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At least 163 records · Page 9Linked to original sources
PubMed1992

Ear3/COUP-TF binds most tightly to a response element with tandem repeat separated by one nucleotide.

Ear3/COUP-TF belongs to the steroid/thyroid hormone receptor superfamily of nuclear transcription factors. Previous studies have identified six target response elements to which the factors of the family bind with various affinities. They are palindromic pair of hexameric half site (Thyroid hormone Response Element; TRE, Estrogen Response Element; ERE, Glucocorticoid Response Element; GRE) or directly repeated half sites [Direct Repeat (DR) 3, DR4, DR5]. In this report, these six elements and three additional putative elements, DR0, DR1, and DR2, which are directly repeated half sites separated by 0, 1 and 2 nucleotides, respectively, were tested for binding to Ear3/COUP-TF protein employing gel retardation method. Among the palindromic series of response elements, TRE showed the highest affinity, ERE showed second highest, and no binding was observed for GRE. Among six direct repeat response elements, DR1 showed the highest affinity. Comparison of binding affinities between DR1 and TRE revealed that the DR1 has the highest affinity of all the nine response elements tested. The consensus sequence of the DR1 element resembles well the COUP sequence found in the upstream promoter of chicken ovalbumin gene, which has been shown to serve as a natural response element for Ear3/COUP-TF. Retinoid X receptor (RXR), another member of the receptor family, reportedly utilizes DR1 sequence as its response element, suggesting that Ear3/COUP-TF could regulate the expression of the same subsets of genes as RXR does.

Base Sequence↗
PubMed2000

C/EBP beta and Elk-1 synergistically transactivate the c-fos serum response element.

BACKGROUND: The serum response element (SRE) in the c-fos promoter is a convergence point for several signaling pathways that regulate induction of the c-fos gene. Many transcription factors regulate the SRE, including serum response factor (SRF), ternary complex factor (TCF), and CCAAT/enhancer binding protein-beta (C/EBPbeta). Independently, the TCFs and C/EBPbeta have been shown to interact with SRF and to respond to Ras-dependent signaling pathways that result in transactivation of the SRE. Due to these common observations, we addressed the possibility that C/EBPbeta and Elk-1 could both be necessary for Ras-stimulated transactivation of the SRE. RESULTS: In this report, we demonstrate that Elk-1 and C/EBPbeta functionally synergize in transactivation of both a Gal4 reporter plasmid in concert with Gal4-SRF and in transactivation of the SRE. Interestingly, this synergy is only observed upon activation of Ras-dependent signaling pathways. Furthermore, we show that Elk-1 and C/EBPbeta could interact both in an in vitro GST-pulldown assay and in an in vivo co-immunoprecipitation assay. The in vivo interaction between the two proteins is dependent on the presence of activated Ras. We have also shown that the C-terminal domain of C/EBPbeta and the N-terminal domain of Elk-1 are necessary for the proteins to interact. CONCLUSIONS: These data show that C/EBPbeta and Elk-1 synergize in SRF dependent transcription of both a Gal-4 reporter and the SRE. This suggests that SRF, TCF, and C/EBPbeta are all necessary for maximal induction of the c-fos SRE in response to mitogenic signaling by Ras.

3T3 Cells↗
PubMed2004

General transcriptional silencing by a Polycomb response element in Drosophila.

Polycomb response elements (PREs) are cis-regulatory sequences required for Polycomb repression of Hox genes in Drosophila. PREs function as potent silencers in the context of Hox reporter genes and they have been shown to partially repress a linked miniwhite reporter gene. The silencing capacity of PREs has not been systematically tested and, therefore, it has remained unclear whether only specific enhancers and promoters can respond to Polycomb silencing. Here, using a reporter gene assay in imaginal discs, we show that a PRE from the Drosophila Hox gene Ultrabithorax potently silences different heterologous enhancers and promoters that are normally not subject to Polycomb repression. Silencing of these reporter genes is abolished in PcG mutants and excision of the PRE from the reporter gene during development results in loss of silencing within one cell generation. Together, these results suggest that PREs function as general silencer elements through which PcG proteins mediate transcriptional repression.

