What are the contributions in this paper?

The link between these processes provides a simple explanation for the frequent and intimate overlap of TADs and degrons ( 3–5 ). Because of the dual role of Ub in transcriptional activation and activator destruction, therefore, the authors propose that Ub “ licenses ” transcription factors by linking their activity to their destruction. This suggests that activator destruction by the proteasome is a natural consequence of ubiquitylation.

(Open Access) Duration of nuclear NF-κB action regulated by reversible acetylation (2001) | Lin Feng Chen

restore LexA-VP16 destruction in Met30-null

cells (Fig. 3A), the fusion did rescue tran-

scriptional activation, restoring wild-type

levels of VP16 TAD activity (Fig. 3B, com-

pare VP16 and Ub-VP16 in the Met30-null

cells). Moreover, both Ub and the VP16 TAD

are required for transcriptional activation in

the Met30-null cells because Ub alone fused

to LexA (Ub-⌬) did not activate transcription

(14 ). The observation that Met30’s role in

transcription can be complemented by fusion

of LexA-VP16 to Ub argues that Met30 co-

activates the VP16 TAD by signaling LexA-

VP16 ubiquitylation. Moreover, the metabol-

ic stability of the Ub-VP16 protein (Fig. 3A)

demonstrates that it is Met30-mediated ubiq-

uitylation, not destruction, that is required for

transcriptional activation.

The requirement of ubiquitylation for VP16

activator function reveals that the degron func-

tion of the VP16 TAD is intimately tied to its

ability to activate transcription. The link be-

tween these processes provides a simple expla-

nation for the frequent and intimate overlap of

TADs and degrons (3–5). This requirement for

ubiquitylation, which has not been observed in

vitro, reveals a function for Ub distinct from its

role in proteolysis (2). Recent evidence has

demonstrated that the 19S subunit of the protea-

some plays an essential role in transcriptional

elongation (15). Given the role of the 19S com-

plex as a Ub binding module (16 ), it is possible

that activator ubiquitylation serves to recruit the

19S complex to promoters, where the chaperone

functions of this complex promote transcription

elongation.

Although our data demonstrate that proteol-

ysis is not required for transcriptional activation,

it is important to note that Met30 does direct

LexA-VP16 destruction. This suggests that ac-

tivator destruction by the proteasome is a natural

consequence of ubiquitylation. Because of the

dual role of Ub in transcriptional activation and

activator destruction, therefore, we propose that

Ub “licenses” transcription factors by linking

their activity to their destruction. We imagine

that non-ubiquitylated activators are stable and

inactive. Interactions of an activator with a Ub-

ligase result in activator ubiquitylation, which

simultaneously activates the transcription factor

and primes it for destruction by the proteasome.

Given the large number of transcription factors

that are targeted for Ub-mediated proteolysis, it

is possible that many transcription factors are

regulated through this mechanism.

References and Notes

1. D. Thomas, M. Tyers, Curr. Biol. 10, R341 (2000).

2. A. Varshavsky, Trends Biochem. Sci. 22, 383 (1997).

3. S. E. Salghetti, S. Y. Kim, W. P. Tansey, EMBO J. 18,

717 (1999).

4. E. Molinari, M. Gilman, S. Natesan, EMBO J. 18, 6439

(1999).

5. S. E. Salghetti, M. Muratani, H. Wijnen, B. Futcher, W. P.

Tansey, Proc. Natl. Acad. Sci. U.S.A. 97, 3118 (2000).

6. S. J. Triezenberg, R. C. Kingsbury, S. L. McKnight,

Genes Dev. 2, 718 (1988).

7. R. Brent, M. Ptashne, Cell 43, 729 (1985).

8. D. Skowyra, K. L. Craig, M. Tyers, S. J. Elledge, J. W.

Harper, Cell 91, 209 (1997).

9. Because the VP16 TAD contains no lysine residues,

LexA-VP16 ubiquitylation probably occurs within the

LexA protein.

10. A. Barberis, et al., Cell 81, 359 (1995).

11. Met30 (–) cells expressing LexA–VP16 grow at an

identical rate to Met30 (⫹) cells expressing this

protein. The indistinguishable growth rates of these

cells support the argument that LexA-VP16 does not

globally suppress (i.e., squelch) transcription in the

Met30-null background.

