The relationship between carbamoylphosphate synthetase-I expression and in vivo occupancy of the response elements was examined by comparing a carbamoylphosphate synthetase-I-expressing
Trang 1the far-upstream enhancer of the carbamoylphosphate
synthetase gene upon glucocorticoid induction
Maarten Hoogenkamp1, Ingrid C Gaemers1, Onard J L M Schoneveld1, Atze T Das1,
Thierry Grange2and Wouter H Lamers1
1 AMC Liver Center, Academic Medical Center, University of Amsterdam, the Netherlands
2 Institut Jacques Monod du CNRS, Universites Paris 6-7, Paris, France
Many of the metabolic functions of the liver are
divi-ded in a complementary fashion among the periportal
and pericentral hepatocytes The expression of enzymes
involved in amino acid degradation and gluconeogene-sis is largely confined to the periportal hepatocytes and regulated by intracellular cAMP levels, in combination
Keywords
carbamoylphosphate synthetase-I; FoxA;
glucocorticoid receptor; in vivo footprinting;
liver
Correspondence
W H Lamers, AMC Liver Center, Academic
Medical Center, University of Amsterdam,
Meibergdreef 69-71, 1105 BK, Amsterdam,
the Netherlands
Fax: +31 205669190
Tel: +31 205665405
E-mail: w.h.lamers@amc.uva.nl
(Received 26 July 2006, revised 12 October
2006, accepted 27 October 2006)
doi:10.1111/j.1742-4658.2006.05561.x
Carbamoylphosphate synthetase-I is the flux-determining enzyme of the ornithine cycle, and neutralizes toxic ammonia by converting it to urea An
80 bp glucocorticoid response unit located 6.3 kb upstream of the trans-cription start site mediates hormone responsiveness and liver-specific expression of carbamoylphosphate synthetase-I The glucocorticoid response unit consists of response elements for the glucocorticoid receptor, forkhead box A, CCAAT⁄ enhancer-binding protein, and an unidentified protein With only four transcription factor response elements, the car-bamoylphosphate synthetase-I glucocorticoid response unit is a relatively simple unit The relationship between carbamoylphosphate synthetase-I expression and in vivo occupancy of the response elements was examined
by comparing a carbamoylphosphate synthetase-I-expressing hepatoma cell line with a carbamoylphosphate synthetase-I-negative fibroblast cell line DNaseI hypersensitivity assays revealed an open chromatin configuration
of the carbamoylphosphate synthetase-I enhancer in hepatoma cells only
In vivo footprinting assays showed that the accessory transcription factors
of the glucocorticoid response unit bound to their response elements in bamoylphosphate synthetase-I-positive cells, irrespective of whether car-bamoylphosphate synthetase-I expression was induced with hormones In contrast, the binding of glucocorticoid receptor to the carbamoylphosphate synthetase-I glucocorticoid response unit was dependent on treatment of the cells with glucocorticoids Only forkhead box A was exclusively present
in hepatoma cells, and therefore appears to be an important determinant
of the observed tissue specificity of carbamoylphosphate synthetase-I expression As the glucocorticoid receptor is the only DNA-binding protein specifically recruited to the glucocorticoid response unit upon stimulation
by glucocorticoids, it is likely to be directly responsible for the transcrip-tional activation mediated by the glucocorticoid response unit
Abbreviations
C ⁄ EBP, CCAAT ⁄ enhancer-binding protein; CPS, carbamoylphosphate synthetase-I; CRU, cAMP response unit; FoxA, forkhead box A;
GR, glucocorticoid receptor; GRE, glucocorticoid receptor response element; GRU, glucocorticoid response unit; LM, ligation-mediated; PEPCK, phosphoenolpyruvate carboxykinase; PFK-2, 6-phosphofructo-2-kinase; PKA, cAMP-dependent protein kinase; TAT, tyrosine aminotransferase.
