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Samuel Lunenfeld Research Institute (M.A., Y.-F.Y., P.G.W.)
and Departments of Medicine, Pediatrics, and Otolaryngology
(P.G.W.) University of Toronto Medical School Divisions
of Head and Neck Oncology and Endocrinology of Mount Sinai
Hospital, Toronto, Ontario M5G 1X5, Canada
The Wistar
Institute (N.A.B., S.L.B.) Philadelphia, Pennsylvania 19104
Centre de Recherche (M.V.G.) Hotel-Dieu de Quebec
Université Laval Quebec G1R 2J6, Canada
LifeSensors
Inc. (T.R.B.) Malvern, Pennsylvania 19355
Department of
Biochemistry & Biophysics (T.R.B.) University of Pennsylvania
School of Medicine Philadelphia, Pennsylvania 19104-6509
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The yeast GCN5 transcriptional adaptor protein has been documented to have in its central structure several highly conserved HAT activational domains and an adjacent essential ADA2 interacting domain. These domains form the HAT catalytic subunit of at least two distinct multisubunit adaptor complexes regulating transcriptional activation by a number of acidic activators (8, 9, 10, 11, 12, 13, 14, 15). The first is the ADA trimeric complex, consisting of ADA2, GCN5, and ADA3 proteins (8, 9, 10, 11, 12, 13, 14). The second is the SAGA complex consisting of SPT, ADA, GCN5, and Acetylation adaptor complex (11, 12, 13). Both adaptor protein complexes were initially identified in yeast model systems. SPT 3, 7, 8, and SPT 20 (or ADA5) and ADA1 (15) are subunits within the SAGA complex (Ref. 13 and references therein). GCN5 provides an essential in vivo HAT function for the SAGA complex. Nucleosomal acetylation in vivo requires that the C terminus bromodomain (BrD) of yGCN5 be intact (Ref. 13 and references therein). Additionally, SPT components of the SAGA complex (13, 16) can interact with TATA-binding protein (TBP) and the TBP-activating factors (TAFIIs). Thus, both ADA and SAGA complexes have multiple, distinct transcription-related functions leading to interactions with the TATA box and facilitating acetylation of nucleosomal histones.
Several mammalian coactivator proteins have been identified through their ability to interact with class I and class II hormone-dependent nuclear receptors (NRs). These NR coactivators function as accessory factors distinct from general factors and transcriptional activators. Bona fide NR coactivator proteins SRC-1 (steroid receptor coactivator 1), TIF2 (transcription intermediary factor 2) GRIP1 (glucocorticoid receptor interacting protein 1) (Ref. 17 and references therein) and CBP interacting protein (pCIP) (18) function as p160 transcriptional coregulatory proteins distinct from general and transcription initiation apparatus factors by acting as hormone-dependent coactivator proteins recruited to NRs. NR coactivators such as SRC-1 (Refs. 19, 20 and references therein) and activator of retinoic acid receptor (ACTR) (21) also have intrinsic HAT activity. Furthermore, studies in mammalian cells have observed that thyroid (T3) hormone receptors (TRs) and retinoic acid receptor (RAR) NRs are activated by binding to the p300/cAMP response element binding protein (CBP), which in turn binds to the p300/CBP-associated factor (PCAF) and the p160 pCIP coactivator (19, 20, 21, 22, 23). The PCAF and p300/CBP components of this complex also have intrinsic HAT activity (19, 20, 21, 22, 23). These observations have favored the conclusion that recruitment of p160 coactivators and CBP/PCAF plays a central role in the regulation of transcriptional activation by RAR and other NRs by acetylation of nucleosomes to unfold chromatin and enhance contact with the general transcriptional machinery (19, 20, 24).
TRs are members of the Class II subclass of NRs. TRs function as hormone-regulated transcription factors that bind to enhancer regions in the promoter of target genes to control growth, development, and homeostasis (Refs. 25, 26 and references therein). TRs modulate gene expression by binding to enhancer regions containing hexameric (AGGTCA) core motifs, designated as T3 response elements (TREs) located at variable distances upstream from the transcription initiation site. TR/TRE interactions are hormone independent and are mediated through the highly conserved zinc fingers in the DNA binding domain (Refs. 25, 26 and references therein). Activation of transcription by T3 hormone and accessory proteins occurs by either direct or indirect interaction of the activation function 2 (AF-2) domain in the C terminus of TRs with downstream components of the transcription initiation apparatus assembled at the TATA-box (27, 28). TRs and RARs interact directly (29, 30) with basal transcription factors TFIIB and TBP/TFIID as well as with specific TAFs (Ref. 28 and references therein). However, the precise signaling pathways whereby DNA bound upstream transcriptional activators, such as TRs, increase the rate of transcription through linkage with accessory factors and the transcription initiation apparatus are not fully understood.
