Binding Sites For Transcription Factors AP-2 Research Articles

HIV-1 Promoter Single Nucleotide Polymorphisms Are Associated with Clinical Disease Severity.

The large majority of human immunodeficiency virus type 1 (HIV-1) markers of disease progression/severity previously identified have been associated with alterations in host genetic and immune responses, with few studies focused on viral genetic markers correlate with changes in disease severity. This study presents a cross-sectional/longitudinal study of HIV-1 single nucleotide polymorphisms (SNPs) contained within the viral promoter or long terminal repeat (LTR) in patients within the Drexel Medicine CNS AIDS Research and Eradication Study (CARES) Cohort. HIV-1 LTR SNPs were found to associate with the classical clinical disease parameters CD4+ T-cell count and log viral load. They were found in both defined and undefined transcription factor binding sites of the LTR. A novel SNP identified at position 108 in a known COUP (chicken ovalbumin upstream promoter)/AP1 transcription factor binding site was significantly correlated with binding phenotypes that are potentially the underlying cause of the associated clinical outcome (increase in viral load and decrease in CD4+ T-cell count).

Progressive Epigenetic Programming during B Cell Maturation Is Reflected in a Continuum of Epigenetic Disease Phenotypes in Chronic Lymphocytic Leukemia

The malignant phenotype combines characteristics that are acquired and inherited from the normal cell of origin. Hematological malignancies and related disease subtypes are thought to arise from diverse cell types that may reflect various developmental stages within the hematopoetic lineage. The contribution of different normal cell states and processes to the biological and clinical features of malignancy is not well understood. In chronic lymphocytic leukemia (CLL), two or three subtypes have been identified by variation in the degree of somatic IGHV mutations and recently uncovered epigenetic differences, respectively, suggesting that these subtypes derive from distinct normal B cell subsets at different stages of maturity. However, in CLL, as well as in most malignancies, the full possible extent of maturity states and the relative contribution of normal versus malignant developmental programs to the malignant phenotype have not been defined in a high-resolution manner.It is widely accepted that epigenetic patterns are important to establish and stabilize cellular phenotypes. Using whole genome bisulfite sequencing and sequence-specific methods, we assessed the dynamic DNA methylation events that occur during the maturation of B cells using six highly purified B cell subsets representing various stages of maturation. We confirmed previous reports that broad epigenetic programming affects about 25% of the genome from naïve to memory B cells, and further revealed that B cell subpopulations of intermediate maturity retained increasing degrees of the maturation program resulting in a singular developmental trajectory. Maturation was driven in part by the activity of a specific set of transcription factors (e.g. AP-1, EBF1, RUNX3, OCT2, IRF4 and NFkB). Using the developmental epigenetic signature defined by transcription factor binding site (TFBS) programming in normal cells to compare to tumor cells of 268 CLL revealed that tumors have the potential to derive from a continuum of possible maturation states that are reflected in the maturation stages of normal cells. Using RNA sequencing to measure gene expression, we found the degree of maturation achieved in tumors closely associates with the acquisition of a more indolent pattern of gene expression, evidenced by progressive downregulation of CLL oncogenes, such as ZAP70, TCL1 and BTK. Further assessment of the level of DNA methylation maturity in an independent sample cohort of 348 CLL cases revealed a quantitative, continuous relationship with increasingly favorable clinical outcomes.Although the majority of methylation differences found between tumor subtypes are naturally present in normal B cells, by identifying changes that are only present in CLL we further uncovered a previously unappreciated pathogenic role of transcription factor dysregulation. Specifically, a blockade in the epigenetic maturation of EBF and AP-1 TFBSs was found to define low-programmed (less mature, poor outcome) CLL cases and was associated with transcriptional and genetic loss of EBF1 and FOS transcription factors in tumor cells. Aberrantly acquired DNA methylation events in CLL were linked to excess activity of specific transcription factor families, namely EGR and NFAT. Intriguingly, we show that recurrent somatic mutations within the DNA binding domain of EGR2 selectively influence the methylation status of its cognate binding sites in mutant cases, establishing a role for this transcription factor in epigenetic dysregulation in CLL.Collectively, this work reveals that a unique epigenetic maturation signature, directed by normal developmental processes, defines individual CLL cases resulting in a spectrum of maturity across tumors. The majority of DNA methylation differences observed between individual CLLs reflects the state of maturity of the founder cell and profoundly influences the disease phenotype. We further propose that in CLL the disease-specific state results, in part, by dysregulation of key transcription factors that imbalance the normal B cell epigenetic program. DisclosuresKipps:Celgene: Consultancy, Honoraria, Research Funding; Gilead: Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Honoraria; AbbVie: Consultancy, Research Funding. Stilgenbauer:AbbVie: Consultancy, Other: travel grants, Research Funding; Amgen: Consultancy, Other: travel grants, Research Funding; Boehringer-Ingelheim: Consultancy, Other: travel grants, Research Funding; Celgene: Consultancy, Other: travel grants, Research Funding; Hoffman-LaRoche: Consultancy, Honoraria, Other: travel grants, Research Funding; Genentech: Consultancy, Other: travel grants, Research Funding; Genzyme: Consultancy, Other: travel grants, Research Funding; Gilead: Consultancy, Other: travel grants, Research Funding; GlaxoSmithKline: Consultancy, Other: travel grants, Research Funding; Janssen: Consultancy, Other: travel grants, Research Funding; Mundipharma: Consultancy, Other: travel grants, Research Funding.