Trang 2with glucocorticoids One of these enzymes is
car-bamoylphosphate synthetase-I (CPS; EC 6.3.4.16),
which mediates the rate-determining step of the
orni-thine cycle that converts ammonia into urea [1]
Hepatocyte-specific expression of CPS is regulated
by a distal enhancer, located 6.3 kb upstream of the
transcription start site, in combination with the
pro-moter region [2] The distal enhancer is composed of
two functional units, i.e an upstream cAMP-response
unit (CRU), covering 150–200 bp, and 100 bp further
downstream, a glucocorticoid response unit (GRU) of
approximately 80 bp Transient transfection
experi-ments have shown that the functions of these two units
are well separated The CPS CRU is the sole mediator
of cAMP-dependent transcriptional activity (O J L M
Schoneveld, M Hoogenkamp, J M P Stallen, I C
Gaemers & W H Lamers, unpublished results) On
the other hand, constructs containing the CPS GRU
and the elements at the promoter show approximately
70% of the maximal induction of reporter gene activity
after addition of dexamethasone alone The
combina-tion of dexamethasone and cAMP induces the
con-struct maximally, whereas such a concon-struct is not
sensitive to cAMP alone [3]
The GRU consists of a response element for the
ubiquitously expressed glucocorticoid receptor (GR)
and three accessory factors These accessory factors
are the liver-enriched transcription factors forkhead
box A (FoxA) and CCAAT⁄ enhancer-binding protein
(C⁄ EBP), whereas the third factor is an unidentified
75 kDa protein denoted P3 [3,4] The importance of
each individual factor has been investigated extensively
in transient transfection experiments [2,4] Mutation
analyses have shown that the presence of each of the
four elements is essential for the glucocorticoid
response
Although there does not seem to be a general rule
for how a GRU is organized, the CPS GRU and the
GRUs of gluconeogenic genes that are expressed in
hepatocytes all contain binding sites for FoxA and
C⁄ EBP [5] Mutation analyses of the CPS GRU have
shown that each of the four elements is essential for
the glucocorticoid response and that changes in
spa-cing, order or orientation of the elements all cause a
strong reduction in gene inducibility [2,4]
Neverthe-less, it is unknown what rules determine their activity
In particular, it is unknown to what extent DNA
accessibility to transcription factors and the sequence
in which the transcription factors bind play a role
Furthermore, it remains unclear whether accessory
fac-tors have to bind first to the GRU, thereby allowing
stable binding of GR, or whether it is binding of GR
that allows access for the accessory factors [6,7]
With only four transcription factor response ele-ments located within close proximity of each other, the CPS GRU is a relatively simple unit Therefore, this GRU is an ideal target with which to establish which factors are constitutively bound and which function as the trigger to initiate liver-specific gene expression In order to investigate the in vivo occupancy of the GRU response elements, we compared CPS-positive FTO-2B hepatoma cells with CPS-negative Rat-1 fibroblasts
We show that the CPS enhancer is in an open confi-guration in FTO-2B cells, whereas the chromatin is not accessible in Rat-1 cells We further show by
in vivo footprinting assays that the accessory factors bind constitutively to their response elements in the CPS-expressing hepatoma cells, but not in CPS-negat-ive fibroblasts Similarly, GR solely binds to its response element in CPS-expressing cells, but does so only after activation by its ligand
Results
Because the expression of CPS in FTO-2B hepatoma cells has previously been shown to be responsive to the hormonal stimuli relevant in vivo [2], these cells were used as a paradigm for CPS-expressing cells, whereas Rat-1 fibroblasts served as CPS-negative control cells (Fig 1A) [8] Western blot analysis showed that the
GR is expressed at a comparable level in both cell lines (Fig 1A) C⁄ EBPa DNA-binding activity was only present in nuclear extract from FTO-2B cells, whereas
C⁄ EBPb DNA-binding activity was present in nuclear extracts of both cell lines (Fig 1B) FoxA1 and FoxA2 DNA-binding activities were found only in FTO-2B nuclear extracts, whereas FoxA3 DNA-binding activity could not be detected in either cell line The P3 protein
is ubiquitously expressed [3]
