letters to nature
solution supplemented with ,20% (w/v) PEG 400. After using the SeMet
crystals to obtain a ¯uorescence curve as a function of the incident X-ray
energy, we collected data sets at 12659.0 eV, 12663.5 eV, 12800.0 eV and
12630.0 eV using inverse-beam geometry. The data sets from the R-Axis4
detector at NSLS X4a and the Quantum 4 detector at CHESS were processed
using DENZO and SCALEPACK22.
Structure determination. Determination and re®nement of the selenium sites
and initial phase calculations were done using the program SOLVE (from
T. Terwilliger; see http://www.solve.lanl.gov and references therein). Density
modi®cation that included solvent ¯attening and histogram matching, using
the program DM23, improved the quality of the electron-density maps
calculated using either the NSLS or the CHESS data. However, the maps
derived from the NSLS or CHESS data were mediocre and could not be used to
trace more than a small fraction of the polypeptide chain. Substantial
improvement of the electron-density maps resulted from combination of the
NSLS and CHESS phases using the program SIGMAA24 and use of jF o j derived
from a selenomethionine data set collected at home (2.3 AÊ resolution, CuKa Xrays) and MAD-combined phases. Phase combination in reciprocal space and
map averaging in real space produced similar results. Using experimental maps
calculated at 2.5 and 2.3 AÊ resolution, we built a structure comprising 86% of
the 279 residues by using the program O (ref. 25). After the ®rst round of Powell
minimization and B-factor re®nement using XPLOR26, the conventional Rvalue was 0.322 and the R-free value was 0.389. At this point, we used native
data measured to 1.9 AÊ resolution for the remainder of the re®nement and
model building until the R values converged, beginning at 2.3 AÊ resolution and
gradually including higher-resolution terms. We then built kainate into an
`omit' map calculated using phases from the re®ned model and F o 2 F c
coef®cients, and carried out another round of Powell minimization and Bfactor re®nement. Unless otherwise noted, CCP4 programs27 were used for
crystallographic calculations. There is either no or weak electron density for
the following side chains and they have therefore been omitted from the
structure and replaced by Ala: Lys 393, Lys 410, Glu 416, Lys 434, Asp 456,
Leu 483, Lys 493, Glu 634, Ser 635, Glu 637, Lys 641, Gln 642, Thr 643, Arg 661,
Lys 663, Glu 678, Lys 695, Lys 697 and Ser 741.
Received 14 August; accepted 18 September 1998.
1. Hollmann, M. & Heinemann, S. Cloned glutamate receptors. Annu. Rev. Neurosci. 17, 31±108 (1994).
2. Rogers, S. W. et al. Autoantibodies to glutamate receptor GluR3 in Rasmussen's encephalitis. Science
265, 648±651 (1994).
3. Stern-Bach, Y. et al. Agonist selectivity of glutamate receptors is speci®ed by two domains structurally
related to bacterial amino acid-binding proteins. Neuron 13, 1345±1357 (1994).
4. Kuusinen, A., Arvola, M. & KeinaÈnen, K. Molecular dissection of the agonist binding site of an AMPA
receptor. EMBO J. 14, 6327±6332 (1995).
5. Paas, Y. The macro- and microarchitectures of the ligand-binding domain of glutamate receptors.
Trends Neurosci. 21, 117±125 (1998).
6. Chen, G.-Q., Sun, Y., Jin, R. & Gouaux, E. Probing the ligand binding domain of the GluR2 receptor
by proteolysis and deletion mutagenesis de®nes domain boundaries and yields a crystallizable
construct. Protein Sci. (in the press).
7. Sommer, B. et al. Flip and ¯op: a cell-speci®c functional switch in glutamate-operated channels of the
CNS. Science 249, 1580±1585 (1990).
8. Hendrickson, W. A. Determination of macromolecular structures from anomalous diffraction of
synchrotron radiation. Science 254, 51±58 (1991).
9. Nakanishi, N., Shneider, N. A. & Axel, R. A family of glutamate receptor genes: evidence for the
formation of heteromultimeric receptors with distinct channel properties. Neuron 5, 569±581 (1990).
10. Sun, Y.-J., Rose, J., Wang, B.-C. & Hsiao, C.-D. The structure of glutamine-binding protein complexed
with glutamine at 1.94 AÊ resolution: comparisons with other amino acid binding proteins. J. Mol. Biol.
278, 219±229 (1998).
11. KeinaÈnen, K., Arvola, M., Kuusinen, A. & Johnson, M. Ligand recognition in glutamate receptors:
insights from mutagenesis of the soluble a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid
(AMPA)-binding domain of glutamate receptor type D (GluR-D). Biochem. Soc. Trans. 25, 835±838
(1997).