12. R. Li et al., Mol. Cell. Biol. 18, 1296 (1998).

13. Y. Marahrens, B. Stillman, Science 255, 817 (1992).

14.

The ﬁnding that the VP16 TAD is still required for

transcriptional activation in this setting demonstrates

that VP16 TAD function is not globally suppressed in the

absence of Met30. Together with the results of the

plasmid stability analysis (Fig. 2C), this ﬁnding demon-

strates that the VP16 TAD retains at least part of its

activity in the Met30-null cells.

15. A. Ferdous, F. Gonzalez, L. Sun, T. Kodadek, S. A.

Johnston, Mol. Cell 7, 981 (2001).

16. D. Voges, P. Zwickl, W. Baumeister, Annu. Rev. Bio-

chem. 68, 1015 (1999).

17. E. E. Patton et al., EMBO J. 19, 1613 (2000).

18. A. Rouillon, R. Barbey, E. E. Patton, M. Tyers, D.

Thomas, EMBO J. 19, 282 (2000).

19. E. S. Johnson, P. C. Ma, I. M. Ota, A. Varshavsky,

J. Biol. Chem. 270, 17442 (1995).

20.

Sequences encoding the VP16, Myc, and Cln3 degrons

were inserted into pRJR238 (10), modiﬁed to include an

in-frame hemagglutinin (HA)-epitope tag ( pRJR238HA).

The resulting LexA-fusion proteins were expressed in

CC849-1B (MAT a, his3, leu2, ura3, trp1, met4::TRP1) and

CC807-7D (MAT a, ade2, his3, leu2, ura3, trp1,

met4::TRP1, met30::LEU2) yeast (17), each carrying an

integrated LexA–␤-galactosidase reporter (10). Pulse-

chase analysis was performed as described (3).

21. LexA fusion proteins were expressed in CC849-1B,

along with either glutathione S-transferase (GST ) or

GST-Met30 (18) under the control of the GAL1 pro-

moter. Log-phase cultures were induced with galac-

tose, proteins were harvested, and GST proteins were

collected on glutathione agarose (18).

22. J. Spence et al., Cell 102, 67 (2000).

23. LexA-VP16 appears to be modiﬁed in Met30 (⫹)

cells. This modiﬁcation is not due to ubiquitylation

because that species of LexA-VP16 does not react

with antibodies to ubiquitin.

24. A. Hecht, M. Grunstein, Methods Enzymol. 304, 399

(1999).

25.

CC894-1B and CC807-7D, which carry the indicated

pRJR238HA plasmids, were transformed with pARS1-

LEXA, which carries a URA3 marker and a modiﬁed

LexA-ARS1 DNA replication origin. Plasmid stability was

measured as described (13).

26. Ub coding sequences were polymerase chain reaction

(PCR)–ampliﬁed from yeast genomic DNA with two

modiﬁcations: (i) the carboxy-terminal glycine resi-

due was changed to alanine to prevent removal of

the Ub moiety by isopeptidases (19), and (ii) the

T7-epitope tag sequence was added at the carboxy

terminus of Ub.

27. For reagents, we thank D. Finley, W. Herr, R. Li, M.

Ptashne, and D. Thomas. We thank S. Grewal, R. Li, A.

Matapurkar, and V. Valmeekam for technical help.

A.A.C. is a George A. and Marjorie H. Anderson Fellow

of the Watson School of Biological Sciences and a

Howard Hughes Medical Institute Predoctoral Fellow.

W.P.T. is a Kimmel Scholar. Supported by the Cold

Spring Harbor Laboratory Cancer Center Support

Grant CA45508 and by USPHS grant CA-13106 from

the National Cancer Institute.

30 April 2001; accepted 2 July 2001

Published online 19 July 2001;

10.1126/science.1062079

Include this information when citing this paper.