Animals↗
PubMed1996

A novel type of retinoic acid response element in the second intron of the mouse H2Kb gene is activated by the RAR/RXR heterodimer.

We have identified and characterized a novel retinoic acid (RA) response element (Hi-RARE) in the second intron of the mouse major histocompatibility H2Kb gene. The Hi-RARE sequence is conserved in all mouse classical and Q class 1 genes, in MHC class 1 genes of the rat, Rhesus macaque, cat and in the vast majority of human classical and non-classical class 1 genes. The Hi-RARE sequence lies within a regulatory element responsible for constitutive expression of a 5' enhancerless H2Kb gene in the Ltk-fibroblasts. Hi-RARE consists of two inverted palindromic RARE consensus sites (5'-PuGGTCA-3') separated by an 8 nt spacer. Mutational analysis revealed that both inverted palindromic hexanucleotide motifs are indispensable functional sites for the 9-cis RA response. The Hi-RARE sequence confers 9-cis RA inducibility to a heterologous promoter. The inducibility is further augmented in embryonal carcinoma cells by the expression of recombinant retinoic acid receptors (PARs) and the retinoid X receptors (RXRs). In vitro, the recombinant RAR/RXR heterodimer creates DNA-protein complex with the Hi-RARE sequence. Treatment of P19 embryonal carcinoma cells with 9C-RA induces the Hi-RARE binding activity of nuclear proteins that proved to be RAR (or RAR-Like)/RXR heterodimer. Thus the Hi-RARE represents a new type of RA response element with a role in the modulation of the expression of MHC class 1 family genes.

Animals↗
PubMed1994

Specificity and flexibility of vitamin D signaling. Modulation of the activation of natural vitamin D response elements by thyroid hormone.

Cellular responsiveness to 1,25-dihydroxyvitamin D3 (VD) is conferred by its intracellular receptor (VDR), which belongs to the nuclear receptor superfamily of ligand-inducible transcription factors. VDRs are known to bind to specific response elements in the promoter region of VD-regulated genes. Two types of natural VD response elements (VDREs) have been identified so far. One is bound by VDR homodimers and is found in the human osteocalcin gene promoter (DR6-type), and the other is bound by heterodimers of VDR with retinoid X receptors (RXRs) as in the mouse osteopontin promoter (DR3-type). Here, we demonstrate that VDRs directly interact in solution not only with RXR but also with retinoid acid receptors (RARs) and, interestingly, with thyroid hormone receptors (T3Rs). All three heterodimeric partners were able to enhance the in vitro DNA binding affinity of VDR as compared to VDR homodimers, but they showed different affinities for the two types of VDREs, as determined by Scatchard analysis. Moreover, functional assays showed that VDR.T3R heterodimers act differently on the two types of VDREs; on the DR3-type we observed highest induction by thyroid hormone (3,5,3'-triiodothyronine, T3) alone, whereas on a DR6-type VDRE VD and T3 together provided maximal gene activity. Our data suggest that the expression levels of VDRs, RXRs, RARs, and T3Rs and their specific ligands regulate the transcription of VD-responsive genes and illustrates the complexity of this interactive network of nuclear receptors.

Animals↗
PubMed1997

A protein kinase C-activating phorbol ester enhances transcription of the human DBH gene through a cyclic AMP response element in SK-N-BE(2)C cells.

