The co-chaperone XAP2 is required for activation of hypothalamic thyrotropin-releasing hormone transcription in vivo.

Abstract

Transcriptional control of hypothalamic thyrotropin-releasing hormone (TRH) integrates central regulation of the hypothalamo-hypophyseal-thyroid axis and hence thyroid hormone (triiodothyronine (T(3))) homeostasis. The two beta thyroid hormone receptors, TRbeta1 and TRbeta2, contribute to T(3) feedback on TRH, with TRbeta1 having a more important role in the activation of TRH transcription. How TRbeta1 fulfils its role in activating TRH gene transcription is unknown. By using a yeast two-hybrid screening of a mouse hypothalamic complementary DNA library, we identified a novel partner for TRbeta1, hepatitis virus B X-associated protein 2 (XAP2), a protein first identified as a co-chaperone protein. TR-XAP2 interactions were TR isoform specific, being observed only with TRbeta1, and were enhanced by T(3) both in yeast and mammalian cells. Furthermore, small inhibitory RNA-mediated knockdown of XAP2 in vitro affected the stability of TRbeta1. In vivo, siXAP2 abrogated specifically TRbeta1-mediated (but not TRbeta2) activation of hypothalamic TRH transcription. This study provides the first in vivo demonstration of a regulatory, physiological role for XAP2.

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The co-chaperone XAP2 is required for activation
of hypothalamic thyrotropin-releasing hormone
transcription in vivo
Marie-Ste ´phanie Clerget Froidevaux1*, Petra Berg2*, Isabelle Seugnet1*, Ste ´phanie Decherf1, Nathalie Becker1,
Laurent M. Sachs1, Patrice Bilesimo1, Maria Nyga ˚rd2, Ingemar Pongratz2& Barbara A. Demeneix1+
1UMR 5166, CNRS/MNHN, Paris, France, and2Department for Biosciences and Nutrition, Karolinska Institute, Huddinge, Sweden
Transcriptional control of hypothalamic thyrotropin-releasing
hormone (TRH) integrates central regulation of the hypothalamo-
hypophyseal-thyroid axis and hence thyroid hormone (tri-
iodothyronine (T3)) homeostasis. The two b thyroid hormone
receptors, TRb1 and TRb2, contribute to T3feedback on TRH,
with TRb1 having a more important role in the activation of TRH
transcription. How TRb1 fulfils its role in activating TRH gene
transcription is unknown. By using a yeast two-hybrid screening
of a mouse hypothalamic complementary DNA library, we
identified a novel partner for TRb1, hepatitis virus B X-associated
protein 2 (XAP2), a protein first identified as a co-chaperone
protein. TR–XAP2 interactions were TR isoform specific, being
observed only with TRb1, and were enhanced by T3both in yeast
and mammalian cells. Furthermore, small inhibitory RNA-
mediated knockdown of XAP2 in vitro affected the stability of
TRb1. In vivo, siXAP2 abrogated specifically TRb1-mediated (but
not TRb2) activation of hypothalamic TRH transcription. This
study provides the first in vivo demonstration of a regulatory,
physiological role for XAP2.
Keywords: XAP2; hypothalamus; TRH; transcription;
TRb1 specificity
EMBO reports (2006) 7, 1035–1039. doi:10.1038/sj.embor.7400778
INTRODUCTION
Hypothalamic thyrotropin-releasing hormone (TRH) is a central
regulator of the hypothalamo-hypophyseal-thyroid axis, with the
final output being the thyroid hormones (TH) tetraiodothyronine
(T4) and triiodothyronine (T3), T3being the biologically active
form. TH homeostasis is vital both during development and
maturity, and is maintained by negative feedback exerted by T3
on TRH and thyroid-stimulating hormone (TSH) production in the
hypothalamus and pituitary, respectively.
