Role of the CCAAT-Binding Protein NFY in SCA17
Li-Ching Lee1., Chiung-Mei Chen2., Hao-Chun Wang1, Hsiao-Han Hsieh1, I-Sheng Chiu1, Ming-Tsan Su1,
Hsiu-Mei Hsieh-Li1, Chung-Hsin Wu1, Guan-Chiun Lee1, Guey-Jen Lee-Chen1*, Jung-Yaw Lin1,3*
1Department of Life Science, National Taiwan Normal University, Taipei, Taiwan, 2Department of Neurology, Chang Gung Memorial Hospital, Chang-Gung University
College of Medicine, Taipei, Taiwan, 3Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan
Spinocerebellar ataxia 17 (SCA17) is caused by expansion of the polyglutamine (polyQ) tract in human TATA-box binding
protein (TBP) that is ubiquitously expressed in both central nervous system and peripheral tissues. The spectrum of SCA17
clinical presentation is broad. The precise pathogenic mechanism in SCA17 remains unclear. Previously proteomics study
using a cellular model of SCA17 has revealed reduced expression of heat shock 70 kDa protein 5 (HSPA5) and heat shock
70 kDa protein 8 (HSPA8), suggesting that impaired protein folding may contribute to the cell dysfunction of SCA17 (Lee et
al., 2009). In lymphoblastoid cells, HSPA5 and HSPA8 expression levels in cells with mutant TBP were also significantly lower
than that of the control cells (Chen et al., 2010). As nuclear transcription factor Y (NFY) has been reported to regulate HSPA5
transcription, we focused on if NFY activity and HSPA5 expression in SCA17 cells are altered. Here, we show that TBP
interacts with NFY subunit A (NFYA) in HEK-293 cells and NFYA incorporated into mutant TBP aggregates. In both HEK-293
and SH-SY5Y cells expressing TBP/Q61,79, the level of soluble NFYA was significantly reduced. In vitro binding assay revealed
that the interaction between TBP and NFYA is direct. HSPA5 luciferase reporter assay and endogenous HSPA5 expression
analysis in NFYA cDNA and siRNA transfection cells further clarified the important role of NFYA in regulating HSPA5
transcription. In SCA17 cells, HSPA5 promoter activity was activated as a compensatory response before aggregate
formation. NFYA dysfunction was indicated in SCA17 cells as HSPA5 promoter activity reduced along with TBP aggregate
formation. Because essential roles of HSPA5 in protection from neuronal apoptosis have been shown in a mouse model,
NFYA could be a target of mutant TBP in SCA17.
Citation: Lee L-C, Chen C-M, Wang H-C, Hsieh H-H, Chiu I-S, et al. (2012) Role of the CCAAT-Binding Protein NFY in SCA17 Pathogenesis. PLoS ONE 7(4): e35302.
Editor: Xiao-Jiang Li, Emory University, United States of America
Received November 7, 2011; Accepted March 13, 2012; Published April 17, 2012
Copyright: ? 2012 Lee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants NSC97-2311-B-003-008-MY3, NSC97-2311-B-182A-001-MY3, NSC98-2321-B-003-004 and NSC100-2325-B-003-001
from National Science Council, Executive Yuan, 96TOP001 and NTNU100-D-02 from National Taiwan Normal University Taipei, and CMRGP38138 from Chang
Gung Memorial Hospital, Taipei, Taiwan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org (G-JL-C); email@example.com (J-YL)
. These authors contributed equally to this work.
Spinocerebellar ataxia type 17 (SCA17) is an autosomal
dominant ataxia caused by an expanded polyglutamine (polyQ)
in a general transcription initiation factor, the TATA-box binding
protein (TBP) [1,2]. As a TATA-box recognition component, TBP
and TAFs (for TBP-associated factors) form general transcription
factor IID (TFIID) for RNA polymerase II to bind its promoter. In
humans, the polyQ tract in TBP normally contains 25,42
glutamine residues  and expanded alleles ranging from 43 to 66
glutamines have been shown to be associated with the disease
[4,5]. Expanded polyQ tracts enhanced the interaction of TBP
with the general transcription factor IIB (TFIIB) . In addition to
progressive gait and limb ataxia, the broad phenotypic spectrum of
this rare disorder includes seizure, cognitive dysfunctions,
psychiatric symptoms, and pyramidal and extrapyramidal features
such as spasticity, dystonia, chorea, and parkinsonism (review in
Protein misfolding and aggregation in the brain have been
implicated as a common molecular pathogenesis of various
neurodegenerative diseases. Our previously proteomics study
using a cellular model of SCA17 has revealed reduced expression
of heat shock 70 kDa protein 5 (HSPA5) , a major endoplasmic
reticulum (ER) chaperone and master regulator of unfolded
protein response (UPR) , suggesting that impaired protein
folding in ER may contribute the cell dysfunction of SCA17. In
lymphoblastoid cells, HSPA5 expression level in cells with mutant
TBP was also significantly lower than that of the control cells .
Moreover, elimination of HSPA5 in Purkinje cells leads to
accelerated cerebellar degeneration in a mouse model . Thus
investigating the regulation of HSPA5 expression in SCA17 cells
may shed light on the pathogenesis of SCA17 and lead to
development of therapeutics for the disease.
