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: email@example.com (D.K.); firstname.lastname@example.org (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.
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
Albright, S.R., and Tjian, R. (2000). TAFs revisited: more data reveal new
twists and confirm old ideas. Gene 242, 1–13.
Cha, J.H. (2000). Transcriptional dysregulation in Huntington’s disease.
Trends Neurosci. 23, 387–392.
Chen, J.L., Attardi, L.D., Verrijzer, C.P., Yokomori, K., and Tjian, R.
(1994). Assembly of recombinant TFIID reveals differential coactivator re-
quirements for distinct transcriptional activators. Cell 79, 93–105.
Conaway, J.W., Shilatifard, A., Dvir, A., and Conaway, R.C. (2000). Con-
trol of elongation by RNA polymerase II. Trends Biochem. Sci. 25, 375–
Courey, A.J., and Tjian, R. (1988). Analysis of Sp1 in vivo reveals multiple
transcriptional domains, including a novel glutamine-rich activation motif.
Cell 55, 887–898.
DiFiglia, M., Sapp, E., Chase, K., Schwarz, C., Meloni, A., Young, C.,
Martin, E., Vonsattel, J.P., Carraway, R., Reeves, S.A., et al. (1995). Hun-
tingtin is a cytoplasmic protein associated with vesicles in human and rat
brain neurons. Neuron 14, 1075–1081.
DiFiglia, M., Sapp, E., Chase, K.O., Davies, S.W., Bates, G.P., Vonsattel,
J.P., and Aronin, N. (1997). Aggregation of huntingtin in neuronal intra-
nuclear inclusions and dystrophic neurites in brain. Science 277, 1990–
Dunah, A.W., Jeong, H., Griffin, A., Kim, Y.M., Standaert, D.G., Hersch,
S.M., Mouradian, M.M., Young, A.B., Tanese, N., and Krainc, D. (2002).
Sp1 and TAFII130 transcriptional activity disrupted in early Huntington’s
disease. Science 296, 2238–2243.
Frejtag, W., Zhang, Y., Dai, R., Anderson,M.G.,and Mivechi, N.F. (2001).
Heat shock factor-4 (HSF-4a) represses basal transcription through inter-
action with TFIIF. J. Biol. Chem. 276, 14685–14694.
Gaiser, F., Tan, S., and Richmond, T.J. (2000). Novel dimerization fold of
Garrett, K.P., Serizawa, H., Hanley, J.P., Bradsher, J.N., Tsuboi, A., Arai,
N., Yokota, T., Arai, K., Conaway, R.C., and Conaway, J.W. (1992). The
carboxyl terminus of RAP30 is similar in sequence to region 4 of bacterial
sigma factors and is required for function. J. Biol. Chem. 267, 23942–
Gerber, H.P., Seipel, K., Georgiev, O., Hofferer, M., Hug, M., Rusconi, S.,
and Schaffner, W. (1994). Transcriptional activation modulated by homo-
polymeric glutamine and proline stretches. Science 263, 808–811.
Hoshino, M., Tagawa, K., Okuda, T., and Okazawa, H. (2004). General
linked to the presence of inclusion bodies. Biochem. Biophys. Res. Com-
mun. 313, 110–116.
Lemon, B., Inouye, C., King, D.S., and Tjian, R.(2001). Selectivity of chro-
matin-remodelling cofactors for ligand-activated transcription. Nature
Li, S.H., Cheng, A.L., Zhou, H., Lam, S., Rao, M., Li, H., and Li, X.J.
(2002). Interaction of Huntington disease protein with transcriptional acti-
vator Sp1. Mol. Cell. Biol. 22, 1277–1287.
Luthi-Carter, R., Hanson, S.A., Strand, A.D., Bergstrom, D.A., Chun, W.,
Peters, N.L., Woods, A.M., Chan, E.Y., Kooperberg, C., Krainc, D., et al.
(2002a). Dysregulation of gene expression in the R6/2 model of polyglut-
amine disease: parallel changes in muscle and brain. Hum. Mol. Genet.
