Cognate Binding Sites Research Articles

Mechanisms of transactivation by retinoic acid receptors

Retinoids play an important role in development and differentiation. Their effect is mediated through nuclear receptors, RAR (alpha, beta and gamma) and RXR (alpha, beta and gamma), which are members of a distinct subclass (hereafter referred to as type II) of the nuclear receptor superfamily that includes the thyroid hormone receptor (T3R), the vitamin D3 receptor (VD3R) and the peroxisome proliferator activated receptor (PPAR). Type II receptors transactivate through binding sites composed of closely related half-sites (consensus sequence AGG/TTCA) arranged as direct repeats and, with the possible exception of RXR, do not bind to their cognate binding sites as homodimers but require RXR for high affinity binding. RXR thus provides a link between biologically distinct ligand induced pathways and is a potential target for cross-regulation. In addition, RAR can utilize alternative routes to enhance transcription initiation mediated through transcriptional co-activators which are expressed in a cell-type specific manner.

Antennapedia homeobox peptide enhances growth and branching of embryonic chicken motoneurons in vitro.

Spinal motoneuron development is regulated by a variety of intrinsic and extrinsic factors. Among these, a possible role for homeoproteins is suggested by their expression in the motoneuron at relatively late stages. To investigate their possible involvement in motoneuron growth, we adapted a novel technique recently developed in this laboratory, based on the ability of the 60 amino acid-long homeobox of Antennapedia (pAntp) to translocate through the neuronal membrane and to accumulate in the nucleus (Joliot, A. H., C. Pernelle, H. Deagostini-Bazin, and A. Prochiantz. 1991. Proc. Natl. Acad. Sci. USA. 88:1864-1868; Joliot, A. H., A. Triller, M. Volovitch, C. Pernelle, and A. Prochiantz. 1991. New Biol. 3:1121-1134). Motoneurons from E5 chicken spinal cord were incubated with pAntp, purified by panning on SC1 antibody and plated on polyornithine/laminin substrata without further addition of pAntp. After 24 h, neurite outgrowth was already extensive in controls. In cultures of motoneurons that had been preincubated with 10(-7) M pAntp, neurite length was doubled; a similar effect was obtained using postnatal muscle extracts. Morphological analysis using a neurofilament marker specific for axons indicated that the homeobox peptide enhances primarily axonal elongation and branching. To test the hypothesis that the biological activity of pAntp involves its specific attachment to cognate homeobox binding sites present in the genome, we generated a mutant of pAntp called pAntp40P2, that was still able to translocate through the motoneuron membrane and to reach the nucleus, but had lost the specific DNA-binding properties of the wild-type peptide. Preincubation of pAntp40P2 with purified motoneurons failed to increase neurite outgrowth. This finding raises the possibility that motoneuron growth is controlled by homeobox proteins.

Determinants for DNA-binding site recognition by the glucocorticoid receptor.

The glucocorticoid receptor binds with high specificity to glucocorticoid response elements, discriminating them from other closely related binding sites. Three amino acids in the recognition alpha-helix of the DNA-binding domain of the receptor are primarily responsible for this specific DNA binding activity. In this study we analyze in detail how these residues determine the specific DNA binding by studying a series of mutant glucocorticoid receptor DNA-binding domains containing all combinations of glucocorticoid and estrogen receptor-specific residues at these positions. Statistical analysis of the results enables us to create models describing the association between amino acids and base pairs. Several strategies appear to be used in accomplishing discrimination between the glucocorticoid and estrogen response elements. Single residues (i.e., Val-443 in the glucocorticoid receptor and Glu-439 in the estrogen receptor) appear to form both positive contacts with specific base pairs in the cognate binding site and negative contacts in the non-cognate site. In the glucocorticoid receptor Ser-440 is pleiotropically negative for all sites tested but the negative effect is stronger for the estrogen response element thus contributing to binding site discrimination. Furthermore, combinations of amino acids appear to act synergistically, most often causing a reduction in binding to non-cognate sites.