Nuclear transcription factor Y (NFY) is a unique evolutionarily
conserved transcription factor that binds to CCAAT motifs in the
promoter regions of a variety of genes. The trimeric NFY is
formed by three subunits, NFYA, NFYB, and NFYC. The
sequence specific interactions of the complex are made by the
NFYA subunit, suggesting a role as the regulatory subunit [12,13].
Recent studies focusing on NFY activity and heat shock 70 kDa
PLoS ONE | www.plosone.org1April 2012 | Volume 7 | Issue 4 | e35302
protein 1A (HSPA1A) expression in the brain of a Huntington’s
disease (HD) mouse model have shown that mutant Huntingtin
(Htt) aggregates sequester NFYA and NFYC leading to the
reduction of HSPA1A gene expression, indicating NFY compo-
nents as modulators of the HD pathological process . Because
NFY has been reported to regulate HSPA5 transcription , we
investigated if NFY activity and HSPA5 expression are altered in
SCA17 cells. We provided evidence to support the postulation that
mutant TBP aggregates sequester NFYA to reduce functional
NFY, leading to the reduction of HSPA5 gene expression.
Considering the role of HSPA5 played in the SCA17 pathological
process, therapeutic interventions for SCA17 may be developed
through modulating NFYA expression.
Incorporation of NFYA into mutant TBP aggregates in
The trimeric NFY binds to CCAAT sequence to regulate gene
transcription. In HD model mouse brain, mutant Htt reduces
HSPA1A expression through sequestration of NFY components
and reduction of NFY binding to HSPA1A promoter . To
determine whether NFYA, the regulatory subunit of NFY,
incorporates into mutant TBP aggregates, HA-tagged TBP
(normal TBP/Q36and expanded TBP/Q61, TBP/Q79) and His-
tagged NFYA constructs were transiently expressed in HEK-293
cells for 48 hr for immunocytochemical staining (TBP and NFYA)
and fluorescence microscopy examination. As shown in Fig. 1A,
while expressed TBP/Q36was seen as diffuse nuclear staining, the
expressed TBP/Q61and TBP/Q79protein formed aggregates and
NFYA co-localized with TBP/Q61 and TBP/Q79 in nuclear
inclusions. When 293-derived cells with inducible TBP/Q36,79
expression for 96 hr and NFYA transfection were examined,
positive nuclei with punctuate inclusions with NFYA co-localiza-
tion were also visible in TBP/Q61,79cells (Fig. 1B).
In vivo interaction between NFYA and TBP/Q36,79
Next, a half-in vivo co-immunoprecipitation was performed to
assess the intracellular association of NFYA with TBP. HEK-293
cells were co-transfected with HA-tagged TBP/Q36,79and His-
tagged NFYA, and the expression of TBP and NFYA proteins
were examined by Western blotting using anti-TBP and anti-
NFYA antibodies. As shown in the left panel of Fig. 2A, the TBP
antibody detected 47,56 kDa HA-tagged TBP/Q36,79proteins
in transfected cells, in addition to a small amount of endogenous
43 kDa TBP protein. While two variants of NF-YA which result
from differential splicing was reported , the NFYA antibody
detected a major 43 kDa His-tagged NFYA in transfected cells.
The cell lysates were subjected to immunoprecipitation with anti-
NFYA antibody and Western blotting with anti-TBP, or vice
versa. As shown in the right panel of Fig. 2A, immunoblotting
analysis showed that transfected HA-tagged TBP carrying 36,79
glutamines were co-immunoprecipitated with NFYA, although
endogenous TBP seems to be more capable of binding to NFYA.
Similarly, transfected His-tagged NFYA was co-immunoprecipi-
tated with TBP carrying 36,79 glutamines. The presence of gel
top bands in TBP/Q61and TBP/Q79-expressing cells, but not in
TBP/Q36-expressing cells, indicates the formation of SDS-
insoluble aggregates by TBP/Q61 and TBP/Q79 and NFYA
forms SDS-insoluble aggregates with mutant TBP. When the
TBP/Q36,79and NFYA co-transfected samples were subjected to
filter trap assay and stained with NFYA or TBP antibody,
incorporation of NFYA into SDS-insoluble aggregates with TBP/
Q61and TBP/Q79was significantly increased (Fig. 2B). The above
data indicated that NFYA interacts with TBP carrying 36,79
glutamines and NFYA is preferentially incorporated into SDS-
insoluble aggregates with TBP/Q61and TBP/Q79in HEK-293
In vitro interaction between NFYA and TBP/Q36,61
Both glutathione S-transferase and thioredoxin soluble protein
tags have been used in E. coli to increase recombinant protein
expression level and/or solubility [17,18]. Thus GST tag and Trx
tag were fused to N-terminal TBP and NFYA respectively, for
recombinant protein production in E. coli. The induced His-tagged
GST-TBP/Q36,61(50,56 kDa) and Trx-NFYA (60 kDa) fusion
proteins were purified to apparent homogeneity from cell extracts
and verified by Western blotting with anti-TBP and anti-NFYA
antibodies (data not shown).
