MOLECULAR AND CELLULAR BIOLOGY, Feb. 2006, p. 1156–1164
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 26, No. 3
Mechanism of Polymerase II Transcription Repression by the Histone
Ce ´cile-Marie Doyen,1,2,6‡ Woojin An,3†‡ Dimitar Angelov,2,6Vladimir Bondarenko,4
Flore Mietton,1Vassily M. Studitsky,4Ali Hamiche,5Robert G. Roeder,3
Philippe Bouvet,2,6* and Stefan Dimitrov1,2*
Institut Albert Bonniot, INSERM U309, 38706 La Tronche cedex, France1; Ecole Normale Supe ´rieure de Lyon, Laboratoire Joliot
Curie, 46 Alle ´e d’Italie, 69007 Lyon, France2; Laboratory of Biochemistry and Molecular Biology, The Rockefeller University,
1230 York Avenue, New York, New York 100213; Department of Pharmacology, University of Medicine and Dentistry of
New Jersey, 675 Hoes Lane, Piscataway, New Jersey 088544; Institut Andre Lwoff, CNRS UPR 9079,
7 rue Guy Moquet, 94800 Villejuif, France5; and Ecole Normale Supe ´rieure de Lyon, LBMC,
CNRS-UMR 5161, 46 Alle ´e d’Italie, 69007 Lyon, France6
Received 29 July 2005/Returned for modification 24 August 2005/Accepted 3 November 2005
macroH2A (mH2A) is an unusual histone variant consisting of a histone H2A-like domain fused to a large
nonhistone region. In this work, we show that histone mH2A represses p300- and Gal4-VP16-dependent
polymerase II transcription, and we have dissected the mechanism by which this repression is realized. The
repressive effect of mH2A is observed at the level of initiation but not at elongation of transcription, and mH2A
interferes with p300-dependent histone acetylation. The nonhistone region of mH2A is responsible for both the
repression of initiation of transcription and the inhibition of histone acetylation. In addition, the presence of
this domain of mH2A within the nucleosome is able to block nucleosome remodeling and sliding of the histone
octamer to neighboring DNA segments by the remodelers SWI/SNF and ACF. These data unambiguously
identify mH2A as a strong transcriptional repressor and show that the repressive effect of mH2A is realized on
at least two different transcription activation chromatin-dependent pathways: histone acetylation and nucleo-
DNA is organized into chromatin in the cell nucleus. Chro-
matin exhibits a repeating structure, and its basic unit, the
nucleosome, is composed of an octamer of the four core his-
tones (two each of H2A, H2B, H3, and H4), around which two
superhelical turns of DNA are wrapped. The structure of the
histone octamer (6) and the nucleosome (25) was solved by
X-ray crystallography. In addition to the conventional core
histones, the cells express a very small amount of their nonal-
lelic isoforms, the so-called histone variants. The small amount
of the histone variants present in the cell suggests that these
proteins may play regulatory roles. Indeed, the incorporation
of the histone variants into the histone octamer brings new
structural properties to the nucleosome, which in turn might be
essential for the regulation of several vital processes of the cell.
For example, the histone variant H2A.Z is implicated in both
gene activation (32) and gene silencing (15). Recently, a role of
H2A.Z in chromosome segregation was also suggested (31).
Another histone variant, H2AX, is essential for repair and the
maintenance of genomic stability (7, 8). Incorporation of the
histone variant H2ABbd into the histone octamer confers
lower stability of the H2ABbd nucleosomes (16). Since the
residues of conventional H2A, which are targets for posttrans-
lational modifications, are mutated in H2ABbd, one could
expect the function of this histone to be regulated in a distinct
way (10, 5).
macroH2A (mH2A) is an unusual histone variant with a size
approximately threefold the size of the conventional H2A (29).
The N-terminal domain of mH2A (H2A-like), which shows a
high degree of homology with the conventional H2A, is fused
to a large nonhistone region (NHR) known as the macro do-
main (1, 24, 29). The immunofluorescence studies indicate that
mH2A is preferentially located on the inactive X chromosome
(9, 12, 13, 27). The mH2A nucleosomes exhibit structural al-
terations in the vicinity of the dyad axis, abrogating the binding
of transcription factors to their recognition sequences when
the sequences are inserted close to the dyad (4). In addition,
the presence of mH2A interferes with SWI/SNF nucleosome
remodeling and movement to neighboring DNA segments (4).
All these data suggest that mH2A could be involved in tran-
scriptional repression, but the mechanism by which mH2A
operates is unknown. Indirect data indicated that the NHR of
mH2A could be responsible for the repression of transcription
(30). It was also recently suggested that macro domains could
possess enzymatic activities [poly(ADP-ribose) formation] and
could bind monomeric ADP-ribose and polymers of poly-
(ADP-ribose) (1, 20). Furthermore, it was recently demon-
strated that the macro domain of macroH2A1.1 but not
macroH2A1.2 was able to bind the SirT1 metabolite O-acetyl-
ADP-ribose (23); however, the consequences of this property
* Corresponding author. Mailing address for Sefan Dimitrov: Insti-
tut Albert Bonniot, INSERM U309, 38706 La Tronche cedex, France.
Phone: (33) 4 76 54 94 73. Fax: (33) 4 76 54 95 95. E-mail: stefan
.firstname.lastname@example.org. Mailing address for Philippe Bouvet: Ecole
Normale Supe ´rieure de Lyon, Laboratoire Joliot Curie, 46 Alle ´e
d’Italie, 69007 Lyon, France. Phone: (33) 4 72 72 80 16. Fax: (33) 4 72
72 80 16. E-mail: email@example.com.
† Present address: Department of Biochemistry and Molecular Bi-
ology, USC/Norris Comprehensive Cancer Center, 1501 San Pablo
Street, ZNI 241, MC 2821, Los Angeles, CA 90089-2821.
