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Department of Physiology University of Maryland School of Medicine Baltimore, Maryland 21201
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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10 min) following
addition of thyroid hormone (T3). In close
agreement with these observations, we find that TR is associated with
potent histone acetyltransferase activity rapidly following
T3-treatment. By contrast, we observe that
formation of TR-TRAP complexes occurs significantly later (3 h) post
T3 treatment. An examination of the kinetics of
T3-induced gene expression in HeLa cells
reveals bimodal or delayed activation on specific
T3-responsive promoters. Taken together, our
data are consistent with the hypothesis that
T3-dependent activation at specific target
promoters may involve the regulated action of multiple TR-coactivator
complexes. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and ß) have
the dual ability to either activate or repress transcription on genes
bearing TR-binding elements (TREs) (2). In general, TRs function as
activators in the presence of thyroid hormone
(T3) and repressors in the absence of
T3, although T3-dependent
repression has also been observed (2, 4, 5). The ability of TRs to
regulate transcription has been linked to their ability to recruit
distinct types of transcriptional coregulatory factors, termed
coactivators and corepressors, to target promoters (6, 7). Coactivators recruited by liganded TR and involved in transcriptional activation include members of the p160/SRC family such as SRC-1/NCoA-1, TIF2/GRIP1, and RAC3/pCIP/AIB1/ACTR/TRAM-1 (for reviews see Refs. 6, 7). While the precise mechanism of action of these proteins is still being defined, their ability to associate with histone acetyltransferases (HATs), such as CREB-binding protein (CBP)/p300 (8, 9, 10, 11, 12, 13) and p300/CBP-associated factor (PCAF) (14), and the presence of intrinsic HAT activity in some family members (9, 15) suggests a functional role in chromatin rearrangement. All members of the p160/SRC family have a centrally located NR-interaction domain containing multiple copies of a consensus leucine-rich motif, LXXLL (also termed NR box) (6, 7). Recent biochemical and crystallographic studies reveal that the surface of a single LXXLL motif directly contacts the highly conserved, ligand-dependent activation domain (AF-2) of NRs, thereby providing a molecular basis for NR-coactivator recruitment (16, 17, 18).
An alternative set of TR coactivators, termed the TR-associated protein (TRAP) complex, was first identified as a large multisubunit group of novel proteins that associate with TR in T3-treated HeLa cells (19). The ability of the TRAP complex to markedly stimulate TR-mediated transcription in vitro on naked DNA templates and in the absence of TATA-binding protein-associated factors suggested that TRAPs mediate a novel TR-coactivator pathway or activation step distinct from those mediated by SRC/p160 proteins and CBP/p300 and possibly involving a more direct influence on the basal transcription machinery (19, 20). Several, if not all, of the subunits of the TRAP complex have been identified in other large transcriptional coregulatory complexes including DRIP (21), NAT (22), SMCC (23), and CRSP (24). Furthermore, several TRAP subunits appear to be human homologs of yeast proteins found within "mediator," a large complex of nuclear proteins that interact with both transcriptional regulatory factors and RNA polymerase II (25). A single subunit of the TRAP complex, TRAP220, has been proposed to target and possibly anchor the entire TRAP complex to TR as well as other ligand-activated NRs (26). Interestingly, and analogous to the SRC/p160 proteins, TRAP220 contains two centrally located LXXLL motifs that are essential for physical and functional interactions with the AF-2 domain of TR (26, 27).
