Palindromic Element Research Articles

Polarity of the ecdysone receptor complex interaction with the palindromic response element from the hsp27 gene promoter.

The functional 20-hydroxyecdysone (20E) receptor is a heterodimer of two members of the nuclear hormone receptors superfamily; the product of the EcR (EcR) and of the ultraspiracle (Usp) genes. As most of the natural 20E-response elements are highly degenerated palindromes, we were interested in determining whether or not such asymmetric elements could dictate the defined orientation of the Usp/EcR complex. We have investigated interaction of EcR and Usp DNA-binding domains (EcRDBD and UspDBD, respectively) with the palindromic response element from the hsp27 gene promoter (hsp27pal). The hsp27pal half-sites contribute differently to the binding of the heterodimer components; the 5' half-site exhibits higher affinity for both DBDs than the 3' half-site. This observation, along with data demonstrating that UspDBD exhibits approximate fourfold higher affinity to the 5' half-site than EcRDBD, suggest that UspDBD locates the EcRDBD/UspDBD heterocomplex in the defined orientation (5'-UspDBD-EcRDBD-3') on the hsp27pal sequence. The binding polarity onto hsp27pal is accompanied by different contribution of the UspDBD and EcRDBD C-terminal sequences to the DNA-binding and heterocomplex formation. This is supported by finding that deletion of the C-terminal of EcRDBD region corresponding to the putative A-helix severely decreased binding of the EcRDBD to the hsp27pal. In contrast, UspDBD in which corresponding residues were deleted exhibited the same hsp27pal binding pattern as the wild type UspDBD. Additional truncation comprising the putative T-box, resulted in a reduced binding of the mutated UspDBD. This truncation however, still allowed effective EcRDBD/UspDBD heterodimer formation. Finally we demonstrated that perfect palindromes, composed of two hsp27pal 5' half-sites (or of the related sequence) contain all of the structural information necessary for the anisotropic UspDBD/EcRDBD heterocomplex formation. However, the perfect palindromes bind isolated homomeric DBDs as well as their heterocomplex with higher affinity than imperfect hsp27pal. This is the first report indicating that natural 20E response elements, which with one exception are degenerated palindromes, may act as functionally asymmetric elements in a manner similar to the action of direct repeats in vertebrates.

Constitutive activation of transcription and binding of coactivator by estrogen-related receptors 1 and 2.

In this report, we demonstrate that, in contrast to most previously characterized nuclear receptors, hERR1 and hERR2 (human estrogen receptor-related protein 1 and -2) are constitutive activators of the classic estrogen response element (ERE) as well as the palindromic thyroid hormone response element (TRE(pal)) but not the glucocorticoid response element (GRE). This intrinsically activated state of hERR1 and hERR2 resides in the ligand-binding domains of the two genes and is transferable to a heterologous receptor. In addition, we show that members of the p160 family of nuclear receptor coactivators, ACTR (activator of thyroid and retinoic acid receptors), GRIP1 (glucocorticoid receptor interacting protein 1), and SRC-1 (steroid receptor coactivator 1), potentiate the transcriptional activity by hERR1 and hERR2 in mammalian cells, and that both orphan receptors bind the coactivators in a ligand-independent manner. Together, these results suggest that hERR1 and hERR2 activate gene transcription through a mechanism different from most of the previously characterized steroid hormone receptors.

Genotyping of Actinobacillus actinomycetemcomitans serotype d isolates based on polymerase chain reaction.