DNA intervention in transcriptional activation

Accurate initiation of eukaryotic mRNA synthesis takes place as a result of the interplay between general transcription factors and RNA polymerase II. Activation of transcription from the basal level involves a number of promoter-specific trans-acting factors which interact with cis elements in the promoter DNA. In this paper we have emphasized the importance of even those portions of the promoter stretch which do not have any identifiable binding sites for regulatory proteins. The length and structure of the DNA between cognate binding sites of trans-acting factors may interfere with the level of transcriptional activation. Depending upon the length of the intervening DNA we describe three cases of transcriptional activation. In addition, based on this classification we propose a new third domain, the other two being DNA binding and transcriptional activation domains, which is involved in bending the intervening DNA so that activation from a distance can take place successfully.

Mutations in the zinc fingers of ADR1 that change the specificity of DNA binding and transactivation.

ADR1 is a yeast transcription factor that contains two zinc fingers of the Cys-2-His-2 (C2H2) class. Mutations that change the specificity of DNA binding of ADR1 to its target site, upstream activation sequence 1 (UAS1), have been identified at three positions in the first zinc finger. Mutations Arg-115 to Gln, His-118 to Thr, and Arg-121 to Asn led to new specificities of DNA binding at adjacent positions 10, 9, and 8 (3'-GAG-5') in UAS1. Arg-115 is at the finger tip, and His-118 and Arg-121 are at positions 3 and 6, respectively, in the alpha helix of finger 1. One double mutant displayed the binding specificity expected from the properties of its constituent new-specificity mutations. Mutations in the second finger that allowed its binding site to be identified through loss-of-contact phenotypes were made. These mutations imply a tail-to-tail orientation of the two ADR1 monomers on their adjacent binding sites. Finger 1 is aligned on UAS1 in an amino-to-carboxyl-terminal orientation along the guanine-rich strand in a 3'-to-5' direction. One of the ADR1 mutants was functional in vivo with both its cognate binding site and wild-type UAS1, but the other two mutants were defective in transactivation despite their ability to bind with high affinity to their cognate binding sites.

Mutations in the zinc fingers of ADR1 that change the specificity of DNA binding and transactivation.

ADR1 is a yeast transcription factor that contains two zinc fingers of the Cys-2-His-2 (C2H2) class. Mutations that change the specificity of DNA binding of ADR1 to its target site, upstream activation sequence 1 (UAS1), have been identified at three positions in the first zinc finger. Mutations Arg-115 to Gln, His-118 to Thr, and Arg-121 to Asn led to new specificities of DNA binding at adjacent positions 10, 9, and 8 (3'-GAG-5') in UAS1. Arg-115 is at the finger tip, and His-118 and Arg-121 are at positions 3 and 6, respectively, in the alpha helix of finger 1. One double mutant displayed the binding specificity expected from the properties of its constituent new-specificity mutations. Mutations in the second finger that allowed its binding site to be identified through loss-of-contact phenotypes were made. These mutations imply a tail-to-tail orientation of the two ADR1 monomers on their adjacent binding sites. Finger 1 is aligned on UAS1 in an amino-to-carboxyl-terminal orientation along the guanine-rich strand in a 3'-to-5' direction. One of the ADR1 mutants was functional in vivo with both its cognate binding site and wild-type UAS1, but the other two mutants were defective in transactivation despite their ability to bind with high affinity to their cognate binding sites.

Interference between pathway-specific transcription factors: glucocorticoids antagonize phorbol ester-induced AP-1 activity without altering AP-1 site occupation in vivo.

Phorbol esters stimulate and glucocorticoid hormones down-regulate a variety of promoters such as that of the collagenase gene through the transcription factor AP-1 (Fos/Jun). We now show by genomic footprinting of the collagenase promoter that phorbol ester treatment of cells results in the binding of AP-1 to its cognate DNA binding site in vivo. The DNA-protein contacts obtained in living cells are also found in vitro using cloned DNA and purified AP-1. Although in vitro synthesized glucocorticoid receptor can disturb the DNA binding of Jun homodimers, it does not interfere with the binding of Fos-Jun heterodimers or of purified AP-1 in vitro. Consistently, fully inhibitory doses of glucocorticoid hormone cause no change in apparent occupation of the AP-1 binding site in vivo. The hormone receptor acts without itself binding to DNA.