Protein kinase C (PKC) activation after treatment of human neuroblastoma SK-N-BE(2)C cells with phorbol 12-myristate 13-acetate (PMA) was found to enhance transcription of the human dopamine beta-hydroxylase (DBH) in those cells. To identify which cis-acting element is responsive to the PMA treatment during DBH gene expression, we employed transient transfection assays with serially deleted constructs of the human DBH gene's 5' upstream region fused to the chloramphenicol acetyltransferase (CAT) gene. Treatment of transfected cells with PMA resulted in an approximate threefold increase in CAT expression for all deletion constructs ranging from -978 bp to -262 bp, while the enhancement did not occur with a construct shortened to -114 bp. The region between -262 and -114 bp from the initiation site of transcription contains several cis-regulatory elements including a cyclic AMP response element (CRE) and putative AP1 and YY1 sequences. Site-directed mutagenesis of those cis-acting elements were performed to identify which of the elements mediated the PMA-induced transcriptional enhancement. Substitution of bases in the putative AP1 site containing in part a putative YY1 sequence did not effect the PMA inducibility. However, specific mutations in the CRE sequence abolished the PMA-inducible effect. Changing the CRE sequence into an authentic AP1 sequence (TGACGTCC --> TGACTCA) did not affect the PMA inducibility, suggesting that AP1 factors might interact with the new AP1 site upon PKC activation. A specific PKC inhibitor, GF109203X, completely inhibited the stimulatory effect of PMA on the expression of the human DBH gene. PMA induced an increase in the DBH mRNA level as detected by Northern blot analysis. Gel retardation showed that the binding of nuclear factors to CRE, putative YY1, and AP1 was sequence specific. Our data suggest that the enhancement of the human DBH gene expression by PMA treatment is mediated by the CRE motif in the 5' upstream region of the gene, and occurs via a PKC-dependent pathway.

Base Sequence↗
PubMed1996

Genetic characterization of transactivation of the human T-cell leukemia virus type 1 promoter: Binding of Tax to Tax-responsive element 1 is mediated by the cyclic AMP-responsive members of the CREB/ATF family of transcription factors.

To achieve a better understanding of the mechanism of transactivation by Tax of human T-cell leukemia virus type 1 Tax-responsive element 1 (TRE-1), we developed a genetic approach with Saccharomyces cerevisiae. We constructed a yeast reporter strain containing the lacZ gene under the control of the CYC1 promoter associated with three copies of TRE-1. Expression of either the cyclic AMP response element-binding protein (CREB) or CREB fused to the GAL4 activation domain (GAD) in this strain did not modify the expression of the reporter gene. Tax alone was also inactive. However, expression of the reporter gene was induced by coexpression of Tax and CREB. This effect was stronger with the GAD-CREB fusion protein. Analysis of different CREB mutants with this genetic system indicated that the C-terminal 92 amino acid residues, which include the basic domain and the leucine zipper, are necessary and sufficient to mediate transactivation by Tax. To identify cellular proteins binding to TRE-1 in a Tax-dependent manner, this strain was also used to screen a library of human cDNAs fused to GAD. Of five positive clones isolated from 0.75 x 10(6) yeast colonies, four were members of the CREB/activating transcription factor (ATF) family: CREB, two isoforms of the cyclic AMP-responsive element modulator (CREM), and ATF-1. Interestingly, these three proteins can be phosphorylated by protein kinase A and thus form a particular subgroup within the CREB/ATF family. Expression of ATF-2 in S. cerevisiae did not activate TRE-1 in the presence of Tax. This shows that in a eukaryotic nucleus, Tax specifically interacts with the basic domain-leucine zipper region of ATF-1, CREB, and CREM. The fifth clone identified in this screening corresponded to the Ku autoantigen p70 subunit. When fused to GAD, the C-terminal region of Ku was able to activate transcription via TRE-1 but this activation was not dependent on Tax.

Activating Transcription Factors↗
PubMed1997

Transcriptional activities of estrogen and glucocorticoid receptors are functionally integrated at the AP-1 response element.