The aims of the study were to determine the genetic diversity of the rare Actinobacillus actinomycetemcomitans serotype d and to compare the ability of the repetitive extragenic palindromic element (REP)-based polymerase chain reaction (PCR) with that of the arbitrarily primed (AP)-PCR to discriminate between and within A. actinomycetemcomitans serotypes. The material included 26 A. actinomycetemcomitans serotype d isolates, 3 reference strains, and 21 A. actinomycetemcomitans isolates, representing the previously described 17 AP-PCR genotypes from 4 serotypes (a, b, c and e). Among A. actinomycetemcomitans serotype d isolates (n = 26), the AP-PCR primer distinguished 2 genotypes, whereas the REP-primer pair (REP1R-I and REP2-I) and the (GACA)4 primer each produced one genotype. Among the total of 50 A. actinomycetemcomitans isolates, REP-primer pair distinguished 6 genotypes, the primer (GACA)4 7 genotypes, and the AP-PCR 19 genotypes. Among A. actinomycetemcomitans serotype a isolates (n = 6), REP-primer pair yielded 3 genotypes and (GACA)4 and AP-PCR primer 4 genotypes, and among serotype e isolates (n = 6) 3 genotypes. All serotype b isolates (n = 7), representing the AP-PCR genotypes 2, 9, 8, 12, 13, 16 and serotype c isolates (n = 5), AP-PCR genotypes 3, 4, 14, 15, belonged to the (REP1R-I and REP2-I)-PCR genotype 4 and to the (GACA)4-PCR genotype 4. In conclusion, based on both the AP-PCR method and the less discriminative REP-PCR methods, the present genotyping results indicated limited genetic diversity among serotype d isolates of A. actinomycetemcomitans.

Site-specific Heterodimerization by Paired Class Homeodomain Proteins Mediates Selective Transcriptional Responses

Alx4 is a paired class homeodomain protein involved in defining anterior/posterior polarity in the developing limb bud. The paired class of homeodomain proteins cooperatively bind palindromic DNA elements as homodimers or as heterodimers with other paired homeodomain proteins. Previous characterization demonstrates that the strength of the cooperativity as well as the preference for targets is dictated largely by the identity of amino acid 50 of the homeodomain. Here we compare and contrast the DNA binding properties of a glutamine 50 paired homeodomain protein, Alx4, and a lysine 50 paired homeodomain protein, Goosecoid. We demonstrate that Alx4 homodimers, Gsc homodimers, and Alx4/Gsc heterodimers each have distinct DNA binding properties, and each can discriminate between highly related palindromic elements. Using reporter gene assays, we show that Alx4 activates transcription in a site-specific manner, and that Gsc is capable of antagonizing Alx4-mediated activation only from promoter elements that support heterodimer formation. These data demonstrate that paired homeodomain proteins with different DNA binding properties are able to form heterodimeric complexes with unique DNA binding and transcriptional activities. Thus, heterodimerization regulates the DNA binding specificity of these transcription factors and may partially explain how paired homeodomain proteins direct specific developmental functions.

Short palindromic repetitive DNA elements in enterobacteria: a survey

We present a survey of short palindromic repetitive elements in enterobacteria. Seven families are presented. Five were already known (RSA, IRU, 29-bp repeats, BIMEs and boxC), and their properties are updated; in particular, a new composite element is shown to include the formerly identified boxC repeats. Two repetitions, YPAL1 and YPAL2, found primarily in Yersinia, are described here for the first time.

The decidualizing effect of progesterone may involve direct transcriptional activation of corticotrophin-releasing hormone from human endometrial stromal cells.

The hypothalamic neuropeptide corticotrophin-releasing hormone (CRH) is also produced by human endometrial cells and is directly involved in the decidualization process as a paracrine inducer. The aim of the present work was to examine the effect of progesterone, the main decidualizing factor, on endometrial CRH, in primary cultures of human endometrial stromal cells. The effect of progesterone was examined by measuring the effects of medroxyprogesterone acetate (MPA) on (i) the concentration of immunoreactive CRH in isolated human endometrial stromal cells and (ii) the activity of the CRH promoter in human endometrial stromal cells transfected with a 0.9 kb fragment of the 5'-flanking region of the human CRH gene coupled to luciferase reporter. The data show that MPA increased the production and secretion of immunoreactive CRH from stromal cells and induced the activity of the CRH promoter, both in a dose-dependent manner. These effects were partially reversed by a molar excess of the antiprogestin RU 486 and were completely abolished in the presence of 100 nmol/l of the cAMP inhibitor, Rp-cAMP. The effect of progesterone on the CRH promoter requires the existence of an intact CRH sequence since experiments carried out with a deleted palindromic cAMP response element (CRE: 5'-TGACGTCA) at -224 bp of the CRH promoter resulted in a complete loss of MPA effect. In conclusion, these data provide evidence that progesterone induces the transcription of CRH gene in human endometrial stroma. This effect coupled with the decidualizing properties of progesterone and CRH may indicate that progesterone and CRH form a decidualizing local pathway within the human endometrium.