Duration of Nuclear NF-␬B

Action Regulated by Reversible

Acetylation

Lin-feng Chen,

Wolfgang Fischle,

Eric Verdin,

1,2

Warner C. Greene

1,2,3

The nuclear expression and action of the nuclear factor kappa B (NF-␬B) tran-

scription factor requires signal-coupled phosphorylation and degradation of the I␬B

inhibitors, which normally bind and sequester this pleiotropically active factor in

the cytoplasm. The subsequent molecular events that regulate the termination of

nuclear NF-␬B action remain poorly deﬁned, although the activation of de novo

I␬B␣ gene expression by NF-␬B likely plays a key role. Our studies now demonstrate

that the RelA subunit of NF-␬B is subject to inducible acetylation and that acety-

lated forms of RelA interact weakly, if at all, with I␬B␣. Acetylated RelA is sub-

sequently deacetylated through a speciﬁc interaction with histone deacetylase 3

(HDAC3). This deacetylation reaction promotes effective binding to I␬B␣ and leads

in turn to I␬B␣-dependent nuclear export of the complex through a chromosomal

region maintenance-1 (CRM-1)– dependent pathway. Deacetylation of RelA by

HDAC3 thus acts as an intranuclear molecular switch that both controls the

duration of the NF-␬B transcriptional response and contributes to the replenish-

ment of the depleted cytoplasmic pool of latent NF-␬B–I␬B␣ complexes.

NF-␬B corresponds to an inducible transcrip-

tion factor complex that plays a pivotal role

in regulating the inflammatory, immune, and

anti-apoptotic responses in mammals (1, 2).

The prototypical NF-␬〉 complex, which cor-

responds to a heterodimer of p50 and RelA

subunits, is sequestered in the cytoplasm by

its assembly with a family of inhibitory pro-

teins termed the I␬Bs (1). Stimulus-induced

phosphorylation of two NH

-terminal serines

in the I␬Bs, mediated by a macromolecular

I␬B kinase complex (IKK) (3), triggers the

R EPORTS

www.sciencemag.org SCIENCE VOL 293 31 AUGUST 2001 1653

rapid ubiquitination and subsequent degrada-

tion of this inhibitor by the 26S proteasome

complex. The liberated NF-␬〉 heterodimer

rapidly translocates into the nucleus, where it

engages cognate ␬〉 enhancer elements and

alters gene expression. The NF-␬B complex

also recruits the p300/CBP and P/CAF coac-

tivators, which participate in the activation of

target gene transcription (4–6 ). Phosphoryl-

ation of an NH

-terminal site in RelA by

protein kinase A facilitates NF-␬B assembly

with CBP/p300 (7 ). Both CBP and p300

contain histone acetyltransferase activity that

has been implicated in the regulation of gene

expression. These effects involve acetylation

of core histones leading to changes in chro-

matin structure (8–13) as well as direct acet-

ylation of select host transcription factors like

Fig. 1. (A) TSA en-

hances TNF-␣–medi-

ated activation of ␬〉-

luciferase gene expres-

sion. 293T cells were

transfected with ␬B-

luciferase reporter

plasmid DNA (0.1 ␮g)

and luciferase activity

was measured (19) af-

ter treatment with

TSA and TNF-␣ (10

ng/ml). Results repre-

sent cumulated data

from three indepen-

dent transfections. (B)

TSA enhances TNF-␣

induction of nuclear

NF-␬B DNA-binding

activity. NF-␬B DNA-

binding activity was

assessed in electro-

phoretic mobility shift

assays (EMSA) with a

P-radiolabeled con-

sensus ␬B enhancer

oligonucleotide (5⬘-

AGTTGAGGGGACT T-

TCCCAGGC-3⬘) (up-

per panel). Supershift-

ing this complex with anti-RelA (SC-109, Santa Cruz Biotechnology,

Santa Cruz, CA) and anti-p50 (SC-1190, Santa Cruz) is shown in lanes

7 and 8. Comparability of the various nuclear extracts was assessed

by EMSA with a

P-radiolabeled Sp1 probe (Promega) (lower panel).

cells in the presence of TSA. HeLa cells were incubated in the presence

(lanes 7 to 12) or absence (lanes 1 to 6) of TSA (400 nM) for 1 hour

before stimulation with TNF-␣ (20 ng/ml) for 30 min. The cells were

chased for the indicated time periods with medium alone (lanes 1

to 6) or medium containing TSA (400 nM) (lanes 7 to 12). Nuclear

extracts from each culture were immunoblotted with anti-RelA and

visualized with an enhanced chemiluminescence reagent (Amersham).

(D) HDAC3, but not HDAC1, 2, 4, 5, or 6, inhibits TNF-␣ activation

of ␬B-luciferase activity. Expression levels of each HDAC determined

by anti-Flag immunoblotting of the cell lysates are shown in the

insets.

