Avidin-biotin Complex DNA-binding Assay Research Articles

Differential recruitment of coactivators to the vitamin D receptor transcriptional complex by 1α,25-dihydroxyvitamin D 3 analogs

To clarify the molecular mechanism for analog potency and selectivity, we investigated the ability of 1,25(OH) 2D 3 analogs to recruit coactivators to the vitamin D receptor (VDR) transcriptional complex. Using a modified version of the avidin–biotin complex DNA binding assay, we discovered that 20 S-analogs enhance the binding of specific coactivators to the transcriptional complex relative to natural hormone and that the enhanced binding occurs independently of vitamin D response element and cell type. With the exception of two of these coactivators, DRIP205 and DRIP240, all proteins were recruited to the transcriptional complex in a dose-dependent manner. While the results do not provide an explanation for tissue selectivity of 2-methylene-19-nor-(20 S)-1,25-dihydroxyvitamin D 3 (2MD), they provide evidence that in the presence of a full-length side chain, the 20 S configuration improves binding of specific proteins to the VDR transcriptional complex while modifications at carbon 2 do not.

A detailed functional and structural analysis of a major thyroid hormone inhibitory element in the human thyrotropin beta-subunit gene.

The first exon of the human thyrotropin-beta (hTSH beta) gene has been demonstrated in our laboratory to contain a major thyroid hormone inhibitory element. In order to characterize fully this element, we have performed a detailed functional and structural scanning mutational analysis of this element. Various -1192 to +37 (base pairs) bp fragments of the hTSH beta gene containing consecutive five deoxythymidine substitution mutations of the first exon were inserted into a luciferase reporter plasmid and transiently transfected into human embryonal cells (293) and stably transfected into rat pituitary cells (GH3). Two domains (domain 1 and 2) were identified by scanning mutations that were essential for function of the thyroid hormone inhibitory element: +3 to +13 bp and +28 to +37 bp. Biotinylated DNA fragments containing -12 to +43 bp of the hTSH beta gene and the identical scanning mutations demonstrate that in vitro synthesized c-erbA-beta binding is disrupted as much as 95% by mutations from -3 to +17 bp and to a lesser extent (20-30%) by mutations from +23 to +27 bp and from +33 to +43 bp. Domain 1 displayed a higher affinity for c-erbA-beta than domain 2 in avidin-biotin complex DNA-binding and gel-mobility assays. Using increasing amounts of in vitro synthesized c-erbA-beta, we were unable to demonstrate more than one protein-DNA complex in gel-mobility assays. However, using the avidin-biotin complex DNA-binding assay and the cross-linking reagent, 1,6-bismaleimidohexane, we were able to demonstrate thyroid hormone receptor dimer formation on domain 1 but not to any significant extent on domain 2. In conclusion, functional and DNA-binding studies suggest that the thyroid hormone receptor binds to two distinct regions in the first exon of the hTSH beta gene. The upstream site (domain 1) binds c-erbA-beta with higher affinity and is capable of binding c-erbA-beta as a dimer under some conditions, while the downstream site (domain 2) appears to bind a single molecule of c-erbA-beta with lower affinity. These results suggest that thyroid hormone receptor, binding to at least two sites in the first exon, act in conjunction to mediate T3 inhibition of hTSH beta expression.

Evidence for direct estrogen regulation of the human gonadotropin-releasing hormone gene.