A Novel Mutation in the α2-Globin Gene in Two Unrelated Iranian Families

Background: α-globin is encoded by two adjacent genes, αl and α2. Evidence suggests that these genes are not expressed equally and that the α2-globin gene encodes the majority of α-globin. This finding predicts that a thalassemic mutation of the α2-globin gene would result in a more severe loss of α-chain synthesis than a similar mutation in the αl-globin gene. Objectives: In the present study we described a novel non-deletion α-thalassemia defect in the 5'UTR region of the α2-globin gene. Materials and Methods: For molecular analysis, genomic DNA was isolated from peripheral blood cells by a salting out procedure. The common alpha deletion mutations were ruled out using the published primers and conditions. The amplification of the entire β and α1 globin genes was also carried out and their DNA was sequenced. No mutation was detected. Results: The mutation under study was located on an AP-1 transcription factor binding site and inherited in two unrelated Iranian families with hypochromic microcytic anemia. Conclusions: The patients in this study had moderate microcytosis and hypochromia without hemolysis, jaundice and splenomegaly. Molecular analysis in these patients revealed a non-deletion type of mutation in the promoter region, which is highly consistent with findings of other studies.

A probabilistic approach to learn chromatin architecture and accurate inference of the NF-κB/RelA regulatory network using ChIP-Seq

Using nuclear factor-κB (NF-κB) ChIP-Seq data, we present a framework for iterative learning of regulatory networks. For every possible transcription factor-binding site (TFBS)-putatively regulated gene pair, the relative distance and orientation are calculated to learn which TFBSs are most likely to regulate a given gene. Weighted TFBS contributions to putative gene regulation are integrated to derive an NF-κB gene network. A de novo motif enrichment analysis uncovers secondary TFBSs (AP1, SP1) at characteristic distances from NF-κB/RelA TFBSs. Comparison with experimental ENCODE ChIP-Seq data indicates that experimental TFBSs highly correlate with predicted sites. We observe that RelA-SP1-enriched promoters have distinct expression profiles from that of RelA-AP1 and are enriched in introns, CpG islands and DNase accessible sites. Sixteen novel NF-κB/RelA-regulated genes and TFBSs were experimentally validated, including TANK, a negative feedback gene whose expression is NF-κB/RelA dependent and requires a functional interaction with the AP1 TFBSs. Our probabilistic method yields more accurate NF-κB/RelA-regulated networks than a traditional, distance-based approach, confirmed by both analysis of gene expression and increased informativity of Genome Ontology annotations. Our analysis provides new insights into how co-occurring TFBSs and local chromatin context orchestrate activation of NF-κB/RelA sub-pathways differing in biological function and temporal expression patterns.

Construction of Iuciferase reporter vectors harboring DC-SIGN promoters without AP-1 or ETS-1 transcription factor binding site and detection of their activity

Objective To detect the role of AP-1 or ETS-1 transcription factor binding sites (TFBS) in activity of DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) pro-moter. Methods Genomic DNA was extracted from peripheral blood. Complete DC-SIGN promoter and those without AP-1 or ETS-1 TFBS were amplified by PCR using designed primers. The PCR products were digested with MLu Ⅰ and Bgl Ⅱ and then ligated into MLu Ⅰ and Bgl Ⅱ digested pGL-3/Basic and pGL-3/En-hancer vectors. Transfection in Hacat and 293 cells was accomplished with Trans Fast liposome. Activity of luciferase was detected after 48 h. Results The DC-SIGN promoters without AP-1 or ETS-1 TFBS and the recombined pGL-3 vectors were correctly constructed. The activity of DC-SIGN promoters without AP-1 was reduced 20% (293) and 10% (Hacat), which was 40%-50% with enhancer. The activity of DC-SIGN pro-moters without ETS-1 was nearly vanished, no matter with or without enhancer. Conclusion ETS-1 TFBS, not AP-1 TFBS, plays an important role in activity of DC-SIGN promoter. Key words: HIV; DC-SIGN; Promoter; Transcription factor binding site; Reporter vector