12. Laube, B., Hirai, H., Sturgess, M., Betz, H. & Kuhse, J. Molecular determinants of agonist
discrimination by NMDA receptor subunits: analysis of the glutamate binding site on the NR2B
subunit. Neuron 18, 493±503 (1997).
13. Paas, Y., Eisenstein, M., Medevielle, F., Teichberg, V. I. & Devillers-ThieÂry, A. Identi®cation of the
amino acid subsets accounting for the ligand binding speci®city of a glutamate receptor. Neuron 17,
979±990 (1996).
14. Hirai, H., Kirsch, J., Laube, B., Betz, H. & Kuhse, J. The glycine binding site of the N-methyl-Daspartate receptor subunit NR1: identi®cation of novel determinants of co-agonist potentiation in the
extracellular M3-M4 loop region. Proc. Natl Acad. Sci. USA 93, 6031±6036 (1996).
15. Swanson, G. T., Gereau, R. W. IV, Green, T. & Heinemann, S. F. Identi®cation of amino acid residues
that control functional behavior in GluR5 and GluR6 kainate receptors. Neuron 19, 913±926 (1997).
16. Jacobson, B. L., He, J. J., Lemon, D. D. & Quiocho, F. A. Interdomain salt bridges modulate ligandinduced domain motion of the sulfate receptor protein for active transport. J. Mol. Biol. 223, 27±30
(1992).
17. Li, F., Owens, N. & Verdoorn, T. A. Functional effects of mutations in the putative agonist binding
region of recombinant a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. Mol.
Pharmacol. 47, 148±154 (1995).
18. Aizenman, E., Lipton, S. A. & Loring, R. H. Selective modulation of NMDA responses by reduction
and oxidation. Neuron 2, 1257±1263 (1989).
NATURE | VOL 395 | 29 OCTOBER 1998 | www.nature.com
19. Sutcliffe, M. J., Wo, Z. G. & Oswald, R. E. Three-dimensional models of non-NMDA glutamate
receptors. Biophys. J. 70, 1575±1589 (1996).
20. Partin, K. M., Bowie, D. & Mayer, M. L. Structural determinants of allosteric regulation in alternatively
spliced AMPA receptors. Neuron 14, 833±843 (1995).
21. Partin, K. M., Fleck, M. W. & Mayer, M. L. AMPA receptor ¯ip/¯op mutants affecting deactivation,
desensitization, and modulation by cyclothiazide, aniracetam, and thiocyanate. J. Neurosci. 16, 6634±
6647 (1996).
22. Otwinowsky, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode.
Enzymol. 276, 307±326 (1997).
23. Cowtan, K. D. & Main, P. Phase combination and cross validation in iterated density-modi®cation
calculations. Acta Crystallogr. D 42, 43±48 (1996).
24. Read, R. J. Model phases: probabilities and bias. Methods Enzymol. 277, 110±128 (1997).
25. Jones, T. A. & Kjeldgaard, M. Electron-density map interpretation. Methods Enzymol. 277, 173±208
(1997).
26. BruÈnger, A. T. X-PLOR. Version 3.1. A System for X-ray Crystallography and NMR 1±382 (Yale Univ.
Press, New Haven, 1992).
27. Collaborative Computational Project Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760±763 (1994).
28. Hollmann, M., Maron, C. & Heinemann, S. N-Glycosylation site tagging suggests a three transmembrane domain topology for the glutamate receptor GluR1. Neuron 13, 1331±1343 (1994).
29. Lomeli, H. et al. Control of kinetic properties of AMPA receptor channels by nuclear RNA editing.
Science 266, 1709±1713 (1994).
8
Acknowledgements. We thank C. Lima and other members of the Hendrickson laboratory for assistance;
J. Lidestri for maintenance of the X-ray laboratory at Columbia University; A. Karlin, J. Javitch and
M. Akabas for advice; S. Heinemann and M. Hartley for glutamate-receptor clones; C. Ogata for help with
MAD data collection at NSLS X4a; T. Terwilliger for advice on the use of Solve; and W. Minor and
Z. Otwinoski for a new version of the HKL suite of programs. Extra MAD data were collected at the
Cornell High Energy Synchrotron Source (CHESS), which is supported by the NSF, using the
Macromolecular Diffraction at CHESS (MacCHESS) facility, which is supported by an award from the
NIH. N.A. was supported by an NIH Ophthalmology training grant. E.G. is an NSF Young Investigator
and the recipient of an Alfred P. Sloan Research Fellowship.