Local chromatin accessibility at the CPS enhancer was determined by DNaseI hypersensitivity analysis Figure 2A shows the position of both SstI restriction sites that were used for digestion, as well as the posi-tion of the probe, directly upstream of the ) 5.3 kb SstI site Untreated samples showed the expected 7.6 kb SstI fragment and an additional, much longer, band, presumably resulting from incomplete SstI diges-tion (Fig 2B, lanes 1, 5, 9 and 13) Both in untreated and in dexamethasone⁄ cAMP-treated FTO-2B cells, fragments of 0.7–1.2 kb could be identified at inter-mediate DNaseI concentrations (Fig 2B, lanes 7 and 15) Thus, chromatin appears to be accessible at the CPS GRU irrespective of hormonal activation In con-trast, Rat-1 fibroblasts did not exhibit such a hypersen-sitive area, regardless of hormone treatment The lack
of accessibility of the GRU enhancer in Rat-1 cells
Trang 3therefore corresponds with the lack of CPS expression
in these cells, as shown in Fig 1A
We analyzed the binding of transcription factors
to the CPS GRU in Rat-1 fibroblasts and FTO-2B
hepatoma cells that were or were not treated with dex-amethasone⁄ cAMP Alterations in the accessibility of DNA sequences were visualized by ligation-mediated PCR (LM-PCR) Previously, we subjected the CPS enhancer to in vitro footprinting using an end-labeled DNA fragment The transcription factors producing the footprints in these assays were identified by com-parison with footprints produced by purified proteins [3] To compare and validate the in vivo footprinting,
we also analyzed in vitro DNaseI-treated DNA by LM-PCR Linearized plasmid containing the CPS enhancer was incubated with either BSA or rat liver nuclear extract prior to DNaseI treatment Analysis of the in vitro footprinted samples for both strands of the GRU region revealed three clear footprints (Fig 3, compare lane 1 with lane 2, and lane 7 with lane 8), in agreement with our earlier observations [3] Very simi-lar results were observed when the in vivo footprinted FTO-2B samples were compared with the Rat-1 sam-ples (Fig 3) The footprint due to C⁄ EBP binding was observed only in the FTO-2B samples at positions 322–343 FoxA binding was apparent in the FTO-2B samples at positions 343–357, and highlighted by a characteristic DNaseI hypersensitivity at position 350
on the upper strand and position 347 on the lower strand [7] Both the in vitro and in vivo experiments showed that this FoxA-specific hypersensitivity was consistently more prominent on the upper strand than
1 3 5
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9
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2
1 3 4 5 6 7 8 9 10 11 12 13 14 15 16
B
Fig 2 DNaseI hypersensitivity of the CPS upstream region (A)
Schematic representation of the upstream region of the CPS gene.
A 130 bp 32 P-labeled probe, located upstream of the SstI site at
) 5.3 kb, was used for hybridization (B) Southern blot of in vivo
DNaseI-digested DNA from Rat-1 fibroblasts and FTO-2B hepatoma
cells Cells were left untreated or were treated with
dexametha-sone and cAMP Under each condition, cells were subjected to
increasing amounts of DNaseI The position of the intact SstI–SstI
fragment and the GRU are indicated.
CPS
160 kDa
GR
95 kDa
Rat-1 FTO-2B Rat-1 FTO-2B
C/EBP probe FoxA probe
P B E /
C α Antiserum: n e C / E B P β n e F x A 1 F x A 2 F x A 3
Pα B E / C S Pβ B E / C S
1 A x F S 2 A x F S 1 A x F
S 1 A x F
Fig 1 Expression of CPS and its regulating transcription factors in Rat-1 and FTO-2B cells (A) CPS and GR were detected by western blot-ting For CPS, 32 lg of total protein from Rat-1 or FTO-2B cells was loaded per lane, whereas for GR, 50 lg of total protein was loaded per lane Amido black staining of the membrane served as loading control (B) The presence of C ⁄ EBP and FoxA family members was visualized
by antibody-mediated supershifts in electrophoretic mobility shift assays In each panel, the first lane corresponds to free probe, whereas the other lanes correspond to probe incubated with Rat-1 or FTO-2B nuclear extracts Where indicated, antibody directed against specific members of the C ⁄ EBP and FoxA families of transcription factors was added The region of the gel showing the supershifted complex with FoxA1 antibody is additionally shown after a longer exposure ‘SS’ indicates observed supershifts.