In this study, we examined the assembly kinetics of distinct types of
TR-coactivator complexes in HeLa cells. We demonstrate that interaction
between TR and members of the p160/SRC family, CBP, and PCAF occurs
rapidly following T3 induction and that the
resulting complexes possess potent levels of HAT activity. By contrast,
formation of TR-TRAP complexes in HeLa cells occurs markedly later
(3 h) post T3-treatment. In examining the
kinetics of T3-induced gene expression in HeLa
cells, we observed that activation on selected
T3-responsive promoters was bimodal or delayed
with regard to T3 treatment. Collectively, these
data suggest that T3-dependent activation of
specific genes in HeLa cells may involve the regulated action of
multiple TR-coactivator complexes.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-2) that
stably expresses a FLAG epitope-tagged human TR and as such, renders
the cells responsive to T3 (19). Previous studies
demonstrated that transcriptionally active TR in association with
specific coactivators could be immunopurified from
T3-treated -2 cells using anti-FLAG antibodies
(19, 20, 23, 28). In this study, we employed the -2 line to examine
the assembly kinetics of distinct types of TR-coactivator complexes as
a function of T3 exposure. Toward this end, TR
was immunoprecipitated from -2 cells cultured with
T3 for different lengths of time ranging from 10
min to 18 h (Fig. 1
).
TRcontaining protein complexes were then transferred to a
membrane and probed by Western blot. We initially probed with specific
antibodies representing the three different subtypes of the SRC/p160
family of coactivators (SRC-1, TIF2, and RAC3) (Fig. 1, A–D).
Interestingly, TR showed rapid interaction kinetics with all three
members of the p160/SRC family as evidenced by significant levels of
associated coactivator 10 min (SRC-1 and RAC3) and 20 min (TIF2) post
T3 treatment (Fig. 1, A–C). Indeed, further
experiments showed that interaction between TR and SRC-1 or RAC3
occurs as quickly as 5 min post T3 induction
(data not shown).
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-TRAP220
interactions. Using the same Western blots initially probed with the
anti-p160/SRC antibodies (Fig. 1
, A–C), we reprobed the membranes with
specific antibodies against TRAP220. In contrast to the TR-p160/SRC
assembly time course, the interaction kinetics between TR and
TRAP220 were significantly slower as evidenced by the appearance of
TRAP220 beginning 1–3 h post T3 treatment (Fig. 1, A–C). To verify that the kinetics of TR-TRAP220 interaction
accurately reflect those for TR-TRAP complex assembly, we repeated
the experiment using antibodies against TRAP100, a subunit of the TRAP
complex that does not directly contact TR. As shown in Fig. 1D, the
pattern of TR coimmunoprecipitation with TRAP100 is nearly identical
to the T3-induced timecourse of TR-TRAP220
interactions (Fig. 1, A–C). The presence of retinoid X receptor (RXR)
within the various TR-coactivator complexes was verified by probing the
immunoblots with specific antibodies against RXR. As evident in Fig. 1C, RXR precipitates with TR in both the presence and absence of
ligand, thus demonstrating that coactivator complexes assemble with
RXR-TR heterodimers.
To confirm that the TR-containing SRC/p160 and TRAP coactivator
complexes observed 3–18 h post T3 treatment are
distinct entities and do not coexist within a single holocomplex, we
performed double coimmunoprecipitation experiments (Fig. 1E). First,
TR was immunoprecipitated from -2 cells cultured in
T3 for different lengths of time. The
precipitated protein complexes were then eluted off the anti-FLAG
immunoaffinity resin using a FLAG peptide and subsequently
reimmunoprecipitated with antibodies against TRAP220 coupled to protein
A-sepharose beads. The resulting doubly precipitated proteins were then
probed by Western blot with antibodies against either TRAP220 or SRC-1.
As shown in Fig. 1E, SRC-1 did not coprecipitate with TRAP220, thus
indicating that the interaction of SRC/p160 proteins vs. the
interaction of the TRAP complex with TR are separable
molecular events that result in distinct TR-coactivator
complexes.
One possible explanation for the observed differential TR-coactivator
interaction kinetics is that continuous treatment of -2 cells with
T3 changes the expression of either TR or its
specific coactivators. To address this possibility, Western blotting of
cellular lysates prepared from -2 cells exposed to
T3 for different lengths of time was performed.
As shown in Fig. 1F, no significant changes in protein expression
levels were observed up to 18 h post T3
treatment for TR, SRC-1, TIF2, RAC3, TRAP220, TRAP100, and PCAF.
Taken collectively, these data indicate that the
T3-induced kinetics of TR interaction with
members of the p160/SRC family are rapid, while TR-TRAP complex
assembly is significantly slower.