As for other target genes, T3 modulates TRH transcription
through its nuclear TH receptors (TRs). Among the TR subtypes,
TRb1 and TRb2 (as opposed to TRa subtypes) are the key
regulators of TRH. Both TRb1 and TRb2 are equally implicated in
T3-dependent repression of TRH (Guissouma et al, 1998, 2002;
Abel et al, 2001), whereas TRb1 has a more marked role in
activation (Dupre et al, 2004).
We addressed the mechanisms of TRb1 activation of TRH by
seeking TRb1-specific partners. We used a yeast two-hybrid assay
based on a library of adult male mice paraventricular nucleus
(PVN) complementary DNA. One of the TRb1 partners identified
was the hepatitis virus B X-associated protein 2 (XAP2), also
known as ARA9 or AIP (Carver & Bradfield, 1997; Ma & Whitlock,
1997; Meyer et al, 1998). XAP2 is known to modulate the
transcriptional activity of the dioxin receptor (AhR) in vitro (Ma
& Whitlock, 1997), associating with hsp90 and regulating the
intracellular localization of AhR (Kazlauskas et al, 2001).
However, no data have shown a physiological role for XAP2
or linked it to TRs.
Our experiments show that TRb1 interacts specifically with
XAP2 and that XAP2 exerts a functional role in modulating TRH
transcription in vivo. By using a small inhibitory RNA (siRNA)
knockdown approach, we show that XAP2 is necessary for
T3-independent activation of TRH transcription mediated by
TRb1. Thus, our results show for the first time a functional and
receptor isoform-specific in vivo role for XAP2.
RESULTS AND DISCUSSION
XAP2 shows TRb1-specific functional interactions
To identify new TRb1 partners, we used a yeast two-hybrid
system. Full-length mouse TRb1, fused to the GAL4 DNA-binding
domain in pGBKT7, was used to screen a mouse PVN cDNA
library fused to the GAL4 activation domain; the library
was screened with and without T3(1mM). XAP2 was isolated in
the presence of T3. TRb1 and XAP2 interactions in yeast
were T3dependent and TR isoform specific (Fig 1A). No other
TR isoform tested (TRb2 and TRa1) interacted with XAP2 with or
Received 30 January 2006; revised 12 July 2006; accepted 12 July 2006;
published online 25 August 2006
*These authors contributed equally to this work
+Corresponding author. Tel/Fax: þ33 1 40 79 36 07;
E-mail: demeneix@mnhn.fr
1UMR 5166, CNRS/MNHN USM 501, 57 rue Cuvier, Bp 32, Paris Cedex 05, France
2Department for Biosciences and Nutrition, Karolinska Institute, 141-57 Huddinge,
Sweden
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without T3, but all TRs tested interacted with a known partner,
RXRb (Fig 1A).
To further analyse TRb1 and XAP2 interactions, we deleted
three conserved domains in the carboxy-terminal end of XAP2, the
so-called tetratricopeptide repeats (TPR; DCtermXAP2 construct).
These domains show high homologies with those of the steroid
hormone receptor-interacting immunophilin FKBP52 (Carver
& Bradfield, 1997; Ma & Whitlock, 1997; Meyer et al, 1998)
implicated in protein–protein interactions (Lamb et al, 1995).
These deletions caused loss of XAP2–TRb1 interactions in yeast
(Fig 1A). To further validate this finding, we tested the T3dose
dependency of the TRb1–XAP2 or the TRb1–DCtermXAP2
interaction in yeast using the b-galactosidase assay. Fig 1B shows
that the strength of the TRb1–XAP2 interaction is T3 dose
dependent. Interaction between full-length XAP2 and TRb1 was
reduced by half as the T3 concentration decreased from 10?6
to 10?7M and no interaction was seen at 10?8M T3.
The TR–XAP2 interactions were tested in mammalian cell lines.
The TRb1 specificity was confirmed, but there was no interaction
with TRa (Fig 2A, lanes 6,7). However, in contrast to the yeast
data, XAP2 still interacted with TRb1 in the absence of T3, but less
than with T3. A similar discrepancy between ligand-dependent
interactions in yeast and ligand-independent interactions in vitro
in mammalian cells was described by Carver & Bradfield (1997)
for XAP2 and AhR interactions.