To examine whether the in vivo interaction between TBP and
NFYA is direct, we conducted a series of GST pull-down assay
using the purified GST-His6-TBP/Q36,61-His6 as bait protein
and Trx-His6-NFYA-His6as prey protein. This bacterial expres-
sion of both bait and prey proteins is commonly used to study
protein-protein interaction [19,20]. GST-His6-TBP/Q36,61-His6
or GST-His6-His6alone (as a negative control) was incubated with
Trx-His6-NFYA-His6. Although the His tag allowed purification
of the TBP and NFYA fusion protein to 90% homogeneity,
undesirable proteolysis by a trace contaminant protease 
generates various amount of cleaved products after GST pull-
down. Apart from a light background band seen with GST alone,
both wild type (36 glutamines) and expansion mutant (45 and 61
glutamines) TBP could pull down NFYA (Fig. 3). The data
indicate that the recombinant Trx-His6-NFYA-His6 directly
interacts with GST-His6-TBP/Q36,61-His6.
NFYA modulation of HSPA5 promoter activity
The proximal region of the human HSPA5 promoter contains
three ER stress response elements consisting of CCAAT-(N)9-
CCACG, in which CCAAT interacts with NFY . Positive
regulation of HSPA5 transcription by NFY has been shown in
HeLa cells by Northern blot analysis . HSPA5 promoter
construct and NFYA cDNA were prepared and tested in HEK-
293 cell transfection assay. As shown in Fig. 4A, luciferase level in
cells co-transfected with NFYA cDNA and HSPA5 reporter
plasmid was 198% (P=0.036) of that in cells transfected with
HSPA5 reporter plasmid alone. RNA interference assay was
performed to examine the role of endogenous NFYA in regulation
of HSPA5 gene transcription. Co-transfection of NFYA siRNA
with HSPA5 reporter construct resulted in reduction in promoter
activity (71%, P=0.005). To ensure the over-expression and knock
down of NFYA, immunoblot analysis was performed to assess the
expression levels of NFYA. As shown in Fig. 4B, while a large
amount (382%, P=0.005) of NFYA was seen in cDNA-transfected
cells, the amounts of NFYA were reduced (74%, P=0.009) in
To further evaluate if NFYA could modulate HSPA5 gene
expression, real-time PCR and immunoblot analysis were
performed to examine endogenous HSPA5 expression in cells
transfected with NFYA cDNA or siRNA. As shown in Fig. 4C and
4D, 125% RNA (P=0.048) and 114% protein (P=0.033) were
observed in cells transfected with NFYA cDNA and 79% RNA
(P=0.037) and 80% protein (P=0.016) were seen in cells
transfected with NFYA siRNA, when respectively compared with
the control. Thus the results suggest that NFYA could modulate
HSPA5 gene expression through the regulation of the promoter
Role of NFY in SCA17
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The incorporation of NFYA into mutant TBP aggregates in
SCA17 cells raises the possibility that NFYA function was altered.
To examine this, luciferase reporter assay to assess the HSPA5
expression was performed in 293-derived cells inducibly expressing
normal TBP/Q36 or expanded TBP/Q61 and TBP/Q79. As
shown in Fig. 4E, when luciferase activity for HSPA5 reporter was
set to 100% in cells expressing TBP/Q36for 2 days, luciferase
activity driven by HSPA5 promoter in cells expressing TBP for
2,6 days were 100,115% for TBP/Q36cells, 130,139% for
TBP/Q61cells, and 187,221% for TBP/Q79cells. Compared to
the cells expressing TBP for 2 days (TBP/Q36, 100%; TBP/Q61,
130%; TBP/Q79, 221%), while no significant change in HSPA5
promoter activity was seen in cells expressing TBP/Q36
P=0.243,0.490) for 4 and 6 days, significant decrease of HSPA5
promoter activity was seen in cells expressing TBP/Q79for 4 days
(188%, P=0.035) and 6 days (187%, P=0.025). Together with
the observed TBP aggregates with NFYA co-localization in 293-
derived TBP/Q79 cells (Fig. 1B), the results suggest that the
activation of HSPA5 transcription is altered in TBP/Q79cells with
NFYA overexpression to reduce TBP aggregation
To determine whether NFYA could suppress aggregation of
mutant TBP, we transiently co-expressed NFYA with TBP/Q36,
TBP/Q61 or TBP/Q79 in HEK-293 cells. Fig. 5A shows the
images of fluorescence microscopy examination after immuno-
staining using TBP antibody (red). Without NFYA co-transfection,
cells built visible aggregates were 7.8% for TBP/Q36 cells.
Significantly increased visible aggregates were seen in TBP/Q61
cells (29.0%, P=0.000) and TBP/Q79cells (23.5%, P=0.000).
Co-transfection of NFYA effectively suppressed aggregate forma-
tion in TBP/Q36(3.3% vs. 7.8%, P=0.046), TBP/Q61(9.4% vs.
29.0%, P=0.003) as well as TBP/Q79 (10.5% vs. 23.5%,
P=0.000) cells (Fig. 5B).