Rapid T3 Induction of TR-Associated HAT
Activity
Members of the SRC/p160 family of coactivators are thought to
function in part by associating with strong HAT coactivators such as
CBP/p300 and PCAF and subsequently recruiting the HAT activity to NRs
in a ligand-dependent manner (6, 7). To examine whether the rapidly
induced TR-p160/SRC complexes (Fig. 1, A–D) are associated with CBP
and PCAF, we again immunoprecipitated TR from -2 cells cultured
with T3, transferred the protein complexes to
membranes, and then probed with specific antibodies against either CBP
or PCAF. Similar to the TR-p160/SRC interaction kinetics (Fig. 1, A–D), significant levels of both CBP and PCAF were associated with
TR rapidly following T3 treatment, thus
suggesting that the kinetics of TR-p160/SRC-CBP/PCAF complex
formation are likewise fast (Fig. 2A).
Furthermore, given that CBP/p300 is capable of direct ligand-dependent
interactions with NRs (11), these data may additionally reflect the
rapid formation of direct TR-CBP/PCAF complexes.
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are
functionally active, we tested the TR-coactivator complexes for
specific HAT enzymatic activity. Using a filter binding assay,
TR-containing complexes immunoprecipitated from
T3-cultured -2 cells were tested for HAT
activity in the presence of 3H-labeled acetyl CoA
and calf thymus histones. Indeed, significant levels of HAT activity
were detected almost immediately (10 min post T3
exposure) with nearly 90% of the maximal HAT activity detected after
only 30 min T3 exposure (Fig. 2B). Thus, in close
correlation with the interaction kinetics of TR with the p160/SRC
and CBP/PCAF proteins, these data demonstrate that TR is associated
with potent HAT activity rapidly following T3
exposure.
Kinetics of T3-Induced Gene
Expression in -2 Cells
The results shown in Figs. 1 and 2 indicate that in
-2 cells, the kinetics of T3-induced
TR-p160/SRC-CBP/PCAF complex assembly are remarkably fast while the
kinetics of TR-TRAP complex assembly are significantly slower. To
begin to examine whether the differential formation of distinct
TR-coactivator complexes might be reflected at the level of gene
expression, we transiently introduced
T3-responsive reporter genes into -2 cells and
measured transcription after exposure to T3 for
different lengths of time. As shown in Fig. 3, A–C, significant activation from
three different luciferase reporters containing either synthetic TREs
(palindromic or DR4) or a natural TRE (chick F2lysozyme element)
was preceded by at least a 6-h lag period following
T3 exposure.
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-2
cells under more physiologically relevant conditions, we measured
endogenous T3-induced gene expression by Northern
blot. Because wild-type HeLa cells express only trace levels of TR and
typically do not exhibit T3-regulated gene
expression, we first needed to identify endogenous genes in the -2
line that are responsive to T3. With this
objective, poly (A)+ mRNA was extracted from -2 cells cultured for
24 h in the absence or presence of T3 and
then hybridized with a panel of 12 different DNA probes representing
genes previously shown to be regulated by T3 in
other tissues (Fig. 4 A and
Materials and Methods). As shown in Fig. 4A, three
genes were identified whose expression was markedly activated by
T3: type 1 iodothyronine deiodinase (dio1),
the proto-oncogene bcl-3, and spot 14.
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-2 cells cultured with T3 for different
lengths of time and then hybridized with either dio1, bcl-3, or spot 14
DNA probes. Activation of dio1 gene expression was relatively weak for
up to 3 h post T3 treatment and then
increased in a linear manner with maximal induction occurring 18 to
24 h post T3 exposure (Fig. 4B).
