Cis-acting DNA Sequences Research Articles

The testis and the adrenal are (transcriptionally) the same

We have been studying the transcriptional regulation of the rat P450c17 gene in both adrenocortical and Leydig cells, to assess which DNA sequences are required for its basal and hormonally stimulated transcription. Comparing the transcriptional regulation in both of these cell types enables us to demonstrate whether specific nuclear factors required for transcriptional regulation of the rat P450c17 gene are tissue-specifically expressed, and whether the same cis-acting DNA elements in the gene are required for transcriptional regulation in both of these two different steroidogenic tissues. Using such an approach, we previously demonstrated that the transcriptional regulation of the rat P450scc gene uses different cis-acting DNA sequences in steroidogenic versus neural tissues, and requires the expression of tissue-specific nuclear factors that are unique to neural tissue. However, in studying the transcriptional regulation of the rat P450c17 gene in cultured mouse adrenocortical Y-1 and mouse Leydig MA-10 cells, we have shown that identical DNA sequences necessary for basal and cAMP-stimulated transcriptional regulation in these two cell types, and that identical nuclear factors from Y-1 and from MA-10 cells bind to these sequences. We have identified four transcriptionally active regions within 500 bp of the transcription initiation start site that are important for basal and/or cAMP-stimulated transcriptional regulation of this gene in Y-1 and MA-10 cells. This paper will discuss two of these regions in greater detail. By studying the regulation of the rat P450c17 gene, we have identified two new members of the orphan nuclear receptor gene family and have discovered new alternative mechanisms by which orphan nuclear receptors activate gene transcription in both mouse adrenocortical Y-1 and Leydig MA-10 cells.

Multiple nuclear regulatory proteins bind a single cis-acting promoter element to control basal transcription of the human alpha 4 integrin gene in corneal epithelial cells.

Expression of the fibronectin-binding integrin alpha 4 beta 1 has been postulated to be an important event in the process of corneal epithelial wound healing. In a previous study, we identified upstream positive and negative cis-acting regulatory elements that are needed to modulate the transcriptional activity of the human alpha 4 integrin subunit gene promoter in primary cultures of rabbit corneal epithelial cells. We have shown that most of the basal activity directed by this promoter was dependent on the presence of a cis-acting DNA sequence designated the alpha 4.1 element, centered at position -45 relative to the human alpha 4 mRNA start site. Here, we demonstrate that five distinct nuclear regulatory proteins (designated Bp1 to Bp5) from rabbit corneal epithelial cells possess the ability to bind the alpha 4.1 element in a specific manner in vitro. However, when they are combined together, only two of them (Bp2 and Bp5) retained their ability to interact with their specific target sequence in in vitro assays. The apparent molecular masses of the Bp1 to Bp5 proteins were determined and found to be of 91, 74, 59, 45, and 39 kD, respectively. Electrophoretic mobility-shift assays (EMSAs) indicated that only Bp2 also possesses the ability to bind the alpha 4.2 element, a site homologous to alpha 4.1 which plays a minor role in alpha 4 gene expression. Despite the presence of three Ets binding sites in the immediate vicinity of alpha 4.1, competition experiments in EMSA clearly indicate that Bp1, Bp2, Bp4, and Bp5 do not belong to the Ets family of transcription factors. Insertion of both alpha 4.1 and alpha 4.2 upstream from the basal promoter of the mouse p12 gene provided evidence that both elements have the ability to modulate basal expression driven from a heterologous promoter. alpha 4.1 was shown to function as an activator, whereas alpha 4.2 acted as a repressor in a manner that is dependent on its orientation, further stressing the critical regulatory function played by these two elements on alpha 4 gene basal expression.