Differential inhibition of the DNA translocation and DNA unwinding activities of DNA helicases by the Escherichia coli Tus protein.

Binding of the Escherichia coli Tus protein to its cognate nonpalindromic binding site on duplex DNA (a Ter sequence) is sufficient to arrest the progression of replication forks in a Ter orientation-dependent manner in vivo and in vitro. In order to probe the molecular mechanism of this inhibition, we have used a strand displacement assay to investigate the effect of Tus on the DNA helicase activities of DnaB, PriA, UvrD (helicase II), and the phi X-type primosome. When the substrate was a short oligomer hybridized to a circular single-stranded DNA, strand displacement by DnaB, PriA, and the primosome (in both directions), but not UvrD, was blocked by Tus in a polar fashion. However, no inhibition of either DnaB or UvrD was observed when the substrate carried an elongated duplex region. With this elongated substrate, PriA helicase activity was only inhibited partially (by 50%). On the other hand, both the 5'----3' and 3'----5' helicase activities of the primosome were inhibited almost completely by Tus with the elongated substrate. These results suggest that while Tus can inhibit the translocation of some proteins along single-stranded DNA in a polar fashion, this generalized effect is insufficient for the inhibition of bona fide DNA helicase activity.

Expression, regulation, and chromosomal localization of the Max gene.

The Max gene encodes a protein that interacts specifically with the Myc protein to form a heterodimer with high affinity for the specific cognate DNA binding site of Myc. Here we examine the expression of Max RNA in comparison to Myc RNA during cell growth and differentiation. Two species of RNA, a major 2.0- and a minor 1.7-kilobase species, hybridized specifically to a Max cDNA probe in all human and murine cell lines that were tested. Unlike Myc, the steady-state level of Max RNA is not significantly modulated with respect to proliferation or differentiation. Max RNA is expressed in quiescent BALB/c 3T3 cells and is modestly increased 3 h after addition of serum to the quiescent cells. In contrast to Myc RNA, Max RNA does not decline immediately upon induction of differentiation of HL60 cells by dimethyl sulfoxide, and only a modest decrease of Max RNA was observed 72 h after induction of differentiation. Unlike Myc RNA, Max RNA is relatively stable with a half-life of greater than 3 h and, therefore, does not exhibit the characteristic short half-life of RNAs encoded by most immediate early genes. The human Max gene was localized to chromosome 14, band q23. With respect to the recurring abnormalities in human tumors, this region of chromosome 14 is involved in deletions in B-cell chronic lymphocytic leukemia and malignant lymphomas and in the 12;14 translocation in uterine leiomyomas.

Histones H2A/H2B inhibit the interaction of transcription factor IIIA with the Xenopus borealis somatic 5S RNA gene in a nucleosome.

A Xenopus borealis somatic 5S RNA gene was assembled with either the complete octamer of histones, (H2A/H2B/H3/H4)2, or the (H3/H4)2 tetramer of histones that comprises the central protein kernel of the nucleosome. Gel-mobility shifts, DNase I protection, and immunoblotting assays demonstrate that the class III transcription factor IIIA (TFIIIA) readily interacts with 5S DNA associated with the tetramer but that little or no binding is detected when 5S DNA is associated with the full octamer of histones. Thus, the presence of histones H2A and H2B in the 5S nucleosome significantly inhibits the interaction of TFIIIA with its cognate binding site within the 5S RNA gene. We propose that either the depletion of histones H2A and H2B from preexisting nucleosomes or the staged assembly of chromatin after replication in which a tetramer of histones H3/H4 associates with DNA before histones H2A/H2B will facilitate the binding of transcription factors to their cognate DNA sequences.

Utilization of heterologous alphavirus junction sequences as promoters by Sindbis virus.