Targeted expression of the DNA binding domain of DRE-binding factor, a Drosophila transcription factor, attenuates DNA replication of the salivary gland and eye imaginal disc.

The promoters of Drosophila genes encoding DNA replication-related proteins contain transcription regulatory elements consisting of an 8-bp palindromic DNA replication-related element (DRE) sequence (5'-TATCGATA). The specific DRE-binding factor (DREF), a homodimer of the polypeptide with 709 amino acid residues, is a positive trans-acting factor for transcription of DRE-containing genes. Both DRE binding and dimer formation are associated with residues 16 to 115 of the N-terminal region. We have established transgenic flies expressing the full-length DREF polypeptide or its N-terminal fragment (amino acid residues 1 to 125) under the control of the heat shock promoter, the salivary gland-specific promoter, or the eye imaginal disc-specific promoter. Heat shock induction of the N-terminal fragment during embryonic, larval, or pupal stages caused greater than 50% lethality. This lethality was overcome by coexpression of the full-length DREF. In salivary glands of the transgenic larvae expressing the N-terminal fragment, this fragment formed a homodimer and a heterodimer with the endogenous DREF. Ectopic expression of the N-terminal fragment in salivary gland cells reduced the contents of mRNAs for the 180-kDa subunit of DNA polymerase alpha and for dE2F and the extent of DNA endoreplication. Ectopic expression of the N-terminal fragment in the eye imaginal discs significantly reduced DNA replication in cells at the second mitotic wave. The lines of evidence suggest that the N-terminal fragment can impede the endogenous DREF function in a dominant negative manner and that DREF is required for normal DNA replication in both mitotic cell cycle and endo cycle.

Description of Cellulophaga baltica gen. nov., sp. nov. and Cellulophaga fucicola gen. nov., sp. nov. and reclassification of [Cytophaga] lytica to Cellulophaga lytica gen. nov., comb. nov.

Phenotypic data indicate that gliding, yellow/orange-pigmented, agar-digesting bacterial strains were members of the Cytophaga-Flavobacterium-Bacteroides (CFB) group. The strains were isolated from the surface of the marine benthic macroalga Fucus serratus L. and the surrounding seawater at three localities in Danish waters. The bacteria were Gram-negative, flexirubin-negative, aerobic, catalase-positive and oxidase-negative and were psychrophilic and halophilic. All strains utilized D-fructose, L-fucose and alpha-ketobutyric acid and degraded alginic acid, carrageenan, starch and autoclaved yeast cells. Amplification with primers specific for repetitive extragenic palindromic elements by PCR divided the strains of this study into two groups. Both groups showed unique PCR amplification patterns compared to reference strains of the CFB group. Phylogenetic analysis of 16S rDNA sequences showed association of these organisms and [Cytophaga] lytica at the genus level. Hybridization of total chromosomal DNA revealed that the new strains and [Cytophaga] lytica ATCC 23178T were clearly distinct from each other and other previously described species of the CFB group. A new genus is described, Cellulophaga gen. nov. comprising two new species, Cellulophaga baltica gen. nov., sp. nov. (NN015840T = LMG 18535T) and Cellulophaga fucicola gen. nov., sp. nov. (NN015860T = LMG 18536T), as well as the emendation of [Cytophaga] lytica to Cellulophaga lytica gen. nov., comb. nov.

Selective activation of the probasin androgen-responsive region by steroid hormones.

