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Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Richard A. Hodin, M.D., Department of Surgery, Massachusetts General Hospital, Gray-Bigelow 504, 55 Fruit Street, Boston, Massachusetts 02114. E-mail: rhodin{at}partners.org.
| ABSTRACT |
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1 and TRß1 were expressed in human colorectal adenocarcinoma Caco-2 cells. Northern blotting detected expression of two IAP transcripts, which were increased approximately 3-fold in response to T3. Transient transfection studies with luciferase reporter plasmids carrying various internal and 5' deletion mutations of the IAP promoter localized a putative thyroid hormone response element (TRE) to a region approximately 620 nucleotides upstream (620) of the ATG start codon. EMSAs using TR
1-retinoid X receptor
(RXR
) on sequential 5' and 3' single nucleotide deletions defined the TRE between 632 and 612 (5'-TTGAACTCAgccTGAGGTTAC-3'). Compared with the consensus TRE, the IAP-TRE is novel in that it contains an everted repeat of two nonamers (not hexamers) separated by three nucleotides. Neither TR
1 nor RXR
binds to the IAP-TRE; however, TRß1 binds to this TRE with minimal affinity. In the presence of TR and RXR
, only the TR-RXR
heterodimer binds to the IAP-TRE. Mutagenesis of either nonamer abolishes the biological activity of IAP promoter. We have thus identified a novel response element that appears to mediate the T3-induced activation of the enterocyte differentiation marker, intestinal alkaline phosphatase. | INTRODUCTION |
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knockout mice display marked hypoplasia in both the crypts and villi, and also have decreased levels of various digestive enzymes (lactase, sucrase, aminopeptidase) and gut transcription factors (Cdx-1 and Cdx-2) (9, 10, 11, 12, 13, 14). Relatively little is known about the molecular mechanisms that govern T3 action within the gut. As such, the present work was designed to elucidate the molecular mechanisms underlying the T3-mediated regulation of one specific target gene, the enterocyte differentiation marker IAP gene. IAP is a membrane-bound glycoprotein that hydrolyzes a wide variety of monophosphate esters at an alkaline pH optimum (15, 16, 17). IAP is exclusively expressed in villus enterocytes and hence serves as an excellent marker of crypt-villus differentiation (18). IAP comprises a major component of the surfactant-like particles that are seen after fat feeding (19). Although the IAP protein is thought to be involved in the process of fat digestion and absorption, a precise physiological role for IAP has not yet been determined. Recent work by Narisawa et al. (20) on IAP knockout mice has shown that IAP limits fat absorption during high-fat feeding. Previously, using both in vitro and in vivo models, we have demonstrated that the IAP gene is a direct target gene for thyroid hormone (21, 22).
T3 action is generally mediated through its nuclear receptors (TR) (23, 24) that belong to a super family of transcription factors, which also includes receptors for retinoic acid, vitamin D, various steroids (androgen, estrogen, glucocorticoids, etc.), and others. Different isoforms of TRs have been isolated. The TR
gene on human chromosome 17 generates TR
1 and TR
2 transcripts, which are the splice variants of a major transcript, and produce 410 and 490 amino acid proteins, respectively (23). In addition, other truncated TR
proteins appear to exist and may be important in modulating T3 action (10). The human TRß gene on chromosome 3 produces 461 and 476 amino acid proteins TRß1 and TRß2, respectively, from two splice variants of a single transcript (23). As bona fide receptors, TR
1, TRß1, and TRß2 contain both DNA- and ligand (T3)-binding domains. The TRs interact with specific DNA sequences termed thyroid hormone response elements (TREs), which generally contain a loosely conserved hexamer (AGGTCA) repeat intervened by four nonspecific nucleotides, also known as direct repeat 4 (DR4) (24, 25, 26, 27). The typical half-sites (AGGTCA) are rarely found to be 100% conserved in actual genes. TR can also bind to other forms of these two hexameric repeats, such as palindromic hexamers (28) and everted repeats (29). TRs require heterodimerization with retinoid X receptor (RXR) for their binding to DR4 and other TREs containing hexamer repeats (24, 30, 31, 32, 33). An octamer TRE (T(A/G)AGGTCA) has been shown to bind to a TR monomer, thus eliminating the necessity of TR-RXR heterodimerization for TR-mediated activity (34, 35).
In this study, we show that T3 induces endogenous IAP gene expression in the human colorectal adenocarcinoma Caco-2 cell line. This IAP gene trans-activation appears to be mediated through an interaction between the TR-RXR complex and a unique TRE located between 632 and 612 in the IAP promoter consisting of an everted repeat of two nonamers spaced by three nonspecific nucleotides. These results provide a molecular description of how thyroid hormone activates a specific enterocyte target gene and will have implications for understanding the myriad effects of T3 on gut development and homeostasis.
| RESULTS |
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1 and TRß1 Are Expressed in Caco-2 Cells
1 cDNA was obtained using primers TRa1.1184F and TRa1.1530R (see Materials and Methods). Similarly, the primers TRb1.121F and TRb1.605R (see Materials and Methods) amplified the expected 485 bp fragment of the TRß1 cDNA. No PCR product was amplified for TR
2 and TRß2. These results indicate that TR
1 and TRß1 are expressed in Caco-2 cells, whereas TR
2 and TRß2 are probably not expressed. The two extra 0.60- and 0.65-kb faint bands that are amplified in the case of TRß1 could represent other isoforms of TR, or artifacts of PCR.
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1 and TRß1 in Caco-2 cells, we performed Western blotting on the whole-cell lysates from untransfected Caco-2 cells as well as from Caco-2 cells transfected with an expression plasmid overexpressing TR
1 or TRß1. We also performed Western blotting on untransfected Caco-2 cell nuclear extract. The results (Fig. 1B
1 (
50 kDa) and TRß1 (
60 kDa) proteins; however, the level of TR
1 is higher than that of TRß1. Overexpression of TR
1 or TRß1 in transfected cells confirms the size and nature of the respective protein in the untransfected cells. Based upon the RT-PCR and Western data, it appears that TR
1 is the predominant TR isoform in Caco-2 cells.