NFYA dysfunction in SCA17 cells
To examine if expression of TBP/Q61,79suppressed the level
of soluble NFYA protein, 293-derived cells with inducible TBP/
Q36,79expression were examined. After six days induction, HA-
tagged TBP-protein levels were first examined by Western blotting
using TBP antibody. As shown in Fig. 6A, in addition to the
43 kDa endogenous TBP protein, the TBP antibody detected
47 kDa TBP/Q36-HA, 50 kDa TBP/Q61-HA and 53 kDa TBP/
Q79-HA proteins in doxycycline induced TBP cells (178%,214%
of the endogenous TBP). In cells expressing TBP/Q61,79-HA
protein, a significant reduction of the endogenous soluble NFYA
protein was observed compared to the non-induced TBP/Q61,79
cells (80.1,74.8% vs. 100%, P=0.007,0.014) or induced TBP/
Q36cells (80.1,74.8% vs. 99.4%, P=0.038,0.014) (Fig. 6B).
The pcDNA5/FRT/TO-TBP constructs containing Q36, Q61
and Q79were also used to generate isogenic SH-SY5Y TBP lines.
After six days induction, TBP and NFYA levels and were
examined by Western blotting using TBP and NFYA antibodies.
Although levels of the induced TBP/Q36,79-HA proteins were
low (10.0%,18.5%) compared to the endogenous TBP (Fig. 7A),
in SH-SY5Y cells expressing TBP/Q61,79-HA protein, a
significant reduction of the endogenous soluble NFYA protein
was observed compared to the non-induced TBP/Q61,79cells
(76.2,74.1% vs. 100%, P=0.028,0.013) or induced TBP/Q36
cells (76.2,74.1% vs. 105.8%, P=0.035,0.023) (Fig. 7B).
Positive nuclei with punctuate inclusions with NFYA co-localiza-
tion were also detected in TBP/Q61(data not shown) and TBP/
Q79cells (Fig. 7C).
Figure 1. Localization of NFYA in TBP/Q36, ,79expressing 293 cells. (A) HEK-293 cells were transiently co-transfected with NFYA and TBP/
Q36,79. After 2 days, cells were fixed and stained with antibodies specific for TBP (red) and NFYA (yellow). (B) Isogenic 293 cells inducibly expressing
TBP/Q36,79were transiently transfected with NFYA. After 4 days, cells were fixed and stained with antibodies specific for TBP (red) and NFYA (yellow).
In both a and b, nuclei were counterstained with DAPI (blue). (The scale bar=8 mm).
Role of NFY in SCA17
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Previous studies have suggested the involvement of transcrip-
tional dysregulation, including increased Cre-dependent transcrip-
tional activity, reduced TFIIB occupancy of the Hspb1 promoter,
and reduced binding of TBP to DNA, in SCA17 pathogenesis
[6,23,24]. In the present study, we identified NFY component A
(NFYA) as a TBP aggregates-interacting protein in HEK-293 cells
(Fig. 1 and Fig. 2). The reporter gene assay and examination of
endogenous HSPA5 expression further indicated the positive
regulation of NFYA on HSPA5 transcription (Fig. 4A,4D). The
result is consistent with the reported mutant TBP binding to NFY
and inhibiting its association with HspA5 promoter to reduces
HspA5 expression in SCA17 knock-in mice . In addition, it
has been shown that mutant Htt caused increased levels of reactive
oxygen species (ROS) in neuronal and non-neuronal cells  and
ER stress caused by ROS activated HSPA5 expression . As
TBP with a polyQ expansion significantly increased ROS
generation and induced oxidative stress in 293 cells (data not
shown), the marked increase of HSPA5 reporter activity in TBP/
Q79cells (221%, P=0.004) (Fig. 4E) may be due to increased
levels of ROS. Along with aggregate formation after 4,6 days
induction of TBP expression (Fig. 1B), HSPA5 transcription in
TBP/Q79cells was significantly decreased (Fig. 4E). These data
suggest that the NFY complex loses its function due to
sequestration of NFYA to mutant TBP aggregates, resulting in
the reduction of HSPA5 gene transcription. The NFYA
dysfunction was also supported by the reduced heat shock
70 kDa protein 8 (HSPA8, with two reverse CCAAT motifs)
promoter activity along with TBP aggregate formation (data not
shown). Indeed, previously reduced expression of HSPA8 has been
observed in SCA17 cells [8,10].
HSPA5 (also known as 78 kDa glucose-regulated heat shock
protein GRP-78) is involved in the folding and assembly of
proteins in the endoplasmic reticulum. Abnormal protein folding
due to decreased rate of HSPA5 hydrolysis and disturbed SIL1-
HSPA5 interaction is the cause of Marinesco-Sjogren syndrome
that is characterized by ataxia, progressive myopathy and cataract
. In UPR pathway, NFY has been reported to control ER
stress-inducible HSPA5 transcription to increase the protein-
folding capacity of ER . De-activation of HSPA5 transcription
Figure 2. Interaction of NFYA and TBP/Q36, ,79in vivo. (A) HEK-293 cells were transiently co-transfected with NFYA and TBP/Q36,79. After 48 h,
cell lysates were prepared (Input, left panel) and immunoprecipitations (IP, right panel) were performed with anti-NFYA or anti-TBP (1TBP18)
antibody. Rabbit or mouse IgG was used as a negative control for IP. Cell lysates and immunoprecipitates were analyzed with anti-TBP or anti-NFYA
antibody. (B) After 48 h, insoluble pellets from NFYA and TBP/Q36,79co-transfected cells were lysed in SDS buffer and the indicated amounts of cell
lysates (5,20 mg) were trapped on an acetate membrane. The filter was probed with antibody against NFYA and TBP, respectively.
