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First published online 14 January 2009
doi: 10.1242/dev.029769
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Rudolf Magnus Institute of Neuroscience, Department of Neuroscience & Pharmacology, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG, Utrecht, The Netherlands.
* Author for correspondence (e-mail: m.p.smidt-2{at}umcutrecht.nl)
Accepted 12 November 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: DA phenotype, Dat, Dopamine, Parkinson's disease, VMAT2, mdDA
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Chung et al., 2002;
Kim et al., 2002;
Olanow et al., 2003;
Andersson et al., 2006;
Hedlund et al., 2008).
Ultimately, stem cells could be exploited as an unlimited source of
transplantable DA neurons. However, in order to engineer stem cells with mdDA
characteristics, the need to obtain the appropriate dopaminergic phenotype
through correct molecular coding has been underlined
(Chung et al., 2005;
Martinat et al., 2006; Smidt
et al., 2007). Therefore, much effort has been put into unravelling the
multi-step process that produces a genuine mdDA neuronal population in vivo,
as this is believed to hold the key to the successful engineering of stem
cells in vitro.
One of the key regulators for development of mdDA neurons in vivo is the
orphan nuclear receptor Nurr1 (Nr4a2), which is crucial for expression of a
set of genes involved in DA metabolism, such as tyrosine hydroxylase
(Th), vesicular monoamine transporter (Vmat2), dopamine
transporter (Dat) and aromatic L-amino acid decarboxylase
(Aadc) (Zetterström et al.,
1997; Saucedo-Cardenas et al.,
1998; Baffi et al.,
1999; Le et al.,
1999; Wallén et al.,
1999; Smits et al.,
2003). Another key factor for the development of mdDA neurons is
the paired-like homeobox gene Pitx3. Because of its highly exclusive
expression in mdDA neurons within the brain, Pitx3 is a selective marker for
the generation of transplantable ES cells
(Zhao et al., 2004;
Chung et al., 2005;
Hedlund et al., 2008). Pitx3
serves a unique function in mdDA neurons, highlighted by the dramatic loss of
neurons of the substantia nigra in Pitx3-deficient mice
(Hwang et al., 2003;
Nunes et al., 2003;
Van der Munckhof et al., 2003;
Smidt et al., 2004), which is
preceded by deficient Th expression during embryonic development
(Smidt et al., 2004;
Maxwell et al., 2005;
Jacobs et al., 2007).
Strikingly, although Pitx3 is expressed in all mdDA neurons, only mdDA neurons
of the substantia nigra are affected by the loss of Pitx3, emphasizing the
complicated role of Pitx3 in mdDA neurons and the diverse properties of
subsets within the mdDA neuronal population.
Despite the fact that both Pitx3 and Nurr1 are crucial for proper
expression of Th in mdDA neurons, it has long been assumed that Pitx3 and
Nurr1 are components of independent developmental pathways
(Smidt et al., 2000;
Chung et al., 2005). However,
there is growing evidence to suggest that these pathways are interconnected at
a functional level. Studies aimed at constructing functional DA neurons from
ES cells, have shown that combined transduction of Nurr1 and
Pitx3 promotes the maturation of ES cells into a dopaminergic
phenotype (Martinat et al.,
2006). Importantly, the recent discovery of aldehyde dehydrogenase
2 (Ahd2; Aldh1a1) as transcriptional target of Pitx3 in mdDA
neurons has highlighted a significant similarity between the phenotype of
Pitx3- and Nurr1-null mice (Jacobs et al.,
2007). Similar to what was observed in
Pitx3-/- embryos, Ahd2 expression is not detected
in the midbrain area of Nurr1-deficient embryos during final differentiation
of mdDA neurons (Wallén et al.,
1999). These observations strongly suggest a functional
relationship between the homeodomain transcription factor Pitx3 and the orphan
nuclear receptor Nurr1 in the development of mdDA neurons. In this study, we
deduce the molecular mechanism of this functional interplay between Pitx3 and
Nurr1 in vivo, with extensive consequences for mdDA development. We establish
Pitx3 as an essential regulator of Nurr1-mediated transcription in mdDA
neurons, which positions Pitx3 as a crucial factor for the specification of
the dopaminergic phenotype both in vivo and in stem cell engineering for
future treatment of Parkinson's disease.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Identification of Pitx3 interacting proteins
MN9D cells were transfected with 0.5 µg
Pitx3-pcDNA3.1(-)myc-His or an equivalent molar amount of
pcDNA3.1(-)myc-His expression vector and His-tagged proteins were purified
using Ni-NTA magnetic agarose beads (Qiagen) according to manufacturer's
protocol. Purified proteins were separated by SDS PAGE and visualized by
protein gel silver-staining. Subsequently, the gel was fixed in 50% methanol
(2x15 minutes) and 5% methanol (10 minutes), rinsed with water
(3x10 seconds), soaked in 10 µM DTT (20 minutes), incubated in 0.1%
AgNO3 (20 minutes), rinsed with water and incubated in developer
solution (3% NaCO3, 0.05% formaldehyde) until protein bands were
visible. The reaction was stopped by adding citric acid and the gel was washed
in water. Protein bands of interest were excised from the gel and subjected to
nanoLC-ESI-MS Mass Spectrometry analysis (Proteome Factory).