We have previously observed that in vivo activation of
transcription by hTRß1 in yeast is relatively weak (31), but could be
markedly enhanced by the presence of the p160 NR coactivator GRIP1
(32). Moreover, the observed reconstitution of
T3-dependent activation of transcription by
hTRß1 and 
1, as well as RARs, in the presence of GRIP1 (32) also
indicated that the yeast general transcription initiation machinery has
been evolutionarily conserved. A search of the Saccharomyces
cerevisiae genome has indicated that yeast is devoid of endogenous
NRs, p160 NR coactivators, and p300/CBP adaptor proteins. Hence, the
eukaryotic cellular context of the Baker’s yeast, S.
cerevisiae, can be used to identify unique putative
p300/CBP-independent adaptor/coactivator protein complexes regulating
transcriptional activation by hTRß1. In the present report, we have
used yeast genetics to examine the in vivo functional role
of several components of the SAGA complex in mediating transcriptional
activation by hTRß1 and a GRIP1 or SRC-1 p160 coactivator in the
presence of T3. Parallel in vitro
protein-protein interaction studies have been undertaken to determine
whether hTRß1 and p160 coactivators could directly bind to
several components of the ADA/SAGA adaptor protein complex. From these
experimental approaches, we show that intact yeast GCN5 and ADA adaptor
proteins are essential for T3-GRIP1/SRC-1
coactivator-dependent activation of transcription by hTRß1. GCN5
protein plays a central coregulatory role in the function of these
adaptor complexes through its highly conserved central HAT catalytic
subunit and a C-terminal BrD sequence. We show that either GRIP1 or
SRC-1 can bind directly to the conserved C terminus of hGCN5 and
enhance the binding of hGCN5 to hTRß1 in the presence of
T3 hormone. We also show that hTRß1 binds to
human ADA2 and that complementation with a LexA/hADA2 fusion protein
can partially restore hTRß1-dependent transcriptional activation in a
ada2 yeast strain knockout. These in vivo and in
vitro experiments suggest that the ADA/SAGA multisubunit adaptor
complex can function as in vivo coregulators of the p160
coactivator/T3-dependent activation of
transcription by hTRß1.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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gcn5 yeast knockout
strains. As we have previously reported,
T3-induced activation of transcription by hTRß1
in wild-type S. cerevisiae yeast is weak (31) but can be
dramatically enhanced by the presence of the p160 coactivator GRIP1
(32). Compared with the wild-type-strain, the gcn5 yeast knockout
abrogated T3-GRIP1-dependent activation of
hTRß1 (see Fig. 1A
).
Complementation with wild-type yeast GCN5 protein almost completely
restored the impaired transcriptional activation responses in the
gcn5 yeast strain (see Fig. 1A). When the p160 hSRC-1 coactivator
was substituted for GRIP1, essentially similar
T3-dependent activation of transcription by
hTRß1 was observed in both the wild-type and gcn5 yeast strains
(Fig. 1B). Interestingly, substitution of the gcn5 knockout for the
wild-type yeast strain detected a slightly greater impairment of
T3-dependent transcription activation by hTRß1
in the presence of the GRIP1 p160 coactivator compared with SRC-1.
These experiments have revealed that maximal
T3/GRIP1/SRC-1 induced gene activation by hTRß1
is dependent upon the presence of the yGCN5 adaptor protein.
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gcn5 yeast
strain, the GY1 mutant (239–241) documented to have the highest HAT
function among the gcn5 HAT mutants studied (34) also had the
greatest in vivo transactivational function, while the
mutant RGY (186–188) retained intermediate HAT function (see Fig. 1C
).
In marked contrast, substitution of the FAE (171–173) or the FKK
(221–223) mutants devoid of HAT function substantially reduced
transcriptional activation (Fig. 1C). These experiments performed in a
yeast model system have demonstrated the essential functional role
played by the conserved yGCN5-HAT domains in regulating of
T3/GRIP1 coactivator-dependent transcriptional
activation by hTRß1.