‡ These authors contributed equally to this work.
on the function of macroH2A and on chromatin structure are
This work summarizes our studies on the effect of mH2A1.2
on transcription. We show that the presence of mH2A inhibits
the Gal4-VP16- and p300-dependent histone acetylation and
transcription from mH2A nucleosomal arrays. Importantly,
this effect was determined only by the NHR of mH2A, since
arrays reconstituted with conventional H2A fused to the
mH2A NHR (H2A-NHR arrays), but not the H2A-like arrays,
exhibited the same behavior. In addition, the chromatin re-
modeling machines SWI/SNF and ACF were unable to both
remodel and mobilize nucleosomes reconstituted with the
H2A-NHR fusion protein. These data suggest that the prop-
erty of mH2A to affect transcriptional regulation resides
mainly in its nonhistone region.
MATERIALS AND METHODS
Preparation of DNA probes and reconstitution of nucleosomes. The 152-bp
EcoRI-RsaI DNA fragments containing the Xenopus borealis 5S RNA gene were
derived from plasmid pXP-10 (17) by PCR amplification. DNA was 3? radiola-
beled at the EcoRI side by [?-32P]ATP and Klenow enzyme. The 255-bp and
241-bp DNA probes, containing the strongly positioning sequence 601 (33) at the
middle or at 8 bp from the 3? end, respectively, were prepared by PCR ampli-
fication of plasmids pGEM3Z-601 and p199-1 (a kind gift from J. Widom and B.
Bartholomew) using[?-32P]ATP-labeled 5? primer. The 154-bp fragment con-
taining the five Gal4-VP16 binding sites was derived from plasmid pG5ML by
PCR amplification using the following primers: 5?-CGA ATC TTT AAA CTC
GAG TGC ATG CCT GCA and 5?-AAA GGG CCA AAT CGA TAG CGA
GTA TAT ATA GGA CTG GGG ATC. All DNA probes were purified on 6%
native polyacrylamide gel electrophoresis.
Nucleosome reconstitutions were performed by salt gradient dialysis as de-
scribed previously (17). Briefly, ?100 ng of radiolabeled DNA (5 ? 105cpm),
was mixed with 2 ?g of nonlabeled ?180-bp average sequence chicken erythro-
cytes DNA in 100-?l volume, together with 0.8% (wt/wt) of preassembled his-
tone octamers in high-salt buffer: 10 mM Tris (pH 7.4), 1 mM EDTA, 5 mM
?-mercaptoethanol, and 2 M NaCl. Then, the solution was stepwise dialysed at
4°C for 2 h/step against decreasing concentrations (1.2 M, 1.0 M, 0.8 M, 0.6 M,
and 0.1 M) of NaCl in the same buffer, followed by dialysis overnight against 10
mM Tris (pH 7.4), 0.25 mM EDTA, and 10 mM NaCl. Nucleosome formation
was assessed by a 5% polyacrylamide electrophoretic mobility shift assay
(EMSA) run in 0.3? Tris-borate-EDTA buffer.
Protein expression and purification. The recombinant Xenopus laevis full-
length histone protein was produced and purified as previously described (26).
For the production of the recombinant H2A-NHR protein, the coding sequences
for the H2A protein (from M1 to P118) and for the NHR domain of human
macroH2A1.2 (from R118 to N371) were individually amplified by PCR and
fused in the pET30a vector to form the coding sequence of the chimera H2A-
After expression in Escherichia coli, the recombinant chimera NHR-H2A was
purified to homogeneity as described for the conventional histones.
Nucleosome mobilization experiments. Nucleosomes (final concentration, 30
to 50 nM) were mixed with SWI/SNF or ACF, as indicated, in buffer containing
10 mM Tris (pH 7.4), 1 mM dithiothreitol, 100 ?g/ml bovine serum albumin, 5%
glycerol, 0.02% (vol/vol) Nonidet P-40, 2.5 mM MgCl2, and 1 mM ATP. After
incubation for 45 min, or the time indicated, the reaction was stopped by 0.05
units of apyrase, 10 mM EDTA (final concentration), and 1 ?g of plasmid DNA.
Nucleosome sliding was analyzed by a 5% polyacrylamide-bisacrylamide (29:1)
EMSA. Nucleosome borders were mapped by limit digestion with exonuclease
III (3 to 5 U/ml) for 15 min, and DNA fragments were analyzed by denaturing
acrylamide-urea gel electrophoresis. The remodeling of the 152-bp 5S nucleo-
somal particles was assessed by DNase I footprinting. Briefly, 0.5 units of DNase
I (Invitrogen) were added to the arrested reaction mixture for 2 min. DNA
digestion was stopped by the addition of 20 mM EDTA (final concentration), 1
?g of proteinase K, and 0.1% sodium dodecyl sulfate (SDS) (final concentra-
tion). DNA partial digests were recovered by phenol extraction and ethanol
precipitation and analyzed by 8% polyacrylamide-8 M urea sequencing gel elec-
trophoresis in 1? Tris-borate-EDTA buffer at a constant power of 65 W.
Transcription experiments. Chromatin arrays containing either conventional
H2A or H2A-like, H2A-NHR histones were assembled using the pG5ML array
template and recombinant Acf1, ISWI, and NAP-1 as previously described (2,
18). The p300 and Gal4-VP16 transcriptions and the histone acetyltransferase
(HAT) assays were performed according to previously described protocols (2).
The chromatin arrays reconstituted with the conventional H2A or the variant
histones were soluble under the conditions used for transcription (data not
shown). For the analysis of the transcription elongation through nucleosomes,
polymerase II (Pol II) elongation complex assembly was ligated to either the
DNA or the conventional or variant nucleosomes, and transcriptions were car-
ried out as described previously (21).
mH2A represses Gal4-VP16- and p300-dependent histone
acetylation and transcription. To analyze the effect of
mH2A1.2 on transcription, we assembled mH2A nucleosomal
arrays according to previously described protocols (18) using
recombinant assembly factors and histone proteins (Fig. 1 and
see Fig. 3). The assembly was carried out on a pG5ML array
plasmid, which contains a promoter sequence flanked by five
208-bp repeats of the 5S RNA sea urchin gene. The reconsti-
tuted chromatins containing conventional H2A or histone vari-
ant mH2A were analyzed by both supercoiling assay (Fig. 1A)
and digestion with micrococcal nuclease (MNase) (Fig. 1B).