Glucocorticoid and androgen receptors have been shown to function through the same palindromic glucocorticoid response element (GRE) and yet have differential effects on gene transcription. In this study, we examined the functional and structural relationship of the androgen and glucocorticoid receptors with the androgen responsive region (ARR) of the probasin (PB) gene containing two androgen receptor binding sites, ARBS-1 and ARBS-2. Transfection studies indicated that one copy of each cis-acting DNA element was essential for maximal androgen-induced chloramphenicol acetyltransferase (CAT) activity and that androgen selectivity was maintained when multiple copies of the minimal wild type (wt) androgen responsive region containing both ARBS-1 and ARBS-2 (-244 to -96) were subcloned in front of the thymidine kinase promoter. Furthermore, replacing the androgen response region with 1, 2 or 3 copies of either ARBS-1 or ARBS-2 restored less than 4% of the biological activity seen with the wt PB ARR. Multiple copies of either ARBS-1 or ARBS-2 did not result in glucocorticoid-induced CAT gene activity. By comparison, 1 or 2 copies of the tyrosine aminotransferase (TAT) GRE, as well as the mouse mammary tumour virus GRE, were strong inducers of CAT activity in response to both androgen and glucocorticoid treatment. In addition, band shift assays demonstrated that although the synthetic glucocorticoid receptor, GR-DNA binding domain (GR-DBD), and the synthetic androgen receptor, AR2, could interact with the TAT GRE (dissociation constants Kd of 63.9 and 14.1 respectively), only AR2 but not GR-DBD binding could be detected on ARBS-1 and ARBS-2. Our findings provide further evidence that androgen-induced regulation of gene transcription can occur through androgen-specific DNA binding sites that are distinct from the common GRE.

A trans-acting enhancer modulates estrogen-mediated transcription of reporter genes in osteoblasts.

The presence of bone-specific estrogen agonists and discovery of the osteoblast-specific transcription factor (TF), Cbfa1, together with the discovery of synergism between a TF Pit-1 and estrogen receptor alpha (ERalpha) on rat prolactin gene, led to investigation of Cbfa1 in the modulation of osteoblast-specific actions of estrogen. Reverse transcribed-polymerase chain reaction demonstrated expression of Cbfa1 in the osteoblastic cell lines, MG63, ROS17/2.8, and MC3T3E1, but not in nonosteoblastic cell lines, MCF7, C3H10T1/2, and HeLa. An ER expression vector and a series of luciferase (Luc) reporter plasmids harboring the Cbfa1 binding site OSE2 (the osteoblast-specific cis element in the osteocalcin promoter) and palindromic estrogen response elements (EREs) were cotransfected into both osteoblastic and nonosteoblastic cells. OSE2 worked as a cis- acting element in osteoblastic cells but not nonosteoblastic cells, whereas EREs were cis- acting in all cell lines. Synergistic transactivation was observed in osteoblastic cells only when both ERE and OSE2 were placed in juxtaposition to the promoter. Forced expression of Cbfa1 in C3H10T1/2 cells also induced synergism. Tamoxifen, a partial agonist/antagonist of estrogen, acted as an osteoblast-specific agonist in cells transfected with a promoter containing ERE and acted synergistically with a promoter containing the ERE-OSE2 enhancer combination. These results support the idea that bone-specific TFs modulate the actions of estrogen in a tissue-specific manner.

Physical and genetic interactions between Alx4 and Cart1.

Alx4 and Cart1 are closely related members of the family of transcription factors that contain the paired-type homeodomain. In contrast to other types of homeodomains, the paired-type homeodomain has been shown to mediate high-affinity sequence-specific DNA binding to palindromic elements as either homodimers or as heterodimers with other family members. Alx4 and Cart1 are co-expressed at several sites during development, including the craniofacial mesenchyme, the mesenchymal derivatives of neural crest cells in the first branchial arch and the limb bud mesenchyme. Because of the molecular similarity and overlapping expression pattern, we have analyzed the functional and genetic relationships between Alx4 and Cart1. The two proteins have similar DNA-binding activity in vitro and can form DNA-binding heterodimers; furthermore, they activate transcription of reporter genes that contain high-affinity DNA-binding sites in cell culture in a similar manner. Therefore, at least by these criteria, the two proteins are functionally redundant. Analysis of double mutant animals reveals several genetic interactions. First, mutation of Cart1 exacerbates Alx4-dependent polydactyly in a manner that is dependent on gene dosage. Second, there are complex genetic interactions in the craniofacial region that reveal a role for both genes in the fusion of the nasal cartilages and proper patterning of the mandible, as well as other craniofacial structures. Third, double mutant mice show a split sternum that is not detected in mice with any other genotype. Interpreted in the context of the biochemical characterization, the genetic analysis suggests that Alx4 and Cart1 are indeed functionally redundant, and reveal both unique and redundant functions for these genes in development.

