In Vitro Analysis of Huntingtin-Mediated
Transcriptional Repression Reveals
Multiple Transcription Factor Targets
Weiguo Zhai,1Hyunkyung Jeong,2Libin Cui,2Dimitri Krainc,2,* and Robert Tjian1,*
1Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, 401 Barker Hall,
Berkeley, CA 94720, USA
2Department of Neurology, Massachusetts General Hospital, Harvard Medical School, MassGeneral Institute for Neurodegenerative
Disease, 114 16thStreet, Charlestown, MA 02129, USA
*Contact: firstname.lastname@example.org (D.K.); email@example.com (R.T.)
Transcriptional dysregulation has emerged
anism in Huntington’s disease, a neurode-
generative disorder associated with poly-
glutamine expansion in the huntingtin (htt)
protein. Here, we report the development
tion assay that is responsive to mutant htt.
We demonstrate that both gene-specific
activator protein Sp1 and selective compo-
nents of the core transcription apparatus,
including TFIID and TFIIF, are direct targets
inhibited by mutant htt in a polyglutamine-
dependent manner. The RAP30 subunit of
TFIIF specifically interacts with mutant htt
both in vitro and in vivo to interfere with for-
mation of the RAP30-RAP74 native com-
plex. Importantly, overexpression of RAP30
in cultured primary striatal cells protects
neurons from mutant htt-induced cellular
hibition of the dopamine D2 receptor gene
by mutant htt. Our results suggest a mutant
htt-directed repression mechanism involv-
ing multiple specific components of the
basal transcription apparatus.
Huntington’s disease (HD) is an autosomal dominant neuro-
degenerative disorder characterized by psychiatric, cogni-
tive, and motor abnormalities. Pathogenesis is associated
with an expansion in the number of glutamine residues
located in the amino (N) terminus of huntingtin (htt), a very
large protein found mainly in the cytoplasm (DiFiglia et al.,
1995; The Huntington’s Disease Collaborative Research
Group, 1993). Polyglutamine (polyQ) expansion in the mu-
tant protein leads to its aberrant proteolytic cleavage, result-
ing in the release of N-terminal fragments that readily enter
the nucleus (DiFiglia et al., 1997). These glutamine-rich mu-
tant htt fragments are thought to contribute to the formation
of nuclear inclusions and neurotoxicity and possibly acti-
vate an apoptotic cascade (Saudou et al., 1998). These pro-
cesses have been experimentally modeled in cell culture and
in transgenic animals expressing mutant htt (Sipione and
Cattaneo, 2001). Importantly, the length of the polyQ tract
in htt has also been correlated with the age of onset and
the severity of symptoms in HD patients (Rubinsztein et al.,
Although a number of different cellular events have been
shown to associate with HD occurrence, the actual patho-
genic mechanisms remain unclear. Recent evidence sug-
gests that mutant htt may disrupt normal transcriptional pro-
grams in susceptible neurons during initial stages of HD
pathogenesis, implicating transcriptional dysregulation as a
potential pathogenic mechanism (Cha, 2000; Sugars and
Rubinsztein, 2003). It has been well established, first in the
case of Sp1 (Courey and Tjian, 1988) and subsequently with
other transcription factors, that activation domains are often
composed of glutamine-rich protein interfaces (Gerber et al.,
1994). Thus, transcription factor interactions with other cel-
lular factors may be disrupted by mutant htt bearing polyQ
expansions. Indeed, mutant htt has been shown to interact
directly with a number of nuclear transcription factors (Oka-
zawa, 2003). Recent DNA microarray studies detected
changes in gene expression profiles in HD transgenic mice
at early stages, suggesting that transcription of select genes
had already been altered even when mice showed only min-
imal abnormalities (Luthi-Carter et al., 2002a; Luthi-Carter
et al., 2002b). Analysis of the affected regulatory sequences
revealed that select Sp1-dependent transcription pathways
were disrupted. This hypothesis is strongly supported by
recent in vivo observations that mutant htt may target Sp1
and its coactivator TAF4 (formerly TAFII130; Chen et al.,
1994; Tanese et al., 1991) through direct protein-protein in-
teractions to disrupt transcription (Dunah et al., 2002). Re-
markably, in primary striatal neurons, coexpression of Sp1
Cell 123, 1241–1253, December 29, 2005 ª2005 Elsevier Inc. 1241 Cell 123, 1241–1253, December 29, 2005 ª2005 Elsevier Inc. 1241
and TAF4 resulted in a significant rescue of mutant htt-
induced inhibition of dopamine D2 receptor gene (D2) pro-
moter activity (Dunah et al., 2002).
Human TAF4 is one of at least 12 TATA binding protein
(TBP)-associated factors (TAFIIs) in TFIID (Albright and Tjian,
2000). The transcription initiation factor TFIID is recruited to
the corepromoterthroughitsinteraction withspecific activa-
tors such as Sp1 and binds to the TATA element at core pro-
moters. A series of transcription factor interactions, involving
TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, CRSP (also called me-
diator), and RNA polymerase II (Pol II) subsequently leads to
tional activation (Ryu et al., 1999). Human TFIIF consists of
two subunits, RAP30 and RAP74, that bind RNA Pol II di-
complex (Conaway et al., 2000).
to the potential relationship between htt, Sp1, and TAF4, it
remains unclear whether mutant htt interferes directly with
Sp1, TFIID, or potentially other components of the PIC to re-
press transcription. To determine the molecular mecha-
nisms employed by mutant htt to repress transcription, the
biochemical properties and specificity of this glutamine ex-
pansion protein may best be revealed by using purified pro-
teins and an integrated in vitro transcription reaction respon-
sive to putative htt targets, such as Sp1 and TFIID.
Here, we report the development of a well-defined
chromatin based in vitro transcription assay consisting of
highly purified factors to study the molecular mechanisms
of mutant htt-mediated transcriptional repression. Various
N-terminal fragments of htt carrying either a normal or an ex-
panded polyQ tract were tested for their ability to repress
transcription in Sp1-dependent versus Sp1-independent
in vitro reconstituted reactions. Using this highly purified
in vitro assay, we identified several specific factors targeted
by mutant htt to repress transcription. Invitro and in vivopro-
tein binding assays revealed a direct interaction between htt
and distinct components of the PIC. Importantly, these inter-
actions were sensitive to the length of the polyQ expansion.
