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Gene and Regulation by Thyroid Hormone in Rat Immortalized Brown Adipocytes1
Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Cientificas-Universidad Autónoma de Madrid (A.M.-H., A.P.-C.), 28029 Madrid; and Departamento de Bioquímica y Biología Molecular, Facultad Medicina, Universidad Complutense de Madrid (A.S.), Madrid 28029, Spain
Address all correspondence and requests for reprints to: Dr. A. Pérez-Castillo, Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Cientificas-Universidad Autónoma de Madrid, 28029 Madrid, Spain. E-mail: aperez{at}iib.uam.es
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and C/EBPß genes is regulated by thyroid
hormone in rat liver during development. The aim of the present study
was to explore the molecular mechanisms underlying the control of
C/EBP gene expression by thyroid hormone. To achieve this goal, we
isolated and characterized a genomic clone containing 1171 bp of the
5'-flanking region of the rat C/EBP gene. This fragment was an
active promoter in MB492 cells, an immortalized brown adipocyte cell
line that expresses the endogenous C/EBP gene in a
T3-dependent manner. Sequence analysis suggested the
presence of three thyroid hormone response elements, TRE-1
(-602/-589), TRE2 (-411/-396), and TRE3 (-376/-350). The results
of deletion, mutagenesis, and gel mobility shift analysis disclosed
that only TRE-1, an ER2-type response element, represented a functional
T3 response element. Our results demonstrate that
T3 is a factor that positively regulates C/EBP gene
expression in a direct fashion. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene was the first member of the family
to be cloned and contains two major in-frame translation start sites
and consequently generates two proteins of 42 and 30 kDa (7, 8). Liver and fat are the major tissues where this gene is
expressed, and overexpression of C/EBP in preadipocytes inhibits
cell proliferation (5) and activates genes characteristic
of differentiated fat cells (3, 9, 10). Conversely,
C/EBP antisense RNA blocks terminal differentiation of
preadipocytes, indicating that this gene is essential for adipocyte
differentiation (11). In the liver, the C/EBP protein
plays a central role in energy metabolism and regulates the
transcription of a number of metabolically important genes, such as
422/aP2, phosphoenolpyruvate carboxykinase (PEPCK), and fatty acid
synthase (12). The important role of C/EBP in fat and
liver development is clearly shown in knockout mice. These animals have
a major reduction in the lipid content of the fat tissue
(6), do not store hepatic glycogen, and die from
hypoglycemia within 8 h after birth (6). Also,
studies with primary hepatocytes from these C/EBP-deficient mice
suggest that this protein influences proliferation, differentiation,
and cell survival in liver (6, 13).
Thyroid hormone (T3) is an important regulator of
growth, differentiation, and metabolic processes (14). The
primary site of initiation of T3 action resides
in the nucleus, where the interaction of this hormone with specific
nuclear receptors can either enhance or repress the rate of
transcription of responsive genes. Thyroid hormone receptors (TRs) are
transcription factors that belong to the superfamily of nuclear
receptors, which also includes, among others, retinoic acid, steroids,
and vitamin D receptors (15). T3
receptors are encoded by two distinct genes, TR (NR1A1) and TRß
(NR1A2). Alternative splicing of the TR primary transcripts at their
3'-extremity generates messenger RNAs (mRNAs) encoding TR1, which
binds T3, as well as the variants TR2 and
TR3, which do not. Alternative promoters and splicing generate two
T3-binding isoforms, TRß1 and TRß2
(16). TR activities are mediated through binding to
specific DNA elements, called thyroid hormone response elements (TREs),
that are present in the promoter of target genes. They are composed of
two hexameric half-sites closely related to the consensus AGGTCA,
arranged either as direct repeats or as palindromic or everted (ER)
palindromic arrays (17, 18).