T3 Activates Endogenous IAP Gene Expression
Northern analysis shows that T3 induces the expression of two IAP transcripts of 2.7 and 3.0 kb in Caco-2 cells (Fig. 1C
). Interestingly, Henthorn et al. (36) identified only one IAP transcript in Caco-2 cells using S1 mapping. The T3 effects are seen as early as 6 h, and maximal levels are seen at 24 h. Densitometric quantitation of bands of Northern autoradigraphs showed that each IAP transcript was increased approximately 3-fold after 24 h treatment with T3 (100 nM). These in vitro results are consistent with our previous data from the intact rat intestine (18, 21) that also showed T3-induced activation of the IAP gene. Hence, these in vitro and in vivo results clearly establish the IAP gene as a T3-responsive target gene.
T3 Activates the IAP-Luciferase Reporter Gene Expression
We constructed an IAP-luciferase reporter plasmid (pIAP-2574/-49) carrying the 2.5 kb 5' flanking region of the human IAP gene proximal to its ATG start codon (see Materials and Methods). To corroborate the Northern data on expression of the IAP gene (Fig. 1C
), we determined the temporal effect of T3-induced activation of the IAP-luciferase reporter gene in Caco-2 cells. We transiently transfected Caco-2 cells with pIAP-2574/-49, and determined luciferase activities after different time periods. After 24-h treatment of the cells with T3 (100 nM), the IAP promoter in pIAP-2574/-49 plasmid showed about 3-fold activation by the endogenous TRs (Fig. 1D
). Minimal effects of T3 were seen as early as 6 h. These results of transfection studies are consistent with that observed by Northern analyses.
The TRE Is Located Approximately at 620 of the 5' Regulatory Region of the IAP Gene
We constructed various derivatives of pIAP-2574/-49 carrying sequential 5' deletions of the IAP promoter (see Materials and Methods). Luciferase activities in Caco-2 cells were determined by transiently transfecting the cells with these pIAP-luciferase reporter plasmids in the presence of T3 (Fig. 2A
). T3 induces the IAP gene about 3-fold in the larger IAP reporter plasmids, but there is a major decrease in activity in the case of the pIAP-616/-49, in which the level of activity was equal to that seen in the case of the vector pGL3-Basic (carrying no insert). Shorter plasmids also showed only basal level of T3 induction, thereby localizing the TRE in between 750 and 616, a 135-bp region (Fig. 2A
). We then constructed several internal deletion mutants of the pIAP-2574/-49, focusing on this 135 bp region (Fig. 2B
). Comparison of the activations of pIAP-2574m1870 and pIAP-2574m1920 indicates that the TRE is localized in the sequence between 661 and 618. Comparison of the sequence between 661 and 618 with the established consensus TRE sequence shows that the sequence around 620, between 631 and 613 (5'-TGAACTCAgccTGAGGTTA-3'), is the likely TRE. Compared with the consensus octamer TRE sequence (T(A/G)AGGTCA) (34, 35), this putative TRE sequence appears to consist of an everted repeat of two octamer half-sites that have approximately 88% homology with the consensus TRE sequence. Most of the TREs identified until now consist of a direct repeat of two hexamers and/or octamers separated by four nonspecific bases (DR4-TRE) (24, 25, 26, 27). Based on the model of DR4-TRE, the putative IAP-TRE could also be assigned as a direct repeat of two octamer half-sites that have approximately 80% homology with the consensus octamer TRE sequence. However, this sequence has only three nucleotides (instead of the expected four) separating its octamer half-sites, indicating that this may be an atypical TRE. We constructed an internal deletion mutant pIAP-2574
TRE, specifically deleting the putative TRE from the plasmid pIAP-2574/-49 (Fig. 2B
). This deletion of the sequence between 631 and 613 completely eliminated T3-induced activation of the IAP-luciferase gene, indicating that this is, indeed, the only biologically active TRE in the human IAP gene within its 2.5-kb proximal promoter region (Fig. 2B
).
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1 and/or RXR
. We also used anti-TR
1 antibody to supershift any TRE-TR
1 complex. Figure 3A
1 nor RXR
alone is capable of binding to the IAP-TRE (lanes 3 and 4, respectively). However, the TR
1-RXR
complex efficiently binds to the TRE thus generating a shifted band (lane 5), which is supershifted by the anti-TR
1 antibody (lane 6). The anti-TRß1 antibodies were used for control purpose and did not supershift the DNA-TR
1-RXR
complex (lanes 7 and 8), indicating the specificity of the anti-TR
1 antibody. Fifty-fold excess of cold competitor oligonucleotides TRE1G and DR4 can compete with TRE1G probe, whereas the mutant mTRE cannot compete (lanes 10, 11, and 12, respectively), thus showing the specificity of binding. Reticulocyte (TnT, transcription and translation in vitro) lysate (lane 2) or anti-TR
1 antibody (lane 9) alone was also used as a control to verify the specificity of the identified band. These results indicate the presence of a genuine TRE sequence in the TRE1G that binds to TR
1-RXR
heterodimer.
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synthesized in vitro. Unlike TR
1, under the present and similar conditions of binding, TRß1 alone can bind to the IAP-TRE, however, the binding affinity is very weak (Fig. 3B
, TRß1 binds to the IAP-TRE only as a TRß1-RXR
heterodimer, generating a major shifted band (lane 4), which is further supershifted by one of the two anti-TRß1 antibodies (lane 8). The minor faint band (lane 4) is of uncertain nature, most probably a nonspecific one. Fifty-fold excess of cold competitor oligonucleotides TRE1G and DR4 can compete with TRE1G probe, whereas the mutant mTRE cannot compete (lanes 5, 6, and 7, respectively), thus showing the specificity of binding. These results indicate that the TRE sequence in the TRE1G also binds to the TRß1-RXR
complex.
Nuclear Extract from Caco-2 Cells Binds to the IAP-TRE
Nuclear extract from Caco-2 cells was incubated with the radiolabeled TRE1G probe, and EMSA was performed. The results are shown in Fig. 3C
. Two shifted bands are observed, which proves that components of nuclear extract are capable of binding to the TRE1G oligonucleotide containing the putative TRE (lane 2). The major band was supershifted with the anti-TR
1 antibody (lane 6) confirming that Caco-2 cells express endogenous TR
1, which is capable of binding to the IAP-TRE and could be recognized by the anti-TR
1 antibody. However, the major shifted bands could not be supershifted with anti-TRß1 antibodies (lanes 7 and 8), even though Caco-2 cells express TRß1 as evidenced by RT-PCR (Fig. 1A
) and Western blotting (Fig. 1B
). The minor shifted band could not be supershifted with the anti-TR
1 or anti-TRß1 antibodies suggesting the nonspecific nature of this band. These data concur with the EMSA results obtained by using the in vitro-synthesized TR
1 and RXR
proteins (Fig. 3A
) and suggest that TR
1 is the major TR isoform in Caco-2 cells.