The role of these factors in transcriptional repression and HD
pathogenesis was further verified in cultured primary striatal
neurons. These studies reveal that specific components of
the core transcriptional apparatus are directly targeted by
soluble forms of mutant htt that operate as a selective re-
pressor or corepressor to disrupt normal gene expression
in HD cells.
Mutant htt Specifically Represses Sp1-Dependent
Transcription In Vitro
We adopted a chromatin-based in vitro transcription system
(Lemon et al., 2001; Naar et al., 1999) to dissect potential
mechanisms by which mutant htt represses Sp1-dependent
transcription. The Sp1 transcription template was first as-
sembled into chromatin using Drosophila S190 and purified
core histones. As expected, high levels of transcription from
the assembled chromatin template were obtained with puri-
fied recombinant Sp1 and a well-defined set of basal tran-
scription factors including purified recombinant TFIIA, TFIIB,
TFIIE, TFIIF and affinity-purified TFIID, TFIIH, RNA Pol II, and
CRSP (Figure 1A, lane 1). To assess the effect of mutant htt
on Sp1-dependent transcription, we expressed the N-termi-
nal portion (1–171 aa) of either wild-type (wt; Htt-23QP) or
mutant (Htt-120QP) htt in E. coli and purified these proteins
using Ni-NTA affinity chromatography (Figure 1D). As shown
in Figure 1A, adding wt htt (up to 0.3 pmol) did not signifi-
cantly alter the levels of transcription (lanes 2–5). In contrast,
adding 0.1–0.3 pmol of mutant htt resulted in a dramatic de-
crease in transcription (Figure 1A, lanes 8 and 9). Thus, pu-
rified recombinant mutant htt fragment is capable of inhibit-
ing Sp1-dependent transcription in a well-defined in vitro
ence of an expanded polyQ in htt.
Next, we examined whether this repression by mutant htt
is specific for Sp1-dependent transcription by assaying its
effect on an unrelated ‘‘acidic’’ activator, Gal4-VP16. Tran-
scription from the Gal4 chromatin template is highly depen-
dent on the presence of Gal4-VP16 (Figure 1B, compare
lanes 1 and 2). In contrast to the results obtained from
Sp1-dependent transcription assays, adding mutant htt to
a level that efficiently repressed Sp1 activation had no signif-
lanes 3–5). However, if much higher amounts of mutant htt
were used, some repression even with VP16 can be ob-
served (data not shown). These findings suggest that the
htt directed repression observed in vitro is rather specific
for certain activators such as Sp1 and not likely due to non-
specific aggregation or global inhibition of activators.
It has been reported that htt protein can bind to the acetyl-
transferase (HAT) domain of CREB binding protein (CBP)
and p300/CBP-associated factor (P/CAF) and inhibit their
HAT activity (Steffan et al., 2001). Therefore, it is possible
that the htt-directed transcriptional repression we observed
was a consequence of mutant htt-mediated interference
with chromatin modifying or remodeling activities present
in our assay system. To address this issue, we performed
in vitro transcription assays using naked DNA templates
and completely purified transcription components. As
shown in Figure 1C, adding purified mutant htt efficiently re-
ked DNA templates while addition of wt htt at these levels
had no effect. Because there is no chromatin modifying
and remodeling required for transcription from naked DNA
templates, the observed repression in vitro is unlikely to re-
sult from its interference with chromatin modifying and/or re-
modeling activities. This is also consistent with our observa-
tion that addition of the histone deacetylase (HDAC) inhibitor
TSA had little or no effect on mutant htt-mediated transcrip-
tional repression in our assay (data not shown).
Rescue of Transcriptional Repression Mediated
by Mutant htt In Vitro
To better understand the mechanisms of transcriptional re-
pression by mutant htt, we decided to systematically test
1242 Cell 123, 1241–1253, December 29, 2005 ª2005 Elsevier Inc.
which components present in our defined in vitro transcrip-
tion system could rescue the inhibition caused by mutant
htt. It has been reported that mutant htt can disrupt the inter-
pression of the D2 promoter activity by mutant htt can be re-
versed by coexpression of Sp1 and TAF4 in cultured striatal
cells (Dunah et al., 2002). Based on these in vivo results, we
anticipated that addition of Sp1 and/or TAF4 (in the form of
TFIID complex) should rescue the transcriptional repression
induced by mutant htt. For these experiments, we chose to
use an amount of mutant htt (Htt-120QP, 0.15 pmol) that
would repress transcription by about 80%–90% (Figure 2A,
compare lanes 1 and 2). We also used the native holo-TFIID
instead of the isolated TAF4 subunit because the purified re-
combinant protein is poorly behaved and has a tendency to
aggregate or becomes proteolyzed into smaller fragments.
To rescue this level of repression, an additional 2- to 4-fold
of each individual basal factor was added to transcription re-
actions containing mutant htt. As expected, addition of puri-
fied Sp1 (Figure 2A, lanes 19 and 20) or the TAF4-containing
TFIID complex (Figure 2A, lanes 7 and 8) was able to effi-
ciently rescue the repression caused by mutant htt. These
findings indicate that our in vitro transcription system largely
recapitulates the transcriptional repression observed for mu-
tant htt in vivo. In addition, they provide evidence that most
likely both Sp1 and TFIID are directly targeted by mutant
htt for repression.
To screen for potential novel targets of mutant htt, we also
attempted to rescue the in vitro repression by adding other
components of the core transcription machinery. Surpris-
ingly, we found that addition of excess TFIIF can reverse
the repression (Figure 2A, lanes 11 and 12), suggesting
that TFIIF may also be targeted by mutant htt for repression.
Addition of the other basal factors and cofactors had little or
no effect on the repression by mutant htt (Figure 2A). As
a control, we also added the same amounts of excess indi-
vidual factors to normal transcription reactions containing no
mutant htt.Asshown inFigure2B,additionofexcessfactors
to our in vitro transcription system had little effect on the
overall transcription level, suggesting that under our stan-
dard transcription condition, the level of basal factors used
are already saturating for transcription initiation.
The Length of polyQ Expansion Correlates
with Repressor Potency
A key observation made in HD patients is that longer polyQ
expansions in htt are associated with more severe symp-
toms and earlier age of onset (Rubinsztein et al., 1993).
