Signaling kinase AMPK activates stress-promoted transcription via histone H2B phosphorylation.
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.
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ABSTRACT: Obesity, diabetes, and related metabolic disorders are major health issues worldwide. As the epidemic of metabolic disorders continues, the associated medical co-morbidities, including the detrimental impact on reproduction, increase as well. Emerging evidence suggests that the effects of maternal nutrition on reproductive outcomes are likely to be mediated, at least in part, by oocyte metabolism. Well-balanced and timed energy metabolism is critical for optimal development of oocytes. To date, much of our understanding of oocyte metabolism comes from the effects of extrinsic nutrients on oocyte maturation. In contrast, intrinsic regulation of oocyte development by metabolic enzymes, intracellular mediators, and transport systems is less characterized. Specifically, decreased acid transport proteins levels, increased glucose/lipid content and elevated reactive oxygen species in oocytes have been implicated in meiotic defects, organelle dysfunction and epigenetic alteration. Therefore, metabolic disturbances in oocytes may contribute to the diminished reproductive potential experienced by women with metabolic disorders. In-depth research is needed to further explore the underlying mechanisms. This review also discusses several approaches for metabolic analysis. Metabolomic profiling of oocytes, the surrounding granulosa cells, and follicular fluid will uncover the metabolic networks regulating oocyte development, potentially leading to the identification of oocyte quality markers and prevention of reproductive disease and poor outcomes in offspring.Cellular and Molecular Life Sciences CMLS 10/2014; · 5.86 Impact Factor
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ABSTRACT: Cystic fibrosis is an inherited multi-organ disorder caused by mutations in the CFTR gene. Patients with this disease exhibit characteristic abnormalities in the levels of unsaturated fatty acids in blood and tissue. Recent studies have uncovered an underlying biochemical mechanism for some of these changes, namely increased expression and activity of fatty acid desaturases. Among other effects, this drives metabolism of linoeate to arachidonate. Increased desaturase expression appears to be linked to cystic fibrosis mutations via stimulation of the AMP-activated protein kinase in the absence of functional CFTR protein. There is evidence that these abnormalities may contribute to disease pathophysiology by increasing production of eicosanoids, such as prostaglandins and leukotrienes, of which arachidonate is a key substrate. Understanding these underlying mechanisms provides key insights that could potentially impact the diagnosis, clinical monitoring, nutrition, and therapy of patients suffering from this deadly disease.International Journal of Molecular Sciences 09/2014; 15(9):16083-16099. · 2.34 Impact Factor
, 1201 (2010);
et al.David Bungard,
Transcription via Histone H2B Phosphorylation
Signaling Kinase AMPK Activates Stress-Promoted
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particles driving the expression of zyxin-GFP,
zyxin-D2/3LIM-GFP, or GFP, respectively (fig.
S11). Delivery of zyxin to the vaginal epithelium
did not result in increased exfoliation of the un-
infected mucosa (fig. S11). However, zyxin, but
not zyxin-D2/3LIM, overexpression blocked the
gonococci (Fig. 4D). The overexpression of zyxin,
CEA-positive cells infected with Ngo OpaCEA
and reestablished the responsiveness of the vagi-
nal mucosa (Fig. 4E and fig. S11). Thus, CEA-
initiated up-regulation of CD105 on superficial
epithelial cells and the resulting delocalization of
zyxin from integrin-rich focal adhesion sites are
binding microorganisms to counteract the exfo-
Although symptomatic gonococcal infection
in humans might be a multistep process orches-
results establish a specific role of OpaCEApro-
teins in promoting mucosal colonization. In vivo
revealed that after infection of the urethra with
non-opaque gonococci, bacteria re-isolated from
these volunteers almost invariably converted to
an Opa protein–expressing phenotype (24,25).In
addition to the urogenital tract, members of the
CEACAM family are present on all mucosal
surfaces including the nasopharynx (26). These
mucosal habitats are colonized by several Gram-
negative bacterial species, which make use of
unrelated protein adhesins to engage human
CEACAMs (27–31). The blockage of epithelial
have driven thisconvergent evolution thatallows
specialized bacteria to transform the mucosa into
a dependable platform for colonization.
References and Notes
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32. We thank M. C. Beckerle for zyxin-deficient fibroblasts
and antibodies; M. Chudakov for the mKate cDNA;
J. W. Greiner for sending the CEAtg mice; T. F. Meyer for
bacterial strains; D. W. Piston for cDNA of mCerulean;
A. J. Schaeffer for hVEC cells; D. Vestweber for providing
antibody; C. Hentschel, J. Scharrer, and R. Mak’anyengo
for assistance with SEM; and B. Planitz for expert animal
care. Supported by Deutsche Forschungsgemeinschaft
grant Ha2856/6-1 (C.R.H.).