Fig. 2. (A) RelA is acetylated in vivo. COS-7 cells were

cotransfected with expression vectors encoding T7-RelA

and HDAC1 or HDAC3. Acetylation levels of RelA deter-

mined by [

H]sodium acetate labeling (19) are shown in

the upper panel. The total amount of T7-RelA in the

individual samples is shown in the lower panel. (B) Endog-

enous RelA is inducibly acetylated after TNF-␣ stimulation.

Acetylation of endogenous RelA was assessed by [

H]so-

dium acetate labeling (19) and is shown in the upper panel.

The level of RelA present in each of the two immunopre-

cipitates is shown in the lower panel. (C) RelA is inducibly

acetylated by p300 in vivo. 293T cells were cotransfected

with T7-RelA and I␬B␣ in the presence or absence of

expression plasmids encoding p300. Cell cultures were

stimulated with TNF-␣ for the indicated time periods, and

acetylation levels of RelA were analyzed by immunoblot-

ting with antibody to acetylated lysine (Cell Signaling,

Beverly, MA). The level of T7-RelA present in each of the

immunoprecipitates is also shown in the lower panel.

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31 AUGUST 2001 VOL 293 SCIENCE www.sciencemag.org1654

p53, GATA-1, and E2F, which alters their

biological function (9, 10).

Although much progress has been made in

understanding the biochemical events that

underlie NF-␬B induction, much less is

known about how activated nuclear forms of

NF-␬B are negatively regulated, ensuring a

transient transcriptional response. Prior stud-

ies have demonstrated that the I␬B␣ gene is

induced by NF-␬B(14–16 ) and that the de

novo expression of I␬B␣ proteins, which dis-

play nucleocytoplasmic shuttling properties,

participates in a negative feedback system

limiting the NF-␬B transcriptional response

(17, 18). We now demonstrate that nuclear

RelA is subject to reversible acetylation and

that this posttranslational modification plays

a pivotal role in NF-␬B regulation by gov-

erning I␬B␣ binding to RelA and the nuclear

export of the NF-␬B complex.

We first observed that trichostatin A

(TSA), a specific inhibitor of the multiple

histone deacetylases (HDACs) (11), en-

hanced tumor necrosis factor (TNF)-␣–in-

duced, but not basal, expression of a ␬B-

luciferase reporter gene (Fig. 1A). In addi-

tion, TSA enhanced nuclear NF-␬B DNA

binding after TNF-␣ stimulation but did not

itself stimulate NF-␬B binding (Fig. 1B).

TSA also did not alter the binding of the

constitutively expressed Sp1 transcription

factor in either the presence or absence of

TNF-␣ (Fig. 1B, lower panel). Immunoblot-

ting of the nuclear extracts with antibodies to

RelA (anti-RelA) revealed sustained nuclear

RelA expression in the presence of TSA (Fig.

1C). Together, these results indicate that TSA

enhances TNF-␣–induced NF-␬B DNA bind-

ing, likely by prolonging intranuclear expres-

sion of RelA.

TSA broadly inhibits the action of the

HDACs. HDACs function not only to deacety-

late core histones leading to repressive changes

in chromatin structure but also to deacetylate

various host transcription factors, altering their

transcriptional activity (10–13). We assessed

potential inhibitory effects of various HDACs

on NF-␬B action by cotransfecting 293T cells

with expression vectors encoding HDAC1, 2, 3,

4, 5, or 6 and a ␬B-luciferase reporter followed

by stimulation with TNF-␣ for 5 hours (Fig.

1D). Although each of the HDACs was com-

parably expressed, only HDAC3 inhibited

TNF-␣–induced ␬B-luciferase activity (lanes 7

and 8). This inhibitory effect of HDAC3 was

abrogated in the presence of TSA, indicating

that the deacetylase function of HDAC3 was

required for these biological effects (Web fig.

1) (19). Consistent with this action of HDAC3,

we found that the RelA subunit of NF-␬Bis

acetylated in vivo (Fig. 2A, lane 2, upper pan-

el). RelA acetylation was abolished in the pres-

ence of HDAC3 (lane 5) but not HDAC1 (lane

3). The addition of TSA to the HDAC3-ex-

pressing cultures was associated with the detec-

tion of acetylated RelA (lane 4). When HeLa

cells were incubated in [

H]sodium acetate and

stimulated with TNF-␣ or medium for 30 min,

signal-induced acetylation of endogenous RelA

was similarly detected (Fig. 2B). Together,

these studies reveal that NF-␬B is subject to

reversible acetylation and that HDAC3 plays a

central role in its deacetylation.