Trang 4on the lower strand Binding of P3 to positions 360–
377 was best detected on the upper strand as
protec-tion around posiprotec-tion 363 and hypersensitivity at
posi-tion 381 Although the in vitro and in vivo footprints
are highly similar to each other, the band patterns are
not identical This difference can be attributed to the
differences in DNaseI accessibility of naked DNA
(in vitro) and DNA in a chromatin context (in vivo) [9]
Interestingly, treatment of the cells with
dexametha-sone⁄ cAMP prior to DNaseI treatment did not result
in any changes in the pattern of bands for either of the
two cell types, showing that binding of C⁄ EBP and
FoxA to the CPS GRU in FTO-2B cells is
independ-ent of these hormonal stimuli
FTO-2B hepatoma cells exhibit a constitutive
cAMP-dependent protein kinase (PKA) activity [10]
In the FTO-2B-derived hepatoma cell line WT-8, PKA
activity is again fully dependent on cAMP [10]
DNa-seI footprinting of the CPS upstream enhancer
gener-ated a near-identical banding pattern in untregener-ated
FTO-2B and WT-8 cells, including the prominent
FoxA-specific hypersensitive band (Fig 4, arrow) As
already shown for FTO-2B (Fig 3), treatment of both
cell lines with dexamethasone⁄ cAMP did not alter the
banding pattern This confirms that the binding of the
accessory factors to the CPS CRU does not result
from PKA activity, but is associated with the hepatic phenotype
As expected [11], DNaseI footprinting was not suit-able for revealing the interaction between the GR and its response element (GRE), but dimethylsulfate foot-printing was (Fig 5) Comparison of the FTO-2B samples with the Rat-1 samples revealed that C⁄ EBP binding to the GRU increased and decreased sensitiv-ity to dimethylsulfate on the upper strand at positions
333 and 326, respectively, whereas changes in reactivity
at positions 331, 336 and 340 were seen on the lower strand FoxA binding resulted in protection of the gua-nines at positions 347 and 351 on the upper strand, and protection at position 352 on the lower strand These footprints in the FTO-2B samples were not influenced by treatment with dexamethasone⁄ cAMP, in accordance with the DNaseI-footprinting experiments
At the position of the GRE, differences between non-treated and hormone-non-treated Rat-1 fibroblasts were not observed on either DNA strand Moreover, there was no difference between the Rat-1 samples and the untreated FTO-2B hepatoma cells After the addition
of hormones to FTO-2B cells, however, the reactivity
of several guanines towards dimethylsulfate was altered These guanines map within the GRE region that was footprinted by the GR in vitro [3] On the
d n a r t s r e p p u
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8 7 Fig 3 DNaseI footprinting of the CPS upstream enhancer in FTO-2B and Rat-1 cells In vitro footprints were obtained by incubating a linea-rized plasmid containing the CPS GRU with 45 lg of BSA or rat liver nuclear extract, after which DNaseI was added The resulting DNA frag-ments were used as template for LM-PCR For in vivo footprints, dexamethasone ⁄ cAMP-treated (+) and untreated (–) Rat-1 fibroblasts and FTO-2B hepatoma cells were permeabilized and incubated with DNaseI After isolation of the DNA, the samples were subjected to LM-PCR.
An arrow indicates the FTO-2B-specific hypersensitive site in the FoxA-binding site Schematic representations of the GRU with its binding sites are included for clarity.
Trang 5upper strand, the bands representing the guanines at
positions 383 and 385 were increased 1.6-fold (± 0.13;
N¼ 4) and 1.7-fold (± 0.18; N ¼ 4), respectively,
whereas the band at position 392 was reduced 1.5-fold
(± 0.05; N¼ 4) On the lower strand, GR binding
resulted in protection over the region covering
posi-tions 386–400 The observed bands corresponding to
the guanines at positions 386, 395 and 400 were
decreased 1.4-fold (± 0.15; N¼ 3), 1.5-fold (± 0.13;
N¼ 3), and 1.4-fold (± 0.08; N ¼ 3), respectively
Elevated levels of cAMP alone did not lead to
altera-tions at the GRE, whereas dexamethasone alone did
(Fig 5C) Five out of six of these changes of reactivity
of guanines map within the consensus GR-binding
sites within the GRE (Fig 5D) All the guanines that
are known to be affected upon GR binding to a
similar GRE [11] showed altered reactivity Altogether,
these findings indicate that these
glucocorticoid-induced footprints were due to GR binding
Discussion
Transcriptional regulation results from the
cooper-ative binding of transcription factors [12] One of the
key determinants of the activation of genes that are
under the control of hormone response units is the order and time at which the transcription factors bind
to their response elements With only four response elements for transcription factors located within a stretch of 80 bp, the CPS GRU is ideal for determin-ing which transcription factor-binddetermin-ing events are pre-requisites and which form the final trigger for GRU activity
DNaseI hypersensitivity analysis showed that the DNA region encompassing the CPS enhancer is in an open chromatin configuration in CPS-expressing hepa-toma cells (Fig 2) An open chromatin configuration
of GRU-containing distal enhancer regions appears to
