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. 2010 Sep 3;329(5996):1201-5.
doi: 10.1126/science.1191241. Epub 2010 Jul 15.

Signaling Kinase AMPK Activates Stress-Promoted Transcription via Histone H2B Phosphorylation

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Free PMC article

Signaling Kinase AMPK Activates Stress-Promoted Transcription via Histone H2B Phosphorylation

David Bungard et al. Science. .
Free PMC article

Abstract

The mammalian adenosine monophosphate-activated protein kinase (AMPK) is a serine-threonine kinase protein complex that is a central regulator of cellular energy homeostasis. However, the mechanisms by which AMPK mediates cellular responses to metabolic stress remain unclear. We found that AMPK activates transcription through direct association with chromatin and phosphorylation of histone H2B at serine 36. AMPK recruitment and H2B Ser36 phosphorylation colocalized within genes activated by AMPK-dependent pathways, both in promoters and in transcribed regions. Ectopic expression of H2B in which Ser36 was substituted by alanine reduced transcription and RNA polymerase II association to AMPK-dependent genes, and lowered cell survival in response to stress. Our results place AMPK-dependent H2B Ser36 phosphorylation in a direct transcriptional and chromatin regulatory pathway leading to cellular adaptation to stress.

Figures

Fig. 1
Fig. 1
AMPK is required for stress-dependent transcription and localizes to stress-responsive genes. (A) Western blot of AMPK activation (AMPKα pThr172) and ACCα phosphorylation (ACCα pSer79) in MEFs in response to UV light, γ irradiation (IR), camptothecin (CPT), adriamycin (Adr), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), or hydrogen peroxide (H2O2). Exposure times were 6 hours for UV, IR, CPT, and Adr and 30 min for MNNG and H2O2. NT, no treatment; Glc, glucose. (B) Viability of wild-type (WT) and ampkα−/− MEFs in response to indicated stresses. Left panel: glucose (Glc) withdrawal; right panel, UV irradiation (UVC). (C) Expression (qPCR) of p21, cpt1c, and gapdh mRNA in wild-type, AMPKα-deficient, or p53 RNAi–expressing MEFs after glucose withdrawal, relative to untreated cells. (D) ChIP of WT or dominant negative (MUT) myc-AMPK after glucose withdrawal in lkb1−/− MEFs cotransfected with either WT or catalytically inactive (MUT) FLAG-LKB1, both able to be immunoprecipitated [see (A), fig. S7, and (5)]. Data represent means ± SEM for n = 3. (E to G) Endogenous AMPKα2 ChIP in untreated MEFs (−), 2-DG (15 min), or UV (6 hours) (E); WT or ampkα−/− MEFs (F); and WT or p53−/− MEFs (G). Data represent means ± SEM for n = 3. #P < 0.06, *P < 0.05, **P < 0.01, ***P < 0.03.
Fig. 2
Fig. 2
H2B is an AMPK target. (A to D) In vitro phosphorylation by myc-AMPK immunoprecipitated from ampkα−/− MEFs of recombinant human histones by beads alone, WT myc-AMPK, or catalytic mutant (D157A) myc-AMPK (right: myc Western blot) (A); recombinant human histones using myc-AMPK from glucose-treated cells (+glucose) or untreated cells (−glucose) (B); H2B peptides (C); and H2B 21–42 and SAMS peptide (D). (E) Western blots of wild-type or ampkα−/− MEFs treated with 2-DG (25 mM for 10 min) or untreated. (F) Western blots of MEFs treated for 1 hour with AICAR (AIC, 2 mM) or phenformin (Ph, 3 mM) or untreated.
fig. 3
fig. 3
AMPK and H2B pS36 are localized to chromatin in response to stress. (A) Western blots showing myc-AMPK immunoprecipitation from WT H2B– or H2B S36A–expressing cells after AICAR treatment (1 mM, 24 hours). WCL, whole-cell lysate. (B) ChIP in WT or ampkα−/− MEFs. Data represent means ± SEM for n = 3. (C and D) ChIP along cpt1c gene (C) and p21 gene (D) with glucose (white bars) or without glucose (black bars). Data represent means ± SEM for n = 3. *P < 0.01, **P < 0.05.
Fig. 4
Fig. 4
H2B Ser36 is essential for transcription and survival in response to metabolic stress. Data for MEF cell lines expressing wild-type H2B or H2B S36A are shown. (A) Relative expression (qPCR) of indicated genes after glucose withdrawal in individual MEF clones (H2B, clones 5 and 6; H2B S36A, clones 4 and 6). (B) Viability of H2B (open squares) or H2B S36A (solid squares) MEFs in no glucose, 2-DG (10 mM), or phenformin (3 mM). (C) ChIP of RNA Pol II across the cpt1c gene. Data represent means ± SEM for n = 3. *P < 0.05, **P < 0.01, #P < 0.03, ##P < 0.08.

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