The potential participation of the p300,

Gladstone Institute of Virology and Immunology,

Department of Medicine,

Department of Microbiol-

ogy and Immunology, University of California, San

Francisco, CA 94141, USA.

*To whom correspondence should be addressed. E-

mail: wgreene@gladstone.ucsf.edu

Fig. 3. (A) HDAC3 and RelA physically assemble in vivo. COS-7 cells were

transfected with the indicated combinations of cDNAs encoding Flag epi-

tope–tagged HDAC1 or HDAC3 and T7-RelA, and anti-T7 immunoprecipi-

tations were performed followed by anti-Flag immunoblotting (19). Levels of

expression of HDAC1-Flag, HDAC3-Flag, and T7-RelA are shown in the lower

two panels. (B) The physical association of endogenous RelA and endoge-

nous HDAC3 in HeLa cells. Whole-cell lysates from HeLa cells were immu-

noprecipitated with polyclonal anti-HDAC1 or anti-HDAC3 followed by

immunoblotting with monoclonal anti-RelA (F-6, Santa Cruz). (C) HDAC3

inhibits TNF-␣–induced nuclear expression of RelA. HeLa cells were trans-

fected with control (lanes 1 to 4) or HDAC3-expression vector DNA and

cultured for 24 hours, followed by stimulation with TNF-␣ (10 ng/ml) for 0, 10, 30, or 90 min. Nuclear extracts were prepared and EMSAs performed

as described in Fig. 1B (panels 1 and 2). TNF-␣–induced degradation of I␬〉␣ was analyzed in the cytoplasmic extracts of these cultures by

immunoblotting with anti-I␬B␣ (C-21, Santa Cruz) (panel 3). Levels of nuclear RelA were assessed by immunoblotting nuclear extracts with speciﬁc

anti-RelA (panel 4). The faster migrating bands reactive with the anti-RelA likely correspond to RelA degradation products and show similar changes

in levels of nuclear expression. (D) HDAC3 stimulates nuclear export of RelA via a leptomycin B–sensitive pathway. GFP-RelA principally localized in

the nucleus when expression plasmid DNA was transfected into HeLa cells. Coexpression of HDAC3 and GFP-RelA resulted in a cytoplasmic expression

pattern of GFP-RelA; HDAC3 and GFP-RelA plasmid DNA was transfected at a 6:1 ratio to ensure that all GFP-RelA–expressing cells also contained

HDAC3. Coexpression of HDAC1 and GFP-RelA did not alter the nuclear pattern of GFP-RelA epiﬂuorescence. Treatment of the GFP-RelA and

HDAC3–expressing cell cultures with TSA (800 nM, 5 hours) resulted in a nuclear pattern of expression of GFP-RelA. Treatment of GFP-RelA

and HDAC3– expressing cultures with leptomycin B (LMB; 20 nM, 2 hours) produced a nuclear pattern of GFP-RelA epiﬂuorescence. Average

percentages of cells displaying the depicted phenotype are shown in the lower right corner of each panel. Results are derived from inspection

of at least 200 transfected cells present in multiple microscopic ﬁelds from two independent experiments.

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www.sciencemag.org SCIENCE VOL 293 31 AUGUST 2001 1655

CBP, and P/CAF acetyltransferases in the

acetylation of RelA was next investigated.

When 293T cells were cotransfected with

RelA, I␬B␣, and p300, and stimulated with

TNF-␣, inducible acetylation of RelA was

detected (Fig. 2C). Coexpression of either

p300 or CBP, but not P/CAF, with RelA

produced a dose-dependent acetylation of T7-

RelA [Web fig. 2 (19)]. Together, these find-

ings demonstrate that endogenous RelA is

acetylated in a signal-coupled manner likely

mediated by p300 and CBP.

Coimmunoprecipitation of T7-RelA and

HDAC3 in COS-7 cells suggests a physical

interaction between these two proteins in

vivo (Fig. 3A). In contrast, no interaction

between RelA and HDAC1 was detected de-

spite comparable levels of both HDACs be-

ing expressed (Fig. 3A). Moreover, anti-

HDAC3 effectively coimmunoprecipitated

endogenous RelA from HeLa cells, whereas

only trace amounts of RelA immunoprecipi-

tated with anti-HDAC1 (Fig. 3B). By using

coimmunoprecipitation assays and the mam-

malian two-hybrid system (20), we found that

-terminal regions of both HDAC3 and

RelA are required for the assembly of these

two proteins in vivo [Web fig. 3) (19)].