A 413 bp region upstream the human 5-HT1A receptor gene is sufficient for its in vitro expression

The regulation of a specific pattern of neuronal 5-hydroxytryptamine-1A (5-HT1A) receptor gene expression in the brain is hardly understood. Identification of cis-acting DNA sequences that control expression of the human 5-HT1A receptor gene has been undertaken by fusion of a 413 base pairs (bp)-long DNA segment upstream the human 5-HT1A receptor gene start codon to the 5-HT1A receptor gene. When this fusion product was inserted into the expression vector pcDNA3 and transfected into Cos-7 cells, it was shown to drive expression of specific [3H] (+)-8-hydroxy-2-(di-n-propylamino)tetralin ([3H] 8-OH-DPAT) binding sites with a Kd of 1.18 nM and a Bmax of 105 fmol/mg protein. The yield of expression of [3H] 8-OH-DPAT binding sites by fusion of the 5-HT1A receptor gene to strong promoters, such as the Rous sarcoma virus long terminal repeat and the cytomegalovirus promoter, was 2.5 to 8 times greater. The pharmacological binding profile of these [3H] 8-OH-DPAT binding sites was similar to that of cloned human 5-HT1A receptors stably expressed in HeLa cells. The importance of an Initiator sequence and regulatory Spl sites in the 413 bp region to direct transcription of the human 5-HT1A receptor gene is discussed.

A 413 bp region upstream the human 5-HT1A receptor gene is sufficient for its in vitro expression

The regulation of a specific pattern of neuronal 5-hydroxytryptamine-1A (5-HT1A) receptor gene expression in the brain is hardly understood. Identification of cis-acting DNA sequences that control expression of the human 5-HT1A receptor gene has been undertaken by fusion of a 413 base pairs (bp)-long DNA segment upstream the human 5-HT1A receptor gene start codon to the 5-HT1A receptor gene. When this fusion product was inserted into the expression vector pcDNA3 and transfected into Cos-7 cells, it was shown to drive expression of specific [3H] (+)-8-hydroxy-2-(di-n-propylamino)tetralin ([3H] 8-OH-DPAT) binding sites with a Kd of 1.18 nM and a Bmax of 105 fmol/mg protein. The yield of expression of [3H] 8-OH-DPAT binding sites by fusion of the 5-HT1A receptor gene to strong promoters, such as the Rous sarcoma virus long terminal repeat and the cytomegalovirus promoter, was 2.5 to 8 times greater. The pharmacological binding profile of these [3H] 8-OH-DPAT binding sites was similar to that of cloned human 5-HT1A receptors stably expressed in HeLa cells. The importance of an Initiator sequence and regulatory Spl sites in the 413 bp region to direct transcription of the human 5-HT1A receptor gene is discussed.

Cis-acting elements that mediate the negative regulation of Moloney murine leukemia virus in mouse early embryos.

We have addressed the question of the nature of Moloney murine leukemia virus (MoMuLV) repression in mouse embryos by assaying for the transient expression of MoMuLV-derived constructs microinjected into early cleavage embryos. We show that the same cis-acting DNA sequences responsible for the block in MoMuLV expression in embryonal carcinoma cell lines operate in early embryos: (i) the MoMuLV long terminal repeat is nonfunctional, and (ii) the +147 to +163 repressor binding site, or negative regulatory element, negatively regulates the expression from an active promoter.

Molecular mechanisms of immunosuppression and anti-inflammatory activities by glucocorticoids.