The control of C/EBP gene expression is poorly understood. It is
known that C/EBP is developmentally regulated in liver and adipose
tissue (19, 20). Also, C/EBP mRNA has been shown to be
regulated by insulin and glucocorticoids in vivo (21, 22), and more recently we reported the regulation of this gene
by thyroid hormone (T3) and retinoic acid during
liver development (19). Therefore, to clarify the
mechanism of T3 regulation of the C/EBP gene,
we have isolated, sequenced, and studied the biological activity
of a genomic fragment encoding 1171 bp of the 5'-flanking region of the
C/EBP gene. Our results show that thyroid hormone activates
transcription from the C/EBP promoter. Functional analysis by
transient transfection of different 5'-flanking region fragments as
well as gel mobility shift assays and mutagenic analysis suggest that
the T3 effect is mediated through a functional
TRE located between nucleotides (nt) -602 and -589 (TRE-1), which is
an ER2-type response element.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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t10
(Stratagene, La Jolla, CA) was screened with a
32P-labeled 400-bp fragment of the 5'-region of
the mouse C/EBP promoter. Duplicate filters were hybridized using
standard conditions previously reported (23). Phage
plaques that hybridized positively were identified and plaque-purified
by secondary and tertiary screening procedures. Two clones were
isolated, and one clone containing a 9.0-kb insert was chosen for
further analysis. An approximately 1.2 fragment extending from nt
-1171 to +23 was sequenced by the dideoxynucleotide chain termination
method in an Applied Biosystem 377 sequencer (Foster City, CA),
amplified by PCR, and subcloned into TA-cloning vector
(Invitrogen, Groningan, The Netherlands). The amplified
-1171/+23 fragment was sequenced again to exclude any possible
mutation.
The 1.2-kb fragment was subcloned in the luciferase reporter vector
pXP-1 (pCEBP1171). The 5'-deletions of C/EBP promoter, -689/+23
(pCEBP
689), -587/+23 (pCEBP587), and -380/+23 (pCEBP380),
were generated by PCR and subcloned into the pXP-1-luc vector, and the
3'-deletions, -689/-290 (pCEBP290) and -689/-220 (pCEBP220),
were subcloned in the pT109-luc plasmid upstream of the thymidine
kinase promoter (-105/+5).
Point mutations of the TRE-1 site in the -1171/+23 fragment were created by PCR using the DNA polymerase PfuTurbo following the instructions of the QuickChange site-directed mutagenesis method (Stratagene). The sequences of the oligonucleotides (coding strand) used for mutagenesis are as follows (mutated nucleotides are underlined): MUT1, 5'-GTA GTG GGG TCG ATA TCA GTT CAG AGA TAA-3'; MUT2, 5'-GGG GTC GCC TGG CA GCT GAG ATA AAG ACG-3'; MUT3, 5'-GTA GTG GGG TCG ATT GGA TAT CAG AGA TAA-3'; MUT4, 5'-GGG GTC GCC TGT GAG TTC AGA GAT-3'; MUT5, 5'-GGG TCG CCT GTC GAG TTC AGA GAT-3'; MUT6, 5'-GGG GTC GCC TGA GTT CAG AGA T-3'; and MUT7, 5'-TGG GGT CGC CTA GTT CAG AGA T-3'.
The PCR product was digested with DpnI, and the
DpnI-treated DNA was transformed into DH5 competent
cells. Plasmids carrying inserts of the appropriate size were sequenced
in both directions.
Cell culture and transfections
MB492 cells were maintained in DMEM (Life Technologies, Inc., Grand Island, NY) supplemented with 10% FBS.
Semiconfluent cells were transiently transfected using Lipofectamine
(Life Technologies, Inc.) according to the manufacturer’s
guidelines. Briefly, 105 cells were plated on
3.5-cm tissue culture dishes in DMEM containing 10% FBS and after
24 h were transfected with the different constructions of the
C/EBP promoter. Typically, cells received 2 µg luciferase reporter
plasmid and 2 µg of an internal control plasmid, pCMVßgal, which
contains the gene for ß-galactosidase enzyme. After 8 h of
exposure to the Lipofectamine/DNA mixture, the cells were incubated in
complete medium for another 18 h. Cells were then washed,
incubated in serum-free medium containing, or not,
T3 (100 nM), and harvested after
24 h for determination of ß-galactosidase and luciferase
activities as previously described (23). Quantification of
luciferase activity was performed with a Berthlod-BioLumat
luminometer Beethold GmBH & Co., Bad Wildbad, Germany. Each
transient transfection experiment was repeated at least three times in
triplicate.