Studies were next performed to define the

molecular basis for HDAC3-mediated inhibi-

tion of TNF-␣ activation of NF-␬B. Expression

of HDAC3 in HeLa cells (⬃50% transfection

efficiency) diminished both NF-␬B DNA-bind-

ing activity (Fig. 3C, panel 1) and levels of

nuclear RelA ( panel 4). In contrast, HDAC3

expression did not alter TNF-␣–induced degra-

dation of I␬B␣ occurring in the cytoplasm

( panel 3), nor did it markedly change the levels

of Sp1 DNA-binding activity ( panel 2). To-

gether with the results presented in Fig. 1C,

these findings raise the possibility that HDAC3

may regulate the intracellular trafficking of

RelA. A precedent for acetylation influencing

the intracellular trafficking of a transcription

factor is provided by recent studies on hepato-

cyte nuclear factor-4 and class II transactivator

(CIITA) (21, 22). To monitor potential effects

of HDAC3 on the intracellular trafficking of

RelA, we expressed green fluorescent protein–

RelA fusion proteins (GFP-RelA) in HeLa cells

in the presence and absence of HDAC3 (Fig.

3D). When expressed alone or with HDAC1,

GFP-RelA localized to the nucleus, whereas the

coexpression of HDAC3 resulted in a predom-

inantly cytoplasmic pattern of GFP-RelA ex-

pression. This cytoplasmic relocalization of

GFP-RelA induced by HDAC3 did not occur in

the presence of TSA. These effects of HDAC3

appeared to result from nuclear export of GFP-

RelA because the addition of leptomycin B, a

known inhibitor of CRM-1/exportin-1– depen-

dent nuclear export (23), preserved the nuclear

pattern of GFP-RelA epifluorescence. These

findings suggest that HDAC3-mediated

deacetylation of RelA promotes its export from

the nucleus to the cytoplasm.

Among the target genes activated by NF-␬B

is the I␬B␣ gene (14–16 ). The resultant de

novo synthesis of I␬B␣ serves to replenish the

intracellular stores of this inhibitor depleted

during the course of NF-␬B activation. I␬B␣

also displays nucleocytoplasmic shuttling prop-

erties and likely retrieves nuclear NF-␬B com-

plexes, thereby contributing to the termination

of the NF-␬B transcriptional response (17, 18).

We next examined whether the acetylation sta-

tus of RelA regulates its assembly with I␬B␣.

The acetylation of RelA induced by the coex-

pression of increasing amounts of p300 was

associated with markedly diminished binding

of RelA to GST-I␬B␣ matrices (Fig. 4A, lanes

3 and 4). However, the coexpression of increas-

ing amounts of HDAC3 in the presence of p300

restored RelA binding to GST-I␬B␣ (Fig. 4A,

lanes 5 and 6). Immunoblotting of the T7-RelA

proteins with antibodies to acetylated lysine

confirmed dose-related increases in RelA acet-

ylation by p300 in lanes 3 and 4 and dose-

related deacetylation of RelA by HDAC3 in

lanes 5 and 6 (24 ). These findings suggest that

acetylation of RelA prevents I␬B␣ binding,

whereas deacetylation of RelA by HDAC3

stimulates I␬B␣ binding.

To test whether the HDAC3-induced nu-

clear export of RelA is dependent on I␬B␣,

we studied the subcellular localization of

GFP-RelA in murine embryo fibroblasts

(MEFs) isolated from wild-type or I␬B␣

–/–

mice produced by targeted gene disruption

Fig. 4. (A) Deacetylated RelA

displays greater I␬B␣-binding

activity than acetylated RelA.

293T cells were cotransfected

with T7-RelA and p300 in the

presence or absence of

HDAC3 expression vector

DNA as indicated. Twenty-

four hours later, 50 ␮l of each

whole-cell lysate was incubat-

ed with GST-I␬B␣ (1.0 ␮g).