Role of NFY in SCA17
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in TBP/Q79cells by TBP aggregates may lead to impairment of
protein folding, reduction of stress response, and induction of cell
death. As HSPA5 overexpression leading to suppression of mutant
TBP aggregation , reduced expression of HSPA5 by NFYA
sequestration may further accelerate aggregation of mutant TBP.
This would also induce incorporation of some other transcriptional
factors into aggregates, which may result in altered expression of
their target genes. These phenomena might be cooperating
together to promote SCA17 progression.
In addition to de-activation of HSPA5 transcription, reduction
of functional NFY may also lead to altered expressions of other
NFY target genes. NFY interacts specifically with the CCAAT
motif, one of the common promoter elements present in the
proximal promoter of numerous mammalian genes transcribed by
RNA polymerase II . By cooperative interactions with other
transcription factors that bind to a specific promoter, NFY
[29,30,31,32,33,34]. Thus expressions of NFY target genes other
than HSPA5 might be also affected. In HD R6/2 mouse brain,
reduced expression of HSP70 and 12 other genes containing more
than two CCAAT sequences in their putative promoter regions
might be caused by NFYA sequestration . Further studies will
be needed to understand the relationship between the NFY
function and the other altered gene expressions in SCA17 models.
Unlike preferential interaction with aggregated form of mutant
Htt in neuro2a cells , NFYA interacts with both soluble and
SDS-insoluble TBP in HEK-293 cells (Fig. 2). Given the proximity
of TATA-box and CCAAT-box, the two recognition-factors for
these motifs are in close distance to each other. This may mimic
direct interaction of TBP and NFYA and explain the advantage of
endogenous TBP binding to NFYA (Fig. 2A). In HeLa cells, it has
been shown that NFY controls ER stress-inducible transcription
through recruitment of both ATF6(N) and TBP . Using in vitro
binding assay, we demonstrated that NFYA and TBP interact
directly (Fig. 3). This NFYA-TBP interaction may facilitate the
incorporation of NFYA into mutant TBP aggregates.
The transcription activation domain of NFYA is rich in
glutamine and hydrophobic residues, and shows amino acid
sequence similarity with the glutamine-rich activation domain of
transcription factor Sp1 . Since NFYA interacts physically
with Sp1 in-vitro  and multiple NFY- and SP1-binding sites
exist within HSPA5 proximal promoter region, SP1 may interact
with NFY to regulate HSPA5 expression. In that context, SP1
could be another direct or indirect target of mutant TBP to induce
the de-activation of HSPA5 promoter activity in SCA17 cells.
Whether binding of SP1 to HSPA5 promoter DNA in SCA17 is
reduced remains to be determined.
Although SH-SY5Y cells with full-length TBP can not be
greatly induced, the SH-SY5Y cells with N-terminal TBP/Q36,79
had been established with several fold transgene expression
compared to endogenous TBP expression (data not shown). Like
TBP, most polyQ disease proteins are widely expressed and some
are critical for cellular function. However, the selective neurode-
generation is paradoxical. How the polyQ domain contributes to
the normal function of proteins and the expanded polyQ induces
selective neuropathology remain unclear. It is important that all
the mechanisms proposed thus far provide explanations for the
acceleration of neuronal dysfunction and/or cell death in specific
neurons. We will continue our attempt to establish SCA17 neuron
model for assessing the potential therapeutic targets underlying
In summary, we identified NFYA as a new TBP aggregate-
binding protein and a probable modulator of the SCA17
pathological process. Further studies of the role of NFY in
SCA17 pathology would reveal novel aspects of neuronal
degeneration, leading to development of pharmacotherapeutics
for the disease.
Materials and Methods
TBP and NFYA cDNA constructs
Polyadenylated RNA (200 ng) isolated from neuroblastoma SK-
N-SH cells (ATCC No. HTB11) was reverse transcribed using the
SuperScriptTMIII reverse transcriptase (Invitrogen). The TBP/
Q36cDNA in pGEM-T Easy vector (Promega) was constructed as
described . The TBP/Q45, TBP/Q61and TBP/Q79cDNAs
were made by ligating Fnu4HI partially digested fragments and the
repeat number was verified by DNA sequencing. The TBP
Figure 3. Interaction of NFYA and TBP/Q36, ,–61in vitro. His-tagged GST-TBP/Q36,61and Trx-NFYA fusion proteins were purified and incubated
with glutathione agarose beads. Before (input) and after GST pull-down, each protein was detected by Western blot using anti-TBP and anti-NFYA
antibodies. The positions of GST-TBP/Q36,61and Trx-NFYA were indicated by stars and an arrowhead, respectively.