Olucocorticoids (OCs) are physiologic inhibitors of inflammation and have been used for decades as immunosuppressive agents (1, 2). Despite their wide therapeutic efficacy in the treatment of a variety of inflammatory diseases, such as asthma, rheumatoid arthritis, systemic lupus erythematosus, and other autoimmune disease (3,4), the molecular mechanisms that underlie the therapeutic affects of OCs were for many years poorly understood. On entering the cell, OCs bind to a cytoplasmic glucocorticoid receptor (OCR), a member of the nuclear receptor superfamily. The hormone-receptor complex then translocates to the nucleus, where it binds to cis-acting DNA sequences, known as hormone response elements (HREs), and modulates transcription of specific target genes (reviewed in reference 5). Binding of the liganded OCR to DNA usually results in activation of transcription through positive HREs (pHREs), but binding to less common so-called negative HREs (nHREs) can result in repression of transcription. In recent years, however, it was found that OCR can modulate transcription in a hormone-dependent manner, not through direct binding to HREs, but through interference with the activity of other transcription factors, such as CREB (6, 7), AP-l (reviewed in reference 5), and NF-KB (8-13), whose activity is regulated in response to extracellular signals received by cell surface receptors. These newly discovered activities of the OCR are extremely important because they provide cross-talk between OCs on one hand and polypeptide hormones and cytokines on the other hand. This form of cross-coupling is likely to be essential for maintenance of cellular homeostasis and provides fine tuning to a variety of physiologic processes, such as differentiation, cell proliferation, and inflammatory and immune responses. Some of the genes that are subject to this crosscoupling include metalloproteinases, such as stromelysin and collagenase, and many cytokines, such as interleukin-2 (lL-2). The repression of such genes by OCs is likely to underlie the antiarthritic and immunosuppressive effects of these hormones (8, 9). The expression of metalloproteinases is the cause for tissue destruction in rheumatoid arthritis and other autoimmune disorders (3), while the expression of cytokines and their receptors (some of which are also the target of cross-coupling, e.g., IL-2 receptors) are essential for initiation and maintenance of immune and inflammatory responses. Deregulated expression of such genes, many of which are activated by transcription factors AP-l (14) and NF-KB (15), occurs during acute inflammation or various immune disorders, and OC provides a very effective means to curtail their expression and thus prevent disease progression. The purpose of the review is to describe the molecular mechanisms that may account for the potent repressiveeffects of OCs on genes that are activated by transcription factors AP-I and NF-KB.

Developmental modulation of a β myosin heavy chain promoter-driven transgene

The molecular mechanisms underlying heart and skeletal muscle-specific gene expression during development and in response to physioloic stimuli are largely unknown. Using a novel immunohistochemical procedure to detect chloramphenicol acetyltransferase (CAT), we have investigated, in vivo at high resolution, the ability of cis-acting DNA sequences within the 5' flanking region of the mouse beta myosin heavy chain (MyHC) gene (beta-MyHC) to direct appropriate gene expression throughout development. A 5.6-kb fragment 5' to the beta-MyHC's transcriptional start site was linked to the reporter gene encoding CAT (cat) and used to generate transgenic mice. The anti-CAT in situ assay described in this report allowed us to define the ability of the promoter fragment to direct appropriate temporal, tissue- and muscle fiber type-specific gene expression throughout early development. In skeletal muscles, the transgene expression profile mimics the endogenous beta-myHC's at all developmental stages and is appropriately restricted to slow (type I) skeletal fibers in the adult. Surprisingly, transgene expression was detected in both the atria and ventricles during embryonic and fetal development, indicating that ventricular specification involves elements outside the 5.6-kb fragment. In contrast, in the adult, hypothyroid conditions led to transgene induction specifically in the ventricles, suggesting that distinct regulatory mechanisms control fetal versus adult beta-MyHC expression in the cardiac compartment.

Pip2p: a transcriptional regulator of peroxisome proliferation in the yeast Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, peroxisomes are the exclusive site for the degradation of fatty acids. Upon growth with the fatty acid oleic acid as sole carbon source, not only are the enzymes of beta-oxidation and catalase A induced, but also the peroxisomal compartment as a whole increases in volume and the number of organelles per cell rises. We previously identified a cis-acting DNA sequence [oleate response element (ORE)] involved in induction of genes encoding peroxisomal proteins. The aim of our investigation was to test whether a single mechanism acting via the ORE coordinates the events necessary for the proliferation of an entire organelle. Here we report the cloning and characterization of the oleate-specific transcriptional activator protein Pip2p (pip: peroxisome induction pathway). Pip2p contains a typical Zn(2)-Cys(6) cluster domain and binds to OREs. A pip2 deletion strain is impaired in growth on oleate as sole carbon source and the induction of beta-oxidation enzymes is abolished. Moreover, only a few, small peroxisomes per cell can be detected. These results indicate that fatty acids activate Pip2p, which in turn activates the transcription of genes encoding beta-oxidation components and acts as the crucial activator of peroxisomes.

Ligand-induced differentiation of glucocorticoid receptor (GR) trans-repression and transactivation.