The supercoiling assay shows a very efficient assembly of the
chromatin samples (Fig. 1A, lanes 3 and 4). The clear 200-bp
repeats observed upon digestion of both samples with micro-
coccal nuclease indicate a proper structural organization of the
nucleosomal arrays (Fig. 1B, lanes 2 to 4). Then, the conven-
tional and mH2A nucleosomal arrays were used for transcrip-
tion. The two templates, however, exhibited completely differ-
ent behaviors in the transcription assay (Fig. 1C). In both
cases, the presence of histones abolished the basal transcrip-
tion (results not shown), and the addition of Gal4-VP16 re-
sulted in a detectable transcription (Fig. 1C, lanes 1 and 3).
The presence of p300 in the reaction mixture leads to a dra-
matic increase of the transcription from the conventional nu-
cleosomal arrays but to a very small increase of the transcrip-
tion from the mH2A arrays (Fig. 1C, compare lane 1 with lane
2 and lane 3 with lane 4). In the different experiments, the
increase of the transcription from conventional arrays was
found to be 10 to 12 times higher than that from mH2A arrays.
These results clearly show that mH2A is a very efficient repres-
sor of transcription. How does mH2A affect transcription?
Since the efficiency of transcription from the chromatin tem-
plates was strongly dependent on the HAT p300 and thus on
histone acetylation (Fig. 1) (2, 3), one obvious reason for the
transcription repression could be the inability of p300 to acet-
ylate histones within the mH2A nucleosomal arrays. This was
tested by HAT assays, and we indeed found that the acetyla-
tion of histones by p300 was considerably reduced within the
mH2A reconstituted chromatin (Fig. 1D, compare lane 2 with
lane 4), thus confirming our hypothesis.
It is well documented that p300 is recruited to the promoter
through Gal4-VP16 (2, 3, 22). In addition, the presence of
mH2A interferes with the binding of the transcription factor
NF-?B when its recognition sequence is inserted in the vicinity
of the nucleosome dyad axis (4). This suggests that part of the
inhibition of both transcription and histone acetylation could
be associated with some possible impairment of Gal4-VP16
binding to its recognition sequence within the mH2A nucleo-
somes. To check this, we have reconstituted conventional and
VOL. 26, 2006 TRANSCRIPTION OF macroH2A NUCLEOSOMAL ARRAYS1157
mH2A nucleosomes using a 154-bp DNA fragment derived
from the pG5ML vector (used for reconstitution of the 5S
nucleosome array transcription template) and containing the
five Gal4-VP16 binding sites. Then, the binding of Gal4-VP16
to both nucleosomal templates and naked DNA was studied by
EMSA (Fig. 2 and unpublished data). In agreement with the
reports in the literature (14), we found that compared to naked
DNA, a much larger amount of Gal4-VP16 was necessary for
its binding to the nucleosomes (results not shown). The effi-
ciencies of the binding of Gal4-VP16 to both conventional and
mH2A nucleosomes were, however, not significantly different
(Fig. 2B to D). Indeed, the quantification of the EMSA results
demonstrated only a relatively small preference of Gal4-VP16
binding to conventional nucleosomes compared to mH2A nu-
cleosomes (Fig. 2D). This could reflect the interference of the
binding of Gal4-VP16 with two of the five binding sites. This
suggests that the strong inhibition of both transcription and
histone acetylation of mH2A nucleosomal arrays could not be
explained by a lack of binding of Gal4-VP16 to the arrays. In
agreement with this, we found that p300 was able to acetylate
only very poorly the histones of the GAL4-VP16-bound mH2A
nucleosomes (Fig. 2E).
The NHR of mH2A is responsible for the repression of
transcription and the inhibition of histone acetylation. A ma-
jor feature of mH2A is the presence of a long C-terminal
extremity (NHR domain) fused to the histone-like domain
(H2A-like) which is highly homologous to H2A (Fig. 3A). To
determine whether the inhibition of transcription and of p300-
dependent histone acetylation observed with mH2A chromatin
templates could be attributed to one specific domain of mH2A,
recombinant histone proteins corresponding to H2A-like,
H2A-NHR (fusion of the NHR domain of mH2A to the con-
ventional H2A) proteins were purified (Fig. 3B) and used for
the reconstitution of chromatin on the pG5ML plasmid. Su-
percoiling (Fig. 3C) and micrococcal nuclease (Fig. 3D) assays
showed an efficient reconstitution and proper structural orga-
nization of the reconstituted H2A-like and H2A-NHR chro-
matin templates. These chromatin templates were then used in
a transcription assay (Fig. 3E). p300-mediated and Gal4-VP16-
dependent transcription from the H2A-like chromatin tem-
FIG. 1. macroH2A interferes with both p300- and Gal4-VP16-dependent transcription and histone acetylation. Chromatin was assembled on
the pG5ML array DNA by using recombinant Drosophila Acf1, ISWI, and the nucleosome assembly protein 1, an equimolar mixture of either
conventional H2A or variant histone macroH2A, and the three remaining core histones H2B, H3, and H4. (A) DNA supercoiling assay for the
assembly of chromatin. The DNA samples were run on 1% agarose gels and stained with ethidium bromide. Lane 1, supercoiled pG5ML DNA
(S); lane 2, topoisomerase I-relaxed pG5ML DNA (R); lanes 3 and 4 show the plasmid DNA isolated from the chromatin samples assembled with
macroH2A or conventional H2A, respectively. “nc” designates nick DNA. (B) Micrococcal nuclease digestion of the assembled macroH2A (lane
2) and conventional H2A (lanes 3 and 4) chromatin samples. The chromatin samples were digested with either 0.2 mU MNase (lanes 2 and 3) or
0.5 mU MNase (lane 4) for 10 min at 22°C, and the DNA was analyzed on 1.2% agarose gels. Lane 1, a 123-bp DNA ladder marker (M). (C) p300-
and Gal4-VP16-dependent transcription of conventional H2A (lanes 1 and 2) and histone variant mH2A (lanes 3 and 4) nucleosomal arrays. The
arrays were incubated with GAL4-VP16 alone or with both Gal4-VP16 and p300. The results from two independent experiments are shown.