T3 Inhibits the Binding of Anti-TR
1 Antibody to the TRE-TR
1-RXR
Complex
Using EMSA, we determined the effect of T3 (100 nM) on the binding of the TR
1-RXR
protein complex to the IAP-TRE. We also determined the effect of T3 (100 nM) on the binding of the TR antibodies to the protein-DNA complex. The results (Fig. 4A
) suggest that T3 has no effect on the binding of the TR
1-RXR
protein complex to the IAP-TRE (lanes 2 and 10). In contrast, comparison between lane 6 and lane 14 shows that T3 inhibits the binding of anti-TR
1 antibody to the TRE-TR
1-RXR
complex. We also determined the effect of T3 on the binding of the TR
1-RXR
protein complex to the IAP-TRE at different concentrations of the cold competitor (Fig. 4B
). The result shows that the TR
1-RXR
protein complex has similar binding affinity in the presence or absence of T3. Taken together, these results suggest that T3 does not affect the binding affinity of the TR for the IAP-TRE, but likely causes a structural change that alters the ability of the anti-TR
1 antibody to bind to TR
1.
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1-RXR
Complex to the IAP-TRE
1 in intestinal development and homeostasis, we focused on delineating the characteristics and composition of the IAP-TRE using TR
1 and RXR
in EMSA. To determine the importance of the nucleotides in the octamer sequences of the IAP-TRE, we performed EMSA using oligonucleotides containing substitution mutations in the IAP-TRE sequence (Table 1
1-RXR
complex to the TRE (lanes 10, 11, and 1320). The oligonucleotide TRE1sm3, carrying substitutions outside the TRE sequence, efficiently competes with the TRE1G (lane 12), thus indicating that the sequences outside the octamer sequences are probably not involved in binding. Homology of the IAP-TRE with the consensus octamer TRE, as well as these preliminary EMSA results, suggested that each half-site of the IAP-TRE is probably an octamer or larger.
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1-RXR
Complex
1-RXR
complex (Fig. 5
15 bp) of the identified octamers could not be ruled out. To determine the existence of any cryptic octamer/hexamer 5' to the identified upstream octamer, we synthesized double-stranded oligonucleotides TRE1K (Table 2
1-RXR
complex (lanes 10, 7, and 4, respectively). These results indicate that there is no hidden octamer/hexamer within the close vicinity of the identified IAP-TRE octamers, and neither octamer alone is capable of binding to the TR
1-RXR
complex.
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1-RXR
complex can efficiently bind to TRE1P (lane 14), yet the complex cannot bind to TRE1E (lane 6). Comparison of the sequences between TRE1E and TRE1P shows that one extra nucleotide T is required 5' to the upstream octamer for efficient binding. Similarly, the results show that the TR
1-RXR
complex can efficiently bind to TRE1Y (lane 20), whereas the complex cannot bind to TRE1H (lane 9). Comparison of the sequences between TRE1Y and TRE1H shows that one extra nucleotide C is required 3' to the downstream octamer for efficient binding. These results define the core TRE in the IAP gene as 5'-TTGAACTCAgccTGAGGTTAC-3', which contains an extra nucleotide at each end compared with the putative IAP-TRE (see Results, section 4). Thus EMSA on oligonucleotides each carrying a 5' or a 3' sequential single nucleotide deletion defines the IAP-TRE as a repeat of two nonamer half-sites separated by three nucleotides.
The Extra Nucleotides in the Nonamer Half-Sites Are Specific
We investigated the specificity of the two extra nucleotides, one at each end of the core IAP-TRE. We performed EMSA (Fig. 6B
) on oligonucleotides carrying all four different types of nucleotides (A, C, G, T) in the extra positions (Table 3
). For the 5' end extra nucleotide, we found that the TR
1-RXR
complex strongly binds to a TRE carrying T or G (Fig. 6B
, lanes 9 and 12, respectively; Table 3
), whereas the protein complex does not bind to a TRE containing A or C (lanes 10 and 11, respectively). Similarly, for the 3' end extra nucleotide, we found that the TR
1-RXR
complex strongly binds to a TRE carrying C (lane 14), whereas the protein complex does not bind to a TRE carrying T (lane 17), and a TRE containing A or G shows weak binding (lanes 15 and 16, respectively). The observed specificity of the extra nucleotides strongly suggests the nonameric nature of the half-sites of the IAP-TRE in these truncated forms of oligonucleotides. We then decided to elucidate the specificity of these two nucleotides in the context of the larger oligo TRE1G using specific point mutations at these extra positions (Table 3
). We synthesized two mutant derivatives of TRE1G (TREMU1 and TREMD9, Table 3
), and used them in EMSA for determining their binding property to TR-RXR complex (Fig. 6C
). The results indicate that TR
1-RXR
protein complex probably binds to these two mutants with less efficiency. However, to determine accurate binding affinity of the TR
1-RXR
protein complex to these mutant oligos, we compared their competing capability with that of wild-type TRE (TRE1G) for TR
1-RXR
using incremental doses of cold oligos (Fig. 6D
, Table 3
). Comparison of intensities of respective bands of wild-type and mutant TREs shows that mutations of the two extra nucleotides reduce the binding affinity of TR
1-RXR
to the IAP-TRE flanked by four to six nucleotides at both ends. These results define the end nucleotide of the core IAP-TRE and strongly suggest the nonameric nature of this TRE. Similar results were obtained from the binding of the TRß1-RXR
heterodimer to the wild-type and these mutant TREs (data not shown).
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1-RXR
to the IAP-TRE
1-RXR
complex (Fig. 7A
1-RXR
protein complex for these mutant oligos, we compared their competing capability with that of wild-type TRE (TRE1G) for TR
1-RXR
using incremental doses of cold oligos (Fig. 7B
1-RXR
protein complex. Similar results were obtained from the binding of the TRß1-RXR
protein complex to the wild-type and mutant TREs (data not shown).