Estrogens and glucocorticoids often act in opposition to regulate physiological responses. We investigated whether this might reflect the opposing actions of hormone-bound receptors on target genes regulated by the AP-1 response element. We performed a series of transfection experiments in which transcriptional activation, mediated by the AP-1 response element, was reflected in reporter gene activity. As previously described, we found that estrogens stimulate, whereas the glucocorticoid dexamethasone (Dex) inhibits, transcription through a model promoter from the collagenase gene (-73 to +63). This promoter bears a consensus AP-1 response element. When HeLa cells were treated with both estradiol and Dex, the steroids counteracted each other's transcriptional effects. The amount of transfected estrogen and glucocorticoid receptors (ER and GR) determined the extent to which Dex blunted estrogen stimulation or estrogen prevented Dex inhibition. The ER/GR interaction was observed both in the presence of estradiol and tamoxifen, which has previously been shown to have estrogen-like action at an AP-1 response element. The AP-1 family member c-Jun enhanced Dex inhibition and estradiol stimulation of transcriptional activation. c-Fos potentiated the effect of cotransfected c-Jun on estradiol stimulation but not Dex inhibition. The pattern of steroid responses was retained in the presence of the c-Jun activator phorbol 12-myristate 13-acetate. However, estradiol stimulation was lost in the presence of the c-Jun activator tumor necrosis factor-alpha. The ER/GR/AP-1 response element interaction was present, not only in a cell line originally derived from a uterine cervical adenocarcinoma (HeLa), but also in a cell line derived from the hypothalamus (GT1-1). Lastly, both progesterone receptor types A and B also interacted with the ER at the AP-1 site. These data indicate that opposing steroid influences can be mediated at the level of transcription through the AP-1 site and suggest that the integration of hormone action at this response element may underlie some of the opposing actions of estrogens and glucocorticoids or progestins on physiological responses.

Dexamethasone↗
PubMed1994

Response element selectivity for heterodimerization of vitamin D receptors with retinoic acid and retinoid X receptors.

The transcription of vitamin D (VD) responsive genes is regulated by three different nuclear signalling pathways mediated by homodimers of VD receptors (VDRs), heterodimers of VDRs and retinoid X receptors (RXRs) and heterodimers of VDRs with retinoic acid receptors (RARs), Here, the in vitro DNA-binding affinity of all three receptor complexes was shown to be enhanced by the presence of VD. However, the specificity of the three pathways was dictated by the differential affinities of the receptor complexes for VD response elements. Potential response elements were distinguished by the sequence, the separation and the relative orientation of the hexameric core binding motifs. It was found that both VDR-RAR and VDR-RXR heterodimers act functionally on all three response element configurations: direct repeats, palindromes and inverted palindromes. With direct repeats, neither heterodimer type showed a preference for any of the three principal core motifs, (A/G)GGTGA, (A/G)GGTCA and (A/G)GTTCA. However, while they did exhibit preferences for core motifs in palindromes, the spacing requirements were identical for both complexes. Inverted palindromes, however, formed the most specific response elements. A simple model explains a steric link between the optimal spacing of direct repeats and that of inverted palindromes. Taken together, the experimental data and the model provide further criteria for the screening of VD responsive genes.

Animals↗
PubMed1994

Novel estrogen response elements identified by genetic selection in yeast are differentially responsive to estrogens and antiestrogens in mammalian cells.

A powerful and versatile system for the identification of novel response elements for members of the intracellular receptor family is presented as applied to the human estrogen receptor. In the past, a limited number of estrogen response elements (EREs) have been functionally identified in the promoter regions of estrogen-regulated genes. From these a consensus ERE has been defined that is identical to the ERE of the Xenopus laevis vitellogenin gene, i.e., 5'-GGTCA NNN TGACC-3'. In order to investigate without bias the range of sequences that could function as EREs in vivo, we have developed a genetic selection in yeast expressing the human estrogen receptor (hER) and transformed with a random oligonucleotide library in a vector where expression of a selectable marker requires insertion of an upstream activating sequence. More than 1,000,000 transformants were screened and of 726 clones that contained activating sequences, 65 were found to be hormone-dependent. Sequencing revealed that the majority contained at least one 4/5 match to a canonical ERE half-site, but only one contained a full consensus ERE as previously defined. Some contained half-sites arranged as direct repeats. Twelve elements were further characterized to compare estrogen activation in yeast and mammalian cells and in vitro binding to hER. The results of these studies reveal that sequences that bind weakly to hER in vitro are fully functional as EREs in yeast and are conditionally responsive to estrogen in mammalian cells. In addition, an element was identified that is more sensitive to the partial agonist activities of tamoxifen and nafoxidine than is the consensus ERE, indicating that not only promoter context but the sequence of the binding site itself can allow distinction between receptor activated by agonist and that activated by antagonist.

