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FIGURE 1. Organization of the ST3 promoter region and location of the putative hormone response elements. A, schematic diagram of ST3 gene. There are 3 putative hormone response elements (DR2, DR4, and DR6) flanking the two transcription start sites (arrows) with the major one at –94 and minor one at +1 (35), among which DR6 and DR2 are upstream of the transcription start sites, while DR4 is in the first intron of the ST3 gene. E, EcoRI recognition site; H, HindIII recognition site; E1 and E2, the first and second exon, respectively. B, sequences of the putative hormone response elements. Bold letters represent the half-site of each element in the direct repeats, and the faded letters represent the spacer sequences. The numbers are the relative position to the transcription start site (+1). C, sequences of the promoter region, exon 1, and 5′-end of intron 1 of X. laevis ST3 gene. The two transcription start sites are indicated by arrows, and the sequence of the first exon is underlined. Boxed sequences are putative TATA boxes, and italic bold letters represent the DR4 element. The GAGA factor binding sites known to be important for promoter function (35) are indicated by asterisks. D, sequences around the junction of intron 1 and exon 2. The first nucleotide of the exon 2 is denoted as 1E2, and the last nucleotide of the first intron is denoted as –1I. The exon sequence is underlined.
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FIGURE 2. DR4 but not DR2 or DR6 element in ST3 gene binds to TR/RXR complex in gel shift assay. A, labeled DR2, DR4, and DR6 elements were incubated with oocyte extract containing overexpressed TR/RXR, and the resulting mixtures was analyzed on a nondenaturing polyacrylamide gel. Note that only DR4 formed a stable complex with TR/RXR. B, specific competition for TR/RXR binding by ST3 DR4 element. Labeled ST3 DR4 element was incubated with TR/RXR extract in the presence of a 0, 5, 25, or 50-fold excess of indicated unlabeled competitors. The resulting mixture was analyzed as above. Note that only the upper band representing the DR4-TR ·RXR complex as seen in A was shown here. TRβ TRE and TRβ mTRE were wild type and mutant TRE (DR4 type) from the T3 regulated X. laevis TRβA gene promoter, respectively (41).
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FIGURE 3. T3 up-regulates the ST3 promoter containing the DR4 element in vivo in the context of chromatin. A, schematic diagram of the reporter construct pW with the firefly luciferase reporter gene under the control of the ST3 promoter containing the putative TRE. The arrows indicate the two transcription start sites. E1, exon 1. B, TR/RXR heterodimer activates ST3 promoter in the presence of T3. The pW construct was coinjected with the internal control plasmid phRG-TK driving the expression of Renilla luciferase into the nuclei of the oocytes with or without prior microinjection of mRNAs for FLAG-tagged TR and RXR (TR/RXR) into the cytoplasm. The oocytes were incubated at 18°C overnight in the presence or absence of 100 nm T3 and then subjected to dual luciferase assay. The firefly luciferase activity (F) over the Renilla luciferase activity (R) for each sample, i.e. F/R, was measured and plotted here with the F/R for the oocytes without TR/RXR mRNA microinjection or T3 incubation set to 1 (the actual firefly luciferase activity ranged from 10,000 to 60,000 in different oocyte samples with the background of about 200). C, RT-PCR demonstrates the up-regulation of the reporter mRNA (firefly luciferase (F-luc)) by TR/RXR plus T3. Some of the oocytes as in B were subjected to total RNA isolation. The RNA was made DNA-free and analyzed by RT-PCR for the mRNA levels of firefly luciferase driven by the ST3 promoter. A pair of primers specific for the Renilla luciferase mRNA (R-luc) driven by the coinjected internal control plasmid were included in these RT-PCR reactions. Plasmid DNA pW (lane 4) and phRG-TK (lane 5) were used in PCR reactions as positive controls for Renilla luciferase and firefly luciferase PCR. Note that although the Renilla luciferase transcripts from the T3-independent TK promoter were similar in the presence or absence of TR/RXR and/or T3 (lane 1-3), the firefly luciferase mRNA driving the ST3 promoter was up-regulated in the presence of T3 and TR/RXR (compare lane 3 to lanes 1 and 2).