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1-RXR
to the IAP-TRE
1-RXR
protein complex, we synthesized TREMSR123 (Table 3
1-RXR
protein complex to the IAP-TRE. To determine precise binding affinity of the TR
1-RXR
protein complex to this mutant oligo, we compared the competing capability of the mutant oligonucleotide with that of the wild-type TRE (TRE1G) for TR
1-RXR
using incremental doses of cold oligos (Fig. 7D
1-RXR
protein complex to the IAP-TRE. Using EMSA, we also determined the effect of the length of the spacer region between the nonamers of the IAP-TRE on the binding of the respective mutant TRE to the TR
1-RXR
heterodimer. We observed that the protein complex shows wild-type binding affinity for the two nonamers having one to five nonspecific nucleotides between them, whereas the complex showed weak binding affinity for the two nonamers having 0, and 610 nonspecific nucleotides between them (data not shown).
Substitution Mutations in Either Nonamer Greatly Reduces Biological Activity of the IAP-TRE
To assess the biological importance of each nonamer we performed site-directed mutagenesis to construct pIAP-luciferase reporter plasmids carrying substitution mutations for each nonamer (Table 4
). Caco-2 cells were transfected with the mutant plasmids, and luciferase activity was determined in the presence of T3 (100 nM). The results are shown in Fig. 8A
and tabulated in Table 4
. All mutations rendered the IAP-TRE biologically inactive.
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The IAP-TRE Is a Novel TRE Consisting of an Everted Repeat of Two Nonamers
We compared the IAP-TRE with the various positively regulated TREs that have been previously described for other genes (37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51) (Table 5
). It is evident that TRs recognize DNA sequences with great diversity; however, all of the positively regulated TREs are composed of a repeat of a loosely conserved hexamer (AGGTCA) sequence. Katz and Koenig (35) have identified that the dinucleotide TG or TA at the 5' end of the above-mentioned hexamer constitutes a part of a TRE. Approximately 50% of the identified TREs contain an octamer half site (Table 5
). Accordingly, to define the space between two half-sites of a TRE we counted the space between the downstream octamer/hexamer and upstream hexamer/octamer (Table 5
). In Fig. 9A
, we summarized the results of in vitro binding of the IAP-TRE to the TR-RXR complex (EMSA) and biological (in vivo) effects of mutations of the IAP-TRE in T3-induced activation of the IAP gene in Caco-2 cells (Fig. 9A
). The data show that nucleotides at or close to the ends of each nonamer are required for biological activity as well as for in vitro binding of the TR-RXR complex. Thus, the data clearly establish that the IAP-TRE consists of two nonamers separated by three nonspecific nucleotides. Comparison of the nucleotide sequence of a consensus octamer TRE [T(G/A)AGGTCA] with the corresponding nucleotides of a nonamer in either strand of the IAP-TRE shows that the upstream nonamer in the antisense strand and the downstream nonamer in the sense strand each has two bases mismatched. Similarly, the downstream nonamer in the antisense strand and the upstream nonamer in the sense strand each has one base mismatched. Hence, to obtain maximum homology of the IAP-TRE with the consensus octamer TRE, we assigned the IAP-TRE as an everted repeat of two nonamers (Fig. 9A
). Until now, no TRE has been identified containing nonamer half-sites. The IAP-TRE does not bind to TR
1 alone; however, it binds to TRß1 alone making it also an atypical TRE. Further, in the presence of RXR, each TR binds to the TRE only as a heterodimer of TR-RXR. In Fig. 9B
, we present the sequence of the proximal 800 bases of the IAP promoter, showing the TRE and other transcription factor binding sites.
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TRE (Fig. 2B
1, TRß1, or TRß2. The results show that all TRs are able to activate the IAP promoter carrying the wild-type TRE (pIAP-2574/-49; see Fig. 2A
TRE) is not responsive to any exogenous TRs (Fig. 10
1, TRß1, and TRß2 show approximately 10-, 16-, and 9-fold activation, respectively, of the IAP promoter in Caco-2 cells. Hence, we conclude that the 2.5-kb proximal IAP promoter contains only one biologically functional TRE, which is also not an isoform-specific TRE.
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| DISCUSSION |
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Alkaline phosphatases are membrane-bound glycoproteins that hydrolyze a wide variety of monophosphate esters at high pH optima and are widely distributed in different tissues (36). Depending on their biochemical nature and tissue distribution, the alkaline phosphatases are classified as placental, placental-like, intestinal (fetal and adult), and liver, bone, kidney (L/B/K) alkaline phosphatases. The human intestinal alkaline phosphatase (IAP) gene maps to chromosome 2q3437 and produces a 528-amino acid polypeptide. The IAP protein has been identified as a content of the surfactant-like particles, the unilamellar secreted membrane associated with the process of lipid absorption and isolated from the apical surface of enterocytes (19). The IAP protein has also been identified as a Hela cell tumor-associated antigen (53). A recent report by Narisawa et al. (20) on IAP knockout mice demonstrated that IAP limits fat absorption during high-fat feeding. The IAP gene is overexpressed in hyperthyroid intestine, especially in the duodenum, thus indicating the T3-mediated activation of this gene (22). Hypothyroidism is associated with villus atrophy and a decrease in the expression of the 3.0-kb IAP mRNA species, further supporting the involvement of T3 in the positive regulation of the IAP gene (54).
The molecular mechanisms by which T3 exerts its effect on the gut are not well understood. There have been few in vitro studies on T3 action in intestinal cells, one exception being a study by Giannella et al. (55), showing that T3 induces Na,K-ATPase gene expression in the Caco-2 colon cancer cell line. However, Na,K-ATPase is a ubiquitous trans-membrane protein mostly expressed in kidney, intestine, skeletal muscle, and cardiac muscle. In addition to IAP, lactase, and Na,K-ATPase, it will be of interest to identify the full range of T3-responsive genes in the gut, similar to what has been done in other tissues such as the pituitary (56). Wu et al. (38) have tabulated the T3-responsive genes that have been studied at the molecular level identifying their respective TREs; however, the status of expression of these genes in the gut is not known.