Therefore, it is important to determine whether mutant htt
with varying polyQ length would also differ in their ability to
we expressed and purified His6-tagged htt exon1 carrying
various numbers of polyQ (Htt-25QP, Htt-46QP, and Htt-
97QP; Figure 3A). Purified proteins that remained soluble af-
ter dialysis were resolved by SDS-PAGE, and their relative
concentrations were estimated by immunoblotting using
antibodies against the His6tag. When these purified htt poly-
peptides were tested in transcription assays, their ability to
Figure 1. Development of an In Vitro
Transcription System Responsive to Mu-
(A) Mutant htt inhibits Sp1-dependent transcrip-
tion in vitro. In vitro transcription was directed
from the Sp1 chromatin template using purified
Sp1 and basal transcription factors either in the
absence or presence of normal htt (Htt-23QP)
or mutant htt (Htt-120QP).
dent transcription from the Gal4 chromatin tem-
plate in vitro.
(C) Mutant htt inhibits Sp1-dependent transcrip-
tion in vitro from naked DNA template.
ing 23 or 120 polyQ (Htt-23QP and Htt-120QP)
were visualized by Coomassie staining.
Cell 123, 1241–1253, December 29, 2005 ª2005 Elsevier Inc. 1243
repress transcription differed substantially. The addition of
normal htt fragment (Htt-25QP; Figure 3B, lanes 2–5) had
no measurable effect on Sp1-dependent transcription, while
100 pmol of mutant htt (Htt-46QP) inhibited transcription
(Figure 3B, lane 9). When the number of polyQ in htt was in-
creased further, its ability to repress transcription increased
dramatically. The addition of 1.0 pmol of Htt-97QP resulted
in a 90% decrease in the levels of transcription (Figure 3B,
lane 12). Even lower amounts of Htt-120QP were sufficient
to repress transcription efficiently (Figure 3B, lanes 13–15).
Next, we determined whether transcriptional repression
by mutant htt carrying different numbers of polyQ targeted
the same components of the transcription apparatus. As
shown in Figure 3C, transcriptional repression by Htt-97QP
(1.0 pmol) can be rescued by adding more TFIIF, TFIID,
and Sp1. Addition of other basal transcription factors was
not able to alleviate the repressive effect of this mutant htt
(Figure 3C). Likewise, repression by Htt-46QP (100 pmol)
can also be rescued by the addition of more TFIIF, TFIID,
and Sp1 but not the other basal transcription factors
(Figure 3D). Thus, the same set of factors (i.e., Sp1, TFIID,
and TFIIF) was capable of reversing the transcriptional re-
pression mediated by mutant htt carrying different numbers
of polyQ. These results suggest that mutant htt with different
numbers of polyQ likely utilizes the same molecular targets
and mechanisms to repress transcription.
Interplay between Mutant htt and TFIIF
The data we obtained thus far strongly suggest that mutant
htt can inhibit Sp1-dependent transcription by interfering
ity of Sp1 and TFIID to rescue the transcriptional repression
was expected since mutant htt can interact with Sp1 and
TAF4 to disrupt this activator-coactivator pair in vivo (Dunah
et al., 2002). Therefore, we sought to determine whether
TFIIF could be targeted by mutant htt in a similar manner.
To investigate whether htt is able to interact with TFIIF
(composed of RAP30 and RAP74) under normal physiolog-
ical conditions, coimmunoprecipitation (coIP) studies were
performed using extracts prepared from mouse striatum.
As shown in Figure 4A, aRAP30 antibody was able to IP
htt protein from knockin (KI) mice (Heterozygous, wt/140Q,
Menalled et al., 2003) striatum extracts (lane 3) but not
from normal (wt) mice striatum extracts (lane 7). In control re-
actions, IgG alone (Figure 4A, lanes 4 and 8) did not bring
down any detectable htt protein. These data suggest that
mutant htt can form a complex with RAP30 in HD striatum
in a manner that is polyQ dependent and stable enough to
coIP from crude cell extracts.
subunit of TFIIF interacts with htt, we cotransfected COS7
cells with constructs encoding myc-tagged normal htt
(Htt480-17, N-terminal 480 aa) or mutant htt (Htt480-68)
Figure 2. Rescue of Mutant htt-Mediated
(A) Select basal transcription factors can rescue
mutant htt-mediated transcriptional repression
in vitro. An extra 2 (odd-numbered lanes)- or 4-
fold (even-numbered lanes) of each factor (as la-
beled) was added to transcription reactions con-
(B) Effect of additional basal transcription factors
on overall transcription in the absence of mutant
1244 Cell 123, 1241–1253, December 29, 2005 ª2005 Elsevier Inc.
together with Flag-tagged RAP30. The expression levels for
wt and mutant htt in transfected cells were comparable (Fig-
ure 4B, input). CoIP with aFlag antibodies showed that
RAP30 interacted strongly with mutant htt (Figure 4B, lane
4) while displaying only minimal affinity for wt htt (lane 3)
over background (lanes 1 and 2). Reverse IP using the
same lysates with amyc antibodies to precipitate htt con-
firmed that the interaction between RAP30 and htt is polyQ
dependent (Figure 4B, lanes 5 and 6). In contrast, RAP74
showed no detectable affinity for either wt or mutant htt in a
similar experiment (Figure 5A, lanes 5 and 6). Surprisingly,
when we cotransfected these cells with expression con-
structs for htt and RAP30 as well as RAP74, the htt-RAP30
interaction was severely reduced even though there was
Figure 3. Correlation of the Length of polyQ Tract in htt with Its Ability to Repress Transcription
(A) His-tagged htt exon 1 containing 25, 46, or 97 polyQ (Htt-25QP, Htt-46QP, and Htt-97QP) were visualized by Coomassie staining.
(B) In vitro transcription reactions were carried out either in the absence or presence of purified Htt-25QP, Htt-46QP, Htt-97QP, or Htt-120QP.
(C) Rescue of transcriptional repression mediated by Htt-97QP.
(D) Rescue of transcriptional repression mediated by Htt-46QP.
Cell 123, 1241–1253, December 29, 2005 ª2005 Elsevier Inc. 1245
more RAP30 protein in cells cotransfected with RAP74
(Figure 5A, compare lanes 3 and 4 to 1 and 2). CoIP using
cell lysates with a nontagged version of RAP74 yielded the
same result (data not shown). Thus, it appears that RAP74
can efficiently compete with htt for binding to RAP30, sug-
gesting that the interactions between RAP30-RAP74 and
RAP30-htt are likely mutually exclusive.