Inhaled glucocorticosteroids (GCs) are the most effective therapy for the control of asthma and are now advocated as first line therapy for most patients. Steroids reduce airway hyper-responsiveness and suppress the airway inflammatory response in asthma, but their exact mechanisms and cellular targets in the lung are uncertain 111. Classically GCs bind to a cytosolic 94kDa receptor protein, that is held in an inactive, non-DNA binding state by a 9OkDa heat shock protein (hsp90). With lioand binding, the DNA binding site is revealed and enhancement of the transcriptional response occurs following the translocation of active receptor to the nucleus. The GR binds to a consensus cis-acting DNA sequence AGAACAnnnTGTTCT (GRE) in the 5’ upstream controlling region of the genes under positive control by GCs causing an increase in gene transcription [2, 31. The mechanisms involved in gene repression are less well understood. In some instances, this may be because GR binding to a negative GRE results in steric hindrance to the binding of transcription factors that may induce the same gene. GR may also form complexes with activating transcription factors in the nucleus to inhibit their effect and so modulate genes which contain no GREs. Pro-inflammatory transcription factors, such as AP-1 and NF-KB, can form complexes with activated GR. causing mutual repression of DNA binding or affects interaction with the basal transcription apparatus thereby reducing steroid responsiveness [4-6]. Furthermore, GCs can upregulate the synthesis of I-KB, the cytosolic inhibitor of NF-KB [7]. Conversely, GCs may inhibit the effects of those cytokines that produce their effects on gene transcription via activation of AP-1 or NF-KB. For example, in human lung and in peripheral blood monocytes, TNFa and phorbol esters increase AP-1 and NF-KB binding to DNA and this is inhibited by dexamethasone [6, 81. Cytokines play an important role in chronic inflammation and the pattern of cytokine expression largely determines the nature and persistence of the inflammatory response. GCs have potent inhibitory effects on cytokine mediated inflammation and inhibit the transcription of several cytokines that are relevant in chronic inflammation, including interleukin-1 (IL-l), TNFa, GM-CSF, 11-3, 11-4, IL-5, 11-6 and IL-8 [3]. Other members of the steroid superfamily also act in an analogous manner and, in the case of retinoids, the ligand is able to modulate the ability of the receptor to bind to DNA and to AP-1 [9]. We have, therefore, investigated the ability of two glucocorticoids, dexamethasone and fluticasone, to influence GR-induced inhibition of NF-KB and AP-1 DNA binding and on the release of GM-CSF, an NF-KB-inducible gene in A549 cells. GM-CSF release was measured following stimulation with lng/ml IL-lp. Furthermore, we examined the ability of GR to bind to its DNA binding motif. Lung epithelium-like A549 cells were cultured in Dulbecco’s Modified Eagles medium (DMEM) in the presence of 2% foetal calf serum (FCS) until 70-80% confluent. FCS was removed from the culture medium and cells cultured for a further 2 days. Equal numbers of A549 cells were incubated with a range of concentrations of dexamethasone (Dex) and fluticasone propionate (FP) for 1-24 hours in the presence or absence of lnglml of IL-lp. Control experiments using cells incubated in serum free media alone were run in parallel. At every time point cells were collected by microcentrifugation and nuclear protein extracted [6]. Double stranded oligonucleotides encoding the consensus target sequence for GRE, AP-1 and NF-KB were end-labelled using t2P]-ATP and T4 polynucleotide kinase. l pg of nuclear protein from each sample was incubated with 50,000 cpm of labelled oligonucleotide. Protein-DNA complexes were separated on 6% polyacrylamide gels. The retarded band was detected by autoradiogaphy and quantified by laser densitometry. All band density measurements were then expressed as a percentage of initial binding. Specificity was determined by addition of excess unlabelled double stranded oligonucleotides. GM-CSF concentrations in culture supernatants were measured using a specific GM-CSF ELISA calibrated with human recombinant GM-CSF (0-2000pg/ml). Sensitivity of the assay was 8pglml. FP and Dex both caused a time and dose dependent inhibition of IL1p-stimulated GM-CSF release. Inhibition was first detected at 4 hrs post-steroid treatment and was at maximal levels at all time points from 10-24 hrs. At lOhrs FP caused a dose-dependent inhibition of IL-1pstimulated GM-CSF release with complete inhibition at lO-”M (EC= 5.7il.8 x lo’”), whilst Dex inhibition of IL-1p-stimulated GM-CSF release was found to be maximal at 10aM (EC= 1.2iO.5 x 10.9. This showed a 21 1-fold shift in responsiveness in spite of there being only an Figure 1. The effects of dexamethasone (Dex) and Fluticasone Proprionate (FP) on GMCSF release into the Supernatant of A549 cells.