ADA/SAGA Adaptor Protein Components Essential for Gene Activation
by hTRß1
In previous studies, yADA2 has been documented to bind to both
yGCN5 and ADA3 to form an essential trimeric complex for maintaining
transcriptional activation by the acidic activator VP16 (Refs. 10, 13 & references therein). Moreover, both yADA2 and yGCN5 have been
shown to be essential for maintaining transcriptional activation by the
acidic activator VP16 (10, 13, 33, 35, 36). To determine the in
vivo functional role of yADA2 in transcriptional activation of
hTRß1, the transcriptional responses of hTRß1 in the wild-type
yeast strain were compared with those of the ada2 knockout. As shown
in Fig. 2A, substitution of the ada2
mutant for wild-type yeast greatly impaired hTRß1 activation by
T3 and GRIP1 coactivator, and the defect in
transactivation could be almost fully restored when complemented with
wild-type yADA2.
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ada3 and ada1
single knockouts were also studied. Deletions of either ADA3 (a
trimeric component of the ADA complex) or ADA1 (a component of the SAGA
complex) also dramatically reduced
T3-dependent-GRIP1 activation of hTRß1 but
could be effectively complemented in the yeast knockout strain by
wild-type yADA3 and yADA1 protein replacements. (see Figs. 2B and 2C,
respectively). Thus, single replacements of either wild-type GCN5,
ADA2, ADA3, or ADA1 components were sufficient to complement
transactivational function of a mutant yeast strain devoid of a
specific adaptor protein. Taken together, these experiments have also
documented that the SAGA adaptor protein complex can function as
postreceptor cofactors that can regulate
T3/GRIP1-dependent transcriptional activation of
target genes by hTRß1.
hGCN5 and hADA2 Adaptor Proteins Do Not Complement Missing Yeast
Homologs
To determine whether coexpression of human homologs of GCN5 and
ADA2 could restore hTRß1 activation in yeast strains deleted of these
adaptor proteins, complementation experiments using wild-type hGCN5 and
hADA2 were performed. Neither a LexA/hGCN5 (Fig. 3A) nor a LexA/hADA2
(Fig. 3B) fusion protein could fully restore transcriptional activation
by substituting a human protein for a deleted yeast homolog. However,
the partial reconstitution of gene activation by the LexA/hADA2 fusion
protein (Fig. 3B) supports the possibility that hADA2 is a potential
regulator of hTRß1-dependent hormone action. In accord with these
observations is the previously reported failure of human homologs to
complement missing yeast GCN5 and ADA2 proteins in gene activation by
VP16 (33). However, the partial restoration of gene activation by human
and yeast GCN5 and ADA2 chimeric proteins (33) indirectly supports the
possibility that human homologs of yeast adaptor proteins could play
similar roles in regulating transcriptional activation. Together, these
results suggest that the absence of essential interacting sequences in
homologous human adaptor proteins accounts for their failure to
maintain transcriptional activation by hTRß1 in a yeast model
system.
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). The preservation of hTRß1 expression in the yeast
knockout strain indicated that the in vivo functional
consequences observed in mutated ada/gcn5 yeast strains were directly
related to the absence of the specific ADA/GCN5 adaptor protein
components. Additionally, the expression in wild-type and mutant yeast
strains of hADA2 and hGCN5 as LexA fusion proteins was also detected
(Fig. 3D), thereby indicating that the failure of these proteins to
complement gene activation by hTRß1 in yeast
ada2 or gcn5 knockout strains was not
caused by defective expression of their yeast plasmids.
hTRß1 Binds to Yeast and Human GCN5 as Well as Human ADA2
To determine which member(s) of the yeast ADA/GCN5 adaptor complex
interacted with hTRß1, glutathione-S-transferase (GST)
pull-down in vitro binding assays were performed. We
observed that a full-length hTRß1-GST fusion protein specifically
bound in the absence of T3 to full-length
yGCN51-439 and the
hGCN51-476 (the shorter spliced variant devoid
of the extended N terminus present in the longer variant) as well as
full-length hADA2 (see Fig. 4). However, we could detect no specific
binding of the hTRß1-GST fusion protein to yADA2 and yADA3 (data not
shown). In contrast to hGCN5 and hADA2, the central core sequences of
GRIP1730-1121 bound to hTRß1 fused to GST only
in the presence of T3 (see Fig. 4, top
lane). Interestingly, the observed stronger protein-protein
interaction between hTRß1 and hGCN5 compared with yGCN5 likely
reflects a greater preference for binding interactions between human
proteins. Nevertheless, our detection of the weaker but specific yGCN5
binding to the hTRß1 receptor is in accord with its discernible
functional role in the regulation of reporter gene activation by
hTRß1 and accounts for the feasibility of demonstrating in a yeast
coexpression system the functional role of yGCN5 as a coregulator of
gene activation by hTRß1.