Immunoprecipitation (IP) and western blotting
MN9D cells or tissue was homogenized in lysis buffer [50 mM Tris HCl (pH
8), 150 mM NaCl, 5 mM MgCl2, 0.5 mM EDTA, 0.2% NP-40, 5% glycerol
and 0.5 mM DTT + Complete Protease Inhibitor Cocktail (Roche)], and subjected
to IP. Two dishes (10 cm in diameter) of MN9D cells or five ventral midbrains
of E14.5 C57Bl6-Jico embryos were used for each IP. Magnetic dynal beads
(Invitrogen) were blocked in 0.5% BSA and incubated with antibodies overnight.
Lysates were incubated with antibody-bound beads for 3 hours at 4°C. Beads
were washed in lysis buffer (five times) and captured using a magnetic
separator. Elution was performed in elution buffer [50 mM Tris (pH 8.0), 10 mM
EDTA, 1% (v/v) SDS and Complete protease inhibitors] at 65°C with frequent
vortexing. Proteins were separated on Nupage 4-12% gradient gels (Invitrogen),
transferred to Hybond C extra (Amersham) and after overnight blocking in 5%
milk powder in PBS at 4°C the membranes were incubated with antibodies in
PBS-T [PBS + 0.05% (v/v) Tween-20]. Antibodies used: anti-Nurr1 [E-20; Santa
Cruz (SC)], 1:100; anti-Pitx3 (Smidt et
al., 2004), 1:10.000; anti-PSF (B92; Sigma), 1:2500; anti-Sin3a
(K-20; SC), 1:1000; anti-Ncor (C-20; SC), 1:1000; anti-SMRT (H300; SC), 1:200;
anti-RXR (sc-774; SC), 1:500; anti-HDAC1 (sc7872; SC), 1:300. Blots were
incubated with SuperSignal West Dura Extended Duration Substrate (Pierce) and
exposed to ECL films (Pierce). When applicable, films were digitized on a
FLA5000 multi imaging system (Fuji), and the amount of co-immunoprecipitated
proteins was determined by densitometry. Ratios were calculated as relative
increase in amount of co-immunoprecipitated proteins in Pitx3-/-
embryos compared with C57BL6 embryos.
Chip-on-chip analysis
Chromatin immunoprecipitation was performed according to the protocol
supplied by Nimblegen. For the in vitro ChIP-on-chip analysis,
MN9D-Nurr1Tet On13N cells were transfected with 0.5 µg
Pitx3-pcDNA3.1(-). Nurr1 expression was induced by 3 µg/ml
doxycyclin and cells were cultured for 48 hours. Two 10 cm plates were used
for each antibody. For the in vivo ChIP-on-chip analysis, five ventral
midbrains from C57Bl6-Jico E14.5 embryos were used for each ChIP. Chromatin
was sonicated to an average length of 500 bp on a Soniprep 150 sonicator
through 15 pulses of 10 seconds at one-third of maximum power. ChIP was
performed with either Nurr1 antibodies, Pitx3 antibodies or pre-immune serum
(Ctrl). ChIP-DNA was amplified using a Whole Genome Amplification kit (Sigma),
according to the manufacturer's protocol and shipped for ChIP-on-chip analysis
(Nimblegen) where Cy5-labelled Pitx3- and Nurr1-ChIP samples were hybridized
to Cy3-labelled input chromatin on a mouse promoter array set (MM8).
Microarray data was analysed using Signalmap software (v1.9). PCR-based
validation was performed on 10 ng of amplified ChIP-DNA using the following
primers: Vip, 5'-AGCGACTGAGTTGGAGATTC-3' and
5'-GTAAGCCATGGCACTAGCAC-3'; Vmat2,
5'-GTGTCTACTGTCTATCACAG-3' and
5'-GTCAAAGTGTCCATGAAGCC-3'; control,
5'-GACCCGTGTCACTGACCTAC-3' and
5'-GCCTGCTAGGAGCAGCCTTG-3'. The amount of amplified DNA was
determined by densitometry on a FLA5000 multi imaging system (Fuji) and
relative enrichment by Pitx3- and Nurr1-ChIP over pre-immune serum (Ctrl)-ChIP
was calculated. Statistical analysis was performed using Student's
t-test, comparing the relative values of enrichment of promoters of
Vip and Vmat2 to the relative values of a non-enriched
region (control) in the Vmat2 promoter, as determined by
ChIP-on-chip.