Correspondence and requests for materials should be addressed to E.G. (e-mail: jeg52@columbia.edu).
The GluR2 S1S2 coordinates have been deposited with the Protein Data Bank and have accession code
1gr2.
Chromatin deacetylation by an
ATP-dependent nucleosome
remodelling complex
Jeffrey K. Tong*, Christian A. Hassig*, Gavin R. Schnitzler²,
Robert E. Kingston² & Stuart L. Schreiber*
* Howard Hughes Medical Institute, Departments of Chemistry and Chemical
Biology, Molecular and Cellular Biology, Harvard University, 12 Oxford Street,
Cambridge, Massachusetts 02138, USA
² Department of Molecular Biology, Massachusetts General Hospital, Boston,
Massachusetts 02114, USA
.........................................................................................................................
The dynamic assembly and remodelling of eukaryotic chromosomes facilitate fundamental cellular processes such as DNA
replication and gene transcription. The repeating unit of eukaryotic chromosomes is the nucleosome core, consisting of DNA
wound about a de®ned octamer of histone proteins1. Two enzymatic processes that regulate transcription by targeting elements
of the nucleosome include ATP-dependent nucleosome remodelling and reversible histone acetylation2,3. The histone deacetylases,
however, are unable to deacetylate oligonucleosomal histones in
vitro4. The protein complexes that mediate ATP-dependent
nucleosome remodelling and histone acetylation/deacetylation
in the regulation of transcription were considered to be different,
although it has recently been suggested that these activities might
be coupled5. We report here the identi®cation and functional
characterization of a novel ATP-dependent nucleosome remodelling activity that is part of an endogenous human histone
deacetylase complex. This activity is derived from the CHD3
and CHD4 proteins which contain helicase/ATPase domains
found in SWI2-related chromatin remodelling factors, and
facilitates the deacetylation of oligonucleosomal histones in
vitro. We refer to this complex as the nucleosome remodelling
and deacetylating (NRD) complex. Our results establish a physical
and functional link between the distinct chromatin-modifying
activities of histone deacetylases and nucleosome remodelling
proteins.
Nature © Macmillan Publishers Ltd 1998
917
letters to nature
We puri®ed an endogenous histine deacetylase complex, HDAC2
complex, from HeLa cell extract by immunoaf®nity chromatography. The antibody-bound complex was speci®cally eluted by its
peptide antigen, separated by SDS±PAGE and visualized by silver
staining (Fig. 1). As expected, the HDAC2-associated proteins
RbAp48 and HDAC1 co-puri®ed with HDAC2. In addition, three
anti-HDAC2
peptide elution
p240
p110
p70
HDAC1
HDAC2
RbAp48
Figure 1 Silver-stained SDS-polyacrylamide gel of the HDAC2 complex. The
HDAC2-associated polypeptides p70, p110 and p240 were immunoaf®nity-
H
DA
C
2
IP
– +
peptide block
– +
peptide block
anti−Mi-2α
2500
2000
1500
1000
C
m
–
6500
–
–
–
–
500
peptide block
anti-HDAC1
0
H
IP:
+
–
peptide block:
anti-HDAC2
–
–
–
–
–
AC
2
Si
n3
C A
H
D
M 3
i-2
C
hu H α
m D
an 4
M
i-2
+
IP
Si
2
DA
C
H
–
n3
A
H
D
3
M
i-2
α
C
H
D
4
hu
m
an
M
i-2
anti-CHD4
c
a
anti-CHD3
anti-Mi-2 (human)
D
+
m
–
Deacetylase activity
(d.p.m.)
+
H
H
DA
C
DA
C
1
2
1
DA
C
H
–
b
IP
IP
a
IP
puri®ed from HeLa cell extract as described in Methods.
previously uncharacterized HDAC-associated polypeptides of
apparent relative molecular masses of 70K, 110K and 240K (referred
to as p70, p110 and p240, respectively) were identi®ed. The puri®ed
proteins were excised from the gel for sequence analysis by peptide
mass spectrometry. These three uncharacterized polypeptides are
also common to an HDAC1 immune complex prepared from
human Jurkat T cells (data not shown). Peptides obtained from
p110 are identical in sequence to a region of the hypothetical protein
KIAA0601 (GenBank accession number, AB011173). This protein
shares a region of homology with a polyamine oxidase, suggesting a
potential role for the NRD complex in redox reactions of straightchain aminoalkane structures, such as those that occur in lysine side
chains and in the chromatin cofactors spermine and spermidine6.
