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Fig. 2. Pitx3 and Nurr1 target the same regions on promoters of Nurr1 target
genes. (A,B) Immunoprecipitation of Pitx3 and Nurr1 from
crosslinked chromatin. ChIP-Eluates from E14.5 mdDA neurons were immunoblotted
for Nurr1 (A) or Pitx3 (B). (C,D) Representation of uniquely and
mutually enriched promoter regions by ChIP for Nurr1 and Pitx3 in MN9D cells
(C) and E14.5 mdDA neurons (D) [false discovery rate (FDR)<0.01].
(E-J) Pitx3 and Nurr1 interact with the same regions within promoters
of a set of Nurr1-regulated genes. Note that high confidence binding sites of
transcription factors are identified as a positive signal for multiple
neighbouring probes (visualised as peaks). Relative enrichment of the
promoters of Vip (E), Vmat2 (F), Ahd2 (G),
Dat (H), Th (I) and D2R (J) by ChIP for Pitx3 and
Nurr1 in MN9D cells and in vivo E14.5 mdDA neurons. Regions enriched by both
Pitx3 and Nurr1 are indicated as series of red peaks. (K,L)
ChIP-PCR validation. Significant enrichment of the regions indicated in red in
E and F within the promoters of Vip (Pitx3-ChIP; n=3;
P=0.002, Nurr1-ChIP; n=3; P=0.012) and
Vmat2 (Pitx3-ChIP; n=3; P=0.006, Nurr1-ChIP;
n=3; P=0.009) by ChIP for Pitx3 and Nurr1, but not
pre-immune serum. No enrichment was observed for a region (Control) in the
Vmat2 promoter that was not enriched by ChIP-on-chip. Data are
represented as mean±s.e.m., *P<0.05,
**P<0.01; IP, immunoprecipitation; Ctrl, Pre-immune
serum; TSS, transcription start site; AB, antibody; E14.5, dissected E14.5
mdDA area.
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). The location
of the mdDA neurons is indicated in red, the broken line represents the area
shown in B-D. (B-D) In situ hybridization on coronal sections of E14.5
Pitx3+/+ and Pitx3-/- littermate
embryos. Homozygous mutant embryos are indicated with an asterisk. (B,C)
Expression of Vmat2 and D2R was decreased throughout the
mdDA area in E14.5 Pitx3-/- embryos. (D) Expression of
Th was deficient in a subpopulation of the mdDA neurons in E14.5
Pitx3-/- embryos. (E) A similar pattern of
GFP-positive neurons in E14.5 Pitx3gfp/+ (E)
and Pitx3gfp/- (E*) reveals the
apparent normal presence of mdDA neurons in Pitx3-deficient
Pitx3gfp/- embryos. (F-H) The FACS sorting gate was
set, using a E14.5 C57Bl6-Jico (Bl6) reference sample (F) to select
GFP-positive mdDA neurons from Pitx3gfp/+ (G)
and Pitx3gfp/- (H) midbrain cultures with a
purity of 98% (data not shown). (I-M) Treatment with the HDAC inhibitor
sodium butyrate restores expression of Nurr1 target genes in
Pitx3gfp/- mdDA neurons. (I) RNA from
FACS-sorted mdDA neurons derived from cultures treated with 0 mM, 0.3 mM or
0.6 mM of sodium butyrate (n=3) were subjected to semi-quantitative
RT-PCR. Relative transcript levels were determined by densitometry and
compared with transcript levels in untreated
Pitx3gfp/+ mdDA neurons. Relative values were
calculated for Vmat2 (J; Pitx3gfp/- 0.3
mM P=0.006, Pitx3gfp/- 0.6 mM;
P=0.007), D2R (K; Pitx3gfp/- 0.3
mM; P=0.0006, Pitx3gfp/- 0.6 mM;
P=0.001), Th (L; Pitx3gfp/- 0.3
mM; P=0.19, Pitx3gfp/- 0.6 mM;
P=0.05) and Tbp (M; Pitx3gfp/- 0.3
mM P=0.5, Pitx3gfp/- 0.6 mM;
P=0.5). Data are represented as mean±s.e.m.,
*P=0.05, **P<0.01.