-TAP was immunoprecipitated with calmodulin beads. The co-immunoprecipitated proteins were detected with indicated antibodies. c Reciprocal Co-IP assay showing that endogenous Glc7 interacts with Sen1. Glc7 was immunoprecipitated with anti-Glc7 antibody. The co-immunoprecipitated proteins were detected with indicated antibodies. IgG was used as a negative control. d Immunoblot analysis of intracellular H3pT11 levels in WT TetO7, TetO7-SEN1, TetO7-NRD1 and TetO7-NAB3 mutants. WT TetO7, TetO7-SEN1, TetO7-NRD1 and TetO7-NAB3 mutants were grown in YPD medium at 30 °C until OD600 of 0.5–0.7 and then treated with 40 μg/mL doxycycline for 2 h to knockdown the expression of SEN1, NRD1 and NAB3, respectively. The knockdown efficiency results were shown in Supplementary Fig. S4b. e The purified Sen1 complex (Sen1-CBP) dephosphorylates H3T11 as determined by in vitro dephosphorylation assay. f, g Analysis the effect of Sen1 knockdown on autophagy activity as determined by GFP-Atg8 processing assay (f) and fluorescence assay (g). The vacuoles of yeast cells were indicated with red arrows. h RT-qPCR analysis of ATG gene transcription in WT TetO7 and TetO7-SEN1 mutant. i, j ChIP-qPCR analysis of the occupancy of Glc7 (i) and H3pT11/H3 (j) at ATG genes in WT TetO7 and TetO7-SEN1 mutant. For f–j, WT TetO7 and TetO7-SEN1 mutant were grown in YPD medium at 30 °C until OD600 of 0.5–0.7 and then treated with 40 μg/mL doxycycline for 2 h to knockdown the expression of SEN1. Data represent the mean ± SE of three biological independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 2: Glc7-Sen1 dephosphorylates histone H3T11 to induce autophagy-related gene transcription and reduce telomere silencing under glucose starvation conditions.a Co-IP showing that the interaction between endogenous Glc7 and Sen1 was increased when cells were grown in SD-C (−Glu) medium for 2 h when compared with cells grown in SD (+Glu) medium. b Distribution of Sen1-FLAG occupancy across each gene through 1 kb upstream of the TSS to 1 kb downstream from the TES when cells were grown in SD (+Glu) medium and SD-C (−Glu) medium, respectively. c Venn diagram showing the overlap of genes co-bound by Glc7-FLAG and Sen1-FLAG when cells were grown in SD (+Glu) medium or SD-C (−Glu) medium as determined by ChIP-seq. d ChIP-seq tracks showing the occupancy of Glc7-FLAG and Sen1-FLAG at representative ATG genes when cells were grown in SD (+Glu) medium or SD-C (−Glu) medium. e Box plots analysis of the effect of glucose starvation on the occupancy of Sen1-FLAG at 39 ATG genes. f ChIP-qPCR analysis of the occupancy of Sen1-FLAG at indicated ATG genes when cells were grown in SD (+Glu) medium and SD-C (−Glu) medium, respectively. g, h ChIP-qPCR analysis of the occupancy of Glc7-FLAG (g) and H3pT11/H3 (h) at ATG genes in WT TetO7 and TetO7-SEN1 mutant when cells were grown in SD (+Glu) medium or SD-C (−Glu) medium supplemented with 40 μg/mL doxycycline for 2 h. i RT-qPCR analysis of ATG gene transcription in WT, glc7-12, WT TetO7 and TetO7-SEN1 mutants when grown in SD (+Glu) medium or SD-C (−Glu) medium. WT and glc7-12 mutant were grown in SD (+Glu) medium or SD-C (−Glu) medium at 37 °C for 2 h. WT TetO7 and TetO7-SEN1 mutant were grown in SD (+Glu) medium or SD-C (−Glu) medium supplemented with 40 μg/mL doxycycline for 2 h. j RT-qPCR analysis of the transcription of telomere-proximal genes in WT, glc7-12, WT TetO7 and TetO7-SEN1 mutants when grown in SD (+Glu) medium or SD-C (−Glu) medium. k Immunoblots of Sir2 in WT TetO7 and TetO7-SEN1 mutant when grown in SD-C medium supplemented with 40 μg/mL doxycycline for 0–6 h. l ChIP-qPCR analysis of Sir2 occupancy at telomere-proximal genes in WT TetO7 and TetO7-SEN1 mutant when grown in SD (+Glu) medium or SD-C (−Glu) medium supplemented with 40 μg/mL doxycycline for 2 h. For f–l, data represent the mean ± SE of three biological independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 3: PPP1CA–SETX dephosphorylates histone H3T11 to regulate autophagy in mammals.a Immunoblot analysis of H3pT11 in HeLa cells transfected with plasmids overexpressing PPP1CA, PPP1CB and PPP1CC. b Immunoblot analysis of H3pT11 in control (shCtrl) and PPP1CA-knockdown (shPPP1CA#1, shPPP1CA#2) HeLa cells. c The purified recombinant PPP1CA dephosphorylates H3T11 as determined by in vitro dephosphorylation assay. d Analysis of autophagy activity in control (shCtrl) and PPP1CA-knockdown (shPPP1CA#1, shPPP1CA#2) HeLa cells. e ChIP-qPCR analysis of PPP1CA occupancy at autophagy-related genes (ULK1, ATG7, ATG12, LC3) in HeLa cells. f ChIP-qPCR analysis of H3pT11/H3 occupancy at autophagy-related genes in control (shCtrl) and PPP1CA-knockdown (shPPP1CA) HeLa cells. g RT-qPCR analysis of the transcription of autophagy-related genes in shCtrl and shPPP1CA HeLa cells. h Co-IP and reciprocal IP showing the interaction between PPP1CA and SETX. i ChIP-qPCR analysis of SETX occupancy at autophagy-related genes in HeLa cells. j ChIP-qPCR analysis of PPP1CA occupancy at autophagy-related genes in control (shCtrl) and SETX-knockdown (shSETX) HeLa cells. k ChIP-qPCR analysis of H3pT11/H3 occupancy at autophagy-related genes in shCtrl and shSETX HeLa cells. The telomere-proximal genes, C1S and CDL163L1 were used as controls. l RT-qPCR analysis of the transcription of autophagy-related genes in shCtrl and shSETX HeLa cells. m Analysis of autophagy activity in shCtrl and shSETX HeLa cells. For b, d–g, i–m, data represent the mean ± SE of three biological independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.
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