T3-induced expression of bcl-3 exhibited an even
more delayed pattern of induction with maximal activation occurring
6–18 h post T3 exposure (Fig. 4C). Consistent
with earlier studies in rat liver (29),
T3-induced activation of spot 14 transcription
was bimodal, exhibiting a very modest peak of activation 1 h post
T3 treatment and a second stronger peak at 18–24
h post T3 treatment (Fig. 4D). Thus, although
TR is associated with potent HAT activity minutes after
T3 exposure (Fig. 2), significant
T3-induced activation from the three endogenous
genes examined here does not begin until several hours
post-T3 treatment. While we cannot conclude that
all T3-regulated genes exhibit similar activation
kinetics, we were unable to identify in HeLa cells any other
T3-responsive gene promoters, either transiently
or endogenously, which exhibited a more rapid
T3-induced activation. In sum, these results
suggest that transcriptional activation from specific
T3-responsive promoters may necessitate multiple
functional steps involving the temporally regulated action of distinct
coactivator complexes. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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as a means of examining the
cellular assembly kinetics of different TR-coactivator complexes
in vivo. We found that interaction of TR with members of the
p160/SRC family, and with the associated coactivators CBP/PCAF, occurs
minutes after exposure to T3 and that the
resulting complexes possess potent levels of HAT activity. By contrast,
formation of TR-TRAP complexes in HeLa cells occurs markedly later
following T3 treatment.
Our findings raise the question as to whether different types of
TR-coactivator complexes might function in cooperation with one
another, possibly during different functional steps of a common TR
activation pathway. Given the temporal order of formation of
TR-p160/SRC-CBP/PCAF complexes followed by TR-TRAP complexes, our
results are suggestive of a sequential model of TR activation (6, 20).
In this scenario, T3-activated TR might first
recruit HAT activity to a promoter facilitating chromatin derepression.
In a subsequent step, TR might recruit cofactors that more directly
interface with the basal apparatus (e.g. TRAPs) and
potentiate transcription initiation. Indeed, despite TR’s association
with strong HAT activity in HeLa cells almost immediately after
T3 exposure, significant
T3-dependent transcription from three selected
promoters did not occur until several hours post
T3 treatment (Fig. 4), possibly implicating a
multistep pathway involving distinct cofactors. While the significance
of these transcription kinetics with regard to the different temporal
assembly of TR-coactivator complexes is only correlative at this point,
it should be noted that we were unable to identify any other
T3-responsive gene promoters, either transiently
or endogenously, that exhibited a more rapid
T3-induced activation.
The kinetics of estrogen receptor (ER)-mediated gene expression in
MCF-7 cells were recently shown to be significantly more rapid (30)
than those reported here for TR. Interestingly, these studies used
chromatin immunoprecipitation assays to demonstrate that rapid and
transient binding of SRC/CBP complexes to specific estrogen-responsive
promoters precisely coincides with rapid ER-mediated transcriptional
activation (30). These studies may therefore reflect temporal
differences in the specific cofactor requirements for ER vs.
TR. Alternatively, the observed contrast in ER vs. TR
activation kinetics may reflect cell-specific differences in MCF-7
vs. HeLa cells or further reveal differences in the higher
ordered chromatin structure of the dio1, bcl-3, and spot 14 gene
promoters in HeLa cells vs. that for specific
estrogen-responsive promoters in MCF-7 cells (30).
In addition to showing an ordered assembly of TR-HAT followed by TR-TRAP complexes between 0 and 3 h post T3 exposure, we also found that both types of complexes coexist in the nucleus between 3 and 18 h post T3 treatment. Thus, while current models of sequential NR activation propose that coactivators are successively exchanged with static chromatin-bound NRs, our data raise the possibility that entire TRcoactivator complexes are dynamically replaced at specific promoters by completely different TR-bound coactivator complexes. Furthermore, our findings are consistent with a TR activation pathway in which p160/SRC, CBP/PCAF, and TRAP coactivator complexes might function simultaneously at the same promoter (6). Indeed, it is interesting to note that the promoter regions for both the dio1 and spot 14 genes contain multiple TR binding elements (TREs) (31, 32). It is thus conceivable that different types of TR-coactivator complexes might be targeted to different TREs at a common promoter in a sequential or temporal fashion. Finally, the possibility also exists that TR-HAT and TR-TRAP complexes might function completely independently of one another via differential and temporal targeting to different T3-responsive promoters.