Here we focus on the characterization of p240. Sequences from two
peptides derived from p240 correspond to sequences found in the
related human proteins CHD3 and CHD4: peptide 1 (FSWAQGTDTILADEMGLGK) is an identical match to a sequence found in both
CHD3 and CHD4; peptide 2 (HDYWLLAGIINHGYAR) is unique
to CHD4. CHD3 and CHD4 have previously been identi®ed as selfantigens recognized by sera from a subset of patients with the
autoimmune connective-tissue disease Mi-2 dermatomyositis7,8.
We investigated whether CHD3 and CHD4 are present in both
Jurkat HDAC1 and HeLa HDAC2 complexes by co-immunoprecipitation and western blotting analysis. HDAC1 and HDAC2 complexes were immunoprecipitated from Jurkat and HeLa extracts,
respectively, and then western-blotted with Mi 2-positive dermatomyositis patient serum, which recognizes both CHD3 and CHD4
(Fig. 2a). The serum speci®cally reacts with p240 in both the
HDAC1 and HDAC2 complexes and no crossreacting band was
seen when HDAC immunoprecipitation was pre-blocked with the
peptide antigen (Fig. 2a). To con®rm the presence of CHD3 and
CHD4 in the complexes, HDAC immunoprecipitates were probed
with two different rabbit antisera raised against CHD3 (anti-CHD3
and anti-Mi-2a) and one other rabbit antiserum raised against
CHD4. These antibodies were generated against regions of CHD3
anti-HDAC1 or anti-HDAC2 antisera, respectively. Bound proteins were
separated by SDS±PAGE and analysed by western blotting with human Mi-2positive serum. As negative controls, parallel immunoprecipitations were done
using antiserum preincubated with the peptide antigen. b, As a, except that
western blots were prepared with two different anti-CHD3 rabbit sera (antiCHD3 and anti-Mi-2a) as well as an anti-CHD4 rabbit serum. c, Immunopreci-
300
200
100
0
1 2002 nM
TPX: 0 nM
CHD3 IP
pitation from HeLa extract using rabbit antisera against CHD3 and CHD4, and
Deacetylase activity
(d.p.m.)
NRD complex. a, Jurkat and HeLa cell extracts were immunoprecipitated using
Deacetylase activity
(d.p.m.)
b
Figure 2 CHD3 and CHD4 are associated with HDAC1 and HDAC2 in vivo in the
4000
3000
2000
1000
TPX:
0
1 2002nM
0 nM
CHD4 IP
human Mi-2-positive dermatomyositis patient serum. mSin3A and HDAC2
immunoprecipitations were tested in parallel as positive controls for HDAC1
Figure 3 CHD3 and CHD4 complexes contain histone deacetylase activity. a,
and HDAC2. Antibody-bound proteins were separated by SDS±PAGE and
Each immunoprecipitate from Fig. 2c was tested for deacetylase activity. b, CHD3
analysed by western blotting for HDAC2 (bottom). The HDAC2 blot was stripped
and CHD4 were immunoprecipitated from HeLa cell extract and the puri®ed
and reprobed for HDAC1 (top). HDAC1 corresponds to the top band of the
complex was preincubated with or without the HDAC inhibitor trapoxin (TPX)
doublet seen in the CHD4 and Mi-2 lanes.
before assaying for histone deacetylase activity. Background was ,100 d.p.m.
918
Nature © Macmillan Publishers Ltd 1998
NATURE | VOL 395 | 29 OCTOBER 1998 | www.nature.com
8
letters to nature
repression, DNA repair and chromosome recombination2. HDAC
and CHD immune complexes were incubated with the radiolabelled
and photoreactive ATP analogue 8-azido-g-32P-ATP to test for ATP
binding. Irradiation of the complexes with ultraviolet light resulted
in the speci®c crosslinking of ATP with a polypeptide of relative
molecular mass 240K, which corresponds to that of the CHD3 and
CHD4 proteins (Fig. 4a). The same ATP-binding activity is present
in both anti-HDAC as well as in anti-CHD complexes and is
competitively inhibited by a 1,000-fold excess of unlabelled ATP.
We next investigated whether CHD3/4- and HDAC-containing
NRD complexes could use the energy of ATP hydrolysis to remodel
chromatin, as we have observed nucleosome-stimulated ATPase
activity from the NRD complex (J.K.T., C.A.H. and S.L.S., unpublished results). Reconstituted, rotationally phased mononucleosome cores were used as a substrate to assay for any changes that
the NRD complex might impart on the structure of the nucleosome
as re¯ected by an altered sensitivity of the DNA to either DNase I or
PstI restriction endonuclease. Mononucleosome cores were treated
with varying concentrations of NRD complex from either HDAC or
CHD immunoprecipitates, with or without ATP, and then digested
with DNase I. Whereas DNA from control nucleosome cores yields a
repeating ,10-base-pair (bp) pattern after digestion with DNase I,
addition of both anti-HDAC and anti-CHD NRD complexes disrupts this pattern in a dose- and ATP-dependent manner (Fig. 4b).