Various forms of TR
and TRß isoforms are expressed in the gut; however, using RT-PCR we found expression of only the TR
1 and TRß1 receptors in the colorectal adenocarcinoma Caco-2 cell line (Fig. 1A
). We observed two 0.60 and 0.65 kb extra bands in the case of TRß1, which were produced by nested PCR, indicating the possible existence of other TRß isoforms in these cells. Western blotting on whole-cell lysates, as well as nuclear extract, confirmed the production of TR
1 and TRß1 receptors by Caco-2 cells, which corroborates with the RT-PCR data. Based upon the Northern blot analyses, the basal level of TRs in Caco-2 cells was capable of approximately 3-fold activation of the endogenous IAP gene after T3 treatment (Fig. 1C
). Using S1 mapping, Henthorn et al. (15) have shown only one IAP transcript in the Caco-2 cells; however, we observed two transcripts in the Northern blots. This apparent inconsistency could be due to subtype variations among the Caco-2 cells or the relatively lower sensitivity of S1 mapping. Similar to the activation of endogenous IAP mRNA expression, transient transfections in Caco-2 cells with IAP-luciferase reporter constructs also showed approximately 3-fold activation of the IAP gene by T3 (Figs. 1D
and 2A
).
Previously identified naturally occurring TREs generally consist of two hexamer half-sites arranged as either DR4, palindrome, inverted palindrome, or everted palindrome (24, 25, 26, 27, 28, 29, 30, 31, 32, 33). The TREs with hexamer half-sites bind only to TR-RXR heterodimer. An octamer TRE [T(A/G)AGGTCA] has been shown to bind to a TR monomer, thus eliminating the necessity of TR-RXR heterodimerization for TR-mediated activity (34, 35). Most previous work on these octamer TREs has been performed in vitro with synthesized oligonucleotides elucidating the precise sequences, spacing, and orientation (34, 35). Recently, Toyoda et al. (40) have described the TRE1 from the human type 1 deiodinase gene (hdio1) as a naturally occurring, biologically functional TRE consisting of two relatively independent octamer sequences (DR10) that do not require the RXR family of proteins for function.
Using transient transfection assays with IAP-luciferase reporter constructs carrying various 5' and internal deletion mutants of the IAP promoter (Fig. 2
), we were able to convincingly localize the TRE to a 135-bp region in the proximal IAP promoter around 620. We compared the sequence of this 135-bp region with consensus DR4 and octamer TREs (24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35). We identified a putative IAP-TRE (5'-TGAACTCAgccTGAGGTTA-3') consisting of two octamer half-sites separated by three nucleotides instead of the expected four nucleotides. With respect to the spacing of three nucleotides between octamer half-sites, the IAP-TRE resembles the rat prestin TRE (Table 5
). The TRE for rat uncoupling protein 1 consists of a downstream hexamer and upstream octamer separated by three nucleotides (Table 5
). In addition, the pTRE for human thyroid hormone receptor ß1 consists of a direct repeat of two hexamers separated by three nucleotides, and the dTRE consists of an everted repeat of a downstream octamer and an upstream hexamer (Table 5
). By internal deletion mutation of the identified TRE, we showed that this TRE was, indeed, the only biologically active TRE in the 2.5-kb proximal promoter region of the IAP gene. However, our result does not exclude the possibility of another TRE outside the region of the IAP promoter examined in this work. Most in vivo work on T3 regulation of the IAP gene has been performed in rodents. However, by sequence comparison, we could not identify a close homology (>70%) of the identified human IAP-TRE within the 2.5-kb proximal promoter region of the mouse IAP gene. It will be interesting to investigate the localization and structure of the mouse IAP-TRE and compare the underlying mechanisms with those found for the human IAP gene.
Toyoda et al. (40) have shown that the hDIO1-TRE, presumably consisting of two independent DR10 octamers (Table 5
), preferentially binds to two monomers (double occupancy) of TR
1 in the absence of T3, whereas in the presence of T3 (100 nM), TR double occupancy is impaired, and binding of a single monomer (single occupancy) is also observed. The authors (40) have also shown that RXR as well as the TR-RXR heterodimer does not bind to the hDIO1-TRE. In contrast to the binding of TR to hDIO1-TRE, our EMSA data (Fig. 3A
) show that TR
1 cannot independently bind to the IAP-TRE, and only the TR
1-RXR
complex can bind to the TRE efficiently. However, our EMSA data show that TRß1 alone can bind to the IAP-TRE although with less affinity (Fig. 3B
), and such binding is abolished in the presence of RXR
, when only the TRß1-RXR
heterodimer binds to the IAP-TRE. We also observed that TRß1 alone binds to the downstream half-site of the IAP-TRE (data not shown), and it will be interesting to further investigate such binding of TRß1 that might reveal the nucleotide composition of any likely isoform-specific TRE. Like the hDIO-TRE, the IAP-TRE also does not bind to RXR. In contrast to the effect of T3 on the hDIO1-TRE, our results (Fig. 4
) show that T3 has no effect on binding of the TR
1-RXR
complex to the IAP-TRE. However, we found that T3 inhibits the binding of the anti-TR
1 antibody to TRE-TR
1-RXR
complex. We presume that binding of T3 to TR
1 changes the conformation of TR
1 thus making it inaccessible to the antibody.
Using EMSA, we compared the binding of in vitro-synthesized TR
1-RXR
and TRß1-RXR
complexes to the IAP-TRE (Fig. 3
, A and B) with the binding of nuclear extract from Caco-2 cells to the IAP-TRE (Fig. 3C
). The TR
1-RXR
complex, as well as the nuclear extract, is capable of binding to the IAP-TRE and can be supershifted with anti-TR
1 antibody (Fig. 3C
). The polyclonal anti-TRß1 antibody from Upstate Biotechnology (Lake Placid, NY) was able to supershift TRE-TRß1-RXR
complex, whereas monoclonal anti-TRß1 antibody from Santa Cruz Biotechnology (Santa Cruz, CA) was not able to bind to the TRE-TRß1-RXR
complex (Fig. 3B
). Although Caco-2 cells express TRß1 (Fig. 1
, A and B) we found that anti-TRß1 antibodies do not generate any supershifted band (Fig. 3C
). We believe that the amount of TRß1 expressed in Caco-2 cells was probably not sufficient to bind to the TRE in competition with TR
1. We also found that T3 has similar effects on binding of in vitro-synthesized protein complex as well as on binding of nuclear proteins to the IAP-TRE (data not shown).