Identification of cis-acting DNA sequences involved in the transcription of the virulence regulatory gene spvR in Salmonella typhimurium.

The SpvR protein is a DNA-binding protein of the LysR family, required for the transcription of the spvABCD virulence operon of Salmonella typhimurium. An alternative sigma factor, sigma S (RpoS), in conjunction with SpvR, controls the transcription of the spvR gene. In this study, we used a combination of primer extension experiments and deletion/fusion analyses of the spvR gene to identify sequences involved in spvR transcription in S. typhimurium. When induced in the stationary phase of growth in rich medium or during carbon starvation, transcription of spvR in S. typhimurium is driven by a single promoter (spvRp1) and initiates 17 nucleotides upstream of the spvR start codon. The level of spvR transcription originating at spvRp1 was 20-fold higher in the wild-type strain than in the rpoS mutant. In both strains, however, transcription at spvRp1 requires the SpvR protein. 5' Deletions up to position -86, relative to the spvR start codon, did not inhibit inducibility by sigma S and/or SPVR. In contrast, 5' deletion up to -75 abolished the activation of spvRp1 by SpvR in both the wild-type strain and rpoS mutant. Within the 11-bp sequence lying between position -86 and position -75, a 10-bp consensus motif TNTNTGCANA, present in both the spvR and spvA promoter regions, was identified and may contain the DNA recognition site for SpvR. In addition, we detected initiation of transcription within the spvR coding region. This finding may have implications for comparative studies of regulation with spvR gene fusions.

Rox3 and Rts1 function in the global stress response pathway in baker's yeast.

Yeast respond to a variety of stresses through a global stress response that is mediated by a number of signal transduction pathways and the cis-acting STRE DNA sequence. The CYC7 gene, encoding iso-2-cytochrome c, has been demonstrated to respond to heat shock, glucose starvation, approach-to-stationary phase, and, as we demonstrate here, to osmotic stress. This response was delayed in a the hog1-delta 1 strain implicating the Hog1 mitogen-activated protein kinase cascade, a known component of the global stress response. Deletion analysis of the CYC7 regulatory region suggested that three STRE elements were each capable of inducing the stress response. Mutations in the ROX3 gene prevented CYC7 RNA accumulation during heat shock and osmotic stress. ROX3 RNA levels were shown to be induced by stress through a novel regulatory element. A selection for high-copy suppressors of a ROX3 temperature-sensitive allele resulted in the isolation of RTS1, encoding a protein with homology to the B' regulatory subunit of protein phosphatase 2A0. Deletion of RTS1 caused temperature and osmotic sensitivity and increased accumulation of CYC7 RNA under all conditions. Over-expression of this gene caused increased CYC7 RNA accumulation in rox3 mutants but not in wild-type cells.

Identification of cis-acting DNA sequences involved in the transcription of the virulence regulatory gene spvR in

Identification of cis-acting DNA sequences involved in the transcription of the virulence regulatory gene spvR in

Characterization of a maize G‐box binding factor that is induced by hypoxia

G-box cis-acting DNA sequence elements are present in the promoter region of a number of signal-inducible plant genes. In many cases this motif is essential for gene expression. Maize nuclear extracts contain a protein complex that binds specifically to the G-box sequence. Previously, a protein called GF14 was described that is physically associated with the G-box binding complex, but is not a DNA-binding factor in and of itself. This paper reports the isolation of a cDNA encoding a maize G-box binding factor (GBF). The deduced amino acid sequence indicates that maize GBF1 is a basic region-leucine zipper protein. GBF1 binds to the G-box element with specificity similar to that of the binding activity in nuclear extracts. Furthermore, maize GBF1 and the factor detected in nuclear extract are identical in their molecular weight and are immunologically related. GBF1 mRNA accumulates rapidly in hypoxically induced maize cells prior to the increase in Adh1 mRNA levels. Taken together with results that indicate that GBF1 binds to the hypoxia-responsive promoter of maize Adh1, these observations suggest that GBF1 may be one of the factors involved in the activation of Adh1.