(D) HAT assays with nucleosomal arrays assembled with either conventional H2A (lanes 1 and 2) or macroH2A (lanes 3 and 4) nucleosomal
arrays. All reaction mixtures contained p300 while Gal4-VP16 was present in mixtures for reactions 2 and 4 only. The positions of the histones are
1158 DOYEN ET AL.MOL. CELL. BIOL.
plates (Fig. 3E, lanes 1 to 2) showed a transcription level
similar to the transcription level from conventional nucleoso-
mal arrays. In contrast, the transcription level from the H2A-
NHR chromatin templates was repressed about 10-fold (Fig.
3E, lanes 3 to 4) compared to the transcription level from
wild-type or H2A-like chromatin templates. This level of re-
pression was similar to what was obtained with the mH2A
chromatin template (Fig. 1C), suggesting that the repression of
the p300-mediated and Gal4-VP16-dependent transcription
observed in the presence of mH2A could be attributed to the
NHR domain of mH2A. Since the repression of transcription
in the presence of mH2A is associated with a reduced level of
p300-dependent acetylation of histones of the chromatin tem-
plates, we next tested whether this effect is associated with the
presence of NHR (Fig. 3F). Indeed, the level of p300-mediated
histone acetylation within the H2A-like chromatin template
was very similar to the level of acetylation of conventional
nucleosomes (Fig. 3F, lanes 1 to 2), whereas the acetylation
level of histones within the H2A-NHR chromatin template
(Fig. 3F, lanes 3 to 4) was very low, suggesting that the repres-
sion of p300 HAT activity within the mH2A chromatin tem-
plate is the consequence of the presence of the NHR domain
The repression of transcription observed in the presence of
mH2A or of the fusion H2A-NHR could be the consequence
of a lower initiation level or because of an inhibition of the
elongation of Pol II transcription through the nucleosome. To
differentiate between these two possibilities, we carried out
transcription elongation experiments by using conventional
H2A-like, H2A-NHR, and mH2A nucleosomes (Fig. 4).
Briefly, the four types of nucleosomes were reconstituted and
ligated to Pol II elongation complexes immobilized on beads as
described previously (21). The transcription elongation reac-
tion was carried out in the presence of 40 mM, 300 mM, or 1
M KCl, and the nascent RNA was pulse-labeled (21). At 40
mM KCl, the nucleosomal templates efficiently blocked the
elongation reaction (Fig. 4, lanes 2, 6, 10, and 14). The 10 to
25% of transcripts observed at this KCl concentration roughly
reflect the presence of free DNA in the different template
solutions (results not shown) (21). The nucleosome-specific
pausing patterns were similar for the four different templates
(Fig. 4, lanes 2, 6, 10, and 14). An increase of the ionic strength
to 300 mM KCl destabilizes the nucleosomes, and a further
increase of the KCl concentration to 1 M results also in a
partial removal of H2A-H2B and mH2A-H2B dimers. This, in
turn, results in much more efficient transcript elongation on
FIG. 2. The presence of mH2A does not affect the binding of
Gal4-VP16 to the nucleosome. (A) Schematic of the nucleosomes used
in Gal4-VP16 binding studies. A 152-bp DNA fragment, derived from
the pG5ML vector and containing the five Gal4-VP16 sites inserted in
the E4 promoter, was PCR amplified and used to reconstitute both
conventional and mH2A nucleosomes. The positions of the five Gal4-
VP16 binding sites and the nucleosome dyad are designated. nt, nu-
cleotide. (B) Binding of Gal4-VP16 to conventional H2A nucleo-
somes. Increasing amounts of Gal4-VP16 were added to the solution
containing conventional nucleosomes, and Gal4-VP16 binding was as-
sessed by EMSA. The positions of free DNA, nucleosomes (nuc), and
Gal4-VP16 nucleosome complexes (cplx.) are designated on the left
part of the figures. (C) Data are presented as described for panel B but
for macroH2A nucleosomes. (D) Quantification of the data presented
in panels B and C. (E) HAT assays with either conventional H2A (lane
2) or macroH2A (lane 4) mononucleosomes. Acetylation of the his-
tone mixtures consisting of conventional histones or containing mH2A
is shown in lanes 1 and 3, respectively. All reaction mixtures contained
p300 and Gal4-VP16. The positions of the histones are indicated.
VOL. 26, 2006 TRANSCRIPTION OF macroH2A NUCLEOSOMAL ARRAYS1159
FIG. 3. The NHR of the histone variant macroH2A is involved in both the repression of p300- and Gal4-VP16-dependent transcription and
the inhibition of histone acetylation. Chromatin was assembled on the pG5ML array DNA as described in the legend for Fig. 1 but with the
histone-like domain of mH2A (H2A-like) or with the fusion (H2A-NHR) of conventional H2A with the NHR of mH2A. (A) Schematic of the
proteins used in the chromatin assembly experiments. (B) 18% SDS electrophoresis of the recombinant conventional core histones, the fusion
H2A-NHR, and the H2A-like nucleosomal template. (C) Supercoiling assay for DNA isolated from chromatin assembled with the H2A-like (lane
3) or H2A-NHR (lane 4) nucleosomal template. (D) Micrococcal nuclease analysis of chromatin assembled with either the H2A-like (lanes 1 and
2) or H2A-NHR (lanes 3 and 4) nucleosomal template. The digestion was performed with 0.1 mU MNase (lanes 1 and 3) or with 0.5 mU MNase
(lanes 2 and 4) for 10 min at 22°C. DNA was then extracted from the digested samples and analyzed on 1.2% agarose gels. (E) p300- and
Gal4-VP16-dependent transcription of H2A-like (lanes 1 and 2) and H2A-NHR (lanes 3 and 4) nucleosomal templates. The results from two
independent experiments are shown. (F) HAT assays of H2A-like (lanes 1 and 2) and H2A-NHR (lanes 3 and 4) nucleosomal arrays. Note the
complete inhibition of histone acetylation in the H2A-NHR templates.
