XB-ART-53862
Sci Rep
January 1, 2017;
7
(1):
6414.
Genome-wide identification of thyroid hormone receptor targets in the remodeling intestine during Xenopus tropicalis metamorphosis.
Abstract
Thyroid hormone (T3) affects development and metabolism in vertebrates. We have been studying intestinal remodeling during T3-dependent Xenopus metamorphosis as a model for organ maturation and formation of adult organ-specific stem cells during vertebrate postembryonic development, a period characterized by high levels of plasma T3. T3 is believed to affect development by regulating target gene transcription through T3 receptors (TRs). While many T3 response genes have been identified in different animal species, few have been shown to be direct target genes in vivo, especially during development. Here we generated a set of genomic microarray chips covering about 8000 bp flanking the predicted transcription start sites in Xenopus tropicalis for genome wide identification of TR binding sites. By using the
intestine of premetamorphic tadpoles treated with or without T3 and for chromatin immunoprecipitation assays with these chips, we determined the genome-wide binding of TR in the control and T3-treated
tadpole intestine. We further validated TR binding in vivo and analyzed the regulation of selected genes. We thus identified 278 candidate direct TR target genes. We further provided evidence that these genes are regulated by T3 and likely involved in the T3-induced formation of adult intestinal stem cells during metamorphosis.
PubMed ID:
28743885
PMC ID:
PMC5527017
Article link:
Sci Rep
Genes referenced:
abhd11
actg1
acvr1b
acy1.1
akap9
aldh2
ankrd17
anp32a
apmapl
arhgap18
arpp19
asah2
atad2b
babam2
bend7
bnip3
brd1
btg3
c14orf119
ca2
camk2g
capza2
ccdc136
ccnf
ccnj
cdc14b
cebpa
celf4
cers2
ciapin1
cited4
cndp1
commd5
cops5
creld2
csnk1g3
ctu1
cul4a
cyct
cyp20a1
dbp
decr1
dhcr24
dll1
dot1l
dpy19l4
dtx2
dusp6
dydc1
echdc2
efnb2
eif2s1
eif5
etfb
etnppl
fam214a
fbxo6
fhod1
flvcr2
foxo3
foxo6
gadd45g
galnt2
gata6
gatm
gca
ghsr
gmppa
gpat4
gpatch8
gsdme
gtdc1
h2ac21
h2az1
h3-3b
hadhb
haghl
hal.2
hbp1
hmgn1
hnf1b
hook1
hoxa4
hoxd13
hps5
hyal1
idh1
idh2
ier5
igfbp1
ikzf2
il4i1
impad1
inpp5j
insig1
irf2bpl
isoc1
ivd
junb
jup
kiaa0232
klf11
klf9
letmd1
lgals4.1
loc100488227
lonrf1
mark3
mbd3
mettl1
mgc75872
mgc89226
mhc1a
mindy2
mospd2
mpc1
mplkip
mrpl46
mrps30
mtfp1
mtmr14
mtrf1
myo19
myo5c
n4bp1
narf
ncbp2
ndufa3
nfib
nfil3
nr4a1
nrg1
ntm
nudt4
oaz1
ociad2
pafah1b1
pcca
pdlim5
pex11a
pgpep1
pias3
pim3
pla2g6
plcb2
plcxd2
plekhg1
pm20d2
pmpcb
polr2e
ppm1a
ppm1b
ppp1cc
ppp1r11
prkaa1
prtfdc1
psmc6
ptch1
pum2
rabl2b
ranbp9
rcbtb2
rhou
rps2
runx1t1
rxrb
sae1
sdha
sec24d
sema3b
sema4g
setd1a
setd1b
sgms1
sh3rf3
siah1
ski
slc25a1
slc25a23
slc25a43
slc25a48
slc2a11
slc2a2
slc3a2
smarcc2
sox4
spag7
spata5
sptlc1
stk10
syap1
tfg
tgfa
thra
thrb
timm23
tiparp
tmem130
tmem185a
tmem241
tmem243
tmem67
tnfrsf10b
tor2a
triap1
trib2
trim63
trpm1
uba52
ubxn2a
ufm1
ugp2
uqcrq
urod
vdac2
vgll1
vmo1
washc5
xpnpep2
xrcc3
zfp36
zmym2
znf420
znf706
znf827
Article Images:
[+] show captions
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Figure 1. ChIP-on-chip assay identified putative TR targets in the tadpole intestine. (A) Schematic representation of a gene in the genomic chips. About 5500 bp upstream and 2500 bp downstream of the 5′-end of the cDNA sequence in the databank for each putative gene in the Xenopus tropicalis genome database were used to design a 60 bp oligonucleotide probe at an average tiling distance of 205 bp, covering the entire 8000 bp. The probes were custom-printed onto the genomic chips. PSS: putative transcription start site. (B) Venn-diagram showing the genes with TR binding sites as identified from the ChIP-on-chip assay with the intestine samples from control and T3-treated stage 54 premetamorphic tadpoles. Note that vast majority of the genes bound by TR in the control tadpoles were also found in the T3-treated animals.