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FIGURE 4. TR/RXR binds to the DR4 element in vivo and recruits corepressors to the promoter in the absence of T3. A, oocytes were injected with or without FLAG-tagged TR (F-TR)/RXR and the reporter plasmids and incubated with or without T3 as in Fig. 3. The oocytes were then subjected to ChIP assay using antibodies against the FLAG tag in TR, endogenous N-CoR, or TBLR1. The immunoprecipitates were subjected to PCR with a primer pair flanking the DR4 region to determine the presence of the DR4 TRE sequence. The PCR products were analyzed by agarose gel electrophoresis and visualized with ethidium bromide staining. Oocytes were injected and incubated with or without T3. Aliquots of DNA before antibody immunoprecipitation were amplified as the Input control to show the amounts of DNA in different samples. B, quantitative PCR analysis of the ChIP samples as shown in A. The immunoprecipitated and the Input control DNA as shown in A was analyzed by real-time PCR. The ratio of the precipitated DNA by each antibody to the corresponding Input control is shown here with the ratio in lane 1 set to 1. The results shown here represent the sum of the data from three independent experiments carried out on different days. Note that TR bound to the promoter constitutively (lanes 2 and 3) and recruited N-CoR and TBLR1 in the absence of T3 (lane 2). The addition of T3 dissociated the corepressors (lane 3).
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FIGURE 5. Intronic TRE mediates T3 regulation of the ST3 promoter. A, schematic diagram of mutant ST3 promoter constructs. Deletions were introduced into the first intron and/or the first exon of the ST3 gene to produce pM1, pM2, and pM3, all of which contained regions flanking the intron 1/exon 2 junction to allow proper splicing of the primary transcripts. The construct pM4 had an internal deletion in exon 1 and truncation of the first intron. The transcripts from constructs pW and pM4 should not undergo splicing. The total 5′-UTR nucleotide lengths (nt) (from the major start site at –94) before and after (if applicable) splicing are shown. N/A, not applicable. B, the 5′-UTR from pW inhibits translation. The constructs in A were co-injected with phRG-TK DNA into the nuclei of the oocytes. The oocytes were incubated at 18°C in an incubator overnight and isolated for dual luciferase assay. The firefly luciferase activity over Renilla luciferase (F/R) from the construct pW was denoted as 1, and the F/R ratios from other constructs were normalized accordingly. Note that all mutated promoter constructs had much higher activity than pW, suggesting that the long 5′-UTR from pW inhibited translation. C, the primary transcripts from pM1, pM2, and pM3 undergo proper splicing in the oocyte. The plasmids pW, pM1, pM2, and pM3 were injected into oocyte nucleus. The oocytes were incubated at 18°C. Total RNA was then isolated from the oocytes and subjected to RT-PCR analysis. The plasmid DNA for each construct was also amplified for comparison. The bands designated as S and US correspond to spliced and unspliced RNA transcripts, respectively. D, DNA template; R, RNA template. D, all promoter constructs containing TRE respond to T3 similarly. The promoter constructs were coinjected with phRG-TK DNA into the nuclei of the oocytes with or without prior microinjection of FLAG-tagged TR (F-TR)/RXR mRNAs into the cytoplasm. The oocytes were incubated at 18°C overnight in the presence or absence of 100 nm T3 and then isolated for dual luciferase assay. The ratio of the firefly luciferase activity (F) over that of the Renilla luciferase (R) for each sample was normalized again from the oocytes without F-TR/RXR mRNA microinjection or T3 incubation for each promoter construct separately (vertical axis). Note that for all constructs TR/RXR had little or a small repressive effect in the absence of T3 but activated strongly and similarly in the presence of T3. The exception was pM4, which had about 4-fold higher activity, possibly due to the fact that the TRE in this construct is the closest to the transcription start sites.
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Figure S1. Figure S1: Comparison of TR mRNA levels in the intestine of metamorphosing tadpoles and the ooyctes with or without F-TR mRNA injection. The intestines were dissected from tadpoles at premetamorphic stage 54 (lane 1) and during metamorphosis (stage 60, the climax of metamorphosis, lane 2), respectively, and subjected to RNA isolation. Oocytes without (lane 3) or with (lane 4) F-TR mRNA injection were collected 2 hours after mRNA microinjection and groups of 8 oocytes each were used for RNA isolation. The RNA was extracted with TRIzol reagent (Invitrogen), made DNA-free with RNA-free DNase I treatment, and re-extracted with TRIzol reagent. 0.5 μg (oocyte RNA) or 2.5 μg (intestinal RNA) of each RNA sample was used for reverse-transcription in 50 μl with a High Capacity Reverse Transcription Kit (Applied Biosystems), and 4 μl of the product were subjected real- time PCR analysis for TR transcripts. The value for the endogenous TR mRNA in oocytes (lane 3) was set to 1.
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