The levels of RelA captured by

the GST-I␬B␣ matrix under

each condition were assessed

by immunoblotting with anti-

T7 (upper panel). The total

amounts of GST-I␬B␣ and

RelA present in the reaction

mixtures are shown in the

lower two panels. (B) HDAC3

does not stimulate GFP-RelA

nuclear export in I␬B␣-deﬁ-

cient MEFs. MEF cells from

wild-type and I␬B␣

–/–

mice

were transfected with GFP-

RelA (0.3 ␮g) ( panels a and c)

or GFP-RelA (0.3 ␮g) and

HDAC3 (1.7 ␮g) ( panels b

and d) expression vector DNA. The I␬B␣

⫺/⫺

MEFs were also recon-

stituted with limiting amounts of I␬B␣ expression vector (0.1 ␮g) in

the absence and presence of HDAC3 ( panels e and f ). Average

percentages of cells displaying the depicted phenotype derived from

inspection of at least 120 cells present in multiple microscopic ﬁelds

from two independent experiments are shown in the lower right

corner of each panel. (C) Schematic model for the role of HDAC3-

mediated deacetylation of RelA as an intranuclear molecular switch

promoting I␬B␣ binding and I␬B␣-dependent nuclear export of the

NF-␬B complex. This deacetylation-controlled response both leads to

the termination of the NF-␬B transcriptional response and aids in

reestablishing latent cytoplasmic forms of NF-␬B bound to I␬B␣,

thereby preparing the cell to respond to the next NF-␬B–inducing

stimulus.

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31 AUGUST 2001 VOL 293 SCIENCE www.sciencemag.org1656

(25) (Fig. 4B). In wild-type MEFs, GFP-

RelA was principally expressed in the nucle-

us ( panel a), whereas the coexpression of

HDAC3 induced a cytoplasmic pattern of

GFP-RelA epifluorescence ( panel b). How-

ever, a very different pattern was obtained in

the I␬B␣

–/–

MEFs. Whereas GFP-RelA also

exhibited a nuclear pattern of epifluores-

cence, the coexpression of HDAC3 in these

I␬B␣

–/–

MEFs failed to induce a cytoplasmic

redistribution of RelA ( panel d). Reconstitu-

tion of these I␬B␣

–/–

cells by transfection

with small quantities of an I␬B␣ expression

vector restored HDAC3-induced cytoplasmic

expression of the GFP-RelA protein (panel

f ). In the absence of HDAC3, GFP-RelA

remained principally nuclear, indicating that

the levels of I␬B␣ expressed were not suffi-

cient on their own to produce cytoplasmic

sequestration of GFP-RelA in these I␬B␣

–/–

MEFs ( panel e). These results indicate that

I␬B␣ is required for the nuclear export of

deacetylated forms of RelA, which display

increased binding of I␬B␣.

These findings reveal a new mechanism

through which nuclear NF-␬B function is regu-

lated (Fig. 4C). RelA is subject to stimulus-

coupled acetylation likely mediated through the

p300 and CBP coactivators. One biological con-

sequence of this modification is that acetylated

RelA becomes a very poor substrate for binding

by newly synthesized I␬B␣. Whether p50 or

perhaps I␬B␣ are similarly subject to biologi-

cally important acetylation/deacetylation reac-

tions remains to be explored. Our studies iden-

tify acetylated RelA as a novel nonhistone sub-

strate of HDAC3. As such, HDAC3-mediated

deacetylation functions as an intranuclear mo-

lecular switch that when activated initiates a

series of events culminating in the termination

of the NF-␬B transcriptional response. The

I␬B␣-dependent nuclear export of the HDAC3-

deacetylated RelA-containing complexes also

serves to replenish the depleted cytoplasmic

pool of latent NF-␬B–I␬B␣ complexes needed

for the next inductive NF-␬B response in these

cells.

References and Notes

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1653/DC1.

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26. We thank R. Goodman (Vollum Institute) and Y.

Nakatani (NIH) for reagents; J. F. Klement ( Jefferson

Medical College) for providing the I␬B␣

⫺/⫺

MEF cells;

and A. O’Mahony for helping establish cultures of

these cells. We also thank J. Carroll for assistance in

the preparation of the ﬁgures and R. Givens for

assistance in the preparation of the manuscript. Sup-

ported in part by funds provided by the J. David

Gladstone Institutes, Pﬁzer, and the University of

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(P30MH59037).