RAP74 (Tan et al., 1995), we reasoned that RAP30 is likely to
use thesameoranoverlapping domain forhttinteraction.To
directly test this idea and map the regions ofRAP30 required
for RAP74 and htt interactions, we assayed a series of Flag-
tagged RAP30 deletion mutants in cotransfection and coIP
experiments. As shown in Figure 5B (top panels), deletion
mutants of RAP30 containing its first 90 or 60 amino acids
interacted with RAP74 as efficiently as the full-length protein
(lanes 1–3). However, further removal of N-terminal amino
acids of RAP30 (1–40 aa) resulted in a severe loss of
RAP74 interaction (Figure 5B, lane 4). As expected, mutants
of RAP30 lacking various regions of the N terminus (40–249
aa and 60–249 aa) were unable to interact with RAP74
(Figure 5B, lanes 5 and 6), suggesting that the first 60 amino
acidsofRAP30are minimallyrequired forRAP74 interaction.
their ability to interact with mutant htt (Htt480-68). As shown
in Figure 5B (bottom panels), truncations of RAP30 contain-
ing its first 90 or 60 amino acids retained strong affinity for htt
aa, Figure 5B, lanes 10 and 11), which had lost their ability to
interact with RAP74, also showed no detectable affinity for
mutant htt. However, a RAP30 mutant lacking the first 40
amino acids of its N terminus but containing the remainder
of RAP30 protein (40–249 aa) showed significant affinity for
mutant htt despite its inability to interact with RAP74 (Fig-
identify the interacting regions of RAP30 are summarized in
nal region of RAP30 (40–60 aa) is required and sufficient for
mutant htt interaction. This same region is also required but
not sufficient for RAP74 interaction. RAP74 appears to inter-
act with more RAP30 N-terminal sequences that lie between
amino acids 1 and 40. Thus, RAP30 appears to use overlap-
ping domains for its interaction with RAP74 and mutant htt,
providing us with a molecular rationale for the observation
that RAP30 binding to htt and RAP74 are mutually exclusive.
To further confirm that the interaction between htt and
RAP30 is direct, we performed GST pull-down experiments
and bacterial lysates expressing RAP30. As shown in Fig-
ure 5D, GST-fusion htt protein can selectively bind RAP30,
whereas GST alone does not (lanes 1–4). As expected, mu-
tant htt is somewhat more efficient in binding RAP30 than
wt htt. In contrast, GST-htt did not pull down any RAP74
from bacterial lysates expressing RAP74 (Figure 5D, lanes
and confirm that the interaction between RAP30 and htt is
Our coIP experiments showed that mutant htt might com-
tion of the TFIIF complex. To test whether mutant htt can in-
terfere with the TFIIF complex, we performed GST pull-down
experiments with highly purified recombinant TFIIF complex
(Figure 5D, lane 9) and GST-htt proteins. As shown in
Figure 5D (bottom panels), GST mutant htt pulled down sig-
nificantly more RAP30 than wt htt (Figure 5D, compare lanes
15 and 16). In contrast, very little RAP74 was pulled down
(Figure 5D). Because a significant proportion of the RAP30
and RAP74 are in complex with each other in our TFIIF prep-
flects binding between htt and a pool of free RAP30 subunits
dissociated from intact TFIIF. Thus, we envision that the way
in which the RAP30-htt interaction could interfere with the
formation of active TFIIF is by sequestering RAP30.
Having found that RAP30 interacts with htt primarily
through its N-terminal RAP74 binding domain, we sought
to determine the features of htt that are important for this
binding. We first tested whether the proline-rich region and
sequences further downstream could affect this interaction.
Figure 4. PolyQ-Dependent Interaction between htt and RAP30
(A) Increased interaction between htt and RAP30 in HD mouse striatum. Extracts prepared from either mutant htt knockin (KI, heterozygous, wt/140Q) mice
striatum or normal (wt) mice striatum were immunoprecipitated with aRAP30 antibody and control IgG and subsequently immunoblotted with ahtt (top) or
aRAP30 antibodies (bottom).
(B) COS7 cells were cotransfected with Flag-tagged RAP30 and myc-tagged htt constructs (1–480 aa, Htt480-17 or Htt480-68). The cell lysates were im-
munoprecipitated (IP) and subsequently immunoblotted (IB) with either amyc or aFlag antibodies. Inputs of htt were detected using amyc antibodies.
1246 Cell 123, 1241–1253, December 29, 2005 ª2005 Elsevier Inc.
As shown in Figure 6, GST fusions that also contain the pro-
line-rich domain (GST-20QP and GST-51QP), and down-
teract efficiently with RAP30. In contrast, their counterparts
without the proline-rich domain (GST-20Q and GST-51Q)
showed little or no affinity for RAP30. The presence of the
residues immediately C-terminal to the proline-rich region
did not significantly enhance the ability of htt to bind these
three target factors. Thus, the proline-rich region in htt ap-
pears to be an important element for the in vitro interaction
with RAP30. Interestingly, the proline-rich domain in htt
also contributes to its interaction with TAF4 and Sp1 (Fig-
ure 6B). The finding that the same polyQ and proline-rich re-
gions play critical roles in the interaction between htt and all
three factors as well as CBP (Steffan et al., 2001) suggests
that mutant htt may utilize similar or overlapping interfaces
to interfere with transcription involving different transcription
pathways. Quantitation of the data revealed that Htt interac-
tions with RAP30 and Sp1 are significantly enhanced by
polyQ expansion, whereas its interaction with TAF4 appears
to be less affected by the number of glutamines.
Rescue of Mutant htt-Mediated D2 Promoter
Inhibition by RAP30
To determine the potential relevance ofRAP30 in mutant htt-
mediated transcriptional repression in vivo, we investigated
the transcriptional rescue of the D2 promoter in striatal
neurons. When a D2 promoter reporter construct was
Figure 5. Mutually Exclusive Interactions between RAP30-RAP74 and RAP30-htt
(A) RAP74 competes with mutant htt for binding to RAP30. Lysates from cotransfected COS7 cells were immunoprecipitated and subsequently immuno-
blotted with amyc and aFlag antibodies. Inputs of myc-htt were detected using amyc antibodies.