Sequences 5′ of the bovine keratin 5 gene direct tissue- and cell-type-specific expression of a lacZ gene in the adult and during development

Expression of keratin K5 (and K14) in multilayered epithelia occurs predominantly in the basal layer of proliferating keratinocytes. When a keratinocyte becomes committed to terminal differentiation, it moves out of the basal layer towards the epithelial surface. As part of this program of terminal differentiation, the expression of K5 (and K14) is downregulated in suprabasal cells, and new pairs of differentiation-specific keratins are expressed. To define the cis-acting DNA sequences required for K5 cell-type- and differentiation-specific expression, chimeric gene fusions between portions of the bovine keratin K5 locus and the Escherichia coli lacZ gene were used to generate transgenic mice. In the genomic fragment consisting of 5.3 kb of 5' flanking sequences, 6.1 kb corresponding to the body of the gene and 4.5 kb of 3' flanking sequences, the subfragment extending from -5300 bp to +138 bp was the smaller region that directed lacZ expression to stratified epithelia in a manner analogous to the endogenous keratin K5. Proximal sequences from -1300 bp to +138 bp were inactive. We also determined the expression pattern of keratin K5 during mouse development using an antiserum specific for mouse keratin K5. Expression was first detected in ectodermal cells of 11.5 days postcoitum embryos, and from day 13.5 postcoitum onwards K5 was detected in the precursors of most epithelia and organs which express K5 at adult stages. This pattern was reproduced, with few differences, by the construct with sequences from -5300 bp to +138 bp fused to the lacZ gene. These findings identify sequences between -5.3 kb and -1.3 kb of the bovine K5 gene as being important for cell-type- and differentiation-specific gene expression both during mouse development and in the adult.

Isolation and characterization of a novel transcription factor that binds to and activates insulin control element-mediated expression.

Pancreatic beta-cell-type-specific transcription of the insulin gene is principally regulated by a single cis-acting DNA sequence element, termed the insulin control element (ICE), which is found within the 5'-flanking region of the gene. The ICE activator is a heteromeric complex composed of an islet alpha/beta-cell-specific factor associated with the ubiquitously distributed E2A-encoded proteins (E12, E47, and E2-5). We describe the isolation and characterization of a cDNA for a protein present in alpha and beta cells, termed INSAF for insulin activator factor, which binds to and activates ICE-mediated expression. INSAF was isolated from a human insulinoma cDNA library. Transfection experiments demonstrated that INSAF activates ICE expression in insulin-expressing cells but not in non-insulin-expressing cells. Cotransfection experiments showed that activation by INSAF was inhibited by Id, a negative regulator of basic helix-loop-helix (bHLH) protein function. INSAF was also shown to associate in vitro with the bHLH protein E12. In addition, affinity-purified INSAF antiserum abolished the formation of the activator-specific ICE-binding complex. Immunohistochemical studies indicate that INSAF is restricted in terms of its expression pattern, in that INSAF appears to be detected only within the nuclei of islet pancreatic alpha and beta cells. All of these data are consistent with the proposal that INSAF is either part of the ICE activator or is antigenically related to the specific activator required for insulin gene transcription.

Early myeloid cell-specific expression of the human cathepsin G gene in transgenic mice.