Base Sequence↗
PubMed1998

Functional analysis of the stress response element and its role in the multistress response of Saccharomyces cerevisiae.

The DDR2 gene of Saccharomyces cerevisiae is a multistress response gene whose transcription is rapidly and strongly induced by a diverse array of xenobiotic agents, and environmental and physiological conditions. The multistress response of this gene requires the pentanucleotide, 5' CCCCT, (C4T;STRE (STress Response Element)) and the zinc-finger transcription factors, Msn2p and Msn4p. A 51bp oligonucleotide (oligo 31/32) containing two STREs from the DDR2 promoter region was previously shown to direct heat shock activation of a lacZ reporter gene. In this work we demonstrate that the same element conferred a complete multistress response to an E. coli galK reporter gene introduced into yeast cells. A variant oligonucleotide in which both the STRE spacing and neighboring sequences were altered responded to the same spectrum of stresses, while substitution of nucleotides within the pentanucleotide completely abolished the multistress response. These results directly demonstrate that STREs are not only necessary but are sufficient for mediating a transcriptional response to a surprisingly diverse set of environmental and physiological conditions.

Base Sequence↗
PubMed1994

Variations in vitamin D receptor transcription factor complexes associated with the osteocalcin gene vitamin D responsive element in osteoblasts and osteosarcoma cells.

Vitamin D responsive transcription of the bone-specific osteocalcin gene differs markedly in osteosarcoma cells and normal diploid osteoblasts. In osteoblasts the osteocalcin gene is transcribed, and upregulated by Vitamin D, only in post-proliferative cells, but in osteosarcoma cells expression is constitutive. This distinction in transcriptional regulation of the osteocalcin gene correlates with striking differences in the relative representation of two principal Vitamin D-dependent protein/DNA complexes designated V1 and V2 at the Vitamin D responsive element in the osteocalcin promoter. Formation of both complexes is Vitamin D dependent and they contain the Vitamin D receptor as well as an RXR related protein. Pore size exclusion and sedimentation velocity analyses suggest that the V1 and V2 complexes represent oligomeric protein assemblies (respectively, tetramers and trimers), and reflect primarily DNA-directed association of the monomeric protein components at the osteocalcin Vitamin D responsive element. UV crosslinking and methylation interference analyses of the V1 and V2 complexes at the osteocalcin Vitamin D responsive element indicate differences in protein/DNA recognition. For example, the V1 complex interacts with both steroid half-elements, whereas the V2 complex appears to recognize the proximal half-element. Our findings suggest variations in protein/protein and protein/DNA interactions of the VDR and RXR related complexes V1 and V2 at the osteocalcin Vitamin D responsive element that reflect unique properties of the osteosarcoma and normal diploid osteoblast phenotype.

Animals↗
PubMed1992

Maximal serum stimulation of the c-fos serum response element requires both the serum response factor and a novel binding factor, SRE-binding protein.