XAP2 colocalizes with TRH and TRb1 in the PVN
We next determined whether XAP2 was expressed in the PVN and
whether it colocalized with TRH and TRb1, as colocalization of
TRH and TRb1 had already been documented by Lechan et al
(1994). Using in situ hybridization (supplementary information
and supplementary Fig S1A online), we found that TRH and XAP2
transcripts were expressed in the same regions of the PVN, and
XAP2 seemed to be more widely expressed than TRH. To assess
the expression of XAP2 protein, we used immunocytochemistry
with antibodies against XAP2 and TRb1 on immediately succes-
sive PVN sections. XAP2 and TRb1 were expressed in the same
neurons (supplementary Fig S1B online). Thus, the localization of
XAP2 is compatible with a functional interaction with TRb1 in the
hypothalamus, and with its role in regulating TRH transcription.
Extrapolating fromthe results
of XAP2 and AhR (Kazlauskas et al, 2000), showing that XAP2
modified the subcellular localization of AhR, we next investigated
whether TRb1 localization was modified in the presence of XAP2.
We used a green fluorescent protein (GFP)–TRb fusion construct
transfected into HeLa cells and followed GFP distribution
by epifluorescence. The presence or absence of XAP2 did not
modify the principally nuclear localization of TRb1 (supplemen-
tary Fig S2 online). Moreover, addition of T3 did not affect
reported oninteractions
TRβ1
TRα1
TRβ2
T3
T3 (M)
XAP2
∆Cterm
RXR
+++
–
10–6
10–7
10–8
10–9
0
0 2.5
β-Gal activity (units)
5.07.510
∆CtermXAP2
XAP2
A
B
Fig 1|Functional interactions between hepatitis virus B X-associated
protein 2 and thyroid hormone receptor in yeast. (A) XAP2: two-hybrid
experiments in yeast show T3-dependent specific interactions of XAP2
with TRb1 but not with TRa1 or TRb2. DCterm: when the carboxy-
terminal region of XAP2 is deleted, the interaction between TRb1 and
XAP2 is lost. RXR: positive control of interaction with each TR.
(B) XAP2 and TRb1 interactions in yeast depend on T3concentration.
Plasmids expressing different Gal4 fusion proteins were used in mating.
Diploids were grown with increasing doses of T3in selection medium.
b-Galactosidase (b-Gal) activity was measured by densitometry.
Each bar represents the average of two different transformants.
T3, triiodothyronine; TR, thyroid hormone receptor; TRa1, TRb1, TRb2,
TR subtypes; XAP2, hepatitis virus B X-associated protein 2.
–
–
–––+
+
–+
1234567
T3
T3
XAP2
TRβ1
TRβ1
β-Actin
–+
T3
TRα
TRα
β-Actin
A
BC
Fig 2|Hepatitis virus B X-associated protein 2–TRb1 interactions in
mammalian cells. (A) XAP2 interacts with TRb1 (but not with TRa) in
mammalian cells. P19 cells were incubated in the absence or presence of
1mM of T3for 2h. Cells were then collected and whole-cell extracts
(WCEs; lane 1) prepared. Extracts were used for immunoprecipitation
using IgG (lane 2) or no antibody (lane 3) as controls, and TRb1 (lanes
4,5) and TRa antibodies (lanes 6,7). The presence of XAP2 was examined
by western blot analysis using XAP2 antibody. (B, C): Both TRb1 (B) and
TRa (C) are present in the P19 WCE. Samples (100mg) of the WCE of the
P19 cells used for the above experiment were fractionated on a 10% SDS–
polyacrylamide gel electrophoresis gel. The levels of TRb1 and TRa were
detected by western blot analysis using TRb1 and TRa antibodies, showing
that both receptors were expressed in these cells. b-Actin was used as
loading control. T3, triiodothyronine; TRa, TRb1, thyroid hormone
receptor subtypes; XAP2, hepatitis virus B X-associated protein 2.