Another intriguing issue raised by our findings is the mechanistic nature of the delay in TR-TRAP complex assembly after T3 exposure. Previous studies have established that the TRAP complex, excluding TR, exists in a preassembled steady state complex (23, 26, 28). Given that the binding of p160/SRC proteins and TRAP220 with TR’s AF2 motif is mutually exclusive, one possible explanation for the delay is an initial greater T3-dependent TR affinity for the p160/SRC proteins than for the TRAP complex (33). Although we fail to detect a significant decrease in TR-p160/SRC complexes at the time when TR-TRAP complex formation is occurring (as might be predicted from this hypothesis), the situation here may be complicated by the steady state turnover of TR and TR cofactors, and by the dynamic formation of new complexes several hours post T3 exposure. In theory, a temporal delay in TR-TRAP assembly might also involve a regulatory posttranslational modification step (e.g. phosphorylation) of either TR, TRAP220, other TRAP subunits, or perhaps other regulatory factors that ultimately control TR interaction with the TRAP complex. Finally, TR-HAT complex assembly and action might be a regulatory prerequisite for TR-TRAP assembly. Future experiments will be aimed at investigating the molecular mechanisms regulating TR-TRAP complex formation and further examining whether different types of TR-coactivator complexes function at specific T3responsive promoters in a temporal fashion.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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cell line -2
(19) was routinely maintained in DMEM supplemented with 10% dialyzed
FCS (Life Technologies, Inc., Gaithersburg, MD). After
addition of T3 (10-7
M) for the duration indicated in the figures, whole cell
lysates were prepared by scraping the cells (1 x
107 cells) in 1 ml of ice-cold buffer A [50
mM Tris-Cl (pH 7.4), 150 mM NaCl, 5
mM EDTA, 0.5% NP-40, 1 mM phenylmethylsulfonyl
fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin]. The lysate was
rotated 3600 for 1 h at 4 C followed by
centrifugation at 12,000 x g for 10 min at 4 C to
clear the cellular debris. Protein concentration was determined by
Bradford assays. Proteins were either resolved directly in
SDS-polyacrylamide gels after boiling in SDS sample buffer or subjected
to immunoprecipitation (as outlined below) with the appropriate
antibodies.
Preparation of nuclear extract was essentially as described previously
(34). Briefly, -2 cells in 15-cm culture dishes were collected for
harvesting by gentle scraping in 1 ml ice-cold PBS and pelleting by
centrifugation at 1200 rpm at 4 C. The PBS was aspirated and the cell
pellet (5 x 10 7 cells) was washed once in
PBS followed by resuspension in 1 ml of lysis buffer [10
mM Tris-Cl (pH 7.4), 10 mM NaCl, 3
mM MgCl2, 0.5% NP-40]. Nuclei were
gently isolated by centrifugation at 4,000 rpm and resuspended in 200
µl of extraction buffer [20 mM Tris-Cl (pH 7.9), 0.42
mM KCl, 0.2 mM EDTA, 10% glycerol, 2
mM dithiothreitol, 0.1 mM
phenylmethylsulfonylfluoride]. The resulting nuclear extracts were
incubated on ice for 10 min and cleared by centrifugation at 10,000
rpm. Protein concentration of nuclear extracts was determined by
Bradford assay.
For coimmunoprecipitation of FLAG-TR-coactivator complexes, 2.5 mg of
whole cell lysate or 1 mg of nuclear extract were incubated with
anti-FLAG antibodies coupled to agarose beads (20 µl packed volume)
(M2 Affinity Resin; Sigma, St. Louis, MO), and the mixture
rotated slowly at 4 C for 6–8 h. The beads were collected by gentle
centrifugation and washed twice with 1.5 ml ice-cold buffer A. After
the final wash, the precipitated TR-coactivator complexes were
resuspended in SDS-sample loading buffer, fractionated by SDS-PAGE, and
transferred to nitrocellulose membrane. Immuno-detection was performed
by first blocking the membranes for 1 h in TBS buffer [20
mM Tris-Cl (pH 7.5), 137 mM NaCl, 0.05%
Tween-20] containing 5% powdered milk followed by addition of the
appropriate antibodies in TBS and incubating for 2 h at room
temperature. Specifically bound primary antibodies were detected with
peroxidase-coupled secondary antibodies and developed by enhanced
chemiluminescence (ECL system, Amersham Pharmacia Biotech,
Arlington Heights, IL) according to manufacturer’s instructions.