Animals
Pitx3-/- and Nurr1-/- embryos were
obtained as described previously (Smits et
al., 2003; Jacobs et al.,
2007). Pitx3-deficient Aphakia (Pitx3-/-) or
C57Bl6-Jico wild-type mice were crossed with
Pitx3gfp/gfp mice to obtain
Pitx3gfp/+ and
Pitx3gfp/- embryos.
Pitx3gfp/- embryos contain both the classical
Ak allele and an allele in which green fluorescent protein (GFP) is knocked-in
the Pitx3 locus (Zhao et al.,
2004; Maxwell et al.,
2005) and are therefore Pitx3 deficient.
Pitx3gfp/+embryos are heterozygous for
wild-type Pitx3 and GFP, and are described to have a normal development of the
mdDA system (Maxwell et al.,
2005).
Genotyping
Genotypes were determined by PCR analysis as described previously
(Saucedo-Cardenas et al.,
1998; Jacobs et al.,
2007).
In situ hybridization
In situ hybridization was performed as described previously
(Smits et al., 2003;
Smidt et al., 2004). The
following digoxigenin (DIG)-labelled probes were used: Aadc; bp
22-488 of the mouse coding sequence (cds)
(Smits et al., 2003),
Vmat2; bp 290-799 of mouse cds
(Smits et al., 2003),
Dat; bp 789-1153 of rat cds, Nurr1; 3' region of rat
Nurr1, En1; 5' region of mouse transcript, D2R; bp
345-1263 of mouse cds.
Tissue culture
Ventral midbrains of Pitx3gfp/+ and
Pitx3gfp/- embryos at stage E13.5 were
dissected in L15 medium (Gibco) and cultured in Neurobasal Medium (Gibco)
supplemented with: 2% (v/v) B-27 supplement (Gibco), 18 mM HEPES-KOH (pH 7.5),
0.5 mM L-glutamine, 26 µM beta-mercapto-ethanol and 100 units/ml
penicillin/streptomycin. Midbrains were cultured and treated with either 0 mM,
0.3 mM or 0.6 mM of sodium butyrate (Sigma) for 48 hours.
FACS sorting
Cultures were dissociated using a Papain dissociation system (Worthington)
and cells were sorted on a Cytopeia Influx Cell sorter. Sort gates were set on
forward scatter versus side scatter (life cell gate), on forward scatter
versus pulse width (elimination of clumps) and on forward scatter versus
fluorescence channel 1 (528/38) filter; GFP fluorescence). Cells were sorted
using a 100 µm nozzle at a pressure of 15 PSI with an average speed of 7000
cells/second.
Semi-quantitative RT-PCR
RNA was isolated in Trizol (Invitrogen), according to manufacturer's
protocol. Semi-quantitative RT-PCR was performed on equal amounts of RNA (0.1
ng) using a one-step RT-PCR kit (Qiagen), according to the supplied protocol.
The following primers were used: Vmat2,
5'-GCAGTCACACAAGGCTACCA-3' and
5'-TGAATAGCCCCATCCAAGAG-3'; D2R,
5'-GCCGAGTTACTGTCATGATC-3' and
5'-ACGGTGCAGAGTTTCATGTC-3'; Th,
5'-GCCACGTGGAATACACAAAG-3' and
5'-GAGGCATGACGGATGTACTG-3'; Tbp,
5'-GAGAATAAGAGAGCCACGGAC-3' and
5'-TCACATCACAGCTCCCCAC-3'. The amount of amplified DNA was
determined by densitometry as described above. Statistical analysis was
performed by two-way unpaired Student's t-test, comparing the
relative transcript levels of sodium butyrate-treated cultures with transcript
levels of untreated cultures.
Statistical analysis
The quantified results represent the average values of experiments
performed in triplicate and presented data indicate means with standard errors
(s.e.m.). Statistical analysis was performed using Student's t-test
(two-way unpaired). P
0.05 is considered significant and are
indicated by asterisks, P<0.01 is indicated with double
asterisks.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Shan et al., 2005). In the
pituitary gland, Pitx1 interacts with the orphan nuclear receptor
`steroidogenic factor 1' (SF1; Nr5a1) and multiple co-regulators to activate
SF1-mediated transcription (Tremblay et
al., 1999; Quirk et al.,
2001). For mdDA neurons, the identification of proteins that
physically interact with Pitx3 might provide clues to the relationship between
Nurr1 and Pitx3 at the level of gene transcription.
|
) (Fig. 1A).