NMDA receptor type 2D gene as target for estrogen receptor in the brain

Although it is well known that estrogen exerts its effect in the brain, the direct target genes transcriptionally regulated by estrogen or rather estrogen receptor (ER) are almost unknown. During the search for estrogen receptor-binding sites from human CpG island library, we found one genomic DNA fragment corresponding to the putative 3′-untranslated region of human NMDA receptor subunit 2D (NR2D) gene. It contained at least four half palindromic estrogen responsive elements (hEREs) within two hundred nucleotides, which was conserved also in the rat. Interestingly, the NR2D mRNA is co-localized with ERα and/or ERβ mRNA in a number of regions of rat brain. We have also demonstrated that NR2D mRNA is up-regulated in rat hypothalamus by estrogen possibly via hEREs identified here. Thus, we suggest that NR2D is one of the direct targets of estrogen receptors which are involved in reproductive as well as non-reproductive actions in the brain.

Typing of Listeria monocytogenes strains by repetitive element sequence-based PCR.

Listeria monocytogenes strains possess short repetitive extragenic palindromic (REP) elements and enterobacterial repetitive intergenic consensus (ERIC) sequences. We used repetitive element sequence-based PCR (rep-PCR) to evaluate the potential of REP and ERIC elements for typing L. monocytogenes strains isolated from humans, animals, and foods. On the basis of rep-PCR fingerprints, L. monocytogenes strains were divided into four major clusters matching origin of isolation. rep-PCR fingerprints of human and animal isolates were different from those of food isolates. Computer evaluation of rep-PCR fingerprints allowed discrimination among the tested serotypes 1/2a, 1/2b, 1/2c, 3b, and 4b within each major cluster. The index of discrimination calculated for 52 epidemiologically unrelated isolates of L. monocytogenes was 0.98 for REP- and ERIC-PCR. Our results suggest that rep-PCR can provide an alternative method for L. monocytogenes typing.

Activation of a CrkL-Stat5 Signaling Complex by Type I Interferons

Type I interferons (IFNalpha and IFNbeta) transduce signals by inducing tyrosine phosphorylation of Jaks and Stats, as well as the CrkL adapter, an SH2/SH3-containing protein which provides a link to downstream pathways that mediate growth inhibition. We report that Stat5 interacts constitutively with the IFN receptor-associated Tyk-2 kinase, and during IFNalpha stimulation its tyrosine-phosphorylated form acts as a docking site for the SH2 domain of CrkL. CrkL and Stat5 then form a complex that translocates to the nucleus. This IFN-inducible CrkL-Stat5 complex binds in vitro to the TTCTAGGAA palindromic element found in the promoters of a subset of IFN-stimulated genes. Thus, during activation of the Type I IFN receptor, CrkL functions as a nuclear adapter protein and, in association with Stat5, regulates gene transcription through DNA binding.

Transcriptional regulation by cyclic AMP is essential for development, reproduction and survival: lessons from the transgenic mice.