We used Sindbis virus, an alphavirus, as a model to study the evolution of the recognition of viral cis-acting sequences. During the life cycle of alphaviruses, a full-length minus-strand RNA is made and serves as a template for both genomic RNA replication and subgenomic mRNA transcription. Transcription initiates at an internal promoter site, the junction sequence, to produce a subgenomic mRNA. The junction sequences of alphaviruses are highly conserved, but they do contain a number of base differences. These could have been essentially neutral mutations during evolution, such that any of the contemporary sequences can be recognized efficiently by any of the alphaviruses. Alternately, the changes could have resulted in significant functional divergence, such that the contemporary viruses can no longer recognize heterologous junction sequences as promoters. To distinguish between these possibilities, we constructed Sindbis virus derivatives with two subgenomic mRNA promoters. One is the wild-type Sindbis virus promoter used for expression of the structural proteins. The other is either the minimal Sindbis virus promoter or the corresponding junction sequences from other alphaviruses, which are placed upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene. RNA analyses were used to determine the relative promoter strengths of the various junction sequences. The results showed that all but two were recognized as promoters by Sindbis virus. CAT enzyme assays were used to measure the accumulation of CAT protein made from mRNAs transcribed by using the heterologous junction sequences as promoters. Most of the viruses expressed amounts of CAT enzyme within 10-fold of each other. The two viruses with junction sequences that were not recognized as promoters did not give significant CAT expression. We conclude that, with respect to Sindbis virus, the junction sequences are functionally conserved; i.e., most of the contemporary nucleotide differences in the junction sequences are neutral or near-neutral mutations. The functional conservation suggests that neither the cis-acting sequence nor the cognate binding site of the transcription factor can change independently. This type of coupled evolution between cis-acting sequences and their cognate viral protein binding sites may be a general phenomenon. For example, it explains the ubiquitous presence of conserved cis-acting sequences in each of the families of RNA viruses. There are implications of this hypothesis for the design of antiviral drugs.

Regulation of Gene Expression by Steroid Hormones

The general model of activation of gene expression by steroid hormones is the conversion of the steroid hormone receptor from a nonDNA-binding complex into a DNA-binding protein. This chapter discusses the different elements that mediate the steroid hormone response. It also discusses different sequences that mediate a positive action of steroid hormones. Sequences that mediate a negative action of steroid hormones have also been classified into various mechanistic groups. Most of these cis-acting elements have been shown to bind the various steroid receptors by in vitro binding studies using “naked” DNA. However, in vivo, the receptor binding sites do not occur on naked DNA, but are nucleosomally packaged. There is, therefore, the major question of the way steroid hormone receptors recognize their cognate binding sites on chromatin to regulate transcription. Experiments with reconstituted chromatin on a short DNA from the mouse mammary tumor virus (MMTV) long terminal repeat (LTR) show that the glucocorticoid receptors and the progesterone receptors bind naked DNA and reconstituted nucleosomes with equivalent affinity. However, nuclear factor I (NFI), a transcription factor essential for MMTV promoter activity, binds tightly to naked DNA, but not at all to the in vitro nucleosomally organized promoter. This has, therefore, led to the notion that hormone induction at the MMTV promoter proceeds through the displacement of a nucleosome at the hormone-response elements by the steroid receptors, allowing transcription factors, such as NFI, to bind to the promoter.

A damage-responsive DNA binding protein regulates transcription of the yeast DNA repair gene PHR1.

The PHR1 gene of Saccharomyces cerevisiae encodes the DNA repair enzyme photolyase. Transcription of PHR1 increases in response to treatment of cells with 254-nm radiation and chemical agents that damage DNA. We report here the identification of a damage-responsive DNA binding protein, termed photolyase regulatory protein (PRP), and its cognate binding site, termed the PHR1 upstream repression sequence, that together regulate induction of PHR1 transcription after DNA damage. PRP activity, monitored by electrophoretic-mobility-shift assay, was detected in cells during normal growth but disappeared within 30 min after irradiation. Copper-phenanthroline footprinting of PRP-DNA complexes revealed that PRP protects a 39-base-pair region of PHR1 5' flanking sequence beginning 40 base pairs upstream from the coding sequence. A prominent feature of the foot-printed region is a 22-base-pair palindrome. Deletion of the PHR1 upstream repression sequence increased the basal level expression of PHR1 in vivo and decreased induction after exposure of cells to UV radiation or methyl methanesulfonate, whereas insertion of the PRP binding site between the CYC1 upstream activation sequence and "TATA" sequence reduced basal level expression and conferred damage responsiveness upon a reporter gene. Thus these observations establish that PRP is a damage-responsive repressor of PHR1 transcription.