AP-1 and ERK1 but not p38 nor JNK is required for CRPV early protein 2-dependent MMP-9 promoter activation in rabbit epithelial cells

Human papillomaviruses (HPV) are the known cause for a variety of cancers including cervical and epithelial cancers. The cottontail-rabbit papillomavirus (CRPV) serves as a suitable animal model to study the development of these cancers in vivo. We have previously demonstrated that CRPV-induced skin carcinomas express high levels of MMP-9, a metalloproteinase contributing to cancer progression by extracellular matrix remodelling. Based on our previously reported finding that CRPV early protein 2 can activate a truncated human 670 bp MMP-9 promoter fragment, we hypothesized that MMP-9 expression in the rabbit carcinomas is a consequence of activation of the rabbit MMP-9 promoter in-trans by CRPV early protein 2. Further elucidation of the mechanism revealed the requirement for both a proximal and distal AP-1 transcription factor binding site in the rabbit MMP-9 promoter and the AP-1 complex as demonstrated by the inhibitory effect of TAM67, a trans-activation deficient c-jun mutant. The characterization of signal-transduction requirements revealed predominantly ERK1 to be required for CRPV early protein 2-dependent MMP-9 promoter activation, but not JNK nor p38. In summary CRPV early protein 2 activates the expression of MMP-9 in-trans through AP-1 and ERK1 and may contribute to cancer development and progression via this mechanism within the animal model.

Regulation of Tcrb recombination ordering by c-Fos-dependent RAG deposition

Antigen receptor variable-(diversity)-joining (V(D)J) recombination at the locus encoding the T cell antigen receptor-beta (Tcrb) is ordered, with D(beta)-to-J(beta) assembly preceding V(beta)-to-DJ(beta) joining. The molecular mechanism underlying this 'preferred' order of rearrangement remains unclear. Here we show that the D(beta) 23-base pair recombination signal sequence (D(beta) 23-RSS) contains a specific AP-1 transcription factor-binding site bound by AP-1 and its component c-Fos expressed at a specific stage. Cell-based recombination assays suggested that c-Fos interacted directly with the RAG recombinase and enhanced its deposition to D(beta) 23-RSSs, thus conferring the priority of DJ(beta) recombination. Loss of c-Fos decreased Tcrb recombination efficiency and disrupted recombination ordering in vivo. Our results show an unexpected function for c-Fos as a direct regulator of Tcrb recombination, rather than its usual function as a transcription regulator, and provide new insight into the mechanisms of recombination ordering.

Synergistic Activation of Human Involucrin Gene Expression by Fra-1 and p300—Evidence for the Presence of a Multiprotein Complex

Involucrin is expressed in the differentiated suprabasal epidermal layers, and an AP1 transcription factor-binding site present in the involucrin promoter distal regulatory region is required for this regulation. This site binds Fra-1, but cofactor interaction at this site has not been adequately characterized. We show that Fra-1 and p300 histone acetyltransferase are present at the AP1 site, as detected by chromatin immunoprecipitation. This interaction is functional, as treating p300 expressing keratinocytes with calcium or 12-O-tetradeconylphorbol-13-acetate, results in a synergistic increase in hINV expression, and this enhanced activation can be reproduced by coexpression of Fra-1 and p300. p300 also co-precipitates with Fra-1, but protein fractionation studies suggest that this interaction requires an additional protein. Fra-1 also interacts with other proteins that interact at the AP1-5 site, including JunD, JunB, Sp1, and P/CAF. Contrary to results in some other systems, Fra-1 functions as a positive transcriptional regulator in human keratinocytes. These studies suggest that a large multiprotein complex, which includes Fra-1, p300, P/CAF, junD, junB, and Sp1 acts at the AP1-5 site to produce a synergistic increase in hINV gene expression.

MicroRNA May Link Inflammation and Cancer

For mammals, inflammatory responses of the innate immune system provide an indispensable protective mechanism against infection. But these signaling mechanisms may also contribute to certain diseases, including cancer. O’Connell et al . investigated the role of microRNAs (noncoding RNAs that regulate gene expression through interaction with cellular mRNAs) in the inflammatory response to viral