(B) The N-terminal region of RAP30 is required for its interaction with both RAP74 and mutant htt. Flag-tagged RAP30 deletion mutants (including amino
acids aslabeled) were cotransfected into COS7cells with eithermyc-RAP74ormyc-Htt480-68. Thecelllysates were immunoprecipitated usingaFlagresin
followed by immunoblotting with amyc antibodies.
(C) A schematic summary of the functional domain mapping results as described in (B).
(D) GST-htt interacts directly with RAP30 and pulls down RAP30 from intact TFIIF. GST-htt was able to pull down RAP30 (top left) but not RAP74 (top right).
The purified recombinant TFIIF (rTFIIF) and GST-fusion proteins used in these pull downs were visualized by Coomassie staining (bottom left). Immunoblot-
ting with aRAP30 and aRAP74 antibodies were used to detect their presence in pull downs (bottom right).
Cell 123, 1241–1253, December 29, 2005 ª2005 Elsevier Inc. 1247
cotransfected together with htt into primary striatal neurons,
wt htt (Htt480-17) had no observable repressive effect
whereas mutant htt (Htt480-68) consistently resulted in
ceptor 1 (NR1) promoter, which is known not affected in HD,
was not repressed under the same experimental conditions
(data not shown). Importantly, when the RAP30 subunit
alone or together with RAP74 was cotransfected into pri-
mary striatal neurons, the D2 promoter inhibition was allevi-
ated (Figure 7A). As a control, expression of RAP30 subunit
alone or together with RAP74 in the absence of mutant htt
had no significant effect on the D2 promoter activity
(Figure 7A). To exclude the possibility that the transcription
rescue we observed at the D2 promoter was caused by an
altered expression level of htt protein as a result of added
RAP30 and RAP74, we performed an immunoblotting assay
to measure the protein levels of transfected htt and TFIIF
subunits under the same assay conditions. As shown in
Figure 7B, RAP30 and RAP74 expression did not change
the protein levels of either wt or mutant htt (compare lanes
1–2 and 3–4). In addition, neither wt nor mutant htt coex-
pression had any significant effect on the expression level
of RAP30/RAP74 in primary neurons (compare lanes 3 and
4 to 5). It should also be noted that no obvious cell toxicity
was observed at the time of reporter activity analysis. Taken
together, these cell-based assays corroborate our in vitro
findings and suggest that TFIIF is a contributing factor in-
volved in transcriptional inhibition mediated by mutant htt.
Rescue of Mutant htt-Induced Cellular
Toxicity by RAP30
To correlate our transcription and protein interaction findings
with possible HD pathogenic mechanisms in vivo, we inves-
tigated the potential relationship of TFIIF subunits and mu-
tant htt in a cellular toxicity assay. Previous experiments
demonstrated that mutant htt is toxic when overexpressed
in cultured primary striatal neurons (Saudou et al., 1998).
As shown in Figure 7C, exon1 of wt htt (25Q) was seen in
both cytoplasm and nucleus as well as in neurites. In con-
trast, exon 1 of mutant htt (103Q) was observed primarily
as nuclear inclusions. In addition, overexpression of mutant
but not wt htt induced cellular toxicity in cultured primary
striatal neurons. However, when RAP30 was cotransfected
of transfected cells expressing both RAP30 and mutant htt
looked normal (Figure 7C). When the number of cells show-
age of total cells expressing the transfected proteins, we
found that mutant htt-mediated cellular toxicity is dramati-
cally abrogated by overexpression of RAP30 (Figure 7D).
Expression of RAP30 in the absence of mutant htt did not in-
duce cellular toxicity. By contrast, overexpression of RAP74
alone resulted in a significant number of cells showing toxic-
ity (Figure 7D). This toxicity level is nearly comparable to that
of RAP74 with mutant htt actually resulted in more cells
showing signs of cellular toxicity (Figure 7D). Thus, the pres-
ence of free RAP74 may exacerbate mechanisms that inhibit
transcription of certain genes, such as the D2 gene, leading
to cell death in the HD striatum. The striking correlation be-
tween these observations in striatal neurons and our in vitro
transcription studies as well as protein-protein interaction
data strongly suggest that TFIIF along with Sp1 and TAF4
(TFIID) represent specific transcriptional components tar-
geted as part of the mechanism contributing to the repres-
sion of transcription and pathogenesis in HD.
Figure 6. Both the polyQ and Proline-Rich Domain in htt Are Required for Its Interaction with RAP30, TAF4, and Sp1
(A) GST and various GST-fusion htt fragments used in pull downs were visualized by Coomassie staining.
(B) GSTpull downsof [35S]-methionine-labeled RAP30, TAF4,and Sp1 were analyzed bySDS-PAGE, developed, and quantitated (shown as percentage of
input) with a PhosphorImager screen.
1248 Cell 123, 1241–1253, December 29, 2005 ª2005 Elsevier Inc.
Reduced Occupancy of RAP30 at the D2 Promoter
in R6/2 Mouse HD Brain
To obtain additional evidence for the involvement of RAP30/
htt at a relevant target promoter, we performed chromatin
immunoprecipitation (ChIP) analysis to investigate the occu-
pancy of RAP30 at either a promoter affected by mutant htt
(such as the D2 promoter) or a control promoter that is not
affected by mutant htt (such as the NR1 promoter) in both
normal and HD brain. HD brain samples were taken from
10-week-old R6/2 mice (Mangiarini et al., 1996) and normal
Figure 7. Mutant htt-Mediated Repression of the D2 Receptor Promoter and Cell Death Are Prevented by Overexpression of
RAP30 in Primary Striatal Neurons
(A) Effect of RAP30 on htt-induced repression of the D2 promoter. Primary striatal neurons were transfected with D2-75Luc and expression constructs for
htt (Htt480-17 and Htt480-68) and TFIIF subunit. Expression of RAP30 alone or both RAP30 and RAP74 can reverse the mutant htt-induced inhibition of
D2 promoter activity. SEM for samples were calculated from triplicates.
(B) RAP30 and RAP74 expression does not interfere with htt expression in transfected primary striatal neurons. The same amount of cell lysates was sep-
arated by SDS-PAGE and probed with amyc antibody for myc-htt (top), aFlag antibody for Flag-RAP30/RAP74 (middle), or aActin antibody as input control
(C) Effect of RAP30 on htt-induced cellular toxicity. Primary striatal neurons were transfected with htt (exon 1-GFP-25Q or 103Q) and/or RAP30, RAP74
expression constructs. A series of fluorescence micrographs is shown of neurons stained for DNA (Hoechst, blue, left), htt (green, middle), and RAP30
or RAP74 (red, right). Arrows indicate transfected neurons.