The pattern of DNase I hypersensitivity that results from treatment
3
D
H
C
H
DA
C
N
I/S
CHD4 IP
hS
W
HDAC2 IP
ba
re
(– DN
)
A
HDAC1 IP
F
b
a
IP
8
1
IP
Pe
pt
H id
DA e
C blo
1 ck
IP e
d
H
DA
C
2
IP
Pe
pt
H id
DA e
C blo
2 ck
IP e
C
d
H
D
4
IP
and CHD4 that are unique to each protein. All anti-CHD3 and antiCHD4 antisera reacted with p240 in an HDAC-speci®c manner
(Fig. 2b). Reciprocal immunoprecipitation using anti-CHD3 or
anti-CHD4 antisera, or Mi-2 human serum, were positive for
HDAC1 and HDAC2 as well (Fig. 2c).
To determine whether HDACs associated with the CHD proteins
in the NRD complex were enzymatically active, we tested the CHD3
and CHD4 immunoprecipitates for histone deacetylase activity. In
vitro activity was measured by quantifying the release of radiolabelled acetyl groups from puri®ed hyperacetylated HeLa histones.
We found that CHD3 and CHD4 immunoprecipitates from HeLa
cell extract had histone deacetylase activity (Fig. 3a), and that
treatment of these immunoprecipitates with the HDAC inhibitor
trapoxin reduces histone deacetylase activity to background levels
(Fig. 3b).
The presence of CHD3 and CHD4 in the NRD complex
prompted us to investigate their possible function in chromatin
regulation. CHD3 and CHD4 are highly related proteins that
contain a number of putative functional domains, including PHD
zinc-®nger domains, chromo domains, and an ATPase/helicase
domain which is similar to ones found in the SWI2/SNF2 superfamily of proteins9. Although no function has yet been ascribed to
CHD3 or CHD4, other members of the SWI2/SNF2-related proteins are known to be involved in several ATP-dependent chromatin
regulatory processes, including transcriptional activation and
+
–
+
(–
c
+
–
–
+
+
–
+
–
+
+
–
–
+
+
–
2 µM 8-azido-γ-32P-ATP
2 mM cold, unlabelled ATP
Peptide competition
– + – ++ + + – + + + – + + – + + + – + + – + + + – + + + –
ATP
DA
C
Pe 1
I
H ptid P
DA e
C b
H 1 loc
DA IP ke
d
Pe C2
I
H ptid P
DA e
b
hS C2 loc
W IP ked
I/S
N
C
F
H
D
3
I
P
C
H
D
4
IP
+
+
–
H
+
–
–
)
CHD3/4
– + – + – + – + – + – + – + – + ATP
155-mer
49-mer
Figure 4 Functional characterization of CHD3/4. a, NRD complexes contain a
weak disruption by CHD3 is consistent with the small amount of CHD3
240K ATP-binding protein(s). NRD complexes prepared from HDAC or CHD
immunoprecipitated. c, HDAC- and CHD-containing complexes facilitate PstI
immunoprecipitates were crosslinked with a radiolabelled, photoreactive ATP
cutting of DNA in an ATP-dependent manner. HDAC and CHD immunoprecipi-
analogue, 8-azido-ATP by irradiation with ultraviolet light. The radiolabelled 240K
tates were incubated with 2.5 ng of mononucleosome cores in the presence or
band is speci®c to the NRD complex: excess, unlabelled ATP speci®cally
absence of 1 mM ATP/MgCl2. Reactions were treated with 20 U of PstI for 1 h and
competes with radiolabelled ATP for binding. IP, immunoprecipitate. b, HDAC-
separated by gel electrophoresis. Percentage cutting (cut=‰cut ‡ uncutŠ) was
and CHD-containing complexes disrupt mononucleosomes in an ATP- and dose-
determined by phosphorimager analysis. The triplets result from incomplete
dependent manner. Black arrowheads indicate bands of increased DNase I
Klenow ®ll-in during labelling.