Role of NFY in SCA17
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containing 36, 61 and 79 glutamines were cloned into pEF-IRES/
hrGFP vector as described . The sense and antisense (His-
tagged) primers used for nuclear transcription factor Y alpha
variant 1 (NFYA, NM_002505) cDNA amplification were
GGCTGGAGCCTCTGATTGGGTTTC and GTGGTGGTG-
GTGGTGGTGGGACACTCGGATGAT (His6sequence under-
lined). The amplified 1.2 kb full-length cDNAs were cloned into
pGEM-T Easy and sequenced. The cDNAs were then excised
with EcoRI and subcloned into pcDNA3 (Invitrogen). The above
TBP and NFYA constructs were used in transient expression
Cell culture and transfection
Human embryonic kidney (HEK)-293 cells (ATCC No. CRL-
1573) were cultivated in Dulbecco’s modified Eagle’s medium
containing 10% fetal bovine serum in a 37uC humidified
incubator with a 5% CO2atmosphere. HEK-293-derived cells
inducibly expressing TBP/Q36,79were constructed as described
 and maintained in medium containing 5 mg/ml blasticidin and
100 mg/ml hygromycin. Doxycycline (10 mg/ml) was used to
induce TBP expression. For transient overexpression, cells were
plated into 6-well (66105/well) or 12-well (on coverslips, 26105/
well) dishes, grown for 20 hr, and transfected using lipofectamine
2000 (Invitrogen) with TBP/Q36,79 and/or NFYA cDNA
plasmids (4 mg each/6-well or 2 mg each/12-well). The cells were
grown for 48–96 hr for the following immunocytochemical
staining (12-well dishes) and immunoprecipitation and dot-blot
studies (6-well dishes).
Cells were washed with phosphate buffered saline (PBS) and
fixed in 4% paraformaldehyde in PBS for 10 min, followed by
Figure 4. NFYA modulation of HSPA5 promoter activity. (A) HEK-293 cells were transiently transfected with HSPA5 reporter plasmid and
luciferase activity measured two days post-transfection. Levels of luciferase activity in NFYA cDNA or siRNA co-transfected cells were expressed as
folds of the activity of the HSPA5 reporter alone in HEK-293 cells (Control). (B) Cell lysates were prepared and analyzed with anti-NFYA or anti-b-
tubulin antibody. (C) Real time PCR quantification of HSPA5 mRNA level relative to HPRT mRNA in HEK-293 cells transiently transfected with NFYA
cDNA or siRNA for two days. Level of HSPA5 mRNA in vector-transfected cells were set as 100%. (D) Immunoblot analysis of HSPA5 protein level in
HEK-293 cells transiently transfected with NFYA cDNA or siRNA for two days. Level of HSPA5 protein in vector-transfected cells were set as 100%. (E)
293-derived TBP/Q36,79cells were induced to express TBP for 2,6 days and transiently express HSPA5 reporter plasmid for 2 days. Levels of
luciferase activity were expressed as folds of the activity of the HSPA5 reporter in cells expressing TBP/Q36for 2 days. All values are the mean 6 SD of
three independent experiments, each of which was performed in duplicate.
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20 min incubation with 0.1% Triton X-100 in PBS to permeate
cells, overnight incubation with 0.5% bovine serum albumin (BSA)
in PBS to block non-specific binding. The primary antibodies TBP
(N-12, Santa Cruz) and NFYA (H-209, Santa Cruz), diluted 1:500
in 1% BSA in phosphate buffered saline (PBS), were used to stain
cells at 4uC overnight. After washing, cells were incubated for 2 hr
at room temperature in Cy5-conjugated secondary antibody
(Zymed) diluted to 1:500 in PBS containing 1% BSA, and washed
with PBS. Nuclei were detected using 49-6-diamidino-2-pheny-
lindole (DAPI). The stained cells were examined for dual
fluorescent imaging using a Leica TCS confocal laser scanning
Immunoprecipitation and Western blotting
Total protein from TBP/Q36,79and NFYA co-transfected cells
was prepared using buffer containing 50 mM Tris-HCl, 150 mM
NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% SDS and 0.5% sodium
deoxycholate, 1% Triton X-100, protease inhibitor cocktail
(Sigma). After sonication and centrifugation at 15000 rpm for
10 min, supernatants were incubated with anti-NFYA, anti-TBP
(1TBP18, Abcam) or rabbit or mouse IgG (2 mg per 200 mg of
total proteins in 200 ml reaction) for 60 min at 4uC, after which the
antibody-protein complex were precipitated with Protein G
Agarose (20 ml, Pierce), separated on 10% SDS-polyacrylamide
gel electrophoresis (PAGE) and blotted onto nitrocellulose
membranes by reverse electrophoresis. After blocking, the
membrane was stained with antibody to TBP (N-12, 1:3000
dilution) or NFYA (1:500 dilution). The immune complexes were
detected using horseradish peroxidase-conjugated goat anti-mouse
or goat anti-rabbit (Jackson ImmunoResearch) IgG antibody
(1:10000 dilution) and chemiluminescent substrate (Millipore).