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. The conserved carboxy terminus
hGCN5387-837 fragment representing the longer
spliced variant devoid of the N terminus sequences homologous to hPCAF
but retaining HAT, ADA2, and the BrD[76% homology to the
C-terminus of PCAF351-832 and 40% to
yGCN520-439 (37, 38, 39)] was selected for in
vitro binding interaction studies. The
hGCN5387-837 fragment immobilized on a Ni
agarose matrix was observed to bind directly to either
35S-labeled GRIP1 or SRC-1 (Fig. 5B, lanes 8 and
9). In agreement with GST pull-down results shown in Fig. 4, hTRß1
bound to the hGCN5387-837 fragment immobilized
on the agarose matrix in the absence of T3
(lane 10). However, using densitometric analysis, we observed
that hTRß1 binding to hGCN5 was enhanced 
7 fold by
co-added T3 (lane 11) and was increased
40 fold by T3 when either GRIP1 or SRC-1 p160
coactivator was present (Fig. 5B, lanes 13 and 15). Importantly,
the 35- to 40-fold increase in the in vitro binding
of the hTRß1 to hGCN5 induced by T3 and either
GRIP1 or SRC-1 p160 coactivators correlated with the in vivo
increase of gene activation in the presence of wild-type yeast
T3/GRIP1 (see Fig. 1A). These observations are in
accord with the previously reported binding of PCAF (the hGCN5 long
spliced variant homolog) to C-terminal sequences of SRC-1 (19, 24) and
to an RAR/RXR heterodimer (23). Additionally, a
PCAF1-428 mutant devoid of the C-terminal
fragment was observed to be defective in 9-cis retinoic
acid-dependent binding to a RAR/RXR heterodimer-RARE complex and
activation of transcription in NIH3T3 cells (23), further supporting
the notion that the carboxy-terminal domain of PCAF and its GCN5
homolog are required for maximal gene activation by TR and RAR.
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BrD
yeast GCN51-350 or the wild-type
GCN51-439 protein, whether deletion of the
highly conserved BrD of GCN5, while retaining intact HAT and ADA2
domains (Fig. 5A), would impair T3-dependent gene
activation by hTRß1. Compared with wild-type yGCN5, the yGCN5 BrD
mutant was only partially active (65–80% less active than wild-type)
in restoring transcriptional activation in a gcn5 yeast knockout
strain when either wild-type GRIP1 (Fig. 5C) or SRC-1 (Fig. 5D) p160
coactivator was present. As previously documented (13, 33), expression
of either wild-type or BrD mutant in the identical yeast expression
plasmids, and the comparable maintenance of yeast colony growth by
their identical auxotrophic nutritional marker, validated their
efficient expression in a yeast model system. These experimental
observations have documented the distinctive functional role of the
yGCN5 BrD domain in mediating gene activation by hTRß1 in the
presence of an intact HAT and ADA2 domain and has identified a novel
functional role for the BrD in regulating
T3/GRIP1-dependent gene activation by hTRß1. Moreover,
these studies demonstrate that the BrD must be preserved to achieve
maximal activation of transcription by intact HAT and ADA2 yGCN5
domains.
GRIP1-NR Box LXXLL Motif Mutants Decrease hTRß1 Binding
to hGCN5
Our studies have established the important role of full-length
wild-type p160 GRIP1/SRC-1 coactivators in the regulation of both
in vivo activation of transcription (Fig. 1, A and B) and
in vitro the formation of a transcriptional activation
complex with the hGCN5 adaptor protein (Fig. 5B). Hence, further
studies were undertaken to elucidate the functional effects induced by
GRIP1 NR Box II and III LXXLL motif double alanine for leucine
substitution mutants previously shown to regulate binding and gene
activation for several hormone-dependent NR transcription activators
(40). We first evaluated the effects of GRIP1/NR Box II and III mutants
in modulating in vivo transcriptional activation using
T3/GRIP1-F2 TRE yeast assay. In agreement with a
previous study that used a yeast two-hybrid assay (40), the GRIP1
IIm mutant retained approximately 50% and the IIIm mutant
approximately 90% of the transcriptional activation by hTRß1
obtained when wild-type GRIP1 was present. However, the substitution of
the NR Box IIm + IIIm mutant for wild-type GRIP1 resulted in a marked
(>90%) loss of in vivo T3-induced
gene activation (Fig. 6A). We also
confirmed that defects in transcriptional activation shown in Fig. 6A
correlated with reductions in T3-dependent
in vitro binding of hTRß1 to the GRIP1/GST fusions of NR
Box II + IIIm >NR Box II > NR Box III (see Fig. 6B).