cleavage, white arrowheads indicate bands of decreased DNase I cleavage. The
NATURE | VOL 395 | 29 OCTOBER 1998 | www.nature.com
Nature © Macmillan Publishers Ltd 1998
919
letters to nature
of mononucleosome cores with the NRD complex is similar to that
obtained after treatment of mononucleosome cores with the human
SWI/SNF complex. There is a background ATP-stimulated
remodelling activity that is at least 25-fold less than that found in
the NRD and human SWI/SNF complexes. We assayed mononucleosome cores under similar reaction conditions for a 49-bp
product after cleavage by PstI. Formation of this product was
stimulated between 10- and 20-fold by ATP in the presence of
NRD complex from either HDAC1/2 or CHD3/4 immunob
0
ATP:
DNA:
D
H
+ – +
– + +
histone
substrate
IP
IP
–
–
2
2
DA
C
4
I/S
IP
N
F
W
I/S
N
hS
hS
W
)
200
F
0
ATP: – + – +
TPX: – – + +
plasmid chromatin
substrate
400
DA
C
200
600
H
400
c
(–
Deacetylase activity
(d.p.m.)
800
C
Deacetylase activity
(d.p.m.)
600
H
a
– – + – – – + + + – – – – – – + + +
– + + + + – + + – + + – + + – + + –
Trapoxin
ATP
Figure 5 Functional interdependence of histone acetylation and nucleosome
remodelling. a, Deacetylation of plasmid chromatin histones is aided by ATPdependent nucleosome remodelling. Chromatinized histones were treated with
NRD complex prepared from HDAC or CHD immunoprecipitates in the presence
precipitates, as measured by phosphorimager quanti®cation
(Fig. 4c). Again, background ATP-stimulated activity was signi®cantly less than that in the NRD and hSWI/SNF complexes. Because
of the similarity in remodelling activities between the NRD complex
and hswI/SNF, we tested the NRD complex for the presence of the
BRG1 catalytic subunit of hSWI/SNF. The trace amount (,0.4 ng)
of BRG1 that associates with protein A±agarose beads, if present in
intact hSWI/SNF complexes, is insuf®cient to generate the disruption we observed (data not shown). We conclude that the NRD
complex possesses a new nucleosome-remodelling factor that is
distinct from that of hSWI/SNF. Our ATP crosslinking data indicate
that this remodelling activity is derived from the CHD3/4 proteins.
The association of both a histone deacetylase activity and a
nucleosome remodelling activity in the NRD complex suggests a
co-dependence model in which one activity prepares the substrate
for the other. Oligonucleosomes in the absence of ATP are ineffective substrates for HDAC immune complexes in vitro, whereas
mononucleosomes are better substrates4. Using a reconstituted
linear-plasmid chromatin substrate, we tested whether ATP-dependent nucleosome remodelling could help the deacetylation of
histones by HDAC. In deacetylation reactions containing the
NRD complex and plasmid chromatin, we observed enhanced
histone deacetylation that was dependent on ATP (Fig. 5a). This
enhanced deacetylation was sensitive to treatment with trapoxin,
indicating that deacetylation was by HDAC and not by any other
factor. The background deacetylation in the absence of ATP could
be explained by deacetylation of more accessible nucleosomes at the
DNA ends. There was no ATP-stimulated deacetylation in similar
reactions using histones, or histones plus DNA (non-nucleosomal)
as substrates, consistent with the idea that ATP hydrolysis supplies
the energy to overcome structural barriers to deacetylation that are
inherent in nucleosomes but not in free histones (Fig. 5b). Incubation of the NRD complex with trapoxin does not prevent this
complex from disrupting the DNase I digestion pattern of mononucleosomes (Fig. 5c), indicating that chromatin remodelling
occurs independently of histone deacetylation.
Genetic and biochemical evidence supports a role for both
histone acetylation/deacetylation and nucleosome remodelling in
altering chromatin structure and gene transcription10,11. We have
shown that a histone deacetylase exists with a nucleosome-remodelling factor in a functionally coupled complex. The HDAC±CHD
interaction is conserved among metazoans in what is probably an
evolutionarily important association5. Heterochromatin co-localizes with hypoacetylated histones, and certain chromo domaincontaining proteins, including HP1 and Polycomb, are components of heterochromatin1. The presence of chromo domains in
CHD proteins raises the possibility that HDACs are targeted to
heterochromatin through their interaction with CHD3/4. That
HDACs function at heterochromatin has previously been suggested by genetic experiments in Drosophila and yeast12. It
remains to be investigated what role associated histone deacetylases and nucleosome remodelling activities have in transcripM
tional regulation and chromatin structure.