EMSA results (Fig. 5
) on the IAP-TRE containing substitution mutations demonstrated that each half-site of the IAP-TRE is at least an octamer or it could be larger than an octamer. Efficient binding of TR
1 alone to an octamer (TAAGGTCA) has been described (34); however, neither octamer in the IAP-TRE can independently bind to the TR
1-RXR
complex (Fig. 6
), thus proving the necessity of both the half-sites for efficient binding to this TRE. Based on the sequence of the antisense strand, the upstream octamer of the IAP-TRE (TGAACTCA) varies by three bases, whereas the downstream octamer (TGAGGTTA) varies by two bases from the above octamer (TAAGGTCA) sequence (Fig. 9A
). Similarly, based on the sequence of the sense strand, the octamers of the IAP-TRE (TAACCTCA and TGAGTTCA) each varies by two bases from the above-mentioned octamer (TAAGGTCA) sequence (Fig. 9A
). It appears that binding to a single octamer TRE probably requires very stringent homology to TAAGGTCA.
Using EMSA on sequential deletion mutations of a large TRE, we show that the half site of the IAP-TRE is a nonamer (Fig. 6A
). We studied the specificity of the extra nucleotides within the nonamers of the IAP-TRE, and we indeed found the specific role of these nucleotides for the binding of the TR
1-RXR
complex to the IAP-TRE (Fig. 6
, BD). The specificity of the extra nucleotides strongly indicates that the IAP-TRE is a repeat of nonamers. It will be interesting to investigate the biological role of these extra nucleotides in regard to the T3 activation of the IAP gene. To further determine the nonameric nature of half-sites of the IAP-TRE, we also performed EMSA on the mutant IAP-TREs carrying internal mutations. These results also showed that nucleotides at or close to the ends of each nonamer are also involved in the binding of the TRE to the TR-RXR complex (Fig. 7
, A and B). However, it remains possible that the extra nucleotides outside each hexameric half-site (AGGTCA) could be parts of the flanking sequences that are involved in the modulation of activity of the response element (57, 58). It will be interesting to perform methylation interference studies (34), which could identify the nucleotides that contact with the TR-RXR protein complex.
The use of EMSA on oligonucleotides carrying sequential deletions of single nucleotides enabled us to determine the upstream and downstream boundaries of the IAP-TRE. Until now, EMSA has not been used to determine the 5' and 3' boundaries of other identified TREs, although this is probably a valid method for determining the boundaries of any cis element. We predict that boundaries of other TREs could be elucidated using this protocol, and subsequent analysis might generate a broader consensus among the TREs.
The in vitro site-directed mutagenesis of the IAP-TRE shows that the dinucleotide TG at the 5' end of the either octamer in the antisense strand is required to confer the wild-type TRE activity in Caco-2 cells (Fig. 8A
and Table 4
). These mutations could also be assigned as the 3' end dinucleotide (CA) of the octamers in the sense strand. Hence the dinucleotide TG at the 5' end of either octamer in the antisense strand must be a part of the TRE. Our EMSA data show that nucleotides at or close to the ends of each nonamer are required for in vitro binding of the TR-RXR
complex (Figs. 6
and 7
). Thus, the possibility that the IAP-TRE is composed of a repeat of two hexamers separated by seven or five nonspecific nucleotides was considered very unlikely. The EMSA results, supported by the mutagenesis data, strongly suggest that the IAP-TRE most likely consists of a repeat of two nonamers separated by three nucleotides. Based on maximum homology of the IAP-TRE with the consensus octamer TRE, we assigned the IAP-TRE as an everted repeat of two nonamers (Fig. 9A
). However, it will require crystallographic data on TRE-TR-RXR complex to conclusively define whether the IAP-TRE is an everted repeat or direct repeat of nonamers, or otherwise. Until now, no TRE with nonamer half-sites has been reported. However, we believe that many of the identified TREs, especially the octamer TREs, could be in fact nonameric TREs if the boundaries of the TREs were determined. We observed that TR
1-RXR
complex can efficiently bind to the nonamers of the IAP-TRE separated by extensively variable spacer regions (data not shown). Such observation probably indicates a generalized principle of mechanism of TR-RXR binding to an everted repeat that allows extensive variability of the length of the spacer region. However, to establish such a principle extensive work will be required to study in vitro and in vivo receptor functions on different direct and everted repeats.
Analyses of TREs (Table 5
) indicate that TRs exert their effects by interaction with TREs of different forms and structures. Our results (Table 4
), as well as other results (34, 35), indicate that the dinucleotide TG or TA at the 5' end of the downstream hexamer half-site of a TRE is indeed a part of a TRE. Thus, we think that the rule of DR4 does not apply to a TRE containing TG or TA at the 5' end of the downstream hexamer half-site. To define the space between two half-sites of a TRE we counted the space between the downstream octamer/hexamer and upstream hexamer/octamer (Table 5
). We predict that the existence of a TG or TA at the 5' end of a hexamer half-site allows the TR
1-RXR
complex to bind to relatively diverse sequences compared with the consensus DR4 TRE sequence.
To determine the existence of another TRE as well as to ascertain whether the identified TRE was an isoform-specific TRE, we examined the effects of exogenous TRs on a mutant of the identified IAP-TRE. Knockout mouse data suggest that TR
1 is the major TR involved in intestinal development and homeostasis (9, 10, 11, 12, 13, 14). However, the wild-type IAP reporter gene was highly activated in response to T3 when the cells were cotransfected with a plasmid expressing a bona fide TR (TR
1, TRß1, or TRß2) that contains both DNA and ligand (T3) binding domains (Fig. 10
). No exogenous TR was able to activate the IAP reporter construct carrying the deletion of the IAP-TRE. Thus, it was confirmed that there was no other TRE within the 2.5-kb proximal promoter region of the IAP gene, and also that the identified TRE was not an isoformspecific TRE. The varying levels of IAP activation by TR
1 (10-fold), TRß1 (16-fold), and TRß2 (9-fold) are of uncertain significance but may reflect different affinities of the TRs for the IAP-TRE or it may also reflect different levels of expression of TR isoforms in the transfected cells.