Protein preparation and gel mobility assays
To generate the rat TRß1, the complementary DNA containing the
coding region of this protein was amplified by PCR and subcloned into
the BamHI and BglII sites of pQE16, which
introduces a histidine tag on the C-terminus (pQE16 vectors;
QIAGEN, Hilden, Germany). The histidine-tagged
receptor was expressed in the XL1-Blue Escherichia coli
strain. After induction for 1.5 h with 0.1
mM isopropylthio-ß-D-galactoside
(IPTG), the histidine-tagged TRß1 was purified. Human RXR
protein was generated by expressing pGEX-RXR in NM522 E.
coli strain and, after induction with 0.1 mM
IPTG, was purified using glutathione-Sepharose 4B
(Amersham Pharmacia Biotech, Europe, GmbH,
Freiburg, Germany). For the binding reactions, 2 µg purified TRß1
and RXR proteins were preincubated for 10 min at room temperature in a
buffer containing 15 mM HEPES (pH 8.4), 60
mM KCl, 1 mM EDTA, 1
mM dithiothreitol, 5 mM
MgCl2, 12% glycerol, and 2 µg poly(dI-dC) in a
total volume of 20 µl. After a further 20-min incubation at room
temperature in the presence of 60,000 cpm labeled probes, the complexes
were separated by electrophoresis on a 5% native polyacrylamide gel.
In competition assays, nonradioactive double stranded oligonucleotides
were added to the reaction mixture in a 100-fold excess of the
radioactive probe. To perform supershift analysis, anti-TRß1 antibody
was added to the reaction mixture and incubated for an additional 15
min. The oligonucleotides used for the assays were as follows
(complementary strands are not shown): site TRE-1, 5'-GTA GTG GGG TCG
CCT GGA GTT CAG AGA TAA-3' (-611/-582); site TRE-2, 5'-CAC GGT AGC
TCA AGA CTA ACA TCC TC-3' (-417/-392); and site TRE-3, 5'-CCC CCA ACC
TTC ACC TCC CCT TGC TCG GCC TCT GGA TG-3' (-380/-343).
Northern blot analysis
Total RNA was extracted by homogenization in guanidinium
thiocyanate as previously described (24). Twenty
micrograms of RNA were electrophoresed on a 2.2 M
formaldehyde/1% agarose gel in 1 x MOPS {20
mM MOPS = (3[N-morpholino]propane-sulfonic
acid; 1 mM EDTA; 50 mM sodium acetate; 90
mM NaOH)} buffer at 100 V for 3–4 h and transferred to
nylon membranes (Nytran, Renner, Darmstadt, Germany). Labeled C/EBP
probe (>108 cpm/µg) was generated using random
primers and [32P]deoxy-CTP (3000 Ci/mmol) and
hybridized with the membranes for 20 h at 42 C (50% formamide,
3 x SSC, and 0.2% SDS). The washing conditions were 2 x
SSC and 0.5% SDS at 65 C for mild washing and 0.2 x SSC and
0.5% SDS at 65 C for stringent washing. Methylene blue staining of the
membranes was used as a loading control. Values in the text are the
average quantification of at least three independent experiments
corresponding to three different samples.
RT-PCR analysis
Two micrograms of total RNA from MB492 cells were reverse
transcribed at 42 C for 1 h with 0.5 µg of the corresponding
reverse primer and 9 U avian myeloblastosis virus reverse transcriptase
(Promega Corp., Madison, WI) in 20 µl of the PCR buffer
[20 mM Tris-HCl, 16 mM
(NH4)2SO4,
2.5 mM MgCl2, and 0.15 mg/ml BSA, pH
8.55]. After inactivation of the reverse transcriptase (65 C, 10 min),
PCR was performed using 2 µl of the reaction mixture and 2.5 U
Taq polymerase (PE Biosystems) for 25 cycles (94 C for 1
min, 55 C for 1 min, 72 C for 2 min, and a final extension at 72 C for
7 min) in a final volume of 50 µl. Aliquots of the PCR reaction were
analyzed by electrophoresis through 0.8% agarose gel and visualized
with ethidium bromide. The oligodeoxynucleotides used for PCR
amplification were as follows: rat TR1 (forward primer, 5'-GAG GAT
CCA TGG AAC AGA AGC CAA GCA AG-3'; reverse primer, 5'-GAA GAT CTG ACT
TCC TGA TCC TCA AAG AC-3') that will amplify a fragment of 1200 bp (nt
354-1554) and rat TRß1 (forward primer, 5'-GAA GAT CTA TGA CAG AAA
ATC GCC TTC CA-3'; reverse primer, 5'-GAA GAT CTG TCC TCA AAG ACT TCC
AAG AAG-3') that will amplify a fragment of 1300 bp (nt 236-1636).