A great number of hormones act at the cellular level by stimulation of the cAMP signalling pathway, after binding to their specific G protein-coupled transmembrane receptor. Alterations of the cAMP pathway have been implicated in several endocrine diseases such as acromegaly, toxic thyroid adenomas or Cushing’s syndrome. One major cellular effect of the cAMP cascade activation is stimulation of transcription after phosphorylation of nuclear factors by the cAMP-dependent kinase, PKA (Fig. 1). A conserved palindromic cAMP response element (CRE) has been identified in the promoter of various genes regulated by cAMP (i.e. somatostatin, vasoactive intestinal polypeptide, alpha chorionic gonadotrophin, enkephalin, fos, parathyroid hormone (PTH) .... genes). Three proteins have been identified that bind the CRE and are phosphorylated by PKA: CREB (CRE binding protein), CREM (CRE modulator) and ATF-1 (activating transcription factor 1) (1). While CREB and ATF-1 stimulate transcription, some isoforms of CREM can negatively regulate CRE activity. A coactivator of CREB that binds, specifically, CREB phosphorylated by PKA in response to cAMP stimulation, has been cloned and termed CREB binding protein (CBP). CBP appears to be a general transcriptional coactivator for numerous signalling pathways. To evaluate the physiological significance of the various components regulating the CRE, a number of transgenic models have been reported and show the major role of cAMP regulated transcription. CREB null mice generated by homologous recombination die just after birth from respiratory distress (2). Analysis of these animals show that they are smaller than the controls and that they have central nervous system alterations (corpus callosum and anterior commissures reduction). It has been observed previously that mice lacking two of the three main splice product isoforms of CREB have a deficient long-term memory (3). It should be noted that in both models of CREB knockout mice (i.e. the partial and the complete models), an overexpression of CREM and ATF-1 is observed. This suggests a compensatory mechanism between the members of the CREB/CREM/ ATF-1 family. The CREB -/- mice also have impaired T cell development. Interestingly, a T cell proliferative defect and a decreased interleukin-2 production are also observed in transgenic mice expressing a dominantnegative mutant of CREB under the control of the thymocyte-specific CD2 promoter (as the use of the CD2 promoter in the construction of the transgene allows specific expression of the mutant in the thymocytes) (4). In this mutant, the PKA phosphorylation site is mutated and therefore the cAMP activation of CREB is abolished. The same approach has demonstrated the major role of CREB in the development of the pituitary somatotroph. Transgenic mice expressing a similar dominantnegative mutant of CREB under the control of the growth hormone (GH) promoter are dwarf and present with pituitary hypoplasia due to a dramatic reduction in the number of GH cells (5). Targeting a dominantnegative mutant of CREB in the heart using the myocyte-specific alpha-MHC promoter leads to dilated cardiomyopathy in transgenic mice (6). Disruption of the CREM gene by homologous recombination leads to infertility in male mice due to deficient spermiogenesis (7, 8), without hormonal alterations of the gonadotroph axis. Mice homozygous for a targeted mutation of the coactivator CBP die during embryonic development and show neural tube defects (9). Numerous transgenic mice models for the study of transcription regulation by cAMP reported to date have given insights into the major developmental role of the various CRE binding proteins and of CBP. Together with the knockout models, the use of dominant-negative mutants targeted to a given tissue allows the identification of the specific role of CRE binding proteins in each tissue and reveals tissue-specific differences for proteins that seem quite ubiquitously expressed. The CREB null mice suggest that compensation occurs in the CRE binding protein family, in keeping with the essential role of these transcription factors. Nevertheless, a specific role for each member of this family does exist. In this view the development of the ATF-1 null mice and of the double knockout mice will help to explain the function of each nuclear component of the cAMP-regulated transcription (2). The use of the conditional knockout technology which allows control of the timing and/or

CAMP responsiveness of the bovine calpastatin gene promoter

Previous studies have shown that transcription of the gene encoding bovine calpastatin, an inhibitor of the calcium-activated cysteine protease calpain, is upregulated following activation of cAMP-dependent signaling pathways. In this study, deletion and site-directed mutagenesis experiments were performed to identify cis elements conferring cAMP responsiveness. Heterologous promoter assays demonstrated that all cAMP-responsive cis elements were located within −102 nucleotides (nt) of transcription initiation. Deletion of an element (GTCA) at nt +13 that is identical to half of the palindromic cAMP-responsive element (TGACGTCA) identified in other cAMP-responsive gene promoters had no effect on the response of the calpastatin promoter to dibutyryl-cAMP, although a 67% reduction in basal promoter activity was observed. In contrast, two point mutations in a cis element at nt −76 (GTCA to aTCt) abolished cAMP responsiveness. These results demonstrate that the calpastatin promoter sequence between nt −1653 and +130 contains a single cAMP-responsive element (GTCA) located at nt −76, and suggest a direct molecular pathway by which activation of cAMP signaling could lead to increased calpastatin gene transcription and reduction in calpain-mediated proteolysis.

PCR methods and plasmid profile analysis for characterisation of Histophilus ovis strains.

The value of polymerase chain reaction (PCR)-based DNA fingerprinting and plasmid profile analysis for differentiation of Histophilus ovis isolates was assessed. Nineteen isolates of H. ovis were typed by PCR-ribotyping, repetitive extragenic palindromic element (REP)-PCR and enterobacterial repetitive intergenic consensus (ERIC)-PCR. These methods distinguished five types by PCR-ribotyping, 11 types by REP-PCR and seven types by ERIC-PCR. The ribotyping method produced a relatively simple pattern and a small number of distinct types and was useful for differentiation of H. ovis from the phenotypically similar organism, Haemophilus somnus. REP- and ERIC-PCR both produced complex banding patterns, but increased the discrimination between strains. Plasmids were found in 12 of the 19 isolates and there were four different plasmid profiles. A combination of the PCR methods and plasmid profile analysis provided a high resolution typing method for H. ovis.