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), we determined the effects on
GCN5387-837 binding when the GRIP1 NR box IIm +
IIIm mutant was substituted. Compared with wild-type GRIP1,
substitution of the GRIP1 NR Box IIm + IIIm mutant was associated with
a marked reduction in direct T3 independent
binding to the hGCN5387-837 fragment as well as
a loss of the expected increase of hTRß1 in vitro binding
to hGCN5 when 10-6 M
T3 was present (see Fig. 6C, lanes 13 and 14).
Taken together, these observations confirm that GRIP1 NR Box II + III
LXXLL motifs must be intact to facilitate maximal
T3-dependent recruitment of the hGCN5 to
hTRß1. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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), our studies have provided
experimental evidence indicating that the yeast GCN5 and ADA/SAGA
proteins can function as important coactivators (adaptors) in the
regulation of gene activation by hTRß1.
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and 2). Mutational
analyses of the HAT (Fig. 1C) and BrD (Fig. 5, C and D) domains of
yeast GCN5 have indicated that preservation of each domain is essential
for maximal transcriptional activation by hTRß1 in a yeast model
system. When the yGCN5 BrD is intact, loss of HAT function
(i.e. required for acetylation of H3 and H4 histones)
impairs transcriptional activation in a yeast model system (Fig. 1C),
whereas, when its HAT and ADA2 domains are intact, deletion of BrD
substantially reduces transcriptional activation (Fig. 5, C and D).
These findings document, for the first time, distinctive but
interdependent functional roles for the HAT and BrD domains of GCN5 in
hormone-induced transcriptional activation (Fig. 7, schematic
illustration).
In this report, we have also observed that yeast and human GCN5 as well
as human ADA2 transcriptional adaptor proteins can bind directly to the
hTRß1 receptor (Fig. 4). Moreover, the conserved carboxy terminus of
hGCN5387-837 (containing sequences with high
homology to the HAT, ADA2, and BrD domain of yGCN5 and hPCAF) could
also bind directly to GRIP1 or SRC1 coactivators (Figs. 5 and 6).
Importantly, the presence of T3 was not only
essential for the binding of GRIP1 to hTRß1 (Figs. 4 and 6) but also
for the in vitro generation of a hGCN5/p160
coactivator/hTRß1 trimeric complex (Fig. 5B). Mutations in LXXLL
motifs of GRIP1 NR box II and III (Fig. 6) and the absence of
T3 (Figs. 5 and 6) impaired in vitro
formation of the trimeric complex. The marked transcriptional synergy
induced by hTRß1 in the presence of T3 and
GRIP1 could be directly correlated with the in vitro
formation of this T3-dependent trimeric complex.
To our knowledge, the observation of a direct in vitro
binding interaction of hGCN5387-837 with hTRß1
that can be greatly augmented by T3 and GRIP1 or
SRC1 has not been previously reported.
The possibility that yeast ADA/GCN5 adaptor proteins could play a role
in p160 coactivator-dependent transcriptional activation by
hormone-bound NRs has not been previously evaluated. Studies performed
in the absence of a coexpressed p160 coactivator had previously shown
that gene activation in yeast by the 
1c N terminus domain of the
glucocorticoid receptor (GR) (41) as well as the full-length estrogen
receptor and RXR (42) could be regulated by ADA adaptor proteins. GR
also interacted with yADA2, and overexpressed hADA2 weakly enhanced
activation of transcription by GR in HeLa cells (41). Additionally, a
positive yeast two-hybrid hormone-dependent hormone-binding
domain interaction of the estrogen receptor and RXR with yADA3 as well
as a weaker binding to TR but not RAR was reported (42). In contrast,
our report documents that the hTRß1 receptor can bind directly to
GCN5 as well as hADA2 (Fig. 4), but not to yADA3 or yADA1 (data not
shown). Since we have shown that TRs and RARs require GRIP1 (32) or
SRC-1 coactivators (Fig. 1, A and B) for optimal hormone-dependent gene
activation in a yeast model system, the current report has extended
through yeast genetics our understanding of gene activation by a Class
II hormone-dependent NR and demonstrated the importance of p160
coactivator interactions with human and yeast GCN5.