Supporting Online Material
Materials and Methods
Figs. S1 to S10
14 April 2010; accepted 30 June 2010
Signaling Kinase AMPK Activates
Stress-Promoted Transcription via
Histone H2B Phosphorylation
David Bungard,1Benjamin J. Fuerth,2,3Ping-Yao Zeng,1,4Brandon Faubert,2,3Nancy L. Maas,1
Benoit Viollet,5,6David Carling,7Craig B. Thompson,8Russell G. Jones,2,3,8* Shelley L. Berger1,9,10*
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
transcription through direct association with chromatin and phosphorylation of histone H2B at serine 36.
AMPK recruitment and H2B Ser36phosphorylation colocalized within genes activated by AMPK-dependent
pathways, both in promoters and in transcribed regions. Ectopic expression of H2B in which Ser36was
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 Ser36phosphorylation
in a direct transcriptional and chromatin regulatory pathway leading to cellular adaptation to stress.
ever, the central kinases in these pathways are
generally not thought to directly modulate tran-
scription. One such signaling kinase, adenosine
monophosphate–activated protein kinase (AMPK)
(1), is activated by high substrate adenosine
monophosphate levels under conditions of ener-
getic stress, such as nutrient deprivation or hy-
poxia. AMPK activation initiates a program of
ignaling pathways often involve cascades
of protein phosphorylation that terminate
metabolic adaptation to preserve cellular energy
(adenosine triphosphate conservation) and main-
tain cellular viability (2).
We investigated possible direct transcrip-
tional mechanisms in the AMPK pathway. First,
we examined whether AMPK broadly responds
to cellular stress. Mouse embryonic fibroblasts
(MEFs) were stressed by ultraviolet (UV) irra-
diation, g irradiation, the topoisomerase inhibitor
camptothecin, the DNA intercalating agent adri-
amycin, the alkylating agent N-methyl-N´-nitro-N-
nitrosoguanidine, or hydrogen peroxide. AMPK
activation loop and phosphorylation of substrate
ACCa (acetyl coenzyme A–carboxylase-a)] by
all agents (Fig. 1A). Liver kinase B1 (LKB1) is
LKB1 also activated AMPK in response to either
genotoxic (UV irradiation) or metabolic stress
[the glycolytic inhibitor 2-deoxyglucose (2-DG)]
1Department of Cellular and Developmental Biology, Univer-
sity of Pennsylvania Medical School, Philadelphia, PA 19104,
USA.2Rosalind and Morris Goodman Cancer Research Centre,
ment of Physiology, McGill University, Montreal, Quebec H3G
1Y6, Canada.4Institutes of Biomedical Sciences Epigenetics Pro-
gram, Mingdao Building, Room 511, Fudan University, Mail Box
281, 138 Yixue Yuan Road, Shanghai 200032, P.R. China.
5Institut Cochin, Université Paris Descartes, CNRS (UMR 8104),
75014 Paris, France.6INSERM U1016, 75014 Paris, France.7Cel-
lular Stress Group, MRC Clinical Sciences Centre, Imperial College,
Center and Abramson Family Cancer Research Institute, Uni-
ment of Genetics, University of Pennsylvania Medical School,
of Arts and Sciences, University of Pennsylvania, Philadelphia,
PA 19104, USA.
*To whom correspondence should be addressed. E-mail:
firstname.lastname@example.org (R.G.J.); email@example.com.
VOL 329 3 SEPTEMBER 2010
on October 11, 2010
Fig. 1. AMPK is required for stress-dependent
transcription and localizes to stress-responsive
genes. (A) Western blot of AMPK activation (AMPKa
pThr172) and ACCa phosphorylation (ACCa pSer79)
in MEFs in response to UV light, g 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
wild-type (WT) and ampka−/−MEFs in response to
indicated stresses. Left panel: glucose (Glc) with-
drawal; right panel, UV irradiation (UVC). (C)
Expression (qPCR) of p21, cpt1c, and gapdh mRNA
in wild-type, AMPKa-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 T SEM for n = 3. (E to G)
Endogenous AMPKa2 ChIP in untreated MEFs (–),
2-DG (15 min), or UV(6 hours) (E);WT or ampka−/−
means T SEM for n = 3. #P < 0.06, *P < 0.05, **P <
0.01, ***P < 0.03.
- 2-DG UV
MUT WT MUT
0 12 24 36 48
% Viable Cells
% Viable Cells
Relative mRNA level
(normalized to +Glc)
Fig. 2. H2B is an AMPK target. (A to D) In vitro
phosphorylation by myc-AMPK immunoprecipi-
tated from ampka−/−MEFs of recombinant human
mutant (D157A) myc-AMPK (right: myc Western
blot) (A); recombinant human histones using myc-
AMPK from glucose-treated cells (+glucose) or un-
treated cells (–glucose) (B); H2B peptides (C); and
of wild-type or ampka−/−MEFs treated with 2-DG
(25 mM for 10 min) or untreated. (F) Western blots
ofMEFstreated for 1 hour with AICAR (AIC, 2 mM)
or phenformin (Ph, 3 mM) or untreated.