Activation of TRH transcription requires XAP2
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the distribution of interactions, with or without XAP2 (supple-
mentary Fig S2 online).
siXAP2 abrogates TRH activation in vivo
The central question that remained was whether XAP2 influences
the effects of TRb1 on activation of TRH transcription. To address
this question, we used siRNA against XAP2 (siXAP2-1) and first
tested their capacity to abrogate XAP2 protein production in HC11
cells in vitro. As shown in Fig 3A, siRNA against XAP2 diminished
XAP2 protein expression after 24h and even more at 48h. The
scrambled sequence (siCT) did not affect XAP2 protein produc-
tion. To test whether XAP2 was implicated in the stability of TRb1,
we knocked down XAP2 protein and followed TRb1 protein
levels. XAP2 knockdown was associated with lowered TRb1
protein levels (Fig 3B), especially in the presence of T3(Fig 3B,
right panel).
Next, we examined the effects of XAP2 knockdown in vivo
at the site of TRH production, the hypothalamus, using a TRH-
specific transcriptional assay and a recently developed, novel
in vivo siRNA delivery technique (Hassani et al, 2005). siRNAs
against XAP2 were complexed with JetSI lipids and injected into
the hypothalamus of newborn mice along with a TRH reporter
gene (TRH-luciferase (TRH-Luc); Guissouma et al, 1998; Dupre
et al, 2004) and a TRb1 expression vector. In this experimental
paradigm, TRH-Luc expression is activated in the absence of T3,
and repressed by more than 50% in the presence of T3(Fig 4B,
left-hand columns). When co-injecting with TRH-Luc and TRb1
into the hypothalamus of hypothyroid newborn mice, siXAP2 (20
and 200nM) abolished the T3-independent TRH-Luc activation
(Fig 4A, middle columns). This effect was sequence specific, as no
significant change in TRH transcription was seen with the control
siRNA (Fig 4A, siCT). However, the effect at 20nM was not
statistically different from the effect seen at 200nM, suggesting
that the maximal level of inhibition was already attained with
20nM. However, at 2nM, no significant effect on TRH transcrip-
tion was observed (data not shown). This inhibition of TRH
activation was confirmed by the use of two different siRNAs
(siXAP2-1 and siXAP2-2; Fig 4B). Given that siRNA against XAP2
abolished activation of TRH-Luc transcription in the short term
(18h post-transfection), we explored whether XAP2 knockdown
affected TH production through modified endogenous TRH
signalling. We found that, in the longer term (48h post-
transfection), siXAP2 increased total T4 levels slightly, but
significantly (supplementary Fig S3 online), probably reflecting
a rebound after the initial inhibition. This result confirms that
XAP2 contributes to endogenous TRH control and that inter-
ference with XAP2 expression affects hypothalamo-hypophyseal-
thyroid axis output.
The main result from these knockdown experiments is that
although siRNA against XAP2 abrogated T3-independent activa-
tion of TRH transcription, it had no effect on TRH repression by T3
(compare means of white bars in Fig 4B). Thus, XAP2 is necessary
for T3-independent TRH activation but not for T3-dependent
repression. Interestingly, TRb1 has a more pronounced role than
TRb2 in T3-independent activation of TRH (Dupre et al, 2004). We
thus tested whether XAP2 knockdown differentially affected TRb1
and TRb2 effects on TRH transcription. As shown in Fig 4C,
siXAP2 effects were specific to TRb1-dependent regulation of TRH
and had no effect if TRb2 was overexpressed. Thus, the in vivo
finding that siRNA against XAP2 specifically abrogated the TRb1-
dependent TRH activation provides a strong physiologic correlate
for the TRb1-specific XAP2 interaction seen in the different in vitro
situations. To further confirm the role of TRb1–XAP2 in T3-
independent activation of TRH transcription, we looked for the
presence of TRb1 on the site 4 thyroid hormone response element
(Satoh et al, 1996) of the TRH promoter. As expected, chromatin
immunoprecipitation experiments on hypothalamus from hypo-
thyroid newborn mice showed the presence of TRb1 on the TRH
promoter (representing about 3% of the input DNA; see
supplementary Fig S4 online).