(D) RAP30 expression protects against mutant htt-induced cellular toxicity. Primary striatal neurons were transfected as in (B). Cell death was scored and
plotted as percent cell death. SEM for samples were calculated from triplicates.
Cell 123, 1241–1253, December 29, 2005 ª2005 Elsevier Inc. 1249
brain samples were taken form their wt littermates (wt). After
isolating chromatin complexes using aRAP30 antibody, the
specifically precipitated DNAwasanalyzedbymultiplexPCR
in reactions using three sets of primers specific for the core
promoter regions of the mouse D2 (Drd2), NR1, and GAPDH
was used to ensure the linearity of the multiplex PCR assays.
As shown in Figure 8A, significantly less RAP30 is present at
the Drd2 promoter in HD brain than in wt brain. By contrast,
the level of RAP30 occupancy at the control NR1 (Figure 8B)
and GAPDH (Figure 8C) promoter is comparable in wt and
HD brain samples. This reduced occupancy of RAP30 in HD
tant htt responsive promoters such as the preproenkephalin
gene promoter(data notshown). Our invivofindings indicate
that the level of RAP30 occupancy at promoters downregu-
involved in repressing transcription at early stages of HD
In this study, we have developed an in vitro transcription as-
say to dissect the potential molecular mechanisms em-
ployed by mutant htt to repress transcription of specific pro-
defined in vitro transcription system, we demonstrate that
specific components (TFIID and TFIIF) of the transcriptional
machinery are directly targeted by mutant htt. Importantly,
these in vitro results correlate very well with the in vivoeffects
of mutant htt, such as the previously reported disruption of
Sp1 and TAF4 interaction by mutant htt at the D2 promoter
(versus NR1 promoter) in primary neurons (Dunah et al.,
2002). Bearing this principle in mind, we may, in the future,
be able to take advantage of this in vitro system to identify
other potential direct targets and mechanisms of transcrip-
tionaldysregulationassociated withother transcription path-
ways in HD. Secondly, this study demonstrates that soluble
rather than aggregated forms of mutant htt may directly dys-
regulate transcription by interfering with specific compo-
nents of the transcriptional preinitiation complex. Our data
of interference by the soluble forms of mutant htt early in dis-
ease before any aggregation is seen. In addition, our work
suggests that mutant htt may act as a special class of tran-
scriptional repressor or corepressor. This is a potentially im-
direct effects of mutant htt on transcription is via specific re-
pressor mechanisms, whereas other documented effects of
htt such as activation of transcription may be compensatory
or secondary. Finally, our work demonstrates that transcrip-
tional repression by mutant htt is polyQ length dependent.
This strongly confirms the observed toxic gain of function
for mutant htt. Progressive expansion of polyQ in mutant
htt appears to lead to more severe repression while little or
no repression is seen with wt htt both in vitro and in vivo.
The strong correlation between polyQ length and the
efficiency of repression we have observed in vitro fits well
with the documented timing and severity of HD onset. This
striking finding further suggests that direct disruption of
Figure 8. Reduced Occupancy of RAP30
at the D2 Promoter in R6/2 Mice
Brain samples isolated from 10-week-old R6/2
mice(HD), and wild-type (wt)littermateswere an-
alyzed by ChIP using aRAP30 antibody or IgG as
a negative control. Promoter-specific occupancy
of RAP30 was analyzed by multiplex PCR. Quan-
titation results on the D2 promoter (A), NR1 pro-
moter (B), and GAPDH promoter (C) were shown
as graph bars. SEM for samples were calculated
from triplicates. The quantitation number for IgG
control ChIP was extremely low and indifferent
between wt and R6/2 samples.
(D) Model of potential mechanisms used by mu-
tant htt to disrupt Sp1-mediated transcription.
1250 Cell 123, 1241–1253, December 29, 2005 ª2005 Elsevier Inc.
transcription integrity via aberrant interactions between mu-
tant htt, Sp1, TFIID, and TFIIF are specific and may be signif-
icant for orchestrating the pathogenesis of HD.
In our current work, we have used a variety of different htt
N-terminalfragment constructsto takeadvantage ofthevar-
ious systems established by other HD researchers. Although
we realized that truncated htt proteins might behave some-
tive. Indeed, our in vitro studies were inspired by previous
findings showing that various truncated versions of mutant
htt bearing different lengths of polyQ expansions are pro-
duced by proteolytic cleavage in vivo, resulting in fragments
largely attempt to recapitulate the situation that is thought to
occur in vivo.
The most striking finding from the in vitro studies was the
identification of TFIIF as a novel direct target in mutant htt-
mediated transcriptional repression. Although there have
been reports linking TFIIF to the function of transcription ac-
tivators and repressors (Frejtag et al., 2001), this study pro-
vides the first direct connection between TFIIF and transcrip-
tional repression induced by a polyQ expansion protein.
RAP30, a subunit of TFIIF, appears to consist of three func-
tional domains. The N-terminal domain of RAP30 is thought
to bind RAP74 (Tan et al., 1995), the central region binds
minal domain binds DNA (Garrett et al., 1992). In this study,
we found that mutant htt had a strong affinity for RAP30. Be-
cause RAP30 lacks a Q-rich domain, its interaction with mu-
tant htt is likely mediated through an alternative interface.
Crystal structure of the N-terminal fragments of RAP30
and RAP74 have been shown to adopt a triple-barrel struc-
ture with multiple b sheets (Gaiser et al., 2000). Since mutant
(Perutz et al., 2002), it is possible that the RAP30 mutant htt
interaction involves contact between b sheet structures.