Purification of PO-B, a protein that has increased affinity for the pro-opiomelanocortin gene promoter after dephosphorylation

The region -15 to -3 of the pro-opiomelanocortin (POMC) gene promoter specifically binds a transcription factor previously designated PO-B. This region of the POMC gene is involved in the control of constitutive POMC gene expression since mutation of the PO-B DNA-binding site severely reduces transcription from the POMC promoter both in vivo and in vitro (Riegel, A. T., Remenick, J., Wolford, R., Berard, D., and Hager, G. (1990) Nucleic Acids Res. 18, 4513-4521). We have now purified PO-B from HeLa cells approximately 25,000-fold to greater than 90% homogeneity by a combination of ion exchange and reversed phase chromatography. In addition we have studied post-translational modifications that alter the affinity of purified PO-B for its cognate DNA binding site. In Southwestern analysis of column fractions, two bands of apparent molecular masses of 54 and 56 kDa bound specifically to the PO-B recognition sequence. The two copurified components have indistinguishable amino acid composition, are highly hydrophobic, and are heat and acid stable. DNA-binding specificity studies suggest that PO-B does not represent any previously described transcription factor. In addition, dephosphorylation of both species with acid phosphatase induced an about 30-fold increase in DNA binding but failed to produce any significant change in electrophoretic mobility. We conclude that the purified PO-B species represent products of the same gene and suggest that the in vivo function of PO-B may be regulated by its phosphorylation status.

Determination of DNA binding specificities of mutated zinc finger domains

By substituting non conserved amino acids present in the postulated α-helical region of zinc finger domains, we demonstrated that Cys 2/His 2 type zinc finger domains could be targeted to new DNA binding sites. The putative α-helical region of the second SPI zinc finger ( R S DE L QR H) was replaced by amino acids ( K S SA L IS H) occurring in analogous zinc finger positions of human zinc finger protein Kox 29. The DNA binding specificity of the FPLC purified chimaeric protein (SPI-Kox 29) was determined by use of the target detection assay (TDA). Chimaeric protein SPI-Kox 29 wax subjected to randomized oligonucleotides (GGG NNNN GGC) that were designed on the basis that each SPI zinc finger interacts with 3–4 nucleotides concerning its cognate target site GGG GCGG GGC. By this analysis the DNA binding specificity of SPI-Kox 29 was shown to have switched from the cognate SPI binding site to GGG GGTG GGC. Structure-function analysis of this type should facilitate the determination of DNA binding specificities for any individual zinc finger of interest.

Co-operation of progestational steroids with epidermal growth factor in activation of gene expression in mammary tumor cells

The progesterone receptor belongs to a class of ligand binding transcription factors that regulate transcription by interacting with specific DNA sequences on hormone regulated genes. In human mammary tumor T47D cells that contain both progesterone and epidermal growth factor (EGF) receptors, the progestin-induced transactivation at various hormone regulated promoters is enhanced by EGF. The effect of EGF is rapid and does not require new protein synthesis. EGF treatment does not alter the DNA binding activity of the progesterone receptor nor does it affect the total ligand-dependent phosphorylation of this receptor. These results suggest that EGF enhances the transactivation property of the progesterone receptor through mechanisms other than those involving a direct interaction of this receptor with its cognate binding sites.

Activity and tissue-specific expression of the transcription factor NF-E1 multigene family.