Statistical analysis
The data were analyzed using the StatWorks software package.
Significant effects were determined using Student’s t test.
A statistically significant difference was considered at P 
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene expression is regulated by
T3 in MB492 cells gene promoter activity was analyzed in an
immortalized brown adipocyte cell line (MB492). This cell line was
originally obtained by cotransfection of brown adipocytes obtained from
21-day-old Wistar rat fetuses with immortalizing simian virus 40 large
T antigen and pMEXneo. This cell line maintains specific properties of
brown adipocytes, such as expression of the uncoupling protein as well
as expression of several adipose genes, including the C/EBP family of
proteins (25).
Most of the established cell lines lose their thyroid hormone
receptors; therefore, we performed RT-PCR analysis to determine whether
MB492 cells express TR1 and TRß1. As shown in Fig. 1A
, MB492 cells express appreciable
amounts of both isoforms (the identities of the PCR products were
confirmed by sequence analysis). The functionality of these receptors
was checked by analyzing the effect of T3 on the
expression of the endogenous C/EBP gene. To this end,
T3 was added to the culture medium of MB492
cells, and at different times after hormone addition, cells were
harvested and C/EBP mRNA levels were measured by Northern blot
analysis. After treatment with 100 nM
T3 for 2 and 24 h, C/EBP mRNA increased
3.5 ± 0.7- and 3.6 ± 0.5-fold, respectively, compared with
that in untreated controls (Fig. 1B). Therefore, this cell line
constitutes an excellent model to study the molecular mechanisms
implicated in the regulation of the C/EBP gene by thyroid
hormone.
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promoter gene, was cloned by screening a gt10 rat genomic library
and was subsequently sequenced bidirectionally by the Sanger method
using sequential oligonucleotides as primers. Computer analysis of this
5'-flanking sequence revealed several consensus sequences for known
regulatory elements, including a TATA box beginning at position -26,
an Sp1 binding site (-150/-143), and binding sites for particular
transcription factors, such as C/EBP (-194/-185), cAMP response
element (-1056/-1049), nerve growth factor I-A (NGFI-A; -262/-254),
and Oct-1 (-790/-778). This fragment contains also three potential
thyroid hormone response elements at nucleotides -602 to -589
(TRE-1), -411 to -396 (TRE-2), and -376 to -350 (TRE-3; Fig. 2).
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promoter, several
fragments of the C/EBP promoter were cloned upstream of the
luciferase reporter gene in the pXP1 vector (constructs pCEBP1171,
pCEBP689, pCEBP587, and pCEBP380) or in the pT109 plasmid,
containing a minimal promoter of the thymidine kinase (constructs
pCEBP290 and pCEBP220), and tested for their expression in MB492
cells. Basal activities are shown in Fig. 3A. Transfection of the pCEBP1171
construct in MB492 cells led to a modest level of luciferase activity
(assigned a relative value of 1). Deletion of 482 bp from the 5'-end of
this fragment resulted in 100-fold enhancement of the activity obtained
with the bigger construct, suggesting the removal of a negative
element. Deletion of a region between -689 and -587 (construct
pCEBP587) again reduced the activity slightly, whereas removal of
nucleotides between -587 and -380 (construct pCEBP380) elevated
the basal activity to an extent closely similar to that of the
pCEBP689 construct. These data suggest the presence of strong
negative and positive cis-elements along the C/EBP gene
promoter.
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promoter, we next
analyzed the responses to thyroid hormone of all of these constructs.
The results are shown in Fig. 3
, B and C. The fragment -1171/+23
(construct pCEBP1171) conferred maximal responsiveness to
T3 (3-fold the basal value). The response to
T3 decreased with the plasmid containing the
region -689/+23 (construct pCEBP689). No T3
stimulation of luciferase activity was observed with constructs
pCEBP587 and pCEBP380. The constructs pCEBP290 and pCEBP220
(Fig. 3C) conferred a 2-fold T3 inducibility to the
thymidine kinase promoter, indicating that the regions -689/-290 and
-689/-220 can confer T3 responsiveness to a
heterologous promoter. All of these data together suggest that the
fragment -689/-587 is the one that mediates regulation by
T3, and that sequences downstream or upstream of
this fragment are not required for induction by this hormone. These
results would rule out a functional role of putative TRE-2 (-411 to
-396) and TRE-3 (-376 to -350) in mediating the regulation of
C/EBP gene promoter by T3.