Interestingly, PSF and Nono are reported to form a heterodimeric complex
involved in repression of nuclear receptor-mediated transcription
(Mathur et al., 2001;
Shav-Tal and Zipori, 2002;
Sewer et al., 2002;
Dong et al., 2007). PSF has
been most extensively studied in relation to nuclear receptor repression and
therefore we focussed on PSF. The interaction of Pitx3 with endogenous PSF in
MN9D cells was confirmed by means of co-immunoprecipitation (Co-IP)
(Fig. 1B). To investigate
whether the interaction of Pitx3 with PSF also exists in vivo, we performed
immunoprecipitation (IP) on E14.5 mdDA neurons. Indeed, endogenous PSF clearly
co-immunoprecipitated with Pitx3 (Fig.
1C), demonstrating the interaction of Pitx3 and the nuclear
receptor co-repressor PSF in developing mdDA neurons.
Most compelling, PSF has been shown to interact with the retinoic acid
receptor RXR (Mathur et al.,
2001), a well-described heterodimerization partner for Nurr1 in
mdDA neurons (Perlmann and Jansson,
1995; Wallen-Mackenzie et al.,
2003). Therefore, we hypothesised that, like Pitx3, Nurr1 might
also interact with PSF. Immunoprecipitation with Nurr1 antibodies was
performed in MN9D cells, and endogenous PSF was indeed detected in Nurr1
immunoprecipitates (Fig. 1D).
Moreover, PSF was also detected in Nurr1 and RXR-immunoprecipitates of E14.5
mdDA neurons, suggesting that besides Pitx3, the nuclear receptors Nurr1 and
RXR also interact with PSF in vivo (Fig.
1E). These observations, together with the previously described
involvement of PSF in repression of nuclear receptors, makes PSF an appealing
candidate for bridging Pitx3 to Nurr1 in mdDA neurons.
Pitx3 and Nurr1 target the same promoter regions in the genome
The identification of PSF as interacting protein of both Pitx3 and Nurr1
provided novel leads to the molecular background of the functional interplay
between Pitx3 and Nurr1. In their role as developmental regulators, both Pitx3
and Nurr1 are crucial for the induction of Th in mdDA neurons of the
substantia nigra (Smidt et al.,
2004; Maxwell et al.,
2005; Jacobs et al.,
2007). In addition, PSF has been shown to repress the
TH-promoter in human CHP-212 cells
(Zhong et al., 2006). Although
the involvement of Pitx3 or Nurr1 has not been investigated in this context,
other studies have independently shown that both Nurr1 and Pitx3 can bind and
activate the Th promoter (Cazorla
et al., 2000; Iwawaki et al.,
2000; Lebel et al.,
2001; Kim et al.,
2003). The finding that both Pitx3 and Nurr1 interact with PSF
could indicate that Nurr1 and Pitx3 are part of the same transcriptional
complex on the Th promoter and possibly other target genes as part of
a general mechanism for transcription regulation of mdDA-associated genes.
In order to investigate the occupancy of Pitx3 and Nurr1 on promoters of a
large and unbiased set of genes, we performed ChIP-on-chip analysis. We chose
two different approaches to obtain DA neurons. For the first approach, we
expressed Pitx3 and Nurr1 in dopaminergic MN9D cells and for the second
approach we used in vivo mdDA neurons derived from E14.5 ventral midbrain.
Chromatin immunoprecipitation (ChIP) was performed with either Pitx3, Nurr1 or
control (pre-immune serum) antibodies, and the IP-efficiency was determined by
western blot analysis (Fig.
2A,B). Amplified ChIP-DNA fragments were labelled with Cy3 and
hybridized to Cy5-labelled input chromatin on a MM8 mouse promoter array
(Nimblegen). The ChIP-on-chip analysis in MN9D cells revealed that 1652 and
541 promoter regions were enriched by ChIP for Nurr1 or Pitx3, respectively
(Fig. 2C). In the in vivo ChIP,
a smaller number of promoter regions was enriched: 208 by Nurr1 and 140 by
Pitx3 (Fig. 2D). This lower
number may reflect the more stringent conditions of promoter occupancy in the
in vivo situation compared with in a cell line. Strikingly, a substantial
amount of promoter regions that were enriched by Nurr1, were also enriched by
Pitx3 (see Tables S1 and S2 in the supplementary material). This was observed
for 
22% and 28% of the Nurr1-enriched promoter regions in MN9D cells and
in vivo, respectively. Of special interest was the enrichment of promoter
regions near genes that have previously been described to be regulated by
Nurr1. The ChIP-on-chip analysis of MN9D cells revealed that the promoter of
the Nurr1 target gene Vip (vasoactive intestinal peptide)
(Luo et al., 2007) was highly
enriched by Nurr1-ChIP (Fig.