In conclusion, this report shows for the first time a central,
regulatory role for XAP2, with XAP2 contributing specifically to
TRb1 activation of hypothalamic TRH transcription in a physio-
logically relevant context.
METHODS
Plasmids. TRH-Luc
by Guissouma et al (2002), pSG5-rTRb2 as described by
Dupre et al (2004) and pSG5-hXAP2 as described by Kazlauskas
et al (2000).
Two-hybrid experiments. Plasmid construction. A Gal4 DBD-
mTRb1 (full-length) expression vector was constructed by insert-
ing an EcoR1–Pst1 fragment encoding full-length mTRb1 into the
pGBKT7 vector (Clontech, Palo Alto, CA, USA). This fragment was
generated by PCR using the following primers to create,
andpSG5-rTRb1 wereasdescribed
12 h
–
+
+
–
24 h
–
+
+
–
48 h
–
+
+
–
siCT
siXAP2-1
XAP2
–T3
–
+
+
–
+T3
–
+
+
–
siCT
siXAP2-1
XAP2
TRβ1
β-Actin
β-Actin
A
B
Fig 3|In vitro hepatitis virus B X-associated protein 2 small inhibitory
RNA experiments. (A) Inhibition of XAP2 by siRNA: HC11 cells were
transiently transfected with siRNA against mouse XAP2 (siXAP2-1). The
cells were then collected after 12, 24 or 48h and whole-cell extracts were
prepared and fractionated on a 10% SDS–polyacrylamide gel
electrophoresis gel. Levels of protein expression were examined by
western blot analysis using XAP2 or b-actin antibodies. (B) The stability
of TRb1 is affected by the absence of XAP2: P19 cells were transiently
transfected with scrambled siRNA (siCT) or siRNA against mouse XAP2
(siXAP2-1) and grown for 48h. The cells were then incubated (þT3) or
not (?T3) with 1mM of T3for 2h. WCEs were prepared and fractionated
on a 10% SDS–polyacrylamide gel electrophoresis gel. The stability of
TRb1 was detected by western blot analysis using TRb1 antibody. XAP2
and b-actin antibodies were used as controls. siRNA, small inhibitory
RNA; T3, triiodothyronine; TRb1, thyroid hormone receptor subtype;
XAP2, hepatitis virus B X-associated protein 2.
Activation of TRH transcription requires XAP2
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respectively, the EcoR1 and Pst1 sites (in bold): 50-CCGGAATTCA
TGACTCCTAACAGTATGACA-3’ and 50-AACTGCAGTTAGTCCT
CAAATACTTCTAA-3’. To obtain Gal4 DBD-mTRb2 and Gal4
DBD-mTRa1 constructs, full-length mTRb2 and mTRa1 were
cloned by the same method using the following primers to
create EcoR1 and BamH1 sites (in bold), respectively: for
mTRb2, 5’-CCGGAATTCATGAACTACTGTATGCCAGAG-3’ and
5’-CGGGATCCTTAGTCCTCAAATACTTCTAA-3’; for mTRa1, 5’-
CCGGAATTCATGGAACAGAAGCCAAGCAAG-3’ and 5’-CGG
GATCCTTAGACTTCCTGATCCTC-3’. The pGADT7-DCtermXAP2
plasmid was made by digesting pGADT7-XAP2 plasmid with AgeI
and XhoI, thus deleting the region corresponding to the C terminus
of XAP2, from base 420 to base 1,032, including the three TPR
motifs and the GIFSH motif (Carver et al, 1998). The pGADT7-
RXR plasmid was isolated in the two-hybrid screen.