The human cathepsin G (CG) gene is expressed only in promyelocytes and encodes a neutral serine protease that is packaged in the azurophil (primary) granules of myeloid cells. To define the cis-acting DNA elements that are responsible for promyelocyte-specific "targeting," we injected a 6-kb transgene containing the entire human CG gene, including coding sequences contained in a 2.7-kb region, approximately 2.5 kb of 5' flanking sequence, and approximately 0.8 kb of 3' flanking sequence. Seven of seven "transient transgenic" murine embryos revealed human CG expression in the fetal livers at embryonic day 15. Stable transgenic founder lines were created with the same 6-kb fragment; four of five founder lines expressed human CG in the bone marrow. The level of human CG expression was relatively low per gene copy when compared with the endogenous murine CG gene, and expression was integration-site dependent; however, the level of gene expression correlated roughly with gene copy number. The human CG transgene and the endogenous murine CG gene were coordinately expressed in the bone marrow and the spleen. Immunohistochemical analysis of transgenic bone marrow revealed that the human CG protein was expressed exclusively in myeloid cells. Expression of human CG protein was highest in myeloid precursors and declined in mature myeloid cells. These data suggest that the human CG gene was appropriately targeted and developmentally regulated, demonstrating that the cis-acting DNA sequences required for the early myeloid cell-specific expression of human CG are present in this small genomic fragment.

The DNA-bending protein HMG-1 enhances progesterone receptor binding to its target DNA sequences.

Steroid hormone receptors are ligand-dependent transcriptional activators that exert their effects by binding as dimers to cis-acting DNA sequences termed hormone response elements. When human progesterone receptor (PR), expressed as a full-length protein in a baculovirus system, was purified to homogeneity, it retained its ability to bind hormonal ligand and to dimerize but exhibited a dramatic loss in DNA binding activity for specific progesterone response elements (PREs). Addition of nuclear extracts from several cellular sources restored DNA binding activity, suggesting that PR requires a ubiquitous accessory protein for efficient interaction with specific DNA sequences. Here we have demonstrated that the high-mobility-group chromatin protein HMG-1, as a highly purified protein, dramatically enhanced binding of purified PR to PREs in gel mobility shift assays. This effect appeared to be highly selective for HMG-1, since a number of other nonspecific proteins failed to enhance PRE binding. Moreover, HMG-1 was effective when added in stoichiometric amounts with receptor, and it was capable of enhancing the DNA binding of both the A and B amino-terminal variants of PR. The presence of HMG-1 measurably increased the binding affinity of purified PR by 10-fold when a synthetic palindromic PRE was the target DNA. The increase in binding affinity for a partial palindromic PRE present in natural target genes was greater than 10-fold. Coimmunoprecipitation assays using anti-PR or anti-HMG-1 antibodies demonstrated that both PR and HMG-1 are present in the enhanced complex with PRE. HMG-1 protein has two conserved DNA binding domains (A and B), which recognize DNA structure rather than specific sequences. The A- or B-box domain expressed and purified from Escherichia coli independently stimulated the binding of PR to PRE, and the B box was able to functionally substitute for HMG-1 in enhancing PR binding. DNA ligase-mediated ring closure assays demonstrated that both the A and B binding domains mediate DNA flexure. It was also demonstrated in competition binding studies that the intact HMG-1 protein binds to tightly curved covalently closed or relaxed DNA sequences in preference to the same sequence in linear form. The finding that enhanced PRE binding was intrinsic to the HMG-1 box, combined with the demonstration that HMG-1 or its DNA binding boxes can flex DNA, suggests that HMG-1 facilitates the binding of PR by inducing a structural change in the target DNA.

The DNA-bending protein HMG-1 enhances progesterone receptor binding to its target DNA sequences.