.........................................................................................................................
or absence of ATP and the extent of deacetylation was determined by scintillation
Methods
counting. Treatment of the same reactions with trapoxin reduces deacetylation to
Large-scale puri®cation of the HDAC2 complex. Cell extract from 2.4 litres
background, irrespective of the presence of ATP. A background of 37 d.p.m. has
been subtracted. b, Deacetylation of non-chromatinized substrates is not
dependent on nucleosome remodelling. The same histones and DNA that
were used to reconstitute the plasmid chromatin as in (a) were incubated with the
NRD complex as either histones alone or histones plus DNA under conditions in
which nucleosomes do not form. No ATP-stimulated deacetylation occurred.
DNA is weakly inhibitory, possibly because of nonspeci®c interaction with
histones. c, Remodelling of mononucleosome cores does not depend on histone
deacetylase. Cores were treated as in (b) in the presence or absence of 200 nM
trapoxin and digested with DNase I. The remodelling activity of these complexes
was not affected by inhibition of histone deacetylase.
920
of HeLa cells (0:5 3 106 per ml) was prepared4. Proteins were immunoprecipitated using af®nity-puri®ed anti-HDAC2 antibody±protein A beads as
described4 and eluted by addition of HDAC2 immunogenic peptide. Proteins
were re-precipitated in 6% trichloroacetic acid/0.04% deoxycholate and then
separated by SDS±PAGE and stained with Colloidal blue (Novex). Bands
corresponding to p70, p110 and p240 were excised and samples were sequenced
by peptide mass spectrometry.
Immunoprecipitation and western blotting. HeLa and Jurkat cell extracts
were prepared as described4. 1.25, 5 and 7.5 mg cell extract were immunoprecipitated for western blotting, azido-ATP crosslinking, and mononucleosome
disruption assays, respectively. For mononucleosome disruption, immunopre-
Nature © Macmillan Publishers Ltd 1998
NATURE | VOL 395 | 29 OCTOBER 1998 | www.nature.com
8
letters to nature
NATURE | VOL 395 | 29 OCTOBER 1998 | www.nature.com
Acknowledgements. We thank W. Wang for unpublished CHD3 and CHD4 antisera and for sharing
unpublished results; I. Targoff for human Mi-2-positive antiserum and rabbit anti-Mi-2a antiserum;
W. Lane and colleagues at the Harvard Microsequencing Facility for peptide microsequencing; O. Rando,
C. Grozinger, D. Ayer, and I. Wilson for discussion; and the NIH Cell Culture Center for technical
assistance. This work was supported by a National Institute of General Medical Sciences grant to S.L.S.
and an NIH grant to R.E.K. G.R.S. is a Helen Hay Whitney Fellow. Predoctoral fellowships from the NSF
and the Harvard-Markey Biomedical Scientist Program to J.K.T. and an NIH predoctoral training grant to
C.A.H. are gratefully acknowledged. S.L.S. is an Investigator of the Howard Hughes Medical Institute.
8
Correspondence and requests for materials should be addressed to S.L.S. (e-mail: sls@slsiris.harvard.edu).
erratum
FGF-mediated mesoderm
induction involves the
Src-family kinase Laloo
Daniel C. Weinstein, Jennifer Marden, Francesca Carnevali
& Ali Hemmati-Brivanlou
Nature 394, 904±908 (1998)
..................................................................................................................................
In Fig. 4d of this Letter, it was wrongly indicated that activin was
added to samples 5±8. Activin was not added to these samples but to
samples 9±12 as shown here.
+ +
+ +
-
-
-
-
+ + + +
-
-
-
-
-
bFGF
Activin
embryo-RT
-
-
uninjected
embryo
-
-
K259E
laloo/K259E
-
-
laloo
-
laloo/K259E
uninjected
d
uninjected
laloo
K259E
Received 24 August; accepted 28 September 1998.
1. Elgin, S. C. R. Chromatin Structure and Gene Expression (eds Hames, B. D. & Glover, D. M.) (IRL,
Oxford, 1995).
2. Pazin, M. J. & Kadonaga, J. T. SWI2/SNF2 and related proteins: ATP-driven motors that disrupt
protein±DNA interactions? Cell 88, 737±740 (1997).
3. Imhof, A. & Wolffe, A. P. Transcription: gene control by targeted histone acetylation. Curr. Biol. 8,
R422±424 (1998).
4. Hassig, C. A. et al. A role for histone deacetylase activity in HDAC1-mediated transcriptional
repression. Proc. Natl Acad. Sci. USA 95, 3519±3524 (1998).