the nucleosomal templates (Fig. 4, lanes 3, 4, 7, 8, 11, 12, 15,
and 16). The elongation efficiencies of polymerase II were,
however, very similar for the four different types of chromatin
templates (Fig. 4, lanes 3, 4, 7, 8, 11, 12, 15, 16). This demon-
strates that the observed inhibition of transcription from
mH2A and H2A-NHR templates is associated with the initia-
tion but not with the elongation of transcription. Therefore,
the NHR domain of mH2A is responsible for the inhibition of
the initiation of the Pol II transcription reaction.
The NHR of mH2A induces structural alterations within the
nucleosome. To further examine how the NHR of mH2A could
affect transcription initiation, we first determined the conse-
quences of the presence of the NHR on the structure of the
nucleosomes. Conventional H2A and H2A-NHR fusion pro-
teins were produced, purified, and used for the reconstitution
of nucleosomes on a radioactively end-labeled 152-bp 5S DNA
gene (Fig. 5A and B). In order to study the structural conse-
quences of the incorporation of the NHR into the reconsti-
tuted particles, we performed DNase I footprinting (Fig. 5C).
The DNase I cleavage patterns of the control and H2A-NHR
nucleosomes show the 10-bp repeat characteristic of the nu-
cleosome particle. Some pronounced alterations were, how-
ever, detected in the DNase I cleavage of the H2A-NHR
essentially around the dyad axis. The DNase I cleavage pattern
of particles reconstituted with the H2A-like domain of mH2A
was essentially the same as that for conventional H2A (for
detail, see Fig. 7 of reference 4). We attribute the observed
alterations in the DNase I cleavage pattern of the H2A-NHR
particle to the presence of the NHR domain. These alterations
would reflect some changes in the DNA structure in proximity
to the nucleosome dyad and/or some inaccessibility of the
FIG. 4. The NHR of mH2A does not affect polymerase II elonga-
tion through nucleosomal templates. A 245-bp DNA fragment was
used to reconstitute nucleosomes with either conventional H2A or the
fusion H2A-NHR. The templates, a mixed population of two posi-
tioned nucleosomes, N1 and N2, were then ligated to the elongation
Pol II complex immobilized on beads as described previously (21). The
Pol II elongation complex was allowed to transcribe the nucleosomal
DNA, and the nascent RNA was pulse labeled. The transcription was
performed in the presence of either 40 mM, 300 mM, or 1 M KCl, and
the labeled RNA was extracted and analyzed. The RNA isolated from
the transcription reactions of conventional H2A (lanes 1 to 4), mH2A
(lanes 5 to 8), H2A-like (lanes 9 to 12), and H2A-NHR (lanes 13 to 16)
nucleosomal templates was analyzed on an 8% denaturing polyacryl-
amide gel. The transcriptions from preformed stalled elongation com-
plexes are also shown (lanes 1, 5, 9, and 13). M, a marker for the
molecular mass of the transcripts.
FIG. 5. The presence of NHR of mH2A results in alterations in the
structure of the H2A-NHR nucleosomes. (A) EMSA of the reconsti-
tuted H2A and H2A-NHR nucleosomes (Nuc). (B) 18% SDS-poly-
acrylamide gel electrophoresis of the histones isolated from H2A (lane
1) and H2A-NHR (lane 2) nucleosomes. The positions of the histones
are shown on the left part of the figure. Note that the H2A and H2B
Xenopus laevis histones comigrate under the electrophoresis condi-
tions. (C) DNase I footprinting of conventional H2A and H2A-NHR
nucleosomes reconstituted on a 152-bp fragment comprising the 5S
DNA Xenopus borealis gene. The digestion products were analyzed on
an 8% denaturing polyacrylamide gel. The bottom strand of the nu-
cleosomal DNA was P32labeled. The diamond designates the dyad axis
of the nucleosome. Stars indicate the alterations of the H2A-NHR
nucleosome DNase I digestion pattern.
VOL. 26, 2006TRANSCRIPTION OF macroH2A NUCLEOSOMAL ARRAYS1161
enzyme to the nucleosomal DNA. Similar perturbations in the
DNase I footprinting were reported for mH2A (4) and
H2ABbd (5) nucleosomes, and these perturbations were asso-
ciated with the inability of the remodeling factors to remodel
these variant particles, suggesting that the presence of H2A-
NHR within the nucleosome could affect its remodeling.