Steroid hormone receptors are ligand-dependent transcriptional activators that exert their effects by binding as dimers to cis-acting DNA sequences termed hormone response elements. When human progesterone receptor (PR), expressed as a full-length protein in a baculovirus system, was purified to homogeneity, it retained its ability to bind hormonal ligand and to dimerize but exhibited a dramatic loss in DNA binding activity for specific progesterone response elements (PREs). Addition of nuclear extracts from several cellular sources restored DNA binding activity, suggesting that PR requires a ubiquitous accessory protein for efficient interaction with specific DNA sequences. Here we have demonstrated that the high-mobility-group chromatin protein HMG-1, as a highly purified protein, dramatically enhanced binding of purified PR to PREs in gel mobility shift assays. This effect appeared to be highly selective for HMG-1, since a number of other nonspecific proteins failed to enhance PRE binding. Moreover, HMG-1 was effective when added in stoichiometric amounts with receptor, and it was capable of enhancing the DNA binding of both the A and B amino-terminal variants of PR. The presence of HMG-1 measurably increased the binding affinity of purified PR by 10-fold when a synthetic palindromic PRE was the target DNA. The increase in binding affinity for a partial palindromic PRE present in natural target genes was greater than 10-fold. Coimmunoprecipitation assays using anti-PR or anti-HMG-1 antibodies demonstrated that both PR and HMG-1 are present in the enhanced complex with PRE. HMG-1 protein has two conserved DNA binding domains (A and B), which recognize DNA structure rather than specific sequences. The A- or B-box domain expressed and purified from Escherichia coli independently stimulated the binding of PR to PRE, and the B box was able to functionally substitute for HMG-1 in enhancing PR binding. DNA ligase-mediated ring closure assays demonstrated that both the A and B binding domains mediate DNA flexure. It was also demonstrated in competition binding studies that the intact HMG-1 protein binds to tightly curved covalently closed or relaxed DNA sequences in preference to the same sequence in linear form. The finding that enhanced PRE binding was intrinsic to the HMG-1 box, combined with the demonstration that HMG-1 or its DNA binding boxes can flex DNA, suggests that HMG-1 facilitates the binding of PR by inducing a structural change in the target DNA.

Negative modulator of the rat D2 dopamine receptor gene

The rat D2 dopamine receptor gene is transcribed from a TATA-less promoter that has an initiator-like sequence and several putative Sp1 binding sites. The main activator of this gene is between nucleotides -75 and -29, and a strong negative modulator is located between bases -217 and -76 (Minowa, T., Minowa, M. T., and Mouradian, M. M. (1992) Biochemistry 31, 8389-8396). In the present investigation, a small deletion series within this negative modulator fused with the reporter gene for chloramphenicol acetyltransferase was used to transfect the D2-expressing cells, NB41A3. Two cis-acting functional DNA sequences were identified: a 41-base pair segment between nucleotides -116 and -76 (D2Neg-B), which decreased transcription from the D2 promoter by about 45%, and a 26-base pair segment between nucleotides -160 and -135 (D2Neg-A), which, in the presence of the downstream negative modulator, reduced transcription down to the level of a promoterless vector. DNase I footprinting, gel mobility shift, and competitive cotransfection experiments suggested that D2Neg-A functions without trans-acting factors, whereas D2Neg-B interacts with nuclear factors at its Sp1 binding sequences. Gel supershift with anti-Sp1 antibody and UV cross-linking experiments revealed that a novel 130-kDa factor as well as Sp1 interact with D2Neg-B in NB41A3 cells. This novel protein recognizing Sp1 binding sequences in the D2 gene negative modulator is also found in nuclear extract from the rat striatum.

Transcription of a nematode U1 small nuclear RNA in vitro. 3'-end formation requires cis-acting elements within the coding sequence.

We have used block-substitution mutagenesis and in vitro transcription assays to identify cis-acting DNA sequence elements important for initiation and 3'-end formation of a U1 small nuclear RNA (snRNA) in the parasitic nematode Ascaris lumbricoides. Efficient initiation of synthesis by RNA polymerase II requires a compact element centered approximately 50 base pairs upstream of the transcriptional start site. Surprisingly, 3'-end formation of U1 snRNAs synthesized in vitro is solely dependent upon elements within the U1 coding sequence. In all other systems studied thus far, 3'-end formation of U snRNAs requires signals present in the 3'-flanking region. We also show that sequence elements that direct 3'-end formation of the A. lumbricoides trans-spliced leader RNA function when RNA synthesis is initiated from the U1 promoter. These results indicate that 3'-end formation of U snRNAs in nematodes is mechanistically distinct from the analogous process in higher eukaryotes.