Fu L et al. (2017), Genome-wide identification of thyroid hormone r...

XB-ART-53862

Sci Rep. November 15, 2017; 7 (1): 6414.

Genome-wide identification of thyroid hormone receptor targets in the remodeling intestine during Xenopus tropicalis metamorphosis.

Fu L , Das B , Matsuura K , Fujimoto K , Heimeier RA , Shi YB .

Abstract

PubMed ID: 28743885
PMC ID: PMC5527017
Article link: Sci Rep.

Genes referenced: abhd11 actg1 acvr1b acy1.1 akap9 aldh2 ankrd17 anp32a apmap 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 h2afz h3f3b hadhb haghl hal.2 hbp1 hist2h2ab 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

	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.
	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).
	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).
	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).
	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.
	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.

References:

Amano, 1999, Pubmed, Xenbase [+]