Such a structure-based interference mechanism is consis-
tent with our finding that expansion of glutamines in mutant
htt enhanced its affinity for RAP30. Thus, mutant htt may tar-
get not only polyQ-containing proteins, but also non-polyQ
proteins with specific b sheet structures. It should be noted
thatadditionof Congo red, ab sheet-reactive reagent, to our
in vitro system did not prevent mutant htt-mediated tran-
scriptional repression, possibly due to its inability to prevent
mutant htt from forming protofibrils in vitro (Poirer et al.,
An important aspect revealed by our study is that mutant
htt has a higher affinity for RAP30 than wt htt and may com-
pete with RAP74 for interaction with RAP30. Because an
intact TFIIF complex is required for efficient initiation and
elongation of transcription at least for some promoters, we
hypothesize that TFIIF dissociation will contribute to tran-
scriptional dysregulation by mutant htt. It is conceivable that
mutant htt, which has a higher affinity for RAP30, when it ac-
cumulates in both the cytoplasm and nucleus could cause
less TFIIF to be formed in the cytoplasm and more TFIIF to
be disrupted in the nucleus. Such a scenario will likely result
in a general decrease of transcription in HD cells, as has
been observed (Hoshino et al., 2004). In several DNA micro-
array studies, the level of RNA Pol II large subunit has been
shown to increase in mutant HD brain (Luthi-Carter et al.,
2002a). Since the role of TFIIF in transcription is dependent
on its interaction with RNA Pol II, we speculate that elevated
levels of RNA Pol II subunits in HD cells may arise as a com-
However, in vitro, adding excess RNA Pol II did not rescue
the htt-mediated repression.
By contrast, our findings showed that overexpression of
RAP30 is able to abrogate transcriptional repression and
rescue the cellular toxicity induced by mutant htt in primary
striatal neurons. There are two potential explanations. One
possibility is for RAP30 to interact with mutant htt and com-
pete it away from other htt-interacting partners. Another
possibility is for RAP30 to drive the formation of more TFIIF
complexes, thereby potentiating transcription of important
genesinvolved inneuronal survival. Anintriguing observation
we made is that overexpression of RAP74 alone could in-
duce significant cellular toxicity in striatal neurons. This sug-
gests that the chronic release of free RAP74 from TFIIF may
contribute to the progressive nature of HD pathogenesis.
Thus, our data favor the mechanism in which RAP30 can
protect the striatal neurons by promoting TFIIF complex for-
mation. To better understand how much the TFIIF-mediated
mechanism contributes to the selective neuronal death
during HD pathogenesis, it will be important to identify
those genes whose transcription in striatal neurons is partic-
ularly sensitive to both mutant htt and RAP74 in future inves-
Taking our in vitro and in vivo observations together with
previous studies, we propose the following model for how
mutant htt represses Sp1-dependent gene expression in
neurons (Figure 8D). In normal cells, Sp1 is recruited to
GC-box-containing promoters through its DNA binding do-
main. Once bound to DNA, Sp1 utilizes its multiple gluta-
mine-rich activation domains to target components of the
basal transcription machinery, one of which is TAF4, a sub-
unit of TFIID. In a multistep recruiting process involving TFIIA,
TFIID, TFIIB, TFIIE, TFIIF, TFIIH, RNA Pol II, and CRSP, the
scription. In HD cells, soluble nuclear mutant htt fragment is
free to bind Sp1 through direct protein interactions, thus se-
questering this key transcriptional activator from binding to
its cognate GCboxes. Furthermore, mutant htt canalso pre-
vent Sp1-mediated recruitment of TFIID through its interac-
tion with TAF4. In the case where there is already an Sp1-
TFIID complex formed at the promoter, mutant htt could
subsequently disrupt the stepwise PIC assembly by target-
ing TFIIF, an essential transcription factor important for initia-
tion, promoter escape, and elongation at certain promoters.
htt will have differential effects because these multiple tran-
scription factor targets may be differentially required for criti-
cal functions and rate-limiting transactions at specific gene
tential mechanism by which mutant htt can selectively target
Cell 123, 1241–1253, December 29, 2005 ª2005 Elsevier Inc. 1251
an activator (Sp1) and multiple components of the core ma-
chinery (TFIID and TFIIF) to interfere with various stages of
the transcription process. We anticipate that this model will
undergo further refinements as more gene regulatory targets
for mutant htt are identified and their molecular consequen-
Plasmids and Reagents
Bacterial expression constructs for His6-tagged N-terminal human htt
(1–171aa; Htt-23QP and Htt-120QP with 23 and 120 polyQ, respectively)
were kindly provided by X.-J. Li (Emory University; Li et al., 2002). Bacte-
rial expression constructs for His6-tagged human htt exon 1 with different
numbers of polyQ (25QP, 46QP, and 97QP with 25, 46, and 97 polyQ,
respectively) were subcloned into pET28a (Novagen) from corresponding
pcDNA3.1-Htt-exon 1-GFP constructs kindly provided by L. Thompson
GST-htt N-terminal fragments were described (Dunah et al., 2002).
cDNAs encoding the first 480 aa of human htt (Htt480-17 and Htt480-
68 with 17 and 68 polyQ, respectively) and TFIIF subunits (RAP30 and
RAP74) were subcloned into pCMV-Tag3B and pcDNA3-Flag vector, re-
spectively. The D2 luciferase construct, D2-75Luc, was generated by
subcloning the D2 promoter region from D2-75CAT into pGL3-basic vec-
tor (Promega). Antibodies against His6-probe (sc-803), and RAP30 (sc-
236) were obtained from Santa Cruz. Anti-myc antibody (9E10) was ob-
tained from ATCC.
Expression, Purification of Recombinant Proteins,
and GST Pull-Down Analysis
His6-tagged htt fragments were expressed and purified as described (Li
et al., 2002). Eluted proteins were dialyzed to HEMG buffer (25 mM
HEPES [pH 7.6], 0.1 mM EDTA, 12.5 mM MgCl2, 10% Glycerol, 0.2
mM PMSF, 1 mM DTT, 1 mM sodium metabisulfite) containing 0.2 M
KCl. After dialysis, precipitated proteins were removed by centrifugation,
and remaining soluble proteins were frozen in liquid nitrogen and kept at
?80ºC in small aliquots. GST-fusion htt fragments were expressed and
purified as described (Scherzinger et al., 1997). [35S]-methionine-labeled
proteins were produced using TNT quick-coupled transcription/transla-
tion system (Promega). GST pull downs were performed in 100 ml
HENG-0.1 M buffer (25 mM HEPES [pH 7.6], 0.1mM EDTA, 10% Glyc-
erol, 0.1% NP40, 0.2 mM PMSF, 1 mM DTT, 1 mM sodium metabisulfite,
0.1 M NaCl) containing 0.2 mg/ml BSA and 200 mg/ml ethidium bromide
for 3 hr at 4ºC. After binding, beads were washed five times, and bound
proteins were analyzed by SDS-PAGE followed by immunoblotting or ex-
posure to PhosphorImager Screen.