Luthi-Carter, R., Strand, A.D., Hanson, S.A., Kooperberg, C., Schilling,
G., La Spada, A.R., Merry, D.E., Young, A.B., Ross, C.A., Borchelt,
D.R., and Olson, J.M. (2002b). Polyglutamine and transcription: gene ex-
pression changes shared by DRPLA and Huntington’s disease mouse
models reveal context-independent effects. Hum. Mol. Genet. 11,
Mangiarini, L., Sathasivam, K., Seller, M., Cozens, B., Harper, A., Hether-
ington, C., Lawton, M., Trottier, Y., Lehrach, H., Davies, S.W., and Bates,
G.P. (1996). Exon 1 of the HD gene with an expanded CAG repeat is suf-
ficient to cause a progressive neurological phenotype in transgenic mice.
Cell 87, 493–506.
McCracken, S., and Greenblatt, J. (1991). Related RNA polymerase-
binding regions in human RAP30/74 and Escherichia coli sigma 70. Sci-
ence 253, 900–902.
Menalled, L.B., Sison, J.D., Dragatsis, I., Zeitlin, S., and Chesselet, M.F.
(2003). Time course of early motor and neuropathological anomalies in
a knock-in mouse model of Huntington’s disease with 140 CAG repeats.
J. Comp. Neurol. 465, 11–26.
Naar, A.M., Beaurang, P.A., Zhou, S., Abraham, S., Solomon, W., and
Tjian, R. (1999). Composite co-activator ARC mediates chromatin-di-
rected transcriptional activation. Nature 398, 828–832.
Okazawa, H. (2003). Polyglutamine diseases: a transcription disorder?
Cell. Mol. Life Sci. 60, 1427–1439.
Perutz, M.F., Finch, J.T., Berriman, J., and Lesk, A. (2002). Amyloid fibers
are water-filled nanotubes. Proc. Natl. Acad. Sci. USA 99, 5591–5595.
Poirer, M.A., Li, H., Macosko, J., Cai, S., Amzel, M., and Ross, C.A.
(2002). Huntingtin spheroids and protofibrils as precursors in polyglut-
amine fibrilization. J. Biol. Chem. 277, 41032–41037.
Rubinsztein, D.C., Barton, D.E., Davison, B.C., and Ferguson-Smith,
M.A. (1993). Analysis of the huntingtin gene reveals a trinucleotide-length
polymorphism in the region of the gene that contains two CCG-rich
stretches and a correlation between decreased age of onset of Hunting-
ton’s disease and CAG repeat number. Hum. Mol. Genet. 2, 1713–1715.
Ryu, S.,Zhou, S.,Ladurner, A.G.,and Tjian, R. (1999). Thetranscriptional
cofactor complex CRSP is required for activity of the enhancer-binding
protein Sp1. Nature 397, 446–450.
Saudou, F., Finkbeiner, S., Devys, D., and Greenberg, M.E. (1998). Hun-
tingtin acts in the nucleus to induce apoptosis but death does not corre-
late with the formation of intranuclear inclusions. Cell 95, 55–66.
Scherzinger, E., Lurz, R., Turmaine, M., Mangiarini, L., Hollenback, B.,
Hasenbank, R., Bates, G.P., Davies, S.W., Lehrach, H., and Wanker,
E.E. (1997). Huntingtin-encoded polyglutamine expansions form amy-
loid-like protein aggregates in vitro and in vivo. Cell 90, 549–558.
Sipione, S., and Cattaneo, E. (2001). Modeling Huntington’s disease in
cells, flies, and mice. Mol. Neurobiol. 23, 21–51.
B.L., Kazantsev, A., Schmidt, E., Zhu, Y.Z., Greenwald, M., et al. (2001).
Histone deacetylase inhibitors arrest polyglutamine-dependent neurode-
generation in Drosophila. Nature 413, 739–743.
Sugars, K.L., and Rubinsztein, D.C. (2003). Transcriptional abnormalities
in Huntington disease. Trends Genet. 19, 233–238.
Tan, S., Conaway, R.C., and Conaway, J.W. (1995). Dissection of tran-
scriptionfactorTFIIF functional domains required for initiation and elonga-
tion. Proc. Natl. Acad. Sci. USA 92, 6042–6046.
Tanese, N., Pugh, B.F., and Tjian, R. (1991). Coactivators for a proline-
rich activator purified from the multisubunit human TFIID complex. Genes
Dev. 5, 2212–2224.
The Huntington’s Disease Collaborative Research Group. (1993). A novel
gene containing a trinucleotide repeat that is expanded and unstable on
Huntington’s disease chromosomes. Cell 72, 971–983.
Cell 123, 1241–1253, December 29, 2005 ª2005 Elsevier Inc. 1253