2E). Remarkably, the same Vip promoter region was clearly
also enriched by Pitx3-ChIP, which suggests that Pitx3 and Nurr1 bind to the
same promoter region of the Nurr1 transcriptional target Vip. The
enrichment of the Vip promoter was validated by PCR on ChIP-DNA,
confirming a significant enrichment of the Vip promoter region by
both Pitx3- and Nurr1-ChIP compared with Control-ChIP in MN9D cells
[Fig. 2K,L; n=3;
P<0.01 (Pitx3-ChIP)/P<0.05 (Nurr1-ChIP)].
|
; Smits et al.,
2003). PCR-based validation confirmed the significant enrichment
of the selected Vmat2 promoter region by ChIP for both Pitx3 and
Nurr1 (Fig. 2K,L; n=3;
P<0.01). Within the tiled regions of the promoters of Ahd2,
Dat, Th and D2R, which are established as Nurr1-regulated genes
in mdDA neurons (Zetterström et al.,
1997; Le et al.,
1999; Castillo et al.,
1998; Wallén et al.,
1999; Smits et al.,
2003), we did not observe mutually enriched regions
(Fig. 2G-J). However, because
on average 5 kb of each promoter is represented on the array, we cannot
exclude the possibility that Nurr1 and Pitx3 targeted sites are outside the
tiled regions. Altogether, these data demonstrate the concerted occupancy of
Pitx3 and Nurr1 on a set of promoters, including those of the Nurr1-regulated
genes Vip and Vmat2.
|
).
Second, the Pitx3 transcriptional target gene Ahd2 is not detected in
the midbrain area of Nurr1-deficient embryos at E15.5
(Wallén et al., 1999)
or E14.5 (F.M.J.J. and M.P.S., unpublished). In order to elucidate the
gene-regulatory function of Pitx3 in relation to Nurr1 in the development of
mdDA neurons, we performed in situ hybridization on sagittal sections of E14.5
Pitx3-/- embryos, and compared them with E14.5
Nurr1-/- embryos (Fig.
3A). The normal expression of En1 and Nurr1 in
Pitx3-/- embryos and the conserved expression of
En1 and truncated Nurr1 transcripts in
Nurr1-/- embryos is indicative of a normal presence of
mdDA neurons in medial sagittal sections
(Fig. 3B,C,H,I). As described
previously, the embryonic expression of Dat and Vmat2 was
completely lost in Nurr1-/- mdDA neurons
(Fig. 3J,K), whereas the
expression of D2R and Aadc was strongly decreased
(Fig. 3L,M). Interestingly,
Dat was not detected in Pitx3-/- mdDA neurons,
whereas it was clearly expressed in Pitx3+/+ littermate
controls (Fig. 3D).
Furthermore, the expression of Vmat2, which is completely absent in
Nurr1-/- mdDA neurons, was drastically reduced in
Pitx3-/- mdDA neurons
(Fig. 3E). Notably,
Vmat2 was abundantly expressed in the hindbrain of Nurr1- and
Pitx3-deficient embryos, clearly demonstrating a mdDA neuron-specific
deficiency of Vmat2 expression. Similarly, D2R was expressed
at barely detectable levels in Nurr1-/- mdDA neurons
(Fig. 3L) and was strongly
reduced in Pitx3-/- mdDA neurons compared with littermate
controls (Fig. 3F). Whereas
Aadc expression was clearly decreased in Nurr1-/-
mdDA neurons (Fig. 3M), we
observed no difference of Aadc expression between
Pitx3-/- and Pitx3+/+ embryos in these
medial sections (Fig. 3G).
Altogether, besides the previously described effects on the expression of
Th and Ahd2, loss of Pitx3 clearly affects the expression of
Vmat2, Dat and D2R in mdDA neurons during development.
Interestingly, next to Nurr1 as essential factor for the dopaminergic
phenotype, Pitx3 is crucial for canonical expression of genes previously
described as Nurr1 downstream targets, strengthening our hypothesis that Pitx3
and Nurr1 constitute a physical and functional complex in transcription
regulation.