be the rule in well-differentiated hepatoma cells [13– 15], including the distal tyrosine aminotransferase (TAT) GRU at ) 5.5 kb, but this does not apply to the more proximal TAT GRU at) 2.5 kb, which needs prior exposure to glucocorticoids to acquire an open conformation [6,9,15] In contrast to its accessibility in hepatoma cells, the CPS GRU is not in an open confi-guration in the Rat-1 cell line, which does not express CPS These findings are in line with the concept that accessibility of an enhancer region to DNaseI corre-lates with expression from that enhancer [16]
The absence of CPS expression in Rat-1 fibroblasts correlates with the absence of FoxA DNA-binding activities (Fig 1) and a nonaccessible CPS GRU (Fig 2) FoxA is one of the relatively few transcription factors that can function, in the absence of ATP-dependent complexes, to open up compacted chroma-tin, thereby allowing access for other transcription factors [17] It is therefore tempting to speculate that these features underlie the absence of binding of tran-scription factors (Fig 3) and the lack of CPS expres-sion in Rat-1 cells In vivo footprinting of FTO-2B hepatoma cells, on the other hand, showed constitutive binding of FoxA and C⁄ EBP to the CPS GRU (Fig 3), whereas binding of GR was conditional, i.e dependent on treatment of the cells with dexametha-sone⁄ cAMP (Fig 5) Treatment of Rat-1 fibroblasts with dexamethasone⁄ cAMP did not result in binding
of GR, even though GR is abundantly present in these cells (Fig 1) In line with experiments showing that
GR binding to the ) 2.5 kb TAT GRU can only be detected by genomic footprinting when the accessory factors are bound to this GRU [11], these data indicate that prior binding of accessory transcription factors
is necessary to stabilize the interaction between GR and the CPS GRU In vitro experiments with the phosphoenolpyruvate carboxykinase (PEPCK) GRU showed that binding of COUP-TF and especially FoxA increased the affinity of the low-affinity PEPCK GRE for GR and decreased its dissociation rate [18]
d a r t s r e p u
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/
C
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B 2 -O T
+
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3
4 3 2 1 Fig 4 DNaseI footprinting of the CPS upstream enhancer in
FTO-2B and WT-8 hepatoma cells For in vivo footprints, FTO-FTO-2B and
WT-8 hepatoma cells, either untreated (–) or treated with
dexa-methasone ⁄ cAMP (+), were permeabilized and incubated with
DNaseI After isolation of the DNA, the samples were subjected to
LM-PCR An arrow indicates the hypersensitive site in the
FoxA-binding site Schematic representations of the GRU with its FoxA-binding
sites are included for clarity.
Trang 6For both the PEPCK and the CPS GRUs, the distance
between the binding sites for GR and FoxA is of
crit-ical importance for GRU activity, which probably
reflects a direct interaction between the two factors
[4,19]
Although it remains to be established to what extent
these findings in cell lines can be extrapolated to
pri-mary hepatocytes, the absence of FoxA from fibroblasts
is in agreement with the concept that FoxA binding is
an early event in the opening of the chromatin of
he-patocytes [17] FoxA binds in a
glucocorticoid-inde-pendent manner on the CPS GRU (Figs 3 and 5), the
PEPCK GRU [20] and the distal TAT GRU at) 5.5 kb
[9,15] Although FoxA binds to the proximal TAT
GRU at ) 2.5 kb in unstimulated FTO-2B cells, its
binding is significantly enhanced by prior exposure to
glucocorticoids and chromatin remodeling [11], whereas
in H4IIE hepatoma cells, its binding to this GRU is
strictly dependent upon GR activation [7] The
subse-quent binding of the accessory transcription factors, in
turn, stabilizes GR binding (see previous paragraph)
[11] The difference in FoxA binding between the
FTO-2B and H4IIE cell lines could be due to the constitutive
PKA activity that is present in the FTO-2B cell line FoxA binding at the) 2.5 kb TAT GRU in WT-8 cells
is, indeed, very low in the absence of glucocorticoids and can be induced by glucocorticoids and PKA activa-tion in an additive manner [11] However, FoxA bind-ing to the TAT GRU at ) 5.5 kb and the CPS GRU was constitutive in both FTO-2B and WT-8 cells, ren-dering the CPS GRU similar to the distal TAT GRU Another study in FTO-2B cells, conducted on the con-stitutively open GRU of the 6-phosphofructo-2-kinase (PFK-2) gene, showed that FoxA binding was largely glucocorticoid-dependent, despite the constitutive PKA activity and the glucocorticoid-independent chromatin remodeling [13] Taken together, these studies show that although these GRUs are all involved in mediating he-patocyte-specific expression and contain binding sites for GR in combination with response elements for the liver-enriched factors FoxA and C⁄ EBP, they differ in their recruitment of these common transcription fac-tors Although it is still unclear what determines these differences in the assembly of the transcription factor complex, transient transfection studies suggest that the precise arrangement of the various binding sites within
1 0 2 5
3 P
383
401
R
A x o
P E / C
4
0
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3’
+
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untreated +dex +forskolin +dex & forsk.