We have previously reported on the presence of a CArG motif at -100 in the Rous sarcoma virus long terminal repeat which binds an avian nuclear protein termed enhancer factor III (EFIII) (A. Boulden and L. Sealy, Virology 174:204-216, 1990). By all analyses, EFIII protein appears to be the avian homolog of the serum response factor (SRF). In this study, we identify a second CArG motif (EFIIIB) in the Rous sarcoma virus long terminal repeat enhancer at -162 and show only slightly lower binding affinity of the EFIII/SRF protein for this element in comparison with c-fos serum response element (SRE) and EFIII DNAs. Although all three elements bind the SRF with similar affinities, serum induction mediated by the c-fos SRE greatly exceeds that effected by the EFIII or EFIIIB sequence. We postulated that this difference in serum inducibility might result from binding of factors other than the SRF which occurs on the c-fos SRE but not on EFIII and EFIIIB sequences. Upon closer inspection of nuclear proteins which bind the c-fos SRE in chicken embryo fibroblast and NIH 3T3 nuclear extracts, we discovered another binding factor, SRE-binding protein (SRE BP), which fails to recognize EFIII DNA with high affinity. Competition analyses, methylation interference, and site-directed mutagenesis have determined that the SRE BP binding element overlaps and lies immediately 3' to the CArG box of the c-fos SRE. Mutation of the c-fos SRE so that it no longer binds SRE BP reduces serum inducibility to 33% of the wild-type level. Conversely, mutation of the EFIII sequence so that it binds SRE BP with high affinity results in a 400% increase in serum induction, with maximal stimulation equaling that of the c-fos SRE. We conclude that binding of both SRE BP and SRF is required for maximal serum induction. The SRE BP binding site coincides with the recently reported binding site for rNF-IL6 on the c-fos SRE. Nonetheless, we show that SRE BP is distinct from rNF-IL6, and identification of this novel factor is being pursued.

3T3 Cells↗
PubMed1994

Nuclear orphan receptor-binding retinoic acid response elements in keratinocytes.

Keratinocytes are responsive cells for retinoic acid (RA) mediated signal transduction. In this study, we demonstrate binding of some important orphan receptors to previously identified retinoic acid response elements (RAREs) regulated by RA in keratinocytes. Using electrophoretic mobility shift assays (EMSAs) we show that in vitro translated ARP1 (apolipoprotein AI regulatory protein 1), EAR3 and EAR2 (v-erb A related genes) bind two RAREs. The RAREs investigated were previously identified in the VL30 retrotransposon (termed VLRRE2) and retinoic acid receptor beta 2 (termed RARE beta) genes, respectively. Furthermore, using an anti-ARP antibody that recognizes both ARP1 and EAR3 we show that these ARP subfamily member(s) present in keratinocytes bind to both RAREs. To our knowledge, this is the first demonstration of the binding of these proteins in keratinocytes to response elements regulated by RA in these cells. Our data suggest that ARP subfamily member(s) may modulate RA mediated transcription in epidermis.

Animals↗
PubMed2001

Cyclic AMP and mitogen-activated protein kinases are required for glutamate-dependent cyclic AMP response element binding protein and Elk-1 phosphorylation in the dorsal striatum in vivo.

Dopaminergic and glutamatergic signalling cascades are integrated in striatal medium spiny neurones by cyclic AMP response-element binding protein and Elk-1 phosphorylation. Phosphorylated cyclic AMP response-element binding protein and phosphorylated Elk-1 contribute to c-fos expression by binding to the calcium and cyclic AMP response-element and the serum response element, respectively, in the c-fos promoter. The role of cyclic AMP and mitogen-activated protein kinase signalling cascades in glutamate-induced cyclic AMP response-element binding protein and Elk-1 phosphorylation and Fos expression was investigated using semiquantitative immunocytochemistry in vivo. Intracerebroventricular infusion of the sodium channel blocker, tetrodotoxin, decreased the glutamate-induced increase in phosphorylated cyclic AMP response-element binding protein, phosphorylated Elk-1, and Fos immunoreactivity. Intracerebroventricular infusion of the mitogen-activated and extracellular signal-regulated kinase inhibitor, PD98059, the p38 mitogen-activated protein kinase inhibitor, SB203580, or the cyclic AMP inhibitor, Rp-8-Br-cAMPS, decreased glutamate-induced phosphorylated cyclic AMP response-element binding protein, phosphorylated Elk-1, and Fos immunoreactivity. Simultaneous infusion of glutamate and Sp-8-Br-cAMPS, a cyclic AMP analogue, augmented induction of Fos immunoreactivity but not phosphorylated cyclic AMP response-element binding protein or phosphorylated Elk-1 immunoreactivity. These data indicate that cyclic AMP and mitogen-activated protein kinase signalling cascades are necessary for glutamate to induce cyclic AMP response-element binding protein and Elk-1 phosphorylation and Fos expression in the striatum. Furthermore, neuronal activity plays an important role in glutamate-induced signalling cascades in vivo.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1996