In Vitro Transcription
Recombinant basal transcription factors TFIIA, TFIIB, TFIIE, and TFIIF
were purified and kindly provided by C. Inouye. Flag-tagged human
Sp1 and Gal4-VP16 were immunopurified using M2 resin. Human TFIID,
TFIIH, RNA Pol II, and CRSP were immunopurified from column fractions
derived from HeLa nuclear extracts using specific monoclonal antibodies.
In vitro transcription assay using chromatin templates had been de-
scribed (Lemon et al., 2001; Naar et al., 1999). The purified N-terminal
htt fragments were mixed with basal transcription factors on ice before
2- to 4-fold extra of each basal factor was added along with purified htt
Neuronal Cell Culture, Transfection, and Immunocytochemistry
Striatal neurons were prepared from embryonic (E17–18) rats and cul-
tured as described (Dunah et al., 2002). Transfection was performed us-
ing Lipofectamine 2000 (Invitrogen). For each transfection in a 12-well
plate, 3.3 ml of Lipofectamine 2000 reagent and 1.8 mg of plasmid DNA
were used. Transfected neurons were scored for cell death as described
(Dunah et al., 2002). For reporter assay, cultured striatal cells were trans-
fected at DIV 7–DIV 9. Luciferase activity was determined using Prome-
ga’s Dual luciferase kit. For immunocytochemistry, cultured striatal neu-
rons were transfected at DIV 4 and analyzed as described (Dunah
et al., 2002). Antibodies used for staining included aGFP (Chemicon),
aFlag (Sigma), aRAP30 (BD Biosciences), and aRAP74 (Santa Cruz,
sc-235). All experiments were performed in triplicates and repeated at
least twice. For immunoblotting, cultured striatal neurons were trans-
fected on DIV 3. At 48 hr posttransfection, cells were rinsed with PBS
and lysed in lysis buffer (50 mM TrisHCl [pH 7.4], 150 mM NaCl, 10
mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, 0.5% DOC, and 1%
Triton X-100). After clearing by centrifugation, 90 mg of proteins were re-
solved by SDS-PAGE and analyzed by immunoblotting using aFlag M2
(Sigma) or amyc (Santa Cruz) antibodies.
Cell Culture and Coimmunoprecipitation
COS7 cells were cultured in DMEM supplemented with 10% fetal bovine
serum. Transfections were performed using Lipofectamine 2000. Cells
were harvested 36–48 hr later and lysed in 100 ml (for each well from
a 6-well cell-culture plate) lysis buffer with complete protease inhibitor
cocktail (Roche). For coIP, 80 ml of cell lysates were diluted with 320 ml
binding buffer (50 mM TrisHCl [pH 7.9], 0.1% Triton X-100, 50 mM
NaCl) and immunoprecipitated using aFlag or amyc affinity resin for 4
hr at 4ºC. Beads were washed five times with wash buffer (50 mM TrisHCl
[pH 7.9], 0.1 M NaCl, 0.5% DOC, 0.5% Triton X-100), boiled in sample
buffer, and analyzed by SDS-PAGE and immunoblotting.
For coIP using mouse brain extracts, three striata from 4-month-old
heterozygous KI mice and wt mice were pooled and homogenized with
Tissuemizer in 1 ml of ice-cold CHAPS lysis buffer (40 mM HEPES [pH
7.5], 1 mM EDTA, 10 mM pyrophosphate, 10 mM b-glycerophosphate,
50 mM NaF,1 mM PMSF, 0.3% CHAPS, and complete protease inhibitor
cocktail). The tissue homogenates were briefly sonicated and cleared by
centrifugation. After preclearing with protein G agarose, 1 mg of superna-
tant was incubated with 20 ml of protein G agarose and 4 mg of either
aRAP30 antibody (BD Transduction Lab) or normal mouse IgG for 4 hr
at 4ºC. Beads were then washed four times with lysis buffer and once
with wash buffer (50 mM HEPES [pH 7.5], 1 mM EDTA, and 150 mM
NaCl). Samples were analyzed by SDS-PAGE and immunoblotting with
Chromatin Immunoprecipitation and Multiplex PCR
ChIP was performed using a ChIP kit (Upstate) accordingtothe manufac-
turer’s recommended protocol. Briefly, fresh brain tissues were weighted
and homogenized by forcing the fresh tissue through a 22 G needle. The
homogenized cell suspension was crosslinked in DMEM medium con-
taining 1% formaldehyde for 10 min at room temperature. Chromatin
was immunoprecipitated using either control serum or aRAP30 antibod-
ies (Santa Cruz, sc-236). Immunoprecipitated chromatin was analyzed
by multiplex PCR with all three sets of primer in one tube. Primers for
D2 core promoter sequences are 50-CTGGAGCCAAAAGCAGTCTG-30
(forward) and 50-TCCTTCAGGTTTCCGACGCC-30(reverse); NR1 core
promoter sequences are 50-CCACACGGATGACTGTCCC-30(forward)
and 50-GCGTTGGCGTAAATGCTTGG-30(reverse); GAPDH core pro-
moter sequences are 50-AAGCAGCATTCAGGTCTCTGG-30(forward)
and 50-TTTCCCCTCCTCCCTCTCTTT-30(reverse). Multiplex PCR was
performed using Multiplex PCR Kit (Qiagen) for 30 cycles, with each cycle
extension at 72ºC. All PCR reactions were performed in triplicates. PCR
products were analyzed and quantified by using DNA 500 Chips and Agi-
lent Bioanalyzer (Agilent Technologies).
We thank X.-J. Li and L. Thompson for various htt constructs and
C. Inouye for purified recombinant transcription factors. We also thank
N. Tanese and members of the Tjian Lab for their comments on the
manuscript. We are grateful to J-.H. Cha and C. Benn for helpful
1252 Cell 123, 1241–1253, December 29, 2005 ª2005 Elsevier Inc.
discussions and sharing of unpublished chromatin immunoprecipitation
data. This work was funded by HHMI (R.T.) and NIH with grants to R.T.
(CA025417), D.K. (R01NS050352, P01NS045242). W.Z. was supported
by a postdoctoral fellowship grant from the Hereditary Disease Founda-
tion, HighQ Foundation, and Cure Huntington’s Disease Initiative.
Received: January 3, 2005
Revised: May 31, 2005
Accepted: October 18, 2005
Published: December 28, 2005
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