5. Wade, P. A., Jones, P. L., Vermaak, D. & Wolffe, A. P. A multiple subunit Mi-2 histone deacetylase from
Xenopus laevis cofractionates with an associated Snf2 superfamily ATPase. Curr. Biol. 8, 843±846
(1998).
6. Tavladoraki, P. et al. Maize polyamine oxidaseÐprimary structure from protein and cDNA
sequencing. FEBS Lett. 426, 62±66 (1998).
7. Seelig, H. P. et al. The major dermatomyositis-speci®c Mi-2 autoantigen is a presumed helicase
involved in transcriptional activation. Arthr. Rheum. 38, 1389±1399 (1995).
8. Ge, Q., Nilasena, D. S., O'Brien, C. A., Frank, M. B. & Targoff, I. N. Molecular analysis of a major
antigenic region of the 240-kD protein of Mi-2 autoantigen. J. Clin. Invest. 96, 1730±1737 (1995).
9. Woodage, T., Basrai, M. A., Baxevanis, A. D., Hieter, P. & Collins, F. S. Characterization of the CHD
family of proteins. Proc. Natl Acad. Sci. USA 94, 11472±11477 (1997).
10. Hartzog, G. A. & Winston, F. Nucleosomes and transcription: recent lessons from genetics. Curr. Opin.
Genet. Dev. 7, 192±198 (1997).
11. Kadonaga, J. T. Eukaryotic transcription: an interlaced network of transcription factors and
chromatin-modifying machines. Cell 92, 307±313 (1998).
12. De Rubertis, F. et al. The histone deacetylase RPD3 counteracts genomic silencing in Drosophila and
yeast. Nature 384, 589±591 (1996).
13. Schnitzler, G., Sif, S. & Kingston, R. E. Human SWI/SNF interconverts a nucleosome between its base
state and a stable remodeled state. Cell 94, 17±27 (1998).
14. Cote, J., Utley, R. T. & Workman, J. L. Basic analysis of transcription factor binding to nucleosomes.
Meth. Mol. Genet. 6, 108±128 (1995).
laloo
K259E
laloo/K259E
cipitated beads were resuspended in 23 ml lysis buffer and aliquoted as
indicated.
Histone deacetylase assays. Assays were done as described4; where indicated, ATP was added to 2.5 mM in the presence of 5 mM MgCl2, and trapoxin
was added to 200 nM.
8-azido-adenosine-g-32P-ATP crosslinking. NRD antibody-immobilized
bead complexes were incubated with 2 mCi 8-azido-adenosine-59-g-32Ptriphosphate (ICN) for 3 min at room temperature in 50 ml buffer containing
20 mM HEPES, pH 7.6, 100 mM KCl, 5 mM MgCl2, 1 mm ZnSO4, 0.1% Tween20. The reaction mixture was irradiated for 5 min with 254-nm UV light
(1,120 mW cm-2 from 3 cm away). Beads were washed three times with 1 ml
20 mM HEPES, pH 7.6, 100 mM KCl, 0.1% Tween-20. Bound proteins were
resolved by SDS±PAGE and imaged using a phosphorimager.
Nucleosome-disruption assays. the 155-bp pTPT MluI/EcoRI fragment was
prepared and footprinted as described13. 0.2, 1.0 and 5.0 ml HDAC/CHD
immunoprecipitates, 1.0 and 5.0 ml peptide-blocked HDAC/CHD immunoprecipitates, and 0.04, 0.16, 0.6 and 2.4 ml of hSWI/SNF were tested for
disruptive activity. 2 mM ATP/MgCl2 was added where indicated; after
45 min, reactions were treated with 0.12 U DNase I (0.012 U for bare DNA).
Plasmid chromatin reconstitution. HeLa cells were radiolabelled as described
previously4 and hyperacetylated histones were puri®ed by hydroxyapatite
chromatography as described14. 16 mg of a 9-kb plasmid was linearized,
puri®ed, and mixed with an equal mass of radiolabelled, hyperacetylated
core histones in 50 ml of 15 mM HEPES, pH 7.5, 1 M NaCl, 0.2 mM EDTA
and 0.2 mM PMSF. Stepwise dilution of salt was done by addition of the same
buffer without NaCl over 3 h at 10-min intervals until the ®nal concentration of
NaCl was 100 mM. Non-nucleosomal histones were then separated by repeat
Centricon-500 (Amicon) ®ltration. Nucleosomal structure was con®rmed by
micrococcal nuclease digestion of the product.
Xbra
Chordin
Xwnt8
EF1-α
1 2 3 4
Nature © Macmillan Publishers Ltd 1998
5
6 7 8 9 10 11 12 13 14
921