Human homologs of yeast ADA2, GCN5 (33) as well as ADA3 (43), have been
identified. The hGCN5387-837 fragment selected
for study in our experiment had 76% homology to hPCAF and 40% to
yGCN5 (38, 39). Except for its N terminus, PCAF has high homology to
yeast GCN5 (38, 39). PCAF interacts directly with RAR (22, 24) and
RAR/RXR heterodimers (20, 23), and its C terminus is essential for
RAR/RXR heterodimer function (23). It has been established that human
and yeast GCN5 have the same H3 and H4 histone substrate specificity in
the acetylation of nucleosomes (37). Similarly, the conserved HAT
domains of mouse GCN5 and rat PCAF have similar substrate specificity
in the acetylation of nucleosomes (43). Recently, the BrD of yGCN5 has
been demonstrated to interact with the N terminus of H3 and H4 histones
(44). Moreover, the hPCAF BrD has been discovered to consist of four
amphipathic -helix bundles with a left-handed twist and a
hydrophobic pocket formed by two loops that can bind lysine-acetylated
peptides of H3 and H4 histones (45). The in vitro
observations in their report are in accord with our in vivo
studies on the functional role of both the HAT and BrD domains of yGCN5
in hormone-dependent activation of transcription by hTRß1 (Figs. 1C and 5, C and D) These experimental findings support the intriguing
possibility that we have identified a novel structure/function role for
the highly conserved BrD of the GCN5/PCAF family of coactivators to
promote maximal gene activation by hormone-bound NRs through essential
protein-protein interactions of the BrD hydrophobic pocket with
acetylated N-terminal lysine tails of H3 and H4 nucleosomal histones to
unfold chromatin (Fig. 7).
The biological significance of the overlapping function of GCN5/PCAF
adaptor complexes is currently unknown. Redundancy of signaling
pathways may represent a protective biological mechanism for ensuring
the preservation of essential functions by closely related proteins. It
is also possible that each adaptor protein complex may be specifically
expressed in different tissues to fulfill time-related transcriptional
functions (39). Thus, alternatively spliced RNA transcripts may not
only be reciprocally expressed in different tissues but could also have
different functions (38, 39). The possibility of interrelated
biological functions of GCN5 and PCAF has been supported by the
detection of their ubiquitous and complementary expression in the mouse
(39). Human and yeast ADA2 have conserved Cys-rich sequences with high
similarity (
40%) to the CBP/p300 mammalian adaptor proteins (33).
However, a search of the complete yeast genome (46) has revealed the
absence of yeast proteins with significant homology to CBP and p300.
Studies in HeLa cells have found that hPCAF and hGCN5 form multimeric
complexes with hADA2 and hADA3, as well as several
TAFIIs and other SAGA complex proteins, but not
p300/CBP (43). Thus, our experimental findings in a yeast model system
that is devoid of the p300/CBP and PCAF coactivators, has indicated
that potent T3/GRIP1 or SRC-1 dependent gene
activation by hTRß1 can be achieved in the absence of these mammalian
coactivators.
We observed, however, that full-length hGCN5 or hADA2 could not
complement transcriptional defects induced by deletion of a homologous
yeast protein (Fig. 3, A and B). Previous observations on the failure
of human homologs of yADA2 and yGCN5 to complement these missing
wild-type yeast adaptor proteins (33), as well as the failure of the
human TBP/TFIID to complement deleted yeast homologs (47, 48), suggest
that interacting residues essential for yeast adaptor protein function
have not been conserved in human homologs. Interestingly, the
significant but only partial increase in transcriptional activation in
a ada2 yeast strain by a LexA-hADA2 fusion protein (see Fig. 3B) is
in accord with the enhanced activation of transcription by GR when
hADA2 was overexpressed in mammalian cells (41). Moreover, the ability
of yeast and human chimeric proteins of GCN5 and ADA2 (33) or TBP (47)
to achieve partial restoration of gene activation supports the
possibility that human homologs of these adaptor proteins could have
adaptor protein functional roles in gene activation by
hormone-dependent NRs in humans. Nevertheless, subtle
structure/function differences between yeast and human GCN5 and ADA
adaptor proteins in the complementation of gene activation mediated by
hTRß1 and p160 coactivators have been detected, and further studies
on the function of these human homologs in mammalian cells systems will
be required to validate their precise functional role in humans.