Library construction, screening and analysis. The MATCH-
MAKER Library Construction and Screening Kit (Clontech) was
used to construct a cDNA library using PVN of adult male mice.
Briefly, after generating double-stranded cDNA, haploid Mata
yeast strain (AH109) was co-transformed with double-stranded
cDNA and pGADT7-Rec vector, whereas the pGBKT7-TRb1
vector served to transform haploid Mata yeast strain (Y187).
Two-hybrid screening was carried out by mating the two
transformed haploid yeast strains, according to the manufacturer’s
protocol. Diploids expressing the interacting proteins were
identified by nutritional selection using three reporters (ADE2,
HIS3 and MEL1) 71mM T3. After analysis, positive clones
were rescued in Escherichia coli, sequenced and compared
with GenBank.
Strength of interaction analysis by b-galactosidase assay. A total
of about 2?108cells were grown in synthetic medium lacking
leucine and tryptophan, in the presence of T3(0, 10?9, 10?8, 10?7
and 10?6M), and then incubated at 301C for 3–4h until cells
reached the mid-log phase. The A600was measured. An aliquot of
each sample was centrifuged and resuspended in Z buffer (60mM
Na2HPO4, 40mM NaH2PO4, 10mM KCl and 1mM MgSO4).
Cells were permeabilized by two snap-freezing cycles in liquid
nitrogen. b-Galactosidase activity was assayed according to
Lee et al (1995).
Cell cultures. HeLa cells were propagated as described by
Kazlauskas et al (2001). HC11 cells were grown according to
Olsen et al (2002). Before transfection, the HC11 culture medium
was changed to filtered RPMI without phenol red, supplemented
with 5% charcoal-treated fetal bovine serum (FBS), L-glutamine
(2mM), gentamycin (1%), insulin (5mg/ml) and epidermal growth
factor (10ng/ml). P19 cells were grown on Nunc plates coated
Fig 4|In the mouse hypothalamus, hepatitis virus B X-associated protein
2 small inhibitory RNA affects only TRb1-mediated activation of
thyrotropin-releasing hormone transcription. (A) Dose-dependent effect
of XAP2 siRNA on basal TRH transcription. TRH-Luc transcription was
followed in hypothyroid 2-day-old mice, 20h after hypothalamic
injection of 1mg TRH-Luc, 100ng TRb1 expression vector and 0, 20 or
200nM of siXAP2-1 or 200nM of scrambled siRNA (siCT) complexed
with JetSI. Values are means7s.e.m., nX30; ***Po0.001. (B) Two
different XAP2 siRNAs affect the activation but not the T3-dependent
repression of TRH transcription. TRH-Luc transcription was measured in
hypothyroid 2-day-old mice, 20h after hypothalamic injection of 1mg
reporter construct, 100ng of TRb1 expression vector and 200nM of
siXAP2-1, siXAP2-2 or scrambled siRNA (siCT) complexed with JetSI.
After transfection, mice were treated with T3(2.5mg/g of body weight,
filled bars) or saline (open bars). Values are means7s.e.m., nX6; ns: not
significant; *Po0.05; **Po0.01. (C) XAP2 siRNA affects activation of
TRH transcription mediated by TRb1 but not by TRb2. TRH-Luc
transcription was measured in hypothyroid 2-day-old mice, 20h after
hypothalamic injection of 1mg reporter construct, 100ng of either TRb1
or TRb2 expression vectors and 200nM of siXAP2-1 or scrambled siRNA
(siCT) complexed with JetSI. After transfection, mice were treated with
T3(2.5mg/g of body weight, filled bars) or saline (open bars). Values are
means7s.e.m., nX14; ns: not significant; **Po0.01; ***Po0.001. Each
experiment was repeated at least three times. siRNA, small inhibitory
RNA; T3, triiodothyronine; TRb1, TRb2, thyroid hormone receptor
subtypes; TRH, thyrotropin-releasing hormone; XAP2, hepatitis virus B
X-associated protein 2.