Glucocorticoid Receptor Transcriptional Activity Determined by Spacing of Receptor and Nonreceptor DNA Sites

The glucocorticoid receptor (GR) displays distinct modes of regulation when bound at glucocorticoid response elements (GREs) bearing different binding sequences and arrangements of binding sites. For example, it has been shown to activate transcription synergistically with itself or with other regulatory factors, such as AP1, when bound to a consensus palindromic element or "simple GRE" that is multimerized or linked tightly with an AP1 site. In contrast, at certain "composite GREs" GR and AP1 bind to nonconsensus sequences, and GR either activates or represses depending on the subunit composition of AP1. To uncouple the contributions to regulatory behavior of binding sequences and binding element arrangements, we examined GR action at "paired elements," combinations of a simple GRE and a consensus AP1 site, separated by different distances. We found that GR synergized with either c-Jun or c-Jun-c-Fos at paired elements with GRE-AP1 site separations of >/=26 base pairs. In contrast, paired elements with separations of 14-18 base pairs mimicked the composite GRE, i.e. GR synergized with c-Jun and repressed c-Jun-c-Fos. In DNA binding studies, GR and AP1 cooccupied the paired elements. We conclude that the arrangement of binding sites within a compound response element can be a major determinant of regulatory factor action.

Protein-Protein Interaction Domains and the Heterodimerization of Thyroid Hormone Receptor Variant α2 with Retinoid X Receptors

Heterodimerization between thyroid hormone receptors (TRs) and retinoid X receptors (RXRs) is mediated by a weak dimerization interface within the DNA- binding domains (DBDs) and a strong interface within the C-terminal ligand- binding domains of the receptors. Previous studies have shown that the conserved ninth heptad in the TR ligand-binding domain appears to play a critical role in heterodimerization with RXR. However, despite lacking the full ninth heptad, TR variant alpha2 (TRv alpha2) can heterodimerize with RXR on specific direct repeat response elements, but not on palindromic elements or in solution. Two possibilities may account for TRv alpha2-RXR heterodimerization on direct repeats. First, the DBD of TRv alpha2 may play a critical role in heterodimerization with RXR. Second, a specific sequence within the unique C terminus of TRv alpha2 may promote the formation of TRv alpha2-RXR heterodimers. In this study, we used receptor chimeras in which the DBD of RXR was replaced by either the TR DBD or an unrelated DBD from the metalloregulatory transcription factor AMT1 to address the role of the DBD dimerization interface in TRv alpha2-RXR heterodimerization. Gel mobility shift analyses showed that whereas TR alpha1 formed heterodimers with these chimeras, TRv alpha2 failed to do so. Deletion of the unique C terminus of TRv alpha2 had only a marginal effect on heterodimerization with RXR. Mutations within the DBD dimerization interface abolished heterodimerization of full-length TRv alpha2 with RXR but only marginally affected heterodimerization of full-length TR alpha1 with RXR. These data support the hypothesis that the TR-RXR DBD dimerization interface plays a critical role in TRv alpha2-RXR heterodimerization. Additional data show that the amino acid residues that make direct TR-RXR contacts within the DBDs also may play a role in receptor monomer binding to DNA, since mutations within these residues severely impair this interaction.

DNA bending determines Fos-Jun heterodimer orientation.

Heterodimeric transcription factors can bind to palindromic recognition elements in two opposite orientations with potentially distinct effects on transcriptional activity. We have determined the orientation of Fos-Jun binding at different AP-1 sites using a novel gel-based fluorescence resonance energy transfer assay. The orientation preference of heterodimer binding varied over a greater than 10-fold range. Single base pair substitutions that alter bending of flanking sequences reversed the orientation of heterodimer binding. Single amino acid substitutions that reduce the difference in DNA bending between Fos and Jun also reduced the orientation preference. Consequently, indirect read-out mediated by differences in DNA structure can contribute to the structural organization of nucleoprotein complexes.