Although most affected families with a resistance to thyroid hormone
(RTH) syndrome have been previously detected to have dominant negative
mutations in the hormone-binding domains of TRß (Ref. 49 and
references therein), approximately 10% of families with an RTH
phenotype have been identified who do not have a discernible mutation
in either TRß or TR1 genes (50, 51). These observations are in
accord with the speculation that postreceptor mutations in
adaptor/coactivator proteins could also cause RTH syndromes (51). The
detection of partial RTH in SRC-1-/-KO mice has demonstrated that
impaired hormone action could be mediated by a mutation in a p160
coactivator and be accompanied by compensatory increases in TIF2/GRIP1
p160 coactivator (52). However, identifiable disturbances in thyroid
function in humans with the Rubinstein-Taybi syndrome due to a p300/CBP
mutation (53) have not been detected (54). Such observations suggest
that either the p300/CBP adaptor protein plays no major physiological
role in the maintenance of thyroid function in humans or that
alternative signaling pathways with overlapping function do exist. Our
experimental observations support the possibility that mutations in
hGCN5 (or hPCAF homolog) as well as human homologs of other SAGA
complex adaptor proteins could be potential postreceptor sites for
defects in T3 hormone action. However, studies
will be required in those RTH patients without detectable hTRß
receptor mutations to determine whether defects in hGCN5 (hPCAF) or
other ADA adaptor proteins could account for syndromes of resistance to
hormone action in humans.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, Ura3–52, Ade2–101, Leu2–3, 112, Lys2,
trp1::his G, his3–200) wild type or ada/gcn5
mutants (10, 35) was selected for these studies. GCN5, ADA2, ADA3, and
ADA1 deletional mutants (10, 35, 36) were produced in the same genetic
background of the PSY316 yeast strain.
Yeast Expression Vectors
Human TRß1 was cloned downstream of a CUP1 promoter
into a 2 µ multicopy yeast vector containing either a Trp1
selectable marker (YEp 46) or a Leu2 selectable marker (YEp
56). GRIP1320-1121 was cloned into a 2 µ
multicopy plasmid under the control of an alcohol dehydrogenase gene
(ADH) promoter (PRS 423 vector) with a His3 auxotropic
marker (32, 55). Full-lengh SRC-1 and
GRIP1563-1121 wild-type or NR box leucine to
alanine substitutions for IIm (L693A, L694A), IIIm (L748A, L749A), or
combined IIm + IIIm as previously described (40) were cloned into a 2
µ yeast plasmid under the control of a ADH promoter (pGAD424 vector)
containing a Leu2 auxotropic selection marker. The F2
enhancer element of the chicken lysozyme promoter was inserted into 2
µ multicopy pC2 reporter plasmid using Ura3 marker using a
unique XhoI site upstream from a cytochrome C promoter
(CYC1) linked to the Escherichia coli lacZ gene expressing
ß-galactosidase (ß-gal) as previously described (31, 32). yADA2
pC98/Leu2 and LexA-hADA2 BTMN/Trp1 were under the
control of an ADH promoter and a suitable Trp1 or
Leu2 marker (33). yGCN5 PRS414/Trp1 and
LexA/hGCN5 BTMN/TRp1 were under the control of an ADH
promoter and Trp1 auxtrophic marker (33). Wild-type and
mutated forms of the ADA/GCN5 complex were constructed in yeast ARS/CEN
expression vectors under control of an ADH promoter and a
Trp1 auxotropic marker as previously described (33). yADA1
and yADA3 proteins were cloned into the PDB20L ARS/CEN yeast expression
vector with an ADH promoter and a Leu2 selectable marker as
previously described (14, 15).