The NHR domain of mH2A inhibits the remodeling of nu-
cleosomes by SWI/SNF and ACF. In a recent study, we have
demonstrated that the H2A-like domain was able to interfere
with SWI/SNF-induced nucleosome mobilization (4). To test
whether the NHR domain of mH2A is also able to affect
nucleosome remodeling, nucleosomes containing the conven-
tional H2A or the H2A-NHR histone were reconstituted on a
255-bp 5?-end-labeled 601 sequence, which, according to the
reported data, should give rise mostly to centrally positioned
nucleosomes if conventional histones are used for reconstitu-
tion (19). Since both particles show different migration prop-
erties, it was important to map precisely the position of the
variant H2A-NHR nucleosomal particle on this DNA frag-
ment. Exonuclease III digestion of conventional (Fig. 6C, lane
3) and H2A-NHR (Fig. 6C, lane 10) nucleosomes clearly in-
dicates that both nucleosomal particles are on the same start-
ing central position on the 601 DNA fragment. The incubation
of these centrally positioned nucleosomes with increasing
amounts of SWI/SNF in the presence of ATP results in a very
clear SWI/SNF-dependent sliding of conventional nucleo-
somes (Fig. 6A, lanes 2 to 5), whereas no mobilization of the
H2A-NHR nucleosomes could be observed (Fig. 6, lanes 7 to
10). The kinetics of SWI/SNF-induced sliding of conventional
H2A and H2A-NHR nucleosomes (Fig. 6B) confirm this re-
FIG. 6. The NHR of mH2A interferes with SWI/SNF and ACF nucleosome mobilization. Conventional H2A and NHR-H2A nucleosomes
were reconstituted by using a 255-bp fragment containing the centrally positioned sequence 601 (19). (A) SWI/SNF mobilization of H2A and
NHR-H2A nucleosomes. Both types of nucleosomes were incubated for 45 min at 30°C in the presence of increasing amounts of SWI/SNF and
ATP. Mobilization of the histone octamer was revealed by EMSA on 5.5% native polyacrylamide gels. The center- and end-positioned nucleo-
somes and free DNA are indicated on the left part of the figure. (B) Time course of the SWI/SNF-induced mobilization of conventional H2A and
H2A-NHR nucleosomes. The nucleosome solutions were supplemented with ATP and 0.5 ?l of SWI/SNF and incubated for the indicated time.
The nucleosome mobilization was arrested by apyrase treatment, and the reaction mixtures were stored on ice until they were loaded on the gel.
The central- and end-positioned nucleosomes and free DNA are indicated. (C) Mapping of H2A and H2A-NHR nucleosome positions after
treatment with SWI/SNF. Both types of particles were incubated for 45 min at 30°C in the presence of increasing amounts of SWI/SNF as indicated.
Then, the mobilization reaction was arrested by apyrase treatment, the samples were digested with exonuclease III, and the digestion products were
run on an 8% denaturating gel. Stars indicate the radioactively labeled end of the DNA used for the reconstitution. (D) ACF is unable to mobilize
H2A-NHR nucleosomes. Conventional H2A and H2A-NHR nucleosomes were reconstituted by using a 241-bp fragment containing the end-
positioned sequence 601. Reconstituted conventional H2A and H2A-NHR nucleosomes were incubated for 45 min at 30°C with increasing
amounts of ACF in the presence of ATP and run on a 5.5% native acrylamide gel. The center- and end-positioned nucleosomes and free DNA
are indicated. nt, nucleotide; M, a marker for the molecular mass of the transcripts.
1162 DOYEN ET AL.MOL. CELL. BIOL.
sult. No mobilization of the H2A-NHR nucleosomes could be
observed after a 54-min incubation period in the presence of
0.5 ?l of SWI/SNF (Fig. 6B; H2A-NHR), whereas most of the
conventional H2A nucleosomes moved to the DNA ends (Fig.
6B; H2A). Exonuclease III digestion of nucleosomal templates
after incubation with different amounts of SWI/SNF (Fig. 6C)
confirms that the H2A-NHR nucleosomes are not mobilized
by SWI/SNF (lanes 11 to 15), whereas the conventional H2A
nucleosomes move to the DNA ends as expected (lanes 4 to 8).
To further characterize the mobility properties of H2A-NHR
nucleosomes, we used the ACF remodeling factor, which pro-
motes histone octamer sliding from the end to the center of
DNA. End-positioned conventional H2A and H2A-NHR nu-
cleosomes reconstituted on the end-positioned sequence 601
241-bp DNA fragment (19) were incubated with increasing
amounts of ACF (Fig. 6D). Conventional nucleosomes were
efficiently mobilized (Fig. 6C, lanes 1 to 7), whereas no sliding
of the H2A-NHR nucleosomes could be detected (lanes 8 to
We then tested whether H2A-NHR nucleosomes could be
remodeled by SWI/SNF. Conventional H2A and H2A-NHR
nucleosomes were formed using a radioactively end-labeled
152-bp DNA fragment containing the Xenopus borealis 5S
RNA gene. The samples were incubated with increasing
amounts of SWI/SNF and digested with DNase I (Fig. 7). The
perturbation of the 10-bp cleavage pattern of the conventional
H2A nucleosomes in the presence of SWI/SNF shows that
these nucleosomes are efficiently remodeled (Fig. 7, lanes 1 to
6). In contrast, no perturbation of the cleavage pattern could
be detected in the presence of SWI/SNF for the H2A-NHR
nucleosome (Fig. 7, lanes 1 to 7), demonstrating that this
particle cannot be remodeled by this complex. Therefore, the
NHR domain of macroH2A interferes with nucleosome re-
modeling by SWI/SNF.
The data reported in this work demonstrate that in vitro
mH2A is an efficient repressor of p300- and Gal4-VP16-de-
pendent Pol II-activated transcription. We found that this
property of mH2A resides mainly in its NHR domain. Indeed,
our experiments show that mH2A and the fusion H2A-NHR
were able to impede both Gal4-VP16-dependent Pol II-acti-
vated transcription and histone acetylation as well as nucleo-
some remodeling by SWI/SNF and ACF. Since the presence of
mH2A was found to affect weakly the efficiency of Gal4-VP16
binding to the mH2A nucleosomes, the contribution of this
effect to the repression of transcription is expected to be small.
Bearing in mind that Gal4-VP16 is responsible for the recruit-
ment of p300 to the promoter (2, 3, 22) and that Gal4-VP16 is
able to invade mH2A nucleosomes, one could hypothesize that
the NHR domain of mH2A is involved in the impediment of
histone tail acetylation by p300. The mechanism of this imped-
iment is presently unknown. NHR does not exhibit histone
deacetylase activity (1), suggesting that the involvement of
NHR in the impediment of histone acetylation is rather steric.
Interestingly, the H2A-like domain of mH2A does not affect
p300- and Gal4-VP16-dependent Pol II-activated transcription
(this work) but interferes with SWI/SNF nucleosome mobili-
zation (4). Thus, mH2A exhibits some redundancy in function
with respect to nucleosome remodeling since each individual
domain of mH2A (either H2A-like or NHR, when fused to
H2A) was able to impair nucleosome remodeling.