Ligand-independent repression of positively controlled T3-responsive genes has been described (59, 60, 61, 62). In the transient transfections, we also observed ligand-independent repression of the IAP gene by approximately 75% with cotransfected TR expression plasmids (data not shown). Ligand-independent repression was abolished in the IAP-TRE mutants (Fig. 8B
). It is worth noting that Caco-2 cells express only small amount of endogenous TRs. As such, to precisely define the TR-mediated repression of the IAP promoter will require studies in a cell line that does not express any endogenous TR. The unliganded TR acts as a repressor as a result of its association with a complex of proteins, including corepressors [e.g.nuclear receptor coactivator protein (N-CoP)/silencing mediator for retinoid and thyroid hormone receptors (SMRT)], mSinA, ski proto-oncoprotein, and histone deacetylase. Upon binding to the ligand, the repressor complex is replaced by the other proteins, including coactivators such as cAMP response element binding protein-binding protein/p300, chromatin assembly factor (CAF), and other proteins associated with histone hyperacetylation resulting in gene activation (59, 60, 61, 62). It is possible that TRs in the Caco-2 cells recognize the atypical IAP-TRE by their interaction with other tissue-specific factors. We have previously shown that the gut-enriched Kruppel-like factor (KLF) 4 activates the IAP gene through a guanine-cytosine (GC)-rich cis element (Fig. 9B
), and cotransfection studies showed that KLF4 and TR/T3 synergistically activate the IAP gene (63). It will be interesting to investigate the role of other possible corepressor and coactivator factors in Caco-2 cells that associate with TRs as well as to characterize the various TREs in other gut-specific genes determining whether any tissue-specific pattern of TREs exists.
In conclusion, we have identified a novel TRE that is involved in T3-mediated positive regulation of the enterocyte differentiation marker IAP gene. Further studies of T3 regulation of this gene in association with other gut-specific transcription factors will undoubtedly shed light on the molecular mechanisms governing the fundamental process of gut development, homeostasis, and differentiation.
| MATERIALS AND METHODS |
|---|
|
|
|---|
1 antibody was purchased from Covance (Richmond, CA). The monoclonal anti-TRß1 antibody was obtained from the Santa Cruz Biotechnology (Santa Cruz, CA), and the polyclonal anti-TRß1 antibody was purchased from the Upstate Biotechnology Inc (Charlottesville, VA).
Plasmids
The pGL3-Basic mammalian promoter-detection vector and the pRL-CMV control plasmid expressing Renilla luciferase were obtained from Promega. The human RXR
, TR
1, TRß1, and TRß2 expression plasmids were the derivatives of the pSG5 vector (Stratagene, Cedar Creek, TX), and were the kind gifts of Anthony Hollenberg (Beth Israel Deaconess Medical Center, Boston, MA).
The 2.5-kb SacI-BamHI fragment from the plasmid IAP2.4CAT (64), carrying the human IAP promoter region (2574 to 49, relative to translation initiation codon ATG), was subcloned into the pGL3-Basic vector digested with SacI and BglII, thus constructing the plasmid pIAP-2574/-49. Various 5' deletion mutants were constructed by digesting the plasmid pIAP-2574/-49 with SacI and another appropriate restriction enzyme followed by generation of blunt-ended fragments, agarose gel purification of the desired band and recircularization. The internal deletion plasmid pIAP-2574
-753/-617 was generated by deleting the 135-bp PflMI-BstEII restriction fragment from pIAP-2574/-49. The other internal deletion mutants were constructed by replacing the above 135 bp PflMI-BstEII restriction fragment of pIAP-2574/-49 with oligonucleotides representing specific sequences of this 135-bp region. The plasmid pIAP-2574
TRE was generated by deleting 631 to 613 bases by PCR-mediated mutagenesis. The novel joint(s) in each plasmid was determined by DNA sequencing (65, 66).
Cell Culture
The Caco-2 human colorectal adenocarcinoma cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA) and was maintained in DMEM (Invitrogen Life Technologies) supplemented with 10% fetal bovine serum (FBS, Sigma, St. Louis, MO), 2 mM L-glutamine, and 100 U/ml each of penicillin and streptomycin (Invitrogen Life Technologies). DMEM containing FBS stripped of thyroid hormone (T3) was prepared by treating FBS with ion-exchange resin and charcoal (67). The cells were grown at 37 C in the presence of 5% CO2 and were split by trypsinization when they reached about 8090% confluence.
RNA Preparation
Total RNA was prepared using Trizol reagent from Invitrogen Life Technologies following the manufacturers instructions. Where indicated, cells were treated with T3 (100 nM) for the specified period of time before RNA extraction.
RT-PCR
First-strand cDNA was synthesized using RNA from Caco-2 cells with poly-deoxythymidine primer and Superscript II reverse transcriptase after the instructions of the manufacturer. PCR was then performed with gene specific primers using Taq DNA polymerase from Promega. The list of primers used in RT-PCR is given below:
1. TRa1.1153F: 5'-ccg cac ttc tgg ccc aag ctg ctg atg-3' 27 (bases)
2. TRa1.1184F: 5'-gtg act gac ctc cgc atg atc ggg gcc tg-3' 29 (bases)
3. TRa1.1530R: 5'-cac act gtc ctt tcc ata gca agt tc-3' 26 (bases)
4. TRa2.1620R: 5'-cca gct ttc agg cac ctc ctg ctc ttg-3' 27 (bases)
5. TRb1.121F: 5'-cgg ctc cag ggc act ggt aat ttg gc-3' 26 (bases)
6. TRb2.151F: 5'-ggc agc aat tgc tac atg cag tcc ac-3' 26 (bases)
7. TRb1.605R: 5'-cag agc tcg tcc ttg tct aag taa ctg c-3' 28 (bases)
8. TRb1.610R: 5'-gtc taa gta act ggg gat gta ccc t-3' 25 (bases)
Western Blotting
Western blotting was performed according the protocols described in Nomura et al. (60). Proteins were extracted from untransfected Caco-2 cells as well as from Caco-2 cells transiently transfected with an expression plasmid overexpressing TR
1 or TRß1. Approximately 15 µg of plasmid DNA were used to transfect 50% confluent Caco-2 cells in a 10-cm culture dish. Forty-eight hours after transfection, cells were lysed in the lysis buffer [50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100] that also contained a complete range of protease inhibitors (Roche Applies Science, Indianapolis, IN); and whole-cell lysates were prepared. Nuclear extract from Caco-2 cells was prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents kit from Pierce (Rockford, IL) according to instructions from the manufacturer (see below). The respective lysates and nuclear extract were analyzed by Western blotting using anti-TR
1 or anti-TRß1 antibodies. The lysate or nuclear extract was mixed in Laemmli denaturing loading buffer [15 mM Tris-HCl (pH 6.8), 0.5% sodium dodecyl sulfate (SDS), 2.5% glycerol, 0.5% betamercaptoethanol, and 0.001% bromophenol blue], boiled for 10 min, and then electrophoresed through a Ready Gel Tris-HCl Gel (10% polyacrylamide resolving gel, 4% polyacrylamide stacking gel; Bio-Rad, Hercules, CA) in Tris-glycine-SDS running buffer (Boston Bioproducts, Ashland, MA). Proteins were electrotransferred onto nitrocellulose membranes, and blocked with 5% (wt/vol) nonfat milk (Bio-Rad) for 1 h at room temperature. The blot was then incubated overnight at 4 C in the presence of the respective antibody (1:1000 dilution). The horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution; Bio-Rad) was added to the blot and incubated for 1 h at room temperature. The protein bands were identified by developing the blot with Immun-Star horseradish peroxidase chemiluminescent kit from Bio-Rad.