This study is an attempt to determine whether estrogen could directly regulate human gonadotropin-releasing hormone (GnRH) gene expression. Human GnRH expression vectors were constructed by fusing various 5' flanking regions of the human GnRH gene upstream of the luciferase reporter gene (LUC) or the thymidine kinase promoter linked to the chloramphenicol acetyltransferase reporter gene (CAT). These constructs were transiently transfected into a human choriocarcinoma cell line (JEG-3) and LUC or CAT activity was measured after either no treatment or treatment with various concentrations of estradiol. A stimulatory estrogen response element (ERE) was localized to a 32-bp region between -547 and -516 bp. To determine whether estrogen receptor bound to this region of the gene, we performed DNase I footprinting using purified calf uterine estrogen receptor. DNase I footprinting demonstrates a strong footprint between -567 and -514 bp of the human GnRH gene. In addition, an avidin-biotin complex DNA-binding assay demonstrated that a biotinylated DNA fragment containing -541 to -517 bp of the human GnRH gene bound 35S-labeled estrogen receptor as well as a biotinylated DNA fragment containing the xenopus vitellogenin ERE. On the other hand, the negative control biotinylated DNA fragment derived from adenovirus 5 bound insignificant amounts of 35S-labeled estrogen receptor. Both the GnRH ERE and vitellogenin ERE bound 35S-labeled estrogen receptor with high affinity (approximately 1 nM). These data indicate that the human GnRH gene contains an ERE sufficient to mediate a stimulatory response to estrogen in heterologous cells. Based upon these data we hypothesize that the human GnRH gene might also be directly regulated by estrogen in the hypothalamus, and that this regulation may explain the GnRH hypersecretion observed at the time of the preovulatory luteinizing hormone (LH) surge.

3,5,3′-Triiodothyronine Receptor Auxiliary Protein (TRAP) Enhances Receptor Binding by Interactions within the Thyroid Hormone Response Element

We have previously demonstrated that binding of in vitro synthesized thyroid hormone receptor (TR) to thyroid hormone response elements (TREs) is enhanced by the addition of nuclear extracts from several different cell types, suggesting that binding of TR is partially dependent on a T3 receptor auxiliary protein (TRAP). We have used the avidin-biotin complex DNA-binding assay to discriminate between regions of TREs that bind TR alone and sites that are influenced by interactions with TRAP. Mutations in the TREs from rat GH and glycoprotein hormone alpha-subunit genes show that a specific DNA sequence is required for TRAP-mediated enhancement of TR binding. Mutations in the B half-site of the rat GH TRE or in similar sequences [(T/A)GGGA] in the alpha-subunit TRE ablate the enhancement of TR binding by TRAP. Furthermore, binding of TR to a natural half-site in the TSH beta-subunit gene (bases -16 to 6), which lacks an additional AGGGA-like sequence, is not enhanced by the addition of TRAP. Binding of TR to TREs was also tested at physiological salt concentrations in the avidin-biotin complex DNA-binding assay. Binding of human TR beta to TREs decreases dramatically at 140 mM KCl compared to binding at 50 mM KCl; however, the addition of TRAP enhances the binding to almost 4-fold of basal binding, suggesting that TRAP may be important for stabilization of TR binding to TREs in the cell.

3,5,3′-Triiodothyronine (T3) Receptor-Auxiliary Protein (TRAP) Binds DNA and Forms Heterodimers with the T3Receptor

Nuclear factors enhance binding of T3 receptors (TR) to DNA, suggesting that T3 action may require a multicomponent complex bound to thyroid hormone response elements (TREs). We refer to the 65,000 Da nuclear protein in GH3 cells that enhances TR binding to DNA as the TR-auxiliary protein (TRAP) and have characterized its interaction with TR. Using a TRE-DNA affinity matrix we show that TRAP is able to bind to DNA, even in the absence of functional TR. We then used carboxyl-terminal truncations of rat TR alpha-1 and human TR beta in the avidin-biotin complex DNA-binding assay to identify regions that are important for interaction with TRAP. Removal of 34 residues of hTR beta abolishes T3-binding activity, but the ability to bind TRAP is retained. Further truncations and point mutations suggest that TRAP interacts with the ligand-binding domain of TR and with an independent region which overlaps a conserved sequence adjacent to the second Zn2+ finger (amino acids 120-149 in rTR alpha-1). A fragment of rTR alpha-1 (alpha C291) which encompasses these two regions inhibits the ability of TRAP to enhance TR binding to DNA. This is due to binding of alpha C291 to TR, demonstrating the ability of TR to form homodimers. The inability of TRAP to interact with TR dimers and the similarity of the locations of the estradiol receptor dimerization domains with the TRAP interaction regions lead us to conclude that TRAP stabilizes TR binding to DNA by formation of TRAP-TR heterodimers with both proteins bound to the DNA. TR bound to the estrogen response element is unable to respond to TRAP and unable to stimulate transcription, possibly due to the absence of TRAP in the TR-estrogen response element complex. In addition, TRAP may interact with a certain subset of the nuclear receptor superfamily, since human retinoic acid receptor-beta and vitamin D receptor show increased binding to TREs in the presence of nuclear extract, but c-erbA alpha-2, a variant TR, does not respond to TRAP.