b
140
120
100
80
60
40
20
0
0 nM200 nM
siCT
200 nM20 nM
siCT
siCT
siXAP2-1
siXAP2-1
siXAP2-1
TRβ1
siCTsiXAP2-1
TRβ2
siXAP2-2
***
***
***
**
*
*
**
**
**
*** ***
*
Percentage of control
140
120
100
80
60
40
20
0
Percentage of control
140
120
100
80
60
40
20
0
Percentage of control
A
B
C
Activation of TRH transcription requires XAP2
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with 0.1% gelatin in Dulbecco’s minimum essential medium
supplemented with 10% FBS, L-glutamine (2mM) and 1% non-
essential amino acids at 371C and 9% CO2.
Cell extracts and immunoblot assay. Immunoprecipitation and
western blots were carried out according to Kazlauskas et al
(2001). HC11 and P19 cells were grown in T3-depleted medium
7T3(1mM). Cells were collected and whole-cell extracts were
prepared. For immunoprecipitation, 250mg of cellular proteins
were incubated with anti-TRb1 or anti-TRa antibodies (Affinity
BioReagents, Ozyme, St Quentin en Yvelines, France). Anti-XAP2
antibody (1:1,000 dilution; Novus Biologica, Littleton, CO, USA)
was used for western blot (1:1,000 dilution). Secondary antibodies
were horseradish peroxidase-conjugated anti-mouse immuno-
globulins (Dako Cytomation, Glostrup, Denmark).
siRNA experiments. Generation of siRNAs. siRNAs against mouse
XAP2 were designed using the HiPerformance Design Algorithm
licensed from Novartis AG (Qiagen, Courtaboeuf, France).
Duplexes targeted against mXAP2 nucleotides 489–509 were
synthesized (siXAP2-1, 50-CACGTAGTCCTGTATCCTCTA-30). A
second siRNA (SI00058562, siXAP2-2) was from Qiagen. Either
a scrambled sequence (50-CGTCACGAGTACTCCGAAGTT-30)
or an siRNA against an irrelevant GFP sequence served as the
control (siCT).
In vitro siRNA transfection. HC11 and p19 cells were transfected
with a final concentration of 20nM of siRNA (siXAP2-1 or siCT)
using Lipofectamin Plus or Lipofectamin 2000, according to the
manufacturer’s instructions (Invitrogen, Cergy, Pontoise, France).
The transfection medium was changed after 4h (see the Cell
cultures section). Cells were collected and used for western
blotting using mouse anti-XAP2 antibody (1:1,000 dilution),
mouse anti-TRb1 antibody (1:1,000 dilution; Affinity BioReagents)
or mouse b-actin antibody (1:5,000; Sigma, St Quentin Fallavier,
France). Secondary antibodies were horseradish peroxidase-
conjugated anti-mouse immunoglobulins.
In vivo siRNA transfection–in vivo gene transfer. Animal studies
were conducted according to the principles and procedures of
Guidelines for Care and Use of Experimental Animals. In vivo
siRNA experiments were performed, as described previously
(Hassani et al, 2005), using JetSITM(Polyplus Transfection,
France). XAP2 or control siRNA (0, 20 or 200nM) was mixed
with TRH-Luc (0.25mg/ml; Plasmid Factory, Germany) and pSG5-
TRb1 or pSG5-TRb2 (25ng/ml). Complexes (2ml) were injected
bilaterally into the hypothalamus of hypothyroid 1-day-old mice,
brains were dissected and luciferase assays were carried out
20h after transfection (Guissouma et al, 1998).
Supplementary information is available at EMBO reports online
(http://www.emboreports.org).
ACKNOWLEDGEMENTS
We thank G. Bolger and M. Plateroti for antibodies and Z. Hassani for
help with siRNA experiments. The excellent technical assistance of the
animal care personnel is gratefully acknowledged. This work was
supported by EU grants Neurocoregulators, Pioneer and Cascade.
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