AMPKα2 WTAMPKα2 D157A
H2A H2B H3 H4H2A H2B H3H4
H2A H2B H3 H4
1µg 0.1µg 10ng
-AIC Ph- AIC Ph
-/ -wild -type
3 SEPTEMBER 2010VOL 329
on October 11, 2010
We next used wild-type or AMPKa1- and
AMPKa2-deficient MEFs (ampka−/−) to exam-
ine whether AMPK influences cellular survival
during stress (4). The ampka−/−MEFs displayed
decreased survival in the presence of metabolic
stressors (Fig. 1B and fig. S2), UV irradiation
ing that AMPK promotes cell survival in re-
Our previous work implicated AMPK and
LKB1 intumor suppressorp53–dependent meta-
bolic and genotoxic stress responses (2, 5). We
examined the involvement of AMPK in tran-
scriptional regulation of p53-responsive genes.
ampka−/−MEFs showed decreased induction of
p21, a well-characterized p53 target gene, in
response to both glucose withdrawal and UV
glucose-regulated gene expression was similar in
cells deprived of glucose, whereas expression of
a control gene, gapdh, was unaffected (Fig. 1C
and fig. S4). A similar defect in stress-dependent
transcription was observed in human cancer cells
downstream of the DNA-damage checkpoint, as
of wild-type controls (fig. S6). Thus, in cell cul-
ture, the LKB1-AMPK pathway responds to a
wide variety of metabolic and genotoxic stresses
and is required for maximal stress-induced tran-
scription of multiple p53-dependent genes that
anisms have remained elusive. We used chroma-
tin immunoprecipitation (ChIP) and quantitative
polymerase chain reaction (qPCR) to investigate
genes in MEFs. We found that LKB1 coimmu-
noprecipitated with AMPK in response to UVir-
radiation, and this interaction was dependent on
AMPK kinase activity (fig. S7). ChIP showed
ectopic myc-AMPK associated with the cpt1c and
p21 gene promoters in response to glucose
withdrawal and UV treatment (Fig. 1D and fig.
S8). Ectopic kinase-deficient myc-AMPKa or
FLAG-tagged LKB1 abrogated the binding of
AMPK to chromatin (Fig. 1D and fig. S8).
FLAG-LKB1 similarly associated with the p21
promoter (fig. S9). Binding of AMPK or LKB1
andfigs.S8 andS9).AMPKwas also detected at
treatment (fig. S10). This AMPK activation ap-
pears to be specific for survival in response to
stress, because AMPK was not detected at the
PUMA promoter, a p53-dependent, pro-apoptotic
gene (fig. S11). The immunoprecipitation and
ChIP results suggest a functional, kinase-dependent
antibody that immunoprecipitated the protein
(fig. S12), showed increased AMPKa2 binding
2-DG and UV light (Fig. 1E; at the p21 promot-
er, fig. S13). The binding was reduced in both
Thus, in response to cellular stress, LKB1 and
AMPK physically localize to chromatin in an in-
terdependent and p53-dependent fashion to reg-
ulate gene transcription.
Posttranslational modifications of core his-
tones, including phosphorylation, function to reg-
ulate transcription (7, 8). To examine histones
as substrates of AMPK, we epitope-tagged the
predominantly nuclear isoform AMPKa2 (9) and
immunoprecipitated wild-type AMPKa2 or a cat-
alytic mutant (Asp157→ Ala) from transfected
ampka−/−MEFs after low glucose treatment to
activate AMPK. The wild-type AMPKa2 com-
plex specifically phosphorylated histone H2B
above the nonspecific background activity of all
four histones observed in the catalytic mutant and
mock immunoprecipitations (Fig. 2A). AMPKa2
from untreated cells exhibited low activity on H2B
relative to AMPKa2 from stressed cells (Fig. 2B
and fig. S15). H2A was also phosphorylated in
in the mock immunoprecipitation and did not re-
spond to stress, which suggested that the activity
is nonspecific. These data indicate that activated
AMPKa2 specifically phosphorylates H2B.
Analysis of histone H2B revealed a poten-
tial AMPK target motif (10, 11) at Ser36in the
N terminus, similar to the AMPK substrates
phofructokinase 2 (PFK2) (H2B, Arg-Ser-Arg-
Lys-Glu-Ser; eNOS, Arg-Ile-Arg-Thr-Gln-Ser;
PFK2, Arg-Met-Arg-Arg-Asn-Ser), with con-
served Arg residues at P–5 and P–3 relative to
the phosphoacceptor Ser36(fig. S14) (12). We
idues 1 to 20 or 21 to 42 of H2B; only the H2B
21-42 peptide was phosphorylated by immuno-
precipitated AMPKa2. Moreover, an H2B 21-42
ChIP: pS36 H2B
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 ampka−/−MEFs. Data represent means T
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 T SEM for n = 3. *P < 0.01, **P < 0.05.