NF-E1, a DNA-binding protein that recognizes the general consensus motif WGATAR, is the first tissue-specific factor to be identified in erythroid cells. Using a probe from the murine GF-1 (NF-E1) cDNA clone, we isolated three homologous chicken cDNAs: One of these corresponds to an mRNA (NF-E1a) that is abundantly and exclusively expressed in erythroid cells; a second mRNA (NF-E1b) is also expressed in all developmental stages of erythroid cells but is additionally found in a limited subset of other chicken tissues; mRNA representative of a third gene (NF-E1c) is expressed only in definitive (adult) erythrocytes within the red cell lineage but is also abundantly expressed in T lymphocytes and brain. All NF-E1 proteins are highly conserved within the DNA-binding domain and bind to the consensus motif with similar affinities in vitro; they are also all stimulatory trans-acting factors in vivo. The factors differ quantitatively in their ability to trans-activate reporter genes in which the number and position of cognate binding sites is varied relative to the transcriptional initiation site. These data suggest that the NF-E1 consensus motif directs a broader and more complicated array of developmental transcriptional regulatory processes than has been assumed and that NF-E1c may play a unique regulatory role in the developing chicken brain and in T lymphocytes.

TGA1a, a tobacco DNA-binding protein, increases the rate of preinitiation complex formation in a plant in vitro transcription system [corrected

We describe here a plant in vitro transcription system for class II promoters using wheat germ extract. In this system transcription is stimulated by TGA1a, a tobacco DNA-binding protein, and the stimulation is dependent on the presence of its cognate binding site upstream of the TATA box. Titration experiments showed that transcription initiation is more sensitive than transcription elongation to low concentrations of sarkosyl (N-lauroylsarcosine). At 0.07% sarkosyl, the formation of initiated complex is inhibited but transcription elongation is not. Under these conditions, events associated with a single round of transcription can be studied. We demonstrate that the time required for completing transcription of a 380-base-pair template is about 10 min. Addition of TGA1a increases the number of preinitiation complexes by approximately 3-fold with no significant effect on the frequency of transcription initiation from a single complex or on the rate of RNA elongation. We anticipate that this in vitro system will be valuable for the elucidation of mechanisms that regulate transcription in plants.

Different regions of the estrogen receptor are required for synergistic action with the glucocorticoid and progesterone receptors.

Estrogen and progesterone or estrogen and glucocorticoid receptors functionally cooperate in gene activation if their cognate binding sites are close to one another. These interactions have been described as synergism of action of the steroid receptors. The mechanism by which synergism is achieved is not clear, although protein-protein interaction of the receptors is one of the favorite models. In transfection experiments with receptor expression vectors and a reporter gene containing estrogen and progesterone-glucocorticoid receptor binding sites, we have examined the effects that different portions of the various receptors have on synergism. N-terminal domains of the chicken progesterone and human glucocorticoid receptors, when deleted, abolished the synergistic action of these receptors with the estrogen receptor. Deletion of the carboxy-terminal amino acids 341 to 595 of the estrogen receptor produced a mutant receptor that could not trans-activate on its own. This mutant receptor did not affect the action of the glucocorticoid receptor but functioned synergistically with the progesterone receptor. We therefore conclude that the synergistic action of the receptors for estrogen and progesterone is mechanistically different from the synergistic action of the receptors for estrogen and glucocorticoid.

Different regions of the estrogen receptor are required for synergistic action with the glucocorticoid and progesterone receptors.

Estrogen and progesterone or estrogen and glucocorticoid receptors functionally cooperate in gene activation if their cognate binding sites are close to one another. These interactions have been described as synergism of action of the steroid receptors. The mechanism by which synergism is achieved is not clear, although protein-protein interaction of the receptors is one of the favorite models. In transfection experiments with receptor expression vectors and a reporter gene containing estrogen and progesterone-glucocorticoid receptor binding sites, we have examined the effects that different portions of the various receptors have on synergism. N-terminal domains of the chicken progesterone and human glucocorticoid receptors, when deleted, abolished the synergistic action of these receptors with the estrogen receptor. Deletion of the carboxy-terminal amino acids 341 to 595 of the estrogen receptor produced a mutant receptor that could not trans-activate on its own. This mutant receptor did not affect the action of the glucocorticoid receptor but functioned synergistically with the progesterone receptor. We therefore conclude that the synergistic action of the receptors for estrogen and progesterone is mechanistically different from the synergistic action of the receptors for estrogen and glucocorticoid.