Loss of Pitx3 expression in vivo increases the interaction of the co-repressor SMRT with Nurr1
Our data demonstrate the concurrent involvement of Pitx3 and Nurr1 in the
regulation of the same set of genes within mdDA neurons. However, the relative
severity of the analyzed molecular phenotypes suggests that, whereas Nurr1 can
be considered a master switch on DA-gene transcription, Pitx3 appears to
regulate the transcriptional activity of Nurr1. Generally, the activity of
nuclear receptors is highly regulated by co-regulating proteins. Upon
activation of nuclear receptors, co-repressors are released and co-activators
are recruited (Fowler and Alarid,
2004; Wolf et al.,
2008). Possibly, recruitment of Pitx3 affects the composition of
the Nurr1 transcriptional complex in relation to the interaction with
co-repressor proteins such as PSF. As we have demonstrated that the
co-repressor PSF interacts with both Pitx3 and Nurr1, the interaction of Nurr1
with PSF might depend on the presence of Pitx3. Another plausible candidate in
this respect is the co-repressor Sin3a, which is recruited by PSF to mediate
nuclear receptor repression (Mathur et
al., 2001). In order to test any alterations of the composition of
the Nurr1 transcriptional complex upon Pitx3 ablation in vivo, we performed IP
for Nurr1 from E14.5 wild-type and Pitx3-/- mdDA neurons.
Importantly, in accordance with what was observed by in situ hybridization,
similar amounts of Nurr1 were immunoprecipitated from wild-type and
Pitx3-/- mdDA neurons
(Fig. 4E). Besides PSF, the
PSF-interacting co-repressor Sin3a also interacted with Nurr1 in vivo.
However, the level of interaction of Nurr1 with either PSF or Sin3a was not
affected by the presence of Pitx3 (Fig.
4A,B; n=3). These data indicate that the physical
interaction of PSF and Sin3a with Nurr1 is unrelated to the potency of Nurr1
to activate its target genes. This is in line with other studies, reporting
that activation of nuclear receptors by ligand binding does not result in
dissociation of Sin3a or PSF (Mathur et
al., 2001). Although no endogenous ligand has been identified for
Nurr1, its ligand-binding domain interacts with the co-repressors Ncor
(nuclear receptor co-repressor 1) and SMRT (silencing mediator of retinoic
acid and thyroid hormone receptor) (Codina
et al., 2004; Lammi et al.,
2008). In general, Ncor and SMRT bind to unliganded nuclear
receptors, and are believed to keep these complexes in a repressed state
(Nishihara et al., 2004).
Ligand binding to the nuclear receptor complex results in dissociation of Ncor
and SMRT, allowing activation of gene transcription. As a possible regulatory
mechanism of Nurr1-mediated transcription in mdDA neurons, the recruitment of
Pitx3 to the Nurr1 transcriptional complex might mimic the effect of ligand
activation of Nurr1, by inducing the dissociation of SMRT or Ncor. To test
this, we examined whether the level of interaction between Nurr1 and the
co-repressors Ncor and SMRT was altered upon ablation of Pitx3 in mdDA
neurons. Although we did not detect an interaction between Ncor and Nurr1
(Fig. 4C), the co-repressor
SMRT was clearly co-immunoprecipitated with Nurr1. Strikingly, we observed a
significant increase in SMRT interaction with Nurr1 in the absence of Pitx3
(Fig. 4D). The SMRT interaction
with Nurr1 was increased approximately threefold in
Pitx3-/- mdDA neurons compared with wild type, as
determined by densitometry (Fig.
4F; n=4; P<0.01). This suggests that in the
absence of Pitx3, the transcriptional activity of Nurr1 is kept in a repressed
state by SMRT, and recruitment of Pitx3 leads to activation of Nurr1, through
release of the SMRT-mediated repression.
|
;
Guenther et al., 2001;
Fischle et al., 2002). The
de-acetylated state of histones near the transcription start site is
responsible for the repression of gene transcription. The potential
involvement of HDACs in the regulation of Nurr1 was further strengthened by
the detection of endogenous HDAC1 in Nurr1 immunoprecipitates from E14.5
dissected ventral midbrains (see Fig. S1 in the supplementary material). Our
data suggest that Nurr1 occupies the promoters of target genes in
Pitx3-/- embryos but is kept in a repressed state owing to
the interaction with SMRT/HDAC complexes. Hypothetically, interference with
HDAC activity in Pitx3-/- embryos would result in
re-activation of Nurr1-mediated transcription, bypassing the necessity for
Pitx3. To test this hypothesis, we focussed on the expression of
Vmat2 and D2R because of the reduced transcript level
throughout the mdDA area in Pitx3-/- embryos
(Fig. 5B,C) and on Th
because of the mdDA subset-specific loss of Th expression in
Pitx3-/- embryos (Fig.