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Figure 2. The ChIP signals across the 8000 bp promoter region for PGPEP1 and TFG1 from the control and T3 treated tadpole intestine. Both the control and T3-treated groups had two independent samples as shown. The arrow points to the putative start site (PSS).
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Figure 3. ChIP confirmation of the TR binding to target sites identified from the ChIP-on-chip assay. The intestine from stage 54 tadpoles treated with or without T3 was isolated and subjected to anti-TR antibody ChIP assay and the region around the putative TR binding sites as identified from the ChIP-on-chip assay was PCR amplified. Note that no TR binding was found in the control gene (exon 5 of the TRβ gene) while all three newly identified target genes had TR binding in the absence of T3 and this binding was enhanced upon T3 treatment, in agreement with the ChIP-on-chip data. * indicates pairs of samples with significant differences (p < 0.05).
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Figure 4. RT-PCR analysis confirming the regulation of newly identified TR targets as T3 response genes. The RNA was isolated from the intestine of stage 54 tadpoles treated with or without T3 was isolated and subjected to RT-PCR analysis for gene expression. Note that all three genes were found to be induced by T3 treatment. *Indicates pairs of samples with significant differences (p < 0.05).
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Figure 5. The putative TREs in the candidate TR target genes can mediate transcriptional activation by T3 in frog oocytes. The luciferase reporter construct containing the TREs of Dot1L, MBD3, PPM1B, PGPEP1, JUNB, BEND7, respectively, was co-injected with the control Renilla luciferase construct phRG-TK into the nuclei of Xenopus oocytes with or without prior cytoplasmic injection of Xenopus laevis TRα and RXRα mRNAs or GFP mRNA as negative control. The oocytes were incubated at 18 °C overnight in the presence or absence of 100 nM T3 and then used for dual luciferase assays. The relative activities of the firefly luciferase to Renilla luciferase were plotted. Note that all the reporters responded to T3 in the presence of TR/RXR.
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Figure 6. Venn diagram showing overlap of the TR target genes identified by ChIP-on-chip assay with known developmentally regulated genes identified from an early expression microarray study. Of the 278 TR target genes identified by ChIP-on-chip assay, 95 or 34% were also found on the microarray used for the expression study (Note that the actually overlap might be higher as most genes may have different names on the microarray chips used in the expression study and on the genomic chips used here, therefore showing up as non-overlapping). Of the 95 genes present in the gene expression microarray, 38 were found to be regulated during metamorphosis in the intestine in either the epithelium (EP, 13 genes), non-epithelium (Non-EP, 14 genes) or both (11 genes), suggesting that about 40% of the genes identified here are regulated by T3 during intestinal metamorphosis.
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