We speculate that in vivo mH2A could contribute to the
repression of transcription by affecting at least two different
pathways: histone acetylation and chromatin remodeling. Since
these two events, i.e., histone acetylation and nucleosome re-
modeling, are essential for the activation of transcription, it
appears that mH2A could be viewed as a major stopper of
transcriptional activation. Interestingly, the efficiencies of Pol
II passage through conventional H2A, mH2A, and fusion
H2A-NHR nucleosomes were essentially the same for the
three types of particles. This suggests that the presence of a
positioned single mH2A nucleosome on the promoter of spe-
cific genes could be sufficient to impede transcription activa-
tion by repressing the initiation of transcription.
Our data suggest that the interference of the NHR domain
with histone acetylation through steric hindrance would be one
of the reasons for this repression. In addition, as shown in a
FIG. 7. The NHR of mH2A interferes with SWI/SNF nucleosome
remodeling. Conventional H2A and H2A-NHR were reconstituted on
a radioactively end-labeled 152-bp DNA fragment containing the Xe-
nopus borealis 5S RNA gene. Increasing amounts of SWI/SNF were
added to the nucleosome (nuc) solutions, and the remodeling reaction
was carried out for 40 min at 30°C. After digestion with DNase I, DNA
was extracted and subjected to an 8% sequencing gel. The position of
the DNase I cleavage repeat is indicated on the left part of the figure.
The DNase I digestion pattern of free DNA is shown in lane 7. The
diamond designates the dyad axis of the nucleosome.
VOL. 26, 2006TRANSCRIPTION OF macroH2A NUCLEOSOMAL ARRAYS1163
recent report, NHR specifically interacts with HDAC1,2 (11).
Consequently, the NHR domain could interfere with the abil-
ity of HAT to acetylate the histones of the promoter associated
with the macroH2A nucleosome, and in addition, it could
recruit histone deacetylase, which further abrogates the possi-
bility of histone acetylation.
One cannot exclude, however, that the presence of several
mH2A nucleosomes, some of which reside on the gene coding
region, would affect transcription more efficiently. Indeed, the
structure of chromatin domains which contain mH2A could be
distinct from the 30-nm fiber canonical structure, which in turn
might be more refractive to transcription.
Our finding that mH2A behaves as a major stopper of Pol II
activation of transcription in vitro raises several questions,
since to fulfill such function in vivo, mH2A should be localized
specifically on the promoter of transcriptionally inactive genes.
The presence of mH2A on such genes would repress transcrip-
tion. For the transcriptional activation of these genes, the re-
pressive function of mH2A should be eliminated. This could be
achieved by the specific removal of mH2A from the promoter
and its replacement by conventional H2A by an mH2A-specific
histone chaperone as recently described for the histone variant
H2A.Z (28). The identification of genes for which expression is
controlled by mH2A as well as the understanding of the mech-
anism of specific deposition and removal of mH2A from these
genes remains a challenge for future studies.
This work was supported by CNRS, INSERM, Re ´gion Rho ˆne-
Alpes, and grants from the Ministe `re de la Recherche (ACI Biologie
cellulaire Mole ´culaire et Structurale, BCM0070, and ACI Interface
Physique-Chimie-Biologie: Dynamique et re ´activite ´ des Assemblages
Biologiques [DRAB], 2004, no. 04 2 136, ANR Projet no. NT05-
D.A. is on leave from the Institute of Solid State Physics, BAS, Sofia,
1. Allen, M. D., A. M. Buckle, S. C. Cordell, J. Lowe, and M. Bycroft. 2003. The
crystal structure of AF1521 a protein from Archaeoglobus fulgidus with
homology to the non-histone domain of macroH2A. J. Mol. Biol. 330:503–
2. An, W., V. B. Palhan, M. A. Karymov, S. H. Leuba, and R. G. Roeder. 2002.
Selective requirements for histone H3 and H4 N termini in p300-dependent
transcriptional activation from chromatin. Mol. Cell 9:811–821.
3. An, W., and R. G. Roeder. 2003. Direct association of p300 with unmodified
H3 and H4 N termini modulates p300-dependent acetylation and transcrip-
tion of nucleosomal templates. J. Biol. Chem. 278:1504–1510.
4. Angelov, D., A. Molla, P. Y. Perche, F. Hans, J. Cote, S. Khochbin, P. Bouvet,
and S. Dimitrov. 2003. The histone variant macroH2A interferes with tran-
scription factor binding and SWI/SNF nucleosome remodeling. Mol. Cell
5. Angelov, D., A. Verdel, W. An, V. Bondarenko, F. Hans, C. M. Doyen, V. M.
Studitsky, A. Hamiche, R. G. Roeder, P. Bouvet, and S. Dimitrov. 2004.
SWI/SNF remodeling and p300-dependent transcription of histone variant
H2ABbd nucleosomal arrays. EMBO J. 23:3815–3824.
6. Arents, G., R. W. Burlingame, B. C. Wang, W. E. Love, and E. N. Moudri-
anakis. 1991. The nucleosomal core histone octamer at 3.1 Å resolution: a
tripartite protein assembly and a left-handed superhelix. Proc. Natl. Acad.
Sci. USA 88:10148–10152.
7. Bassing, C. H., H. Suh, D. O. Ferguson, K. F. Chua, J. Manis, M. Eckers-
dorff, M. Gleason, R. Bronson, C. Lee, and F. W. Alt. 2003. Histone H2AX:
a dosage-dependent suppressor of oncogenic translocations and tumors. Cell
8. Celeste, A., S. Difilippantonio, M. J. Difilippantonio, O. Fernandez-Ca-
petillo, D. R. Pilch, O. A. Sedelnikova, M. Eckhaus, T. Ried, W. M. Bonner,
and A. Nussenzweig. 2003. H2AX haploinsufficiency modifies genomic sta-
bility and tumor susceptibility. Cell 114:371–383.