Antibodies against TRAP220 and TRAP 100 were described earlier (37).
Anti-TR1 (FL-408), anti-RXR (sc-774), anti RAC3 (C-20), and PCAF
(C-16) were all obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against CBP (catalog no.
06–294) were obtained from Upstate Biotechnology, Inc.
(Lake Placid, NY). Anti-SRC-1 (catalog no. MA1–840) was obtained from
Affinity BioReagents, Inc. (Golden, CO). Anti-TIF2
was generously provided by Pierre Chambon (35).
For double immunoprecipitation assays,
M2-agaroseimmunoprecipitated TR-coactivator complexes were
resuspended in 50 µl of BC100 buffer [20 mM Tris-Cl (pH
7.9), 20% glycerol, 100 mM KCL, 0.2 mM EDTA]
containing 0.2 µg/µl FLAG peptide (N-DYKDDDDK–C), and the mixture
was incubated at 4 C for 1 h. The beads were collected by
centrifugation, and supernatant, containing the eluted
receptorcoactivator complexes, was then subjected to a second
round of immunoprecipitation using anti-TRAP220 antibody. Reactions
containing anti-TRAP220 antibodies were further incubated with protein
A-sepharose beads for an additional 1 h at 4 C. The resultant
double immunoprecipitates were then pelleted by gentle centrifugation,
washed four times with lysis buffer A, resuspended in SDS-sample
loading buffer, fractionated by SDS-PAGE, transferred to nitrocellulose
membrane, and probed with specific antibodies.
For immunoprecipitation of HAT activity, nuclear extract was prepared as described above and incubated with either M2-affinity beads (20 µl packed volume) or anti-CBP antibodies (50 ng) for 2 h at 4 C with rocking. Reactions containing anti-CBP antibodies were further incubated with protein A-agarose beads for an additional 1 h at 4 C. The HAT complexes were then pelleted by gentle centrifugation, washed four times with lysis buffer A, and subjected to HAT filter assay (see below).
HAT Assay
HAT activity was assayed as described by Brownell and Allis
(36). -2 Cells were grown in 15-cm culture dishes and
T3 was added as indicated in the figure legends.
Nuclear extract preparation and immunoprecipitation of HAT complexes
were performed as described above. The immune complexes were first
equilibrated in HAT reaction buffer [50 mM Tris.Cl, pH
8.0, 10% (vol/vol) glycerol, 1 mM dithiothreitol, 1
mM phenylmethylsulfonyl fluoride, 0.1 mM EDTA,
10 mM butyric acid]. The reaction was initiated in a final
volume of 30 µl HAT buffer containing 25 mg of calf thymus histones
(Sigma type II A) and
[3H]Acetyl-coenzyme A (50 nCi; 4.10
Ci/mmol, 152 GBq/mmol (Amersham Pharmacia Biotech), final
concentration of 0.5 µM[rsqb]. The
reactions were incubated for 30 min at 30 C, spotted onto P-81 filters
(Whatman, Clifton, NJ), and then washed extensively with
50 mM sodium carbonate buffer (pH 9.2).
[3H]Acetate incorporation was quantitated by
liquid scintillation counting.
Transient Transfections
Transient transfections were performed using the Lipofectin
reagent (Life Technologies, Inc.) as recommended by the
manufacturer. One day before transfection, -2 cells were seeded in
12-well plates at a density of 2 x 105
cells per well in DMEM containing 10% dialyzed FBS. A transfection
mixture containing 0.3 µg T3-reporter plasmid
[either 2xT3RE-tk-Luc, 2xDR4-tk-Luc, or
F2Lys-tk-Luc] and 0.1 µg of the internal control plasmid
pSV-ß-gal, together with Lipofectin reagent, was added to each well
and incubated at 37 C in 5% CO2 for 3 h.