VOL 3293 SEPTEMBER 2010
on October 11, 2010
peptide harboring a Ser36→ Ala (S36A) substitu-
tion was not phosphorylated, whereas S32A and
S38A peptides were phosphorylated (Fig.2C).In
addition, myc-AMPKa2 phosphorylated the H2B
21-42 peptide more efficiently than did SAMS
Leu-Val-Lys-Arg-Arg) peptide, a canonical AMPK
targetsequence(Fig.2D)(13).Thus,H2B Ser36is a
robust in vitro AMPK substrate.
We next examined whether AMPK is in-
volved in the regulation of H2B Ser36phospho-
an H2B pS36 peptide and extensively charac-
terized the affinity-purified antibody in vitro and
by increased phosphorylation of AMPKa and
ACCa (Fig. 2E), whereas other histone marks,
including H3, K9me3 H3, and pS10 H3, did not
H2B pS36 did not increase after 2-DG treatment
(Fig. 2E). We detected similar induction of H2B
activators, aminoimidazole carboxamide ribonu-
cleotide (AICAR) and phenformin (Fig. 2F). In
addition, H2B pS36 was not detected in lkb1−/−
was present (fig. S18). These results suggest that
H2B Ser36phosphorylation is downstream of the
to a broad range of metabolic stresses, and that
AMPK is likely the direct kinase in this pathway.
This global increase of H2B pS36 may not be lim-
levels were not reduced in p53−/−cells (fig. S19).
We found that transfected myc-AMPK and
FLAG-H2B associate in 293T cells in response
to the AMPK agonist AICAR (Fig. 3A). This
interaction was ablated by an H2B S36A muta-
tion (Fig. 3A), suggesting that Ser36is critical for
the interaction of AMPK and H2B. We next per-
MEFs to determine whether H2B pS36 is as-
sociated with AMPK-dependent genes, specifi-
to both 2-DG and UV treatment, and the ChIP
signal was reduced to background levels in
AMPKa-deficient cells (Fig. 3B).
To compare the locations of endogenous
AMPK and H2B pS36, we performed ChIP at
cpt1c under normal and glucose-free conditions,
using primers along the promoter, transcribed
region, and upstream or downstream nontran-
scribed regions (Fig. 3C, primer sets 1 to 7).
downstream of the gene (primer set 7) (Fig. 3C).
Notably, H2B pS36 associated throughout the
transcribed region of cpt1c (primer sets 5 and 6)
(Fig. 3C). We then used the AMPKa2 antibody
and discovered a positive correlation between
locations of endogenous AMPK and H2B pS36,
region (Fig. 3C). The p21 gene showed a similar
positive correlation between H2B pS36 and en-
dogenous AMPK localization at the p53 binding
sites and along the transcribed region, but not
(Fig. 3D). As a control, we tested the gapdh gene,
the stressed and unstressed cells (fig. S20). Similar
results were observed at the cpt1c gene in response
to UV treatment (fig. S21). Ectopic myc-AMPK
also localized along the p21 transcribed gene in
response to low glucose and required AMPK cat-
alyticactivity(fig.S22).Thiscorrespondence in the
location of AMPK and H2B pS36 suggests that
the role of AMPK in transcriptional regulation is
linked to the phosphorylation of H2B along tran-
scribed regions of target genes.
We generated clonal MEF cell lines with sta-
ble ectopic expression of FLAG-H2B or mutant
FLAG–H2B S36A, and, as shown above, only
FLAG-H2B was detected by the H2B pS36
antibody (fig. S16). The level of FLAG-tagged
wild-type or mutant histone H2B was lower than
blot (fig. S23); however, both ectopic wild-type
and H2B S36A similarly incorporated into chro-
matin, as determined by ChIP and qPCR at the
enous H3 and H2B with FLAG-H2B as shown
by Western blot (fig. S25). We isolated several
H2B S36A–expressing clonal lines (fig. S26).
The cell lines treated with 2-DG showed com-
the S36A mutation. In response to glucose with-
drawal, elevated transcription of the AMPK target
FLAG–H2B S36A–expressing cells showed signif-
Expression of the control genes gapdh and hadh
was unaffected by expression of the S36A mutant
(fig. S28). Thus, although expression of FLAG-
p21cpt1creprimo cyclin G
Relative mRNA level
(normalized to +Glc)
% Viable Cells
% Viable Cells
RNA Pol II
Fig. 4. H2B Ser36is 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
(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 T SEM for n = 3.
*P < 0.05, **P < 0.01, #P < 0.03, ##P < 0.08.
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