Dominant Negative Transcriptional Regulation by a Mutant Thyroid Hormone Receptor-β in a Family with Generalized Resistance to Thyroid Hormone

An abnormal human thyroid hormone beta-receptor (hTR beta-Mf), which has a glycine to arginine substitution in the hormone-binding domain, has been identified in affected members of one family with generalized resistance to thyroid hormone. To better understand the mechanism by which this mutation produces the observed abnormality, expression vectors for the wild-type and mutant thyroid hormone receptors (TRs) were prepared to test hormone-binding activity and trans-activation function. Nuclear extracts of COS-7 cells transfected with wild-type TRs showed specific T3-binding activity, while mutant receptor-transfected COS-7 nuclear extract failed to bind T3. On the other hand, in a avidin-biotin complex DNA-binding assay, in vitro translated hTR beta-Mf showed high binding activity to the thyroid hormone response element, which was indistinguishable from that of wild-type TRs. In a transient expression study, only the wild-type TRs activated a rat GH gene promoter-chloramphenicol acetyltransferase fusion gene in a T3-dependent manner. Additionally, when wild-type TR and hTR beta-Mf were cotransfected, hTR beta-Mf inhibited gene activation regulated by wild-type TRs. From these results we conclude that 1) hTR beta-Mf has no demonstrable T3 binding and appears to have minimal, if any, ability to activate a thyroid hormone-responsive gene in spite of its preserved ability to bind to a TRE in DNA; 2) hTR beta-Mf inhibits the transcriptional activation of a thyroid hormone-responsive gene by the wild-type TRs in a dominant manner; and 3) the dominant negative regulatory function of hTR beta-Mf appears to explain the clinical manifestations of thyroid hormone resistance produced by this mutation when present in the heterozygous state.

Interaction of thyroid hormone receptors with strong and weak cis-acting elements in the human alpha-myosin heavy chain gene promoter.

The alpha-MHC gene is under positive regulation by 3,5,3'-triiodo-L-thyronine (T3), however, the mechanism by which T3 modulates its transcription is not clearly understood. We have used an avidin-biotin complex DNA binding assay and footprint analysis employing dimethyl sulfate interference and hydroxyl radical protection to characterize the interaction of T3 receptors with target sequences located in the 5'-flanking region of the human alpha-myosin heavy chain (MHC) gene. The results indicate that liver T3 receptors and in vitro transcribed-translated beta c-erbA bind with high affinity to a site (TRE1) located at positions -138/-158 base pairs upstream from the CAP site. The Kd of TRE1 for nuclear liver T3 receptors (0.82 nM) was less than that determined for the rat growth hormone gene (1.78 nM). An additional site (TRE2) located at positions -111/-129 was found which bound T3 receptors with considerably lower affinity (Kd = 23 nM). Methylation interference experiments demonstrated that T3 receptors interact with guanines, in TRE1 on the sense and antisense strands, within two octameric imperfect direct repeats (underlined) 5'-TCTGGAGGTGACAG-GAGGACA-3' (antisense strand sequence) containing the consensus sequence 5'-C(T/A)GGAGG(T/A)-3'. By contrast, methylation interference and hydroxyl radical footprinting demonstrate that the T3 binding element of TRE2 is not structurally similar to TRE1 except for a purine-rich octameric cluster (underlined, 5'-ATCAAAGGAGGAGGAGCCA-3') containing six guanines on the sense strand. These results suggest that differences in nucleotide sequences involved in T3 receptor-DNA complex formation determine the binding affinities of TRE1 and TRE2.