Yeast GCN5 Mutants
Using site-directed mutagenesis, yGCN5 HAT domain mutants were
constructed within the highly conserved residues 95–280, which
consisted of three adjacent amino acid alanine substitutions as
previously described (34). Mutants selected for study and their
corresponding sites of three adjacent amino acid alanine substitutions
in yGCN5 were: FAE (171–173), RGY (186–188), FKK (221–223), and GYI
(239–241). A yeast GCN5 truncation mutant devoid of the highly
conserved C-terminal BrD350-439 sequences
(yGCN51-350 BrD) was constructed as
previously described (13) and compared with wild-type
yGCN51-439.
Analysis of NR Transcriptional Activation
The yeast transformants were isolated and grown in the
appropriate minimal medium with added supplements as required. Cells
were grown overnight with T3 at a final
concentration of 1 µM, harvested, washed, resuspended in
Z buffer, and lysed with glass beads (425–600 µm) before
centrifugation. The supernatant was collected, and the protein
concentration was determined by the Lowry method (56) using BSA as a
standard. Twenty micrograms of protein were used for ß-gal assay, and
transcriptional activities were expressed as Miller units/mg of protein
(57). Data shown were pooled from three independent experiments and
calculated as a mean ± SE.
Protein-Protein Interactions in Vitro
GST/full-length TRß1 (gift from C. Glass) and
GST/GRIP1730-1121 and
GST/GRIP1653-1121 wild-type and GRIP1 NR box
double mutant fusion proteins (36) representing alanine for leucine
substitutions in NR box IIm, NR box IIIm, or NR box II + IIIm (gifts
from M. R. Stallcup) were used for in vitro pulldown
interaction studies. GST fusion proteins bound to glutathione-Sepharose
beads were analyzed for binding with 35S-labeled
full-length proteins as previously described (32). In other in
vitro protein-protein interaction studies, full-length
GRIP11-1462 and
GRIP1563-1121 wild-type and NR box II and III
mutants, as well as full-length TRß1 and SRC-1 (gift from B. W.
O’Malley) were [35S]methionine labeled by
transcription and translation using the TNT kit (Promega Corp., Madison, WI). The human long GCN5 spliced variant cDNA
SP64 construct (gift from Y. Nakatani) was digested with BglII
restriction enzyme to isolate the GCN5387-837 C
terminus fragment. This fragment was cloned in frame using the
pRSETA vector (Invitrogen) for the synthesis of
recombinant 6xHis tagged GCN5387-837 protein in
a BL21p Lys host cell. Protein expression was under controlled
conditions (30 C, 200 rpm) and induced with 0.25
mM IPTG for 4 h. To facilitate
[35S]-Met labeling of the
GRIP420-1232 fragment, a 5'-end ACCATG sequence
was added by mutagenesis. 35S-labeled hTRß1,
GRIP1, and SRC-1 were synthesized in vitro in a final volume
of 12.5 µl. After synthesis, the lysate was diluted with 87.5 µl
incubation buffer (10% glycerol, 100 mM KCl, 20
µM Tris/HCl, pH 7.9, 0.5% NP40, and 1% BSA without EDTA
and dithiothreitol). The crude extract of
hGCN5387-837 was thawed on ice and 10 µl of
aliquots of the extracts were incubated with 10 µl of diluted
[35S]methionine-labeled proteins in a final
volume of 25 µl at 4 C for 10 min. The bound and free proteins were
separated using Ni Agarose matrix (QIAGEN, Chatsworth,
CA). Ten microliters of the packed volume of Ni Agarose were added to
the reaction mixture and incubated at 4 C for 30 min with occasional
agitation before washing three times with the incubation buffer
containing 20 µM imidazole. The Ni Agarose-bound proteins
were released by incubation with 25 µl of buffer containing 500
mM imidazole. Nonspecific
[35S] protein binding was monitored using Ni
Agarose matrix devoid of added hGCN5387-837. The
eluate was diluted 1:1 with SDS sample buffer, and the samples were
resolved on 10% or 12% acrylamide gels. Gels were treated with
Enhance (NEN Life Science Products, Boston, MA), dried,
and analyzed by autoradiography.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by grants (to P.G.W.) from the Medical Research Council of Canada Grant MT-14798, The Samuel Lunenfeld Research Institute of Mount Sinai Hospital, The Mount Sinai Hospital Foundation, a Thyroid Foundation of Canada Fellowship (S. Mori), The Mount Sinai Hospital Department of Medicine Research Fund, and the Temmy Latner/Dynacare Foundation.
Received for publication April 30, 1999. Revision received January 26, 2000. Accepted for publication January 28, 2000.
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