Figure 5. NFYA overexpression in SCA17 transient cell models. (A) HEK-293 cells were co-transfected with plasmids encoding TBP/Q36, TBP/
Q61or TBP/Q79, and plasmid with (+NFYA) or without (2NFYA) NFYA cDNA. After 2 days, cells were fixed and stained with TBP antibody (red). Nuclei
were detected with DAPI (blue). (The scale bar=40 mm) (B) The percentage of aggregate formation counted among five random fields.
Role of NFY in SCA17
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Dot-blot filter retardation assay
Transfected cells were lysed on ice for 30 mins in buffer
containing 50 mM Tris-HCl pH8.8, 100 mM NaCl, 5 mM
MgCl2, 0.5% (w/v) NP40, 100 mM EDTA, and protease
inhibitors cocktail. After centrifugation for 5 min at 14000 rpm,
insoluble pellets were resuspended in buffer (20 mM Tris-HCl
pH8.0, 15 mM MgCl2) containing DNase I (0.5 mg/ml). After
37uC incubation for 1 hr, the reaction was terminated by adjusting
the mixture to 20 mM EDTA, 2% SDS and 50 mM DTT,
followed by heating at 98uC for 5 min. Protein concentration was
determined (Bio-Rad Protein Assay) using BSA as a standard.
Extracted protein (5,20 mg) were diluted into 2% SDS and
filtered on a BRL dot-blot filtration unit through a cellulose
acetate membrane (0.2 mm pore size, Schleicher and Schuell) pre-
equilibrated with 2% SDS. Filters were washed twice in 0.1%
SDS, blocked in TBS containing 3% nonfat dried milk and stained
with the TBP (N-12) or NFYA antibody (1:500 dilution). The
immune complexes on the filter were detected as described above.
Trx-tagged NFYA and GST-tagged TBP expression
To generate Trx (thioredoxin)-tagged NFYA, the NFYA cDNA
in pGEM-T Easy was excised with EcoRI and subcloned into
EcoRI-digested pET-32b(+) (Novagen) for Trx-His6-NFYA-His6
expression in BL21(DE3)pLysS.
To generate GST (glutathione S-transferase)-tagged TBP, the
N-terminal TBP (160 amino acids for TBP/Q36) was PCR
amplified using the cloned TBP/Q36,61cDNA as a template and
synthetic 59 and 39 primers GAACACCATGGATCAGAACAA-
CAGCCTGCCAC and GGCTCGAGTGGCGTGGCAG-
GAGTGATGGGGGTC (NcoI and XhoI restriction sites under-
lined). After cloned into pGEM-T Easy vector and sequenced, the
N-terminal TBP/Q36,61cDNAs were excised with NcoI and XhoI
and subcloned into NcoI- and XhoI-digested pET-32b(+). In
advance, the NdeI fragment containing Trx tag in pET-32b(+)
was replaced with a NdeI fragment containing GST which was
amplified by PCR using pGEX-5X-3 as a template and synthetic
TATTGG and GGCATATGACGACCTTCGATCAGAT (NdeI
restriction site underlined).
Protein purification and in vitro binding assay
Bacteria transformed with recombinant TBP/Q36,61or NFYA
were grown in a liquid culture to an A600of 0.6, and expression
was induced with 0.1 mM isopropyl-b-D-thiogalactopyranoside
(IPTG) for 2 hr at 37uC. Bacterial cells were then harvested and
the His-tagged GST-TBP/Q36,61and Trx-NFYA were purified
using His-Bind resins (Novagen) according to supplier’s instruc-
In GST pull-down experiments, GST-tagged TBP/Q36–61and
Trx-tagged NFYA (10 mg each) were incubated with GST-BindTM
resin (50 ml, Novagen) in 600 ml of PBS containing 0.5% NP-40
(vol/vol) for 2 hr at 4uC. After centrifugation at 5006g for 5 min,
pellets were washed twice with 1 ml of binding buffer. The
proteins associated with the beads were separated on 10% SDS-
PAGE and Western blotted with anti-TBP (1:500 dilution) and
anti-NFYA (1:500 dilution) antibodies. The immune complexes
were detected using horseradish peroxidase-conjugated goat anti-
rabbit IgG antibody and chemiluminescent substrate as described.
HSPA5 promoter construct and dual luciferase assay
The sense and antisense primers used for HSPA5 promoter
GG. The amplified promoter fragment was cloned into pGEM-
T Easy and sequenced. The cloned promoter fragments were
inserted upstream of the firefly luciferase reporter gene in a dual
luciferase reporter plasmid .
In experiments examining the role of NFYA in HSPA5
transcription, HEK-293 cells were plated into 6-well (66105/well)
dishes, grown for 20 hr, and transfected with the HSPA5 dual
luciferase reporter plasmid (4 mg). For cDNA co-transfection,
Figure 6. NFYA expression in TBP/Q36, ,79expressing 293 cells. (A) Western blot analysis of induced TBP protein level relative to endogenous
TBP after 6 days induction (+Dox). (B) Western blot analysis of endogenous NFYA relative to endogenous b-tubulin after 6 days induction (+Dox).