5D). In order to isolate mdDA neurons for gene expression analysis
by means of FACS sorting, we set up a breeding scheme to obtain
Pitx3-deficient Pitx3gfp/- and heterozygous
Pitx3gfp/+ embryos
(Zhao et al., 2004;
Maxwell et al., 2005).
Importantly, the expression of GFP was highly similar in
Pitx3gfp/- and
Pitx3gfp/+ embryos at stage E14.5
(Fig. 5E), showing the normal
presence of mdDA neurons in Pitx3-deficient mdDA neurons during embryonic
development. Ventral midbrains of Pitx3gfp/+
and Pitx3gfp/- embryos were dissected at stage
E13.5 and cultured for 2 additional days. To interfere with HDAC activity, we
treated the ventral midbrains with different concentrations of the a-specific
HDAC inhibitor sodium butyrate, after which GFP-positive mdDA neurons were
selected by FACS sorting (Fig.
5F-H). Importantly, similar numbers of GFP-positive neurons were
collected from all treated and untreated
Pitx3gfp/- and
Pitx3gfp/+ cultures. Equal amounts of RNA from
GFP-positive mdDA neurons were subjected to semi-quantitative RT-PCR to
determine the relative transcript levels of Vmat2, D2R and Th.
Tbp (TATA-box binding protein) was taken along as control, which showed
no difference in transcript levels in mdDA neurons of both genotypes and upon
treatment with sodium butyrate (Fig.
5I,M; n=3). In heterozygous
Pitx3gfp/+ mdDA neurons, no significant
increase in relative amounts of Vmat2, D2R and Th
transcripts was observed upon sodium butyrate treatment
(Fig. 5I-L; n=3). In
agreement with what was observed for Vmat2 expression by in situ
hybridization, a significant lower level of Vmat2 transcripts was
observed in untreated Pitx3gfp/- mdDA neurons,
at 7% compared with the level of Vmat2 in untreated
Pitx3gfp/+ mdDA neurons
(Fig. 5I,J; n=3).
Strikingly, treatment with 0.3 mM sodium butyrate resulted in a significant
increase in Vmat2 transcripts, restoring levels to 56% (n=3;
P<0.01). Vmat2 levels were even restored to 84% in
cultures treated with 0.6 mM sodium butyrate (n=3;
P<0.01). Similarly, D2R levels were restored from 53% in
untreated Pitx3gfp/- mdDA neurons to 98% in 0.6
mM-treated cultures (Fig. 5I,K;
n=4; P<0.01) and levels of Th were increased
from 60% in untreated Pitx3gfp/- mdDA neurons
to 94% in cultures treated with 0.6 mM sodium butyrate
(Fig. 5I,L; n=3;
P=0.05). These data clearly demonstrate that inhibition of
HDAC-mediated repression in Pitx3-/- embryos efficiently
restores the transcriptional activation of a set of Nurr1 target genes,
bypassing the necessity for Pitx3. These observations signify our proposed
mechanism that in mdDA neurons, full activation of Nurr1 target genes depends
on the release of the SMRT/HDAC-mediated repression of the Nurr1
transcriptional complex through the regulatory action of Pitx3.
|
|
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
; Wallen-Mackenzie et al.,
2003; Smits and Smidt,
2006; Alavian et al.,
2008).
Interestingly, another member of the paired like homeobox transcription
factors has been described to functionally and physically interact with
nuclear receptors in the pituitary. Pitx1 enhances the transcriptional
activity of the orphan nuclear receptor SF1
(Tremblay et al., 1999;
Quirk et al., 2001;
Melamed et al., 2002), and
Pitx1, together with the estrogen receptor, modify the repressive androgen
receptor/SF1 complex on the LHβ promoter
(Jorgensen and Nilson, 2001).
Moreover, SF1 has also been shown to interact with PSF
(Sewer et al., 2002). The
apparent analogy between the regulation of an orphan nuclear receptor by Pitx1
and our data are indicative of a more general mechanism involving PSF, in
which homeodomain transcription factors modulate nuclear receptor-mediated
transcription. Most likely, more components are involved in the proposed
mechanism. The release of the co-repressor SMRT from Nurr1 could be
accompanied by the recruitment of activating proteins, although co-activators
of Nurr1 have yet to be identified (Castro
et al., 1999; Wang et al.,
2003; Volakakis et al.,
2006; Lammi et al.,
2008). Notably, β-catenin has been reported to affect the
transcriptional activity of both Pitx factors and SF1
(Vadlamudi et al., 2005;
Hossain and Saunders., 2003)
which makes β-catenin an interesting potential component of the
Nurr1/PSF/Pitx3 complex.
|
).