8 May 2001; accepted 3 July 2001

Allosteric Activation of a

Spring-Loaded Natriuretic

Peptide Receptor Dimer by

Hormone

Xiao-lin He, Dar-chone Chow, Monika M. Martick,

K. Christopher Garcia*

Natriuretic peptides (NPs) are vasoactive cyclic-peptide hormones important in

blood pressure regulation through interaction with natriuretic cell-surface re-

ceptors. We report the hormone-binding thermodynamics and crystal struc-

tures at 2.9 and 2.0 angstroms, respectively, of the extracellular domain of the

unliganded human NP receptor (NPR-C) and its complex with CNP, a 22–amino

acid NP. A single CNP molecule is bound in the interface of an NPR-C dimer,

resulting in asymmetric interactions between the hormone and the symmet-

rically related receptors. Hormone binding induces a 20 angstrom closure

between the membrane-proximal domains of the dimer. In each monomer, the

opening of an interdomain cleft, which is tethered together by a linker peptide

acting as a molecular spring, is likely a conserved allosteric trigger for intra-

cellular signaling by the natriuretic receptor family.

The natriuretic peptides (NPs) are three ho-

mologous peptide hormones that play impor-

tant roles in the maintenance of cardiovascu-

lar homeostasis, blood pressure, and body

fluid regulation (i.e., natriuresis) (1). Collec-

tively, these hormones function as an endog-

enous counterbalance to the renin-angioten-

sin/aldosterone system, as well as the hypo-

thalamic/pituitary/adrenal axis. The three

members of this family are atrial (ANP) and

brain (BNP) natriuretic peptides, which are

produced by the heart; and CNP, which is

expressed in endothelial cells (1). ANP and

BNP are thought to be the primary regulators

of peripheral natriuretic activity; CNP is

present mainly in the brain. ANP, BNP, and

CNP are highly homologous (⬃70% identi-

cal) and share as a common motif a 17–amino

acid loop formed by a disulfide bond (Fig. 1).

The lack of defined structure(s) in solution

and the questionable relevance of “lowest

energy” solution conformations to the recep-

tor-bound conformation of peptide hormones

mean that assessing the bioactive conforma-

tions of these peptides remains a general

problem (2).

The actions of the NPs are mediated by

three homologous single-transmembrane,

glycosylated cell-surface receptors (NPR-A,

-B, and -C) (3–5). These receptors possess

about 30% homologous extracellular ligand-

binding domains (ECDs) (⬃450 amino acids)

with conserved topologies but possess differ-

ent downstream activation mechanisms.

NPR-C is the most promiscuous of the recep-

tors, binding to all NPs with high affinity,

whereas NPR-A and NPR-B are more specif-

ic for ANP and CNP, respectively (6 ). In the

cases of NPR-A and NPR-B, hormone bind-

ing to the ECDs results in the production of

intracellular cyclic guanosine 3⬘,5⬘-mono-

phosphate by a guanylyl-cyclase activity that

resides in the intracellular domains (5, 7 ). For

NPR-C, which represents over 95% of NPR

in vivo, ligand binding results in both inter-

nalization and degradation (i.e., clearance), as

well as signaling by heterotrimeric GTP-

R EPORTS

Duration of nuclear NF-κB action regulated by reversible acetylation

Figures

Citations

Signaling to NF-kappaB.

NF-kappaB regulation in the immune system.

Missing Pieces in the NF-κB Puzzle

Anticancer activities of histone deacetylase inhibitors.

Modulation of NF-κB-dependent transcription and cell survival by the SIRT1 deacetylase

References

THE NF-κB AND IκB PROTEINS: New Discoveries and Insights

NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses

Acetylation of Histones and Transcription-Related Factors

Roles of histone acetyltransferases and deacetylases in gene regulation

Phosphorylation of NF-κB p65 by PKA Stimulates Transcriptional Activity by Promoting a Novel Bivalent Interaction with the Coactivator CBP/p300

Related Papers (5)

Phosphorylation of NF-κB p65 by PKA Stimulates Transcriptional Activity by Promoting a Novel Bivalent Interaction with the Coactivator CBP/p300

Modulation of NF-κB-dependent transcription and cell survival by the SIRT1 deacetylase

Activation of p53 Sequence-Specific DNA Binding by Acetylation of the p53 C-Terminal Domain

Missing Pieces in the NF-κB Puzzle

NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses

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