One milliliter of DMEM containing 15% dialyzed FBS was added to the
transfection mixture, and the cells were incubated at 37 C for 12
h. Transfected cells were then further incubated at 37 C for 24 h;
during this period, T3 was added
(10-7 M final) for the duration
indicated in the figures. Cells were harvested with a cell lysis buffer
supplied in a kit (Luciferase Assay System, Promega Corp.,
Madison, WI), and luciferase activity was determined by adding a
commercial assay solution according to the manufacturer’s instructions
(Promega Corp.) and then measuring in a Lumat LB 9507
luminometer (EG & G Wallace, Inc., Gaithersburg, MD). The
ß-galactosidase activity of the lysed transfected cells (as above)
was determined using a kit (ß-galactosidase Enzyme Assay System,
Promega Corp.) according to the manufacturer’s
instructions. The luciferase activity was normalized to the ß-gal
activity and expressed as relative luciferase light units. The reporter
constructs 2xDR4-tk-Luc or F2Lys-tk-Luc were kindly provided by Anthony
Hollenberg; the 2xT3RE-tk-Luc plasmid has been described previously
(37).
Preparation of mRNA and Northern Blots
Total RNA was prepared from -2 cells using TRIZOL Reagent
(Life Technologies, Inc.) following the instructions
provided. The isolation of poly (A)+ RNA from purified total RNA was
performed using Message maker reagent assembly (Life Technologies, Inc.) as instructed by the manufacturer. Two
micrograms of poly (A)+ RNA were loaded per well and fractionated by
electrophoresis in a 1% agarose-formaldehyde gel and subsequently
transferred onto a positively charged nylon membrane (BrightStar-Plus,
Ambion, Inc., Austin, TX) DNA probes were radiolabeled
with -32P-dCTP using a Random Primed DNA
Labeling Kit (Roche Molecular Biochemicals, Indianapolis,
IN). Northern blot hybridization contained 2 x 10
6 cpm/ml of radiolabeled probe and was performed
using the Northern Max Northern blotting kit (Ambion, Inc.) following instructions by the manufacturer. Blots were
exposed to scientific imaging film (Kodak, Rochester, NY)
at -70 C.
The DNA probes used in the Northern blot assays were derived as
follows: the human type 1 deiodinase gene probe is a 2.2-kb
XhoI fragment excised from pL5Xhor (provided by P. Reed
Larsen); the human mdm-2 probe is a 1-kb
XbaI/BamHI fragment excised from pCGT-T7-hmdm2
(provided by Robert Freund); the probe for SERCA2 is a 1.6-kb
EcoRI fragment excised from the pSERCA2 cDNA (provided by
Wolfgang Dillman); the probe for malic enzyme is a 1-kb
EcoRI fragment excised from pME6 (provided by Vera Nikodem);
the probe for spot 14 is a 450-bp fragment excised from the 5'-end of
the spot 14 cDNA (provided by Cary Mariash); the probe for human TRß
is a 395-bp PCR product amplified from the 3'-end of the hTRß cDNA;
probes for Na/K ATPase 1 and Na/K ATPase 2 were 280-bp and 230-bp
PCR fragments, respectively, amplified from plasmids pGem9-MR1 and
pBSKS-MR2 (provided by Dr. Shawn Robinson); the probes for
glucose-6-phosphatase, bcl-3, and -2,3-sialyl transferase genes were
provided by Paul Yen as described previously (38); the ß-actin probe
was provided by Dr. David Pumplin.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by NIH Grant DK-54030–02 (to J.D.F.).