GRE
P M A c / x e
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3
7
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P
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/
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D
A
’ G G A t TG T T t c 3’
C C
-3 G c a C AG c a - ’
8
Fig 5 Dimethylsulfate footprinting of the CPS upstream enhancer in Rat-1 and FTO-2B cells Dexamethasone ⁄ cAMP-treated (+) and untreated (–) Rat-1 fibroblasts and FTO-2B hepatoma cells were incubated with 0.1% dimethylsulfate After DNA isolation, the samples were subjected to LM-PCR (A, B) Upper and lower strands, respectively Closed diamonds and circles indicate guanines showing, respectively, increased and decreased sensitivity towards dimethylsulfate in hormone-treated FTO-2B cells, whereas open diamonds and circles indicate hormone-independent decreased and increased sensitivity to dimethylsulfate in FTO-2B compared to Rat-1 cells (C) Upper strand analysis, showing that dexamethasone treatment alone is sufficient to promote GR binding in FTO2B cells (D) Sequence of the CPS GRE showing the guanine residues that showed altered reactivity to dimethylsulfate following hormonal treatment of FTO2-B cells The consensus palin-dromic GR-binding site is indicated in capital letters The guanines in bold letters are those that showed altered reactivity towards dimethyl-sulfate upon GR binding in vitro to a similar GRE [11].
Trang 7the GRUs and the relative affinities for their cognate
factors play a role, presumably in combination with the
chromatin structure established at the GRUs prior to
glucocorticoid activation [4,19]
Even though the assembly of the transcription
fac-tor complex at the CPS GRU seems to be similar to
that of the distal TAT GRU at ) 5.5 kb, there
appears to be an important difference The CPS
GRU is sufficient to enhance the activity of the basal
promoter [4], whereas the distal TAT GRU has to
cooperate with the proximal TAT GRU at ) 2.5 kb
[15] This difference may be more apparent than real,
however, because we recently showed that the CPS
GRU needs to interact with a GRE directly upstream
of the core CPS promoter to transactivate this
pro-moter [5,21]
For both the proximal TAT GRU and the PFK-2
GRU, it is not clear which transcription factor is
directly responsible for transcriptional activation, as
GR allows recruitment of FoxA and C⁄ EBP, which
could be the factors interacting with the transcription
machinery Therefore, GR could play only an indirect
role by allowing the recruitment of these factors The
distal TAT GRU, where GR is the only
DNA-bind-ing protein interactDNA-bind-ing specifically in the presence of
glucocorticoids, does not provide a clear answer to
this question, as this GRU does not activate
scription on its own Our present analysis of
tran-scription factor recruitment at the CPS GRU
therefore provides the first clear evidence that the
transcription activation domains of GR play a key
role in transcriptional activation mediated by a GRU,
as we show that it is the only DNA-binding protein
of the triad GR, FoxA and C⁄ EBP that is specifically
recruited upon glucocorticoid stimulation The
acces-sory factors FoxA and C⁄ EBP presumably allow
sta-bilization of GR binding to the GRU, and thereby
stabilization of the interaction of the coregulators
interacting with the nucleoprotein complex formed at
the GRU This raises the possibility that the
acces-sory factors play a similar role in GRUs where they
are recruited in a glucocorticoid-dependent manner,
and that the differences that are seen in the
modali-ties of recruitment do not reflect fundamental
differ-ences in their contribution to the function of the
GRUs
Experimental procedures
Cell culture
Rat-1 fibroblasts [22], FTO-2B hepatoma cells [23], and
WT-8, an FTO-2B-derived cell line overexpressing the R1a