Analysis of receptor binding to TRE-1, TRE-2, and TRE-3
To determine whether the three potential response elements of the
C/EBP gene were able to bind TR, we performed gel mobility shift
assays with recombinant receptors and oligonucleotides containing
TRE-1, TRE-2, and TRE-3 sequences, and the complexes formed resolved in
a native acrylamide gel. The results presented in Fig. 4 show that TRE-2 and TRE-3 are able to
specifically bind TRß1 receptor; however, these receptors bind to
these sites only as monomers. The retarded bands were supershifted by a
TRß1-specific antibody, confirming the presence of this receptor. On
the contrary, when RXR was added to the reaction mixture, only TRE-1
was able to specifically bind TR/RXR heterodimers. The retarded bands
were competed with unlabeled TRE-containing oligonucleotides. In most
of the cases known to date, it is thought that the functional
T3-responsive element binds TRs as heterodimers
with RXR proteins. Therefore, as neither TRE-2 nor TRE-3 was able to
bind TR/RXR heterodimers, although they do bind the TRß1 receptor,
these results further suggest that the only functional TRE in the
C/EBP promoter is TRE-1.
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promoter by
T3, we generated several point mutation,
insertion, and deletion constructs, using the QuickChange site-directed
mutagenesis (see Materials and Methods) and
examined their ability to confer T3
responsiveness (Fig. 5). The constructs
were then transfected into MB492 cells, and luciferase activity
was measured 24 h after addition of T3.
Mutations in either of the half-sites of TRE-1 totally eliminated
C/EBP-TRE-1 function (MUT1-MUT3). Addition of one nucleotide (MUT4)
or deletion of one (MUT6) or two nucleotides (MUT7) in the spacing
region between the half-sites also prevented the 3-fold increase in
response to T3. However, addition of two
nucleotides to this spacing region, and therefore changing the response
element to an everted repeat separated by four nucleotides (MUT5, ER4),
did not abrogate the response to T3 of C/EBP
promoter. These results combined with the data from the deletion and
band-shift analysis indicate that TRE-1 is the functional
T3-responsive element in this promoter.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene expression in liver.
In this paper we show that the C/EBP gene promoter contains a TRE
within its 5'-flanking region that appears to mediate the induction of
C/EBP gene expression by T3 via interaction
with TR/RXR heterodimers.
Our earlier studies showed a complex regulation of C/EBP gene
expression by T3 (19). Discrepancies
in the kinetics of C/EBP transcripts and protein accumulation in
response to T3 in hypothyroid neonates prompted
us to postulate a translational and/or posttranslational mechanism of
regulation of this gene by thyroid hormone. However, and in contrast to
hypothyroid animals, the injection of T3 to
euthyroid neonates elicited a very rapid increase in C/EBP mRNA
levels (unpublished results from our laboratory), suggesting the
existence of a direct transcriptional mechanism. This possibility is
reinforced by the rapid response of the C/EBP gene to
T3 in MB492 cells. Here we have cloned 1171 bp of
the C/EBP promoter region and characterized its regulation by
thyroid hormone.
First, we studied the basal activity of the C/EBP promoter and, in
agreement with Rana et al. (26), we found that
this promoter has strong positive and negative cis-acting
elements. The induction of luciferase activity with constructs
pCEBP689 and pCEBP380 strongly suggests the existence of two
potent silencer elements in the regions -1171/-689 and -587/-380.
In addition, the decrease in luciferase activity obtained with the
construct pCEBP587 points to the existence of a strong positive
element between -689 and -587. The presence of such elements is
relatively common in other promoters (27, 28).