Alpha and beta thyroid hormone receptors bind immediately adjacent to the rat growth hormone gene TATA box in a negatively hormone-responsive promoter region.

We report that alpha and beta type rat thyroid hormone receptors bind specifically and with high affinity to the 10-base pair sequence immediately 3' of the rat growth hormone TATA box (positions -25 to -16) in a region of the rat growth hormone promoter which can be negatively hormone responsive (nTRE). The receptors have approximately 7-fold lower affinity in vitro for the nTRE than for the thyroid hormone-responsive enhancer of the rat growth hormone gene (TRE). Proteins extracted with high salt concentration from rat pituitary cell nuclei enhance binding of the receptors to both the TRE and nTRE. A modification of the avidin-biotin complex DNA binding assay which enhances the sensitivity of the assay approximately 100-fold was used in these studies. The immediate proximity of a receptor binding site to the rat growth hormone TATA box suggests that direct interaction between receptor and TFIID (the TATA binding protein) mediates nTRE activity.

A nuclear factor that enhances binding of thyroid hormone receptors to thyroid hormone response elements.

Recent studies from this laboratory have demonstrated the presence of thyroid hormone response elements (TREs) in the 5'-flanking region of the rat alpha and TSH beta subunit genes. Using an avidin-biotin complex DNA binding assay, we have shown that these TREs bind the thyroid hormone (T3) receptor present in nuclear extracts of GH3 cells, as well as the in vitro synthesized Hc-erbA beta, which has been identified as a member of the family of T3 receptors. The binding of Hc-erbA beta to the alpha subunit TRE can be enhanced 3-4-fold by including GH3 nuclear extract in the binding assay. Binding to the TRE present in the TSH beta gene or the rat growth hormone gene was similarly enhanced, although to a lesser degree. The enhanced binding activity is trypsin-sensitive and heat labile, and is not reproduced by the addition of histones, bovine serum albumin, or cytosol instead of nuclear extract. Gel exclusion chromatography suggests a molecular size of approximately 65,000 Da. This protein, which is present in several different cell types, is also able to complement binding of the rat erbA alpha-1 and the pituitary-specific erbA beta-2 forms of the receptor. These data suggest that the binding of the T3 receptor to a TRE is augmented by another nuclear protein, which may be involved in the mechanism of action of thyroid hormone.

Binding of thyroid hormone receptors to the rat thyrotropin-beta gene.

Negative regulation of rat TSH beta gene expression by thyroid hormone is mediated largely by decreased transcription of the gene. This is apparently mediated by a cis-acting element which has been localized to a 57-basepair fragment spanning the second transcriptional start site of the rat TSH beta gene. We have investigated whether thyroid hormone receptors bind specifically to DNA sequences in this region of the gene. We compared binding of native T3 receptor to the TSH beta gene sequences and to the rat GH (rGH) gene T3 response element (TRE), and examined the ability of two different forms of in vitro synthesized T3 receptor to bind to the TSH beta gene. The avidin-biotin complex DNA binding assay was used to examine sequence-specific binding of the receptor. [125I]T3-labeled receptor in GH3 cell nuclear extracts bound to a site within the first exon of TSH beta and also to a region immediately upstream of the second transcriptional start site of the gene. In addition, the Hc-erbA beta and r-erbA alpha-1 forms of the T3 receptor each bound to TSH beta and rGH sequences, demonstrating that both alpha- and beta-forms of T3 receptor can bind to TREs exerting either positive or negative transcriptional regulation. Competition experiments showed that both native and in vitro synthesized T3 receptor bound to the first exon of TSH beta with an affinity slightly less than that for the rGH TRE. The two receptor-binding sites of the rTSH beta gene show sequence similarity to adjacent regions of the rGH TRE. These data indicate that negative regulation of rat TSH beta gene transcription may be effected by direct binding of the T3-receptor complex to one or both of the binding sites flanking the second transcriptional start site.