9. Chadwick, B. P., C. M. Valley, and H. F. Willard. 2001. Histone variant
macroH2A contains two distinct macrochromatin domains capable of direct-
ing macroH2A to the inactive X chromosome. Nucleic Acids Res. 29:2699–
10. Chadwick, B. P., and H. F. Willard. 2001. A novel chromatin protein, dis-
tantly related to histone H2A, is largely excluded from the inactive X chro-
mosome. J. Cell Biol. 152:375–384.
11. Chakravarthy, S., S. K. Gundimella, C. Caron, P. Y. Perche, J. R. Pehrson,
S. Khochbin, and K. Luger. 2005. Structural characterization of the histone
variant macroH2A. Mol. Cell. Biol. 25:7616–7624.
12. Costanzi, C., and J. R. Pehrson. 1998. Histone macroH2A1 is concentrated
in the inactive X chromosome of female mammals. Nature 393:599–601.
13. Costanzi, C., and J. R. Pehrson. 2001. MACROH2A2, a new member of the
MACROH2A core histone family. J. Biol. Chem. 276:21776–21784.
14. Cote, J., J. Quinn, J. L. Workman, and C. L. Peterson. 1994. Stimulation of
GAL4 derivative binding to nucleosomal DNA by the yeast SWI/SNF com-
plex. Science 265:53–60.
15. Dhillon, N., and R. T. Kamakaka. 2000. A histone variant, Htz1p, and a
Sir1p-like protein, Esc2p, mediate silencing at HMR. Mol. Cell 6:769–780.
16. Gautier, T., D. W. Abbott, A. Molla, A. Verdel, J. Ausio, and S. Dimitrov.
2004. Histone variant H2ABbd confers lower stability to the nucleosome.
EMBO Rep. 5:715–720.
17. Hayes, J. J., and K. M. Lee. 1997. In vitro reconstitution and analysis of
mononucleosomes containing defined DNAs and proteins. Methods 12:2–9.
18. Ito, T., M. E. Levenstein, D. V. Fyodorov, A. K. Kutach, R. Kobayashi, and
J. T. Kadonaga. 1999. ACF consists of two subunits, Acf1 and ISWI, that
function cooperatively in the ATP-dependent catalysis of chromatin assem-
bly. Genes Dev. 13:1529–1539.
19. Kagalwala, M. N., B. J. Glaus, W. Dang, M. Zofall, and B. Bartholomew.
2004. Topography of the ISW2-nucleosome complex: insights into nucleo-
some spacing and chromatin remodeling. EMBO J. 23:2092–2104.
20. Karras, G. I., G. Kustatscher, H. R. Buhecha, M. D. Allen, C. Pugieux, F.
Sait, M. Bycroft, and A. G. Ladurner. 2005. The macro domain is an ADP-
ribose binding module. EMBO J. 24:1911–1920.
21. Kireeva, M. L., W. Walter, V. Tchernajenko, V. Bondarenko, M. Kashlev,
and V. M. Studitsky. 2002. Nucleosome remodeling induced by RNA poly-
merase II: loss of the H2A/H2B dimer during transcription. Mol. Cell 9:541–
22. Kundu, T. K., V. B. Palhan, Z. Wang, W. An, P. A. Cole, and R. G. Roeder.
2000. Activator-dependent transcription from chromatin in vitro involving
targeted histone acetylation by p300. Mol. Cell 6:551–561.
23. Kustatscher, G., M. Hothorn, C. Pugieux, K. Scheffzek, and A. G. Ladurner.
2005. Splicing regulates NAD metabolite binding to histone macroH2A. Nat.
Struct. Mol. Biol. 12:624–625.
24. Ladurner, A. G. 2003. Inactivating chromosomes: a macro domain that
minimizes transcription. Mol. Cell 12:1–3.
25. Luger, K., A. W. Mader, R. K. Richmond, D. F. Sargent, and T. J. Richmond.
1997. Crystal structure of the nucleosome core particle at 2.8 Å resolution.
26. Luger, K., T. J. Rechsteiner, and T. J. Richmond. 1999. Expression and
purification of recombinant histones and nucleosome reconstitution. Meth-
ods Mol. Biol. 119:1–16.
27. Mermoud, J. E., C. Costanzi, J. R. Pehrson, and N. Brockdorff. 1999. His-
tone macroH2A1.2 relocates to the inactive X chromosome after initiation
and propagation of X-inactivation. J. Cell Biol. 147:1399–1408.
28. Mizuguchi, G., X. Shen, J. Landry, W. H. Wu, S. Sen, and C. Wu. 2004.
ATP-driven exchange of histone H2AZ variant catalyzed by SWR1 chroma-
tin remodeling complex. Science 303:343–348.
29. Pehrson, J. R., and V. A. Fried. 1992. macroH2A, a core histone containing
a large nonhistone region. Science 257:1398–1400.
30. Perche, P. Y., C. Vourc’h, L. Konecny, C. Souchier, M. Robert-Nicoud, S.
Dimitrov, and S. Khochbin. 2000. Higher concentrations of histone
macroH2A in the Barr body are correlated with higher nucleosome density.
Curr. Biol. 10:1531–1534.
31. Rangasamy, D., I. Greaves, and D. J. Tremethick. 2004. RNA interference
demonstrates a novel role for H2A.Z in chromosome segregation. Nat.
Struct. Mol. Biol. 11:650–655.
32. Santisteban, M. S., T. Kalashnikova, and M. M. Smith. 2000. Histone
H2A.Z regulates transcription and is partially redundant with nucleosome
remodeling complexes. Cell 103:411–422.
33. Thastrom, A., P. T. Lowary, H. R. Widlund, H. Cao, M. Kubista, and J.
Widom. 1999. Sequence motifs and free energies of selected natural and
non-natural nucleosome positioning DNA sequences. J. Mol. Biol. 288:213–
1164 DOYEN ET AL.MOL. CELL. BIOL.