Role of NFY in SCA17
PLoS ONE | www.plosone.org8April 2012 | Volume 7 | Issue 4 | e35302
equal amounts of HSPA5 reporter plasmid and NFYA cDNA
plasmid (4 mg each) were employed. For siRNA co-transfection,
120 pmol of human NFYA siRNA (sc-29947, Santa Cruz) were
used along with HSPA5 reporter plasmid (4 mg). The cells were
grown for 48 h. Cell lysates were prepared and luciferase activity
was measured with a luminometer using a dual luciferase assay
system (Promega). The activity of each promoter was directly
measured and expressed as the ratio of the firefly luciferase level to
the Renilla luciferase level. Three independent transfection
experiments were performed and difference in luciferase activity
was tested using the two-tailed Student’s t-test. To assess the
expression and knock-down of NFYA, Western blotting using
NFYA and b-tubulin (1:5000 dilution, Sigma) antibodies was
performed as described above.
In experiments examining the role of NFYA in SCA17 cells,
293-derived TBP/Q36,79 cells were plated in 24-well dishes
(56104/well). Next day, doxycycline was added for 2, 4 and 6 days
to induce TBP expression. Two days before harvesting cells for
luciferase assay, cells were transfected with HSPA5 reporter
plasmid (1.5 mg).
Real-time PCR and immunoblot analysis of HSPA5
HEK-293 cells were plated into 6-well dishes and transfected
with NFYA cDNA or siRNA as described. Forty-eight hours later,
total RNA was extracted from cells using the Trizol (Invitrogen).
The RNA was DNase treated, quantified, and reverse-transcribed
to cDNA using the SuperScriptTMIII reverse transcriptase
(Invitrogen). Using ABI StepOneTMReal-Time PCR System
Figure 7. Inducible expression of TBP/Q36, ,79 in SH-SY5Y cells. (A) Western blot analysis of induced TBP protein level relatively to
endogenous TBP after 6 days induction (+Dox). (B) Western blot analysis of endogenous NFYA relatively to endogenous b-tubulin after 6 days
induction (+Dox). (C) Isogenic SH-SY5Y cells were inducibly expressed TBP/Q36,79for 6 days. Cells were fixed and stained with antibodies specific for
TBP (red) and NFYA (yellow), and nuclei were counterstained with DAPI (blue). (The scale bar=10 mm).
Role of NFY in SCA17
PLoS ONE | www.plosone.org9April 2012 | Volume 7 | Issue 4 | e35302
(Applied Biosystems), real-time quantitative PCR was performed
on a cDNA amount equivalent to 12.5 ng total RNA with
TaqMan fluorogenic probes Hs99999174_ml for HSPA5 and
4326321E for HPRT1 (endogenous control; Applied Biosystems).
Fold change was calculated using the formula 2DCt, DCT=CT
(HPRT1)2CT(HSPA5), in which CTindicates cycle threshold.
Statistical analysis of differences between the groups was carried
out using one-way analysis of variance (ANOVA).
For HSPA5 protein analysis, total protein was prepared
48 hours after NFYA cDNA or siRNA transfection. Protein was
separated on 10% SDS-PAGE and blotted onto nitrocellulose
membranes as described. After blocking, the membrane was
stained with antibody to HSPA5 (1:500 dilution, Santa Cruz) or
actin (1:5000 dilution, Millipore). The immune complexes were
detected as described.
SCA17 SH-SY5Y cell lines generation
The Flp-InTMT-RExTMSystem (Invitrogen) was used to
generate stably induced SH-SY5Y cell lines exhibiting tetracy-
cline-inducible expression of TBP/Q36,79. Firstly SH-SY5Y-
derived FIp-In host cells were generated from independent
integration of plasmids pcDNA6/TR (a plasmid expressing the
Tet repressor; selected with 5 mg/ml blasticidin) and pFRT/
lacZeo (a plasmid containing the Flp Recombination Target (FRT)
site; selected with 100 mg/ml Zocin) into the genome of SH-SY5Y
cells (ATCC No. CRL-2266). Then the SH-SY5Y host cells were
co-transfected with pOG44 plasmid (constitutively expressed the
Flp recombinase) and pcDNA5/FRT/TO-TBP/Q36,79plasmid
 according to the supplier’s instructions. The repeats in these
TBP cell lines were examined by PCR. These cell lines were
grown in medium containing 5 mg/ml blasticidin and 100 mg/ml
hygromycin. Doxycycline (dox, 5 mg/ml) was added to induce
HA-tagged TBP expression for six days. The proteins were
prepared for Western blotting using antibody to TBP (1TBP18,
1:3000 dilution, Abcam), NFYA or b-tubulin as described.
We thank the Molecular Imaging Core Facility of National Taiwan
Normal University for the technical assistance. We also gratefully
acknowledge Dr. Kuo-Hsuan Chang for his helpful opinions and
Conceived and designed the experiments: L-CL C-MC G-JL-C J-YL.
Performed the experiments: L-CL H-CW H-HH I-SC. Analyzed the data:
L-CL G-JL-C. Contributed reagents/materials/analysis tools: M-TS H-
MH-L C-HW G-CL. Wrote the paper: C-MC G-JL-C J-YL.
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