Northern Hybridization
Northern hybridization was performed following the protocol described in Hodin et al. (18). Twenty micrograms of total RNA were mixed with glyoxal loading dye and electrophoresed through an agarose gel prepared in Northern Max Gly Gel-Prep/Running Buffer (Ambion, Austin, TX). Capillary transfer was performed to transfer RNA onto a nylon membrane (Amersham Pharmacia Biotech) and this RNA was cross-linked to the nylon membrane using the Stratalinker 1880 UV hybridization oven (Stratagene, Cedar Creek, TX) according to the manufacturers instructions. About 50 ng of 32P-labeled DNA probe was prepared using Ready-To-Go DNA Labeling Beads from Amersham Pharmacia Biotech, typically achieving a specific activity of about 5 x 108 cpm/µg DNA. The IAP probe was a 1.9-kb PstI fragment derived from human IAP cDNA (15) and was obtained from ATCC. The actin probe was a 1.0-kb PstI fragment derived from the mouse ß-actin cDNA (68). Hybridization was performed in Rapid-hyb buffer (Amersham Pharmacia Biotech, Buckinghamshire, UK) for overnight at 65 C. The membrane was then washed twice with low stringency wash buffer containing 2x saline sodium citrate (SSC) and 0.1% SDS at 65 C for 30 min each followed by high stringency wash with 0.1x SSC/0.1% SDS (Boston Bioproducts) at 65 C for 30 min. The membrane was then subjected to autoradiography.
DNA Sequencing
DNA sequencing of each plasmid was determined by the Sequencing Core Facility at the Department of Molecular Biology, Massachusetts General Hospital (Boston, MA), using dye-labeled dideoxy nucleotide chain-terminators.
Transient Transfection and Luciferase Reporter Assays
Transient transfection and luciferase reporter assays were performed according to the protocols described in Siddique et al. (63). Caco-2 cells were plated at a density of 300,000 cells per well of a six-well plate in DMEM containing FBS stripped of T3. Cells were grown overnight and transient transfections were performed using SuperFect reagent from QIAGEN. The test plasmid DNA (1.5 µg/well) was cotransfected with control plasmid pRL-CMV DNA (0.5 µg/well), and the total amount of DNA was kept the same for each transfection by adding nonspecific plasmid TF12 DNA. The control Renilla luciferase activity (pRL-CMV) was used to determine transfection efficiency as well as to normalize the firefly luciferase activity data. After transfection, the cells were grown for a further 24 h in DMEM containing FBS stripped of T3. The cells were then maintained in stripped media and treated ± T3 (100 nM) for another 24 h, except the experiments requiring T3 treatment for a specific period of time. Firefly and Renilla luciferase assays were then performed on cell lysates using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturers instructions.
In Vitro Protein Synthesis
TNT T7 Quick Coupled Transcription/Translation System (Promega) was used for in vitro synthesis of human TR
1, TRß1, and RXR
from the derivatives of pSG5 (Stratagene) carrying the relevant coding sequences under the control of the T7 promoter.
Preparation of Nuclear Extract
Nuclear extract was prepared from 2 x 106 Caco-2 cells using NE-PER Nuclear and Cytoplasmic Extraction Reagents kit from Pierce (Rockford, IL) according to instructions from the manufacturer. Halt Protease Inhibitor Cocktail (Pierce) was added to the extract, and stored at 80 C.
EMSA
EMSA was performed according to the protocol described in Giralt et al. (37). The complementary oligonucleotides were annealed and radiolabeled by the kinasing reaction with T4 polynucleotide kinase in the presence of [
-33P]ATP. The radiolabeled probe was purified twice by passing it through Micro Bio-Spin 6 chromatography columns (Bio-Rad), followed by determination of the specific activity, which usually measured about 108 cpm/µg DNA. Approximately 10 ng of radiolabeled probe were incubated at room temperature for 20 min with 1 µl of synthesized TR and/or RXR
each in 10 µl of binding buffer containing 20 mM HEPES (pH 7.7), 50 mM NaCl, 1 mM dithiothreitol, 0.1 mM EDTA, 5 µM nonspecific oligo, 10% glycerol, and 2 µg of polydeoxyinosinic-deoxycytidylic acid. The samples were electrophoresed in a 5% polyacrylamide gel in a cold room (4 C), followed by drying of the gel and autoradiography.
In Vitro Mutagenesis
PCR mutagenesis was performed to introduce a mutation(s) in the putative TRE for functional analysis as described in Wilber and Xu (69). PCR primers were synthesized with a specific mutation followed by PCR amplification and ligation of the PCR product into an appropriate plasmid. Each mutant was sequenced to verify the mutation(s).
| FOOTNOTES |
|---|
Abbreviations: DR, Direct repeat; FBS, fetal bovine serum; hdio1, human type 1 deiodinase gene; IAP, intestinal alkaline phosphatase; KLF, Kruppel-like factor; mTRE, mutant IAP-TRE; RXR, retinoid X receptor; TR, thyroid hormone receptor; SDS, sodium dodecyl sulfate; TRE, thyroid hormone response element.
Received for publication September 12, 2003. Accepted for publication May 3, 2004.
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and TRß in the control of thyroid hormone production and post-natal development. EMBO J 18:623631[CrossRef][Medline]
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