Although PSF is expressed in mdDA neurons (data not shown) and forms a complex
with Pitx3 and Nurr1, the role of PSF in the Nurr1 transcriptional complex is
unclear. The interaction of Nurr1 with PSF is unchanged in
Pitx3-/- embryos, which makes it unlikely that Pitx3 is
involved in the recruitment or dissociation of PSF to the Nurr1
transcriptional complex. Importantly, besides PSF, we showed that Nurr1 also
interacts with Sin3a, which is involved in transcriptional repression by
recruiting SMRT and HDACs (Wolffe,
1997). This may indicate that the effect of Pitx3 on dissociation
of SMRT from the Nurr1 transcriptional complex also involves PSF and
Sin3a.
The shared interaction of Pitx3 and Nurr1 with PSF suggests that Pitx3 and
Nurr1 are part of the same transcriptional complex. The results from the
ChIP-on-chip analysis further strengthen this hypothesis, as we identified a
number of promoter regions that were enriched by ChIP for both Pitx3 and
Nurr1. Most intriguingly, both Pitx3 and Nurr1 interact with the promoter of
Vmat2, which is crucial for DA metabolism and is regulated by Nurr1
in vivo (Smits et al., 2003).
These observations strongly suggest that Pitx3 and Nurr1 are recruited in
concert with a number of promoters, including some that have been previously
associated with Nurr1.
The shared physical interaction of Pitx3 and Nurr1 with the promoter of
Vmat2 is translated to function in vivo, as expression analysis of
E14.5 Pitx3-/- embryos revealed an essential role for
Pitx3 for the proper expression of a set of described Nurr1 target genes,
including Vmat2. These observations shed new light on the complicated
role of Pitx3 in mdDA neurons, as it has been puzzling for a long time that
although Pitx3 is expressed in all mdDA neurons, only the subpopulation of the
substantia nigra is dependent on Pitx3 for induction of Th and
subsequential maintenance into adulthood. This discrepancy indeed holds true
for the expression of Th, underlining the existence of mdDA subsets
with differential reliance on Pitx3. However, based on analysis of the
expression patterns of Vmat2, Dat and D2R, the entire mdDA
population is affected by Pitx3 deficiency. Our observations clearly indicate
that in addition to the previously described subset-specific role for Pitx3 in
the substantia nigra, Pitx3 is crucial for the correct molecular phenotype of
mdDA neurons in general. This finding could be an explanation for the aberrant
electrophysiological properties of surviving Th-positive neurons in
Pitx3-deficient mice, which has remained unexplained until now
(Smits et al., 2005).
Interestingly, in vivo expression analysis revealed a striking difference between the relative severity of the Nurr1 target gene deficit between Nurr-/- and Pitx3-/- embryos, which is most evident for the expression of Vmat2. It appears that although Nurr1 is crucial for the expression of its target genes, Pitx3 is essential for the level of Nurr1-mediated transcription. The increased interaction of Nurr1 with the co-repressor SMRT in the absence of Pitx3 is a suitable molecular explanation for this observation. The regulatory effect of Pitx3 on the physical interaction of Nurr1 with the co-repressor SMRT strongly suggests that Pitx3 has the potency to induce the release of SMRT-mediated repression from the Nurr1 transcriptional complex. Indeed, interference with the repressive activity of HDACs, which are recruited to unliganded nuclear receptors by SMRT, efficiently restores the transcript levels of the Nurr1 target genes Vmat2, D2R and Th in cultured Pitx3gfp/- midbrains. This clearly indicates that, in the absence of Pitx3, the Nurr1 transcriptional complex is kept in a repressed state by SMRT/HDAC, which signifies the regulatory role of Pitx3 on the expression of Nurr1 target genes as we proposed in our model.
Altogether, as part of a proposed general modulatory effect of Pitx factors on nuclear receptor activity, our data provide evidence for the existence of a transcriptional complex involving Pitx3 and Nurr1, positioning Pitx3 as an essential activator of the potency of Nurr1 to drive expression of genes essential for mdDA neurons. In conclusion, our data significantly contribute to a better understanding of mdDA neuron development, as we have shown that Nurr1 and Pitx3 act in concert to induce the mdDA characteristics of neurons, which has large consequences for stem-cell engineering as a future treatment for Parkinson's disease.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/4/531/DC1
|
Footnotes |
|---|
|
REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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