Sequence analysis of the promoter region located between
nucleotides -1171 and +23 revealed the presence of three potential
TR-binding sites: TRE-1 (-602/-589, TCGCCTggAGTTCA), which resembles
the TR-binding site referred to as an everted repeat with two
nucleotides of separation between the two hexamers: TGACCTnnAGGTCA;
TRE-2 (-411/-396), which resembles a TRE of the type everted repeat
with an spacing of three nucleotides; and TRE-3 (-376/-350), which is
similar to a composite site, containing several core motifs found in
the PEPCK promoter (29). All of our data indicate that
only TRE-1 is responsible for the induction of C/EBP gene promoter
by T3. Serial deletions mapped the boundaries of
the elements required for T3 induction of
C/EBP promoter activity to the interval between nucleotides -689
and -587, and consequently, only the constructs pCEBP1171 and
pCEBP689 respond to T3 in the transfection
experiments. Point mutations in either of the half-sites of the
palindromic sequence of the TRE-1 abolished
trans-activation, indicating that these two modules play a
primary role in C/EBP-TRE-1 function. Only MUT5, with four
nucleotides forming the spacing region, instead of two, was also
responsive to T3. Gel mobility shift assays
demonstrate that the only sequence bound with high affinity by TR/RXR
heterodimers is the one that includes the TRE-1 element. This sequence
is unable to bind the receptor monomers; this suggests that the
heterodimer is the molecular element that mediates thyroid hormone
action. To our knowledge, the TRE-1 of the C/EBP gene promoter is
the first ER2 proposed as a positive TRE in a native promoter.
Previously, ERs with 6 nt of spacing also have been found to be
positive TREs in natural promoters such as the promoters of the chicken
lysozyme and rat myelin basic protein genes (30, 31). In
the case of the chicken lysozyme promoter, the TRE is part of the core
nuclei of a silencer sequence, but, as indicated, this element elicits
a positive response to thyroid hormone. Elements of the palindromic
type seem to be less common, but synthetic versions of such sites
confer potent T3 responses.
Also, the C/EBP promoter has binding sites for other transcription
factors, such as C/EBP (-194/-185) and NGFI-A (-262/-254), which
mediate their actions on this gene (32, 33, 34). Both
transcription factors, C/EBP and NGFI-A, have been shown to interfere
with T3 induction (29, 35, 36) of
other genes. Our results showed that the removal of these binding sites
(constructs pCEBP290 and pCEBP220) did not affect the inductive
effect of T3 on C/EBP promoter, suggesting
that the effect of thyroid hormone is independent of the actions of
C/EBP and NGFI-A proteins.
Both factors, C/EBP and thyroid hormone, are involved in the
regulation of carbohydrate and lipid metabolism in liver and
adipose tissue, mainly through the regulation of genes involved in
these processes. C/EBP has been implicated in the transcriptional
activation of several genes, such as PEPCK, acetyl-coenzyme A
carboxylase, Glut 4, insulin receptor, and stearoyl-CoA desaturase
1 (9, 10, 29, 37, 38). Many of these genes are also
regulated by thyroid hormone, such as PEPCK, acetyl-coenzyme A
carboxylase (38, 39), and Glut4 (40, 41). In
addition, and as we have commented previously, it has been shown that
the C/EBP-binding sites present in the promoters of PEPCK and S14 are
required for T3 stimulation of these genes
(29, 35). Therefore, our findings showing a
transcriptional regulation of C/EBP gene by T3
could represent an important mode of action of thyroid hormone on
carbohydrate and lipid metabolism in liver and adipose tissue.
In summary, this report describes a direct transcriptional
regulation of C/EBP gene expression by T3.
This effect requires a TRE that binds TR/RXR heterodimers, is located
between nucleotides -602/-589, and resembles an everted repeat with
two nucleotides of separation (ER2). However, taking into account the
results obtained in vivo (19),
nontranscriptional mechanisms cannot be ruled out. Therefore, it seems
that in this particular gene, transcriptional and posttranscriptional
mechanisms act in concert to amplify the effect of thyroid hormone.
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Acknowledgments |
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plasmid, respectively. |
Footnotes |
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2 Fellow from the Fondo de Investigaciones Sanitarias de la Seguridad
Social.
Received May 25, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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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L. A. Jurado, S. Song, W. J. Roesler, and E. A. Park Conserved Amino Acids within CCAAT Enhancer-binding Proteins (C/EBPalpha and beta ) Regulate Phosphoenolpyruvate Carboxykinase (PEPCK) Gene Expression J. Biol. Chem., July 26, 2002; 277(31): 27606 - 27612. [Abstract] [Full Text] [PDF]
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M. Cortes-Canteli, M. Pignatelli, A. Santos, and A. Perez-Castillo CCAAT/Enhancer-binding Protein beta Plays a Regulatory Role in Differentiation and Apoptosis of Neuroblastoma Cells J. Biol. Chem., February 8, 2002; 277(7): 5460 - 5467. [Abstract] [Full Text] [PDF]
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