subunit of PKA [10], were grown at 37C in DMEM⁄ F12 ⁄ 5% CO2⁄ 10% fetal bovine serum
Nuclear extracts
Nuclear extracts from rat livers were prepared as previ-ously described [24] To prepare nuclear extracts from cell lines, cells were detached with trypsin, washed in NaCl⁄ Pi containing 0.25 mm phenylmethanesulfonyl fluoride, and lysed by resuspension in 1.5 mL of 10 mm Hepes (pH 7.6),
10 mm KCl, 1.5 mm MgCl2 and 0.5 mm dithiothreitol per
2· 107cells After 8 min on ice, nuclei were pelleted by centrifugation in an Eppendorf 5417C centrifuge at
20 800 g for 30 s at 4C The pellets were resuspended in
100 lL of 20 mm Hepes (pH 7.6), 20% glycerol, 420 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA and 0.5 mm dithio-threitol, and incubated on ice for 20 min Nuclear debris was spun down at 20 800 g for 2 min at 4C (Eppendorf 5417C centrifuge)
Western blotting
Whole cell extracts were prepared by lysis of cells in 20 mm Tris (pH 7.5), 150 mm NaCl, 1% NP40, 0.5 mm dithiothrei-tol and 0.2 mm phenylmethanesulfonyl fluoride Insoluble debris was spun down in an Eppendorf centrifuge at
20 800 g for 20 s at 4C (Eppendorf 5417C centrifuge) Western blotting was performed as previously described [25]
Electrophoretic mobility shift assays
Electrophoretic mobibility shift assays were performed as previously described [4], except that the binding reaction contained 20 mm Hepes (pH 7.6), 500 mm KCl, 12% gly-cerol (v⁄ v), 1 mm EDTA, 1 mm dithiothreitol, 1 mm sper-midine, 0.5 lg of double-stranded poly(dIdC)Ælg)1 nuclear extract, and 0.3 lgÆlL)1BSA Double-stranded probes were designed on the basis of the rat CPS GRU sequence (Table 1)
Antibodies
Rabbit polyclonal antibodies against C⁄ EBPa (sc-61),
C⁄ EBPb (sc-746) and GR (sc-1003), and goat polyclonal antibodies against FoxA1 (sc-6553), FoxA2 (sc-6554) and FoxA3 (sc-5360), were obtained from Santa Cruz Biotech-nology (Santa Cruz, CA, USA) Rabbit polyclonal anti-body against CPS has been previously described [26]
DNaseI treatment
Cells were grown to 70% confluence and supplemented, where indicated, with 100 nm dexamethasone, 1 mm dibuty-ryl-cAMP and 0.1 mm 3-isobutyl-1-methylxanthine (IBMX)
Trang 82 h before the start of the experiment DNaseI treatment
was performed exactly as previously described [27]
Dimethylsulfate treatment
Cells were grown to 70% confluence and exposed, where
indicated, to 100 nm dexamethasone, 1 mm
dibutyryl-cAMP and 0.1 mm IBMX for 2 h before the start of the
experiment All solutions used for hormone-treated cells
contained 100 nm dexamethasone After exposure for
4 min at room temperature to 0.1% dimethylsulfate in
NaCl⁄ Pi, cells were washed three times with NaCl⁄ Pi and
lysed in 2.5 mL of 50 mm Tris (pH 8.0), 20 mm EDTA,
1% SDS and 100 lgÆmL)1 proteinase K per 80 cm2 flask,
digested overnight at 55C, and further processed as
des-cribed [28]
In vitro footprinting
In vitrofootprinting was performed as previously described
[29] As matrix, linearized plasmid DNA containing the
CPS enhancer region was used Protein binding was
per-formed using 45 lg of rat liver nuclear extract or BSA
DNaseI hypersensitivity analysis
Thirty micrograms of DNaseI-treated DNA was cut with
SstI, separated on a 1% agarose gel, blotted [25], and
hybridized to a 130 bp [a-32P]ATP-labeled PCR probe
LM-PCR
The starting material consisted of 1 lg of genomic DNA
for in vivo footprints or 2 ng of plasmid DNA for in vitro
footprints LM-PCR was performed as described [30], except that the linker–ligation mix contained 5% poly-ethyleneglycol-6000 The primers used are described in Table 1
Acknowledgements
The research presented in this article was financially supported by ZonMW grant 902-23-250 and by grants
to TG of the Association pour la Recherche sur le Cancer and the Ligue contre le Cancer
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Lower strand
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