Received for publication July 10, 2000. Revision received August 18, 2000. Accepted for publication August 30, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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D. Sharma, J. Blum, X. Yang, N. Beaulieu, A. R. Macleod, and N. E. Davidson Release of Methyl CpG Binding Proteins and Histone Deacetylase 1 from the Estrogen Receptor {alpha} (ER) Promoter upon Reactivation in ER-Negative Human Breast Cancer Cells Mol. Endocrinol., July 1, 2005; 19(7): 1740 - 1751. [Abstract] [Full Text] [PDF]
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 P. Maruvada, N. I. Dmitrieva, J. East-Palmer, and P. M. Yen Cell Cycle-dependent Expression of Thyroid Hormone Receptor-{beta} Is a Mechanism for Variable Hormone Sensitivity Mol. Biol. Cell, April 1, 2004; 15(4): 1895 - 1903. [Abstract] [Full Text] [PDF]
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 K. De Bosscher, W. Vanden Berghe, and G. Haegeman The Interplay between the Glucocorticoid Receptor and Nuclear Factor-{kappa}B or Activator Protein-1: Molecular Mechanisms for Gene Repression Endocr. Rev., August 1, 2003; 24(4): 488 - 522. [Abstract] [Full Text] [PDF]
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K. C. Lee, J. Li, P. A. Cole, J. Wong, and W. L. Kraus Transcriptional Activation by Thyroid Hormone Receptor-{beta} Involves Chromatin Remodeling, Histone Acetylation, and Synergistic Stimulation by p300 and Steroid Receptor Coactivators Mol. Endocrinol., May 1, 2003; 17(5): 908 - 922. [Abstract] [Full Text] [PDF]
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 F. Liang, P. Webb, A. Marimuthu, S. Zhang, and D. G. Gardner Triiodothyronine Increases Brain Natriuretic Peptide (BNP) Gene Transcription and Amplifies Endothelin-dependent BNP Gene Transcription and Hypertrophy in Neonatal Rat Ventricular Myocytes J. Biol. Chem., April 18, 2003; 278(17): 15073 - 15083. [Abstract] [Full Text] [PDF]
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L. Jackson-Hayes, S. Song, E. N. Lavrentyev, M. S. Jansen, F. B. Hillgartner, L. Tian, P. A. Wood, G. A. Cook, and E. A. Park A Thyroid Hormone Response Unit Formed between the Promoter and First Intron of the Carnitine Palmitoyltransferase-Ialpha Gene Mediates the Liver-specific Induction by Thyroid Hormone J. Biol. Chem., February 28, 2003; 278(10): 7964 - 7972. [Abstract] [Full Text] [PDF]
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Q. Wang, D. Sharma, Y. Ren, and J. D. Fondell A Coregulatory Role for the TRAP-Mediator Complex in Androgen Receptor-mediated Gene Expression J. Biol. Chem., November 1, 2002; 277(45): 42852 - 42858. [Abstract] [Full Text] [PDF]
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Y. Maeda, C. Rachez, L. Hawel III, C. V. Byus, L. P. Freedman, and F. M. Sladek Polyamines Modulate the Interaction between Nuclear Receptors and Vitamin D Receptor-Interacting Protein 205 Mol. Endocrinol., July 1, 2002; 16(7): 1502 - 1510. [Abstract] [Full Text] [PDF]
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 D. Sharma and J. D. Fondell Ordered recruitment of histone acetyltransferases and the TRAP/Mediator complex to thyroid hormone-responsive promoters in vivo PNAS, June 11, 2002; 99(12): 7934 - 7939. [Abstract] [Full Text] [PDF]
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 P. M. Yen Physiological and Molecular Basis of Thyroid Hormone Action Physiol Rev, July 1, 2001; 81(3): 1097 - 1142. [Abstract] [Full Text] [PDF]
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A. Warnmark, T. Almlof, J. Leers, J.-A. Gustafsson, and E. Treuter Differential Recruitment of the Mammalian Mediator Subunit TRAP220 by Estrogen Receptors ERalpha and ERbeta J. Biol. Chem., June 22, 2001; 276(26): 23397 - 23404. [Abstract] [Full Text] [PDF]
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D. B. Jump, A. P. Thelen, and M. K. Mater Functional Interaction between Sterol Regulatory Element-binding Protein-1c, Nuclear Factor Y, and 3,5,3'-Triiodothyronine Nuclear Receptors J. Biol. Chem., September 7, 2001; 276(37): 34419 - 34427. [Abstract] [Full Text] [PDF]
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