Thyroid Hormone Regulation of the Rat Glycoprotein Hormone α-Subunit Gene Promoter Activity

Abstract An important physiological control of the glycoprotein hormone alpha-subunit is the negative feedback by thyroid hormones in the thyrotrope. A region of the rat glycoprotein hormone alpha-subunit gene that is involved in transcriptional regulation by thyroid hormone has been identified by transient transfection studies, and sequence-specific binding of the thyroid hormone receptor to a site within this region has been demonstrated. Deletion-mutation studies using plasmid expression vectors containing either 246, 170, or 80 base pairs of the 5'-flanking region of the rat alpha-subunit gene fused to the coding region of the bacterial chloramphenicol acetyltransferase gene demonstrate 3,5,3'-triiodo-L-thyronine (T3)-regulated expression in GH3 cells, a T3-responsive somatotrophic cell line. In order to investigate the possibility of thyroid hormone receptor interaction with this segment of the rat alpha-subunit gene, the binding of the thyroid hormone receptor to synthetic oligodeoxynucleotides was analyzed using an avidin-biotin complex DNA binding assay. An oligodeoxyribonucleotide representing a fragment of the alpha-subunit gene from -74 to -38, relative to the transcriptional start site, shows significant binding to [125I]T3-receptor complex present in nuclear extracts of GH3 cells. This fragment binds receptor to a degree similar to that seen with a fragment of the rat growth hormone gene which contains a putative thyroid hormone-responsive element. In addition, this fragment of the rat alpha-subunit gene binds to the in vitro synthesized human c-erbA beta protein, which has been identified as a member of the family of putative T3 receptors. These data demonstrate that a cis-active thyroid hormone-responsive element resides in the 5'-flanking region of the rat alpha-subunit gene and that the mechanism involved in the suppression of expression of this gene by T3 could involve specific binding of the thyroid hormone receptor to this region of the gene.

A c-erb-A binding site in rat growth hormone gene mediates trans-activation by thyroid hormone.

The substance 3,5,3-triiodothyronine (T3) stimulates growth hormone gene transcription in rat pituitary tumour cells. This stimulation is thought to be mediated by the binding of nuclear T3 receptors to regulatory elements 5' to the transcriptional start site. Understanding of the mechanism by which thyroid hormone activates gene transcription has been limited by failure to purify nuclear T3 receptors because of their low abundance, and by the absence of defined T3 receptor-DNA binding sites affecting T3 regulation. Recently, human and avian c-erb-A gene products have been shown to bind thyroid hormone with high affinity and to have a molecular weight and nuclear association characteristic of the thyroid hormone receptor. In the present report, we describe the development of an avidin-biotin complex DNA-binding assay which can detect specific, high-affinity binding of rat pituitary cell T3 receptors to the sequence 5'CAGGGACGTGACCGCA3', located 164 base pairs 5' to the transcriptional start site of the rat growth hormone gene. An oligonucleotide containing this sequence transferred T3 regulation to the herpes simplex virus thymidine kinase promoter in transfected rat pituitary GC2 cells, and specifically bound an in vitro translation product of the human placental c-erb-A gene. The data provide supporting evidence that the human c-erb-A gene product mediates the transcriptional effects of T3 and also that GC2 cell nuclear extracts contain additional factors that modify the binding of pituitary T3 receptors to the rat growth hormone gene T3 response element.