Intracellular receptors use a common mechanism to interpret signaling information at response elements.

The glucocorticoid receptor (GR) activates transcription in certain glucocorticoid response element (GRE) contexts, and represses or displays no activity in others. We isolated point mutations in one GRE, plfG, at which GR activated transcription under conditions in which the wild-type element was inactive or conferred repression, implying that GREs may carry signals that are interpreted by bound receptors. Consistent with this notion, we identified a mutant rat GR, K461A, which activated transcription in all GRE contexts tested, implying that this residue is important in interpretation of GRE signals. In a yeast screen of 60,000 GR mutants for strong activation from plfG, all 13 mutants isolated contained substitutions at K461. This lysine residue is highly conserved in the zinc-binding region (ZBR) of the intracellular receptor (IR) superfamily; when it was mutated in MR and RARbeta, the resulting receptors similarly activated transcription at response elements that their wild-type counterparts repressed or were inactive. We suggest that IR response elements serve in part as signaling components, and that a critical lysine residue serves as an allosteric "lock" that restricts IRs to inactive or repressing configurations except in response element contexts that signal their conversion to transcriptional activators. Therefore, mutation of this residue produces altered receptors that activate in many or all response element contexts.

Animals↗
PubMed2002

Structural basis of VDR-DNA interactions on direct repeat response elements.

The vitamin D receptor (VDR) forms homo- or heterodimers on response elements composed of two hexameric half-sites separated by 3 bp of spacer DNA. We describe here the crystal structures at 2.7-2.8 A resolution of the VDR DNA-binding region (DBD) in complex with response elements from three different promoters: osteopontin (SPP), canonical DR3 and osteocalcin (OC). These structures reveal the chemical basis for the increased affinity of VDR for the SPP response element, and for the poor stability of the VDR-OC complex, relative to the canonical DR3 response element. The homodimeric protein-protein interface is stabilized by van der Waals interactions and is predominantly non-polar. An extensive alpha-helix at the C-terminal end of the VDR DBD resembles that found in the thyroid hormone receptor (TR), and suggests a mechanism by which VDR and TR discriminate among response elements. Selective structure-based mutations in the asymmetric homodimeric interface result in a VDR DBD protein that is defective in homodimerization but now forms heterodimers with the 9-cis retinoic acid receptor (RXR) DBD.

Crystallography, X-Ray↗
PubMed1991

Developmental regulation of a serum response element binding activity in amphibian embryos.

As part of our studies of transcriptional control during early development in vertebrates, we have examined embryos of the amphibian Xenopus laevis for the presence of sequence-specific DNA-binding proteins, using gel electrophoresis mobility-shift assays. Our analysis has focused on sequence elements in the cytoskeletal actin gene, whose embryonic transcription is initially activated at the gastrula stage, approximately 16 hours after fertilization. We detect activities capable of specific binding to two known transcriptional regulatory elements, the serum response element and the GC-box, located in the 5'-flanking region of the cytoskeletal actin gene. Binding activity specific for a region downstream of the transcriptional startsite is also detected, in a region which may be involved in controlling developmental activation of this gene. Serum response element-binding activity, as well as the downstream binding activity, is enriched in extracts from gastrula and neurula stage embryos, compared to egg extracts, suggesting that increased levels of one or both of these activities might play a role in developmentally timed transcriptional activation of the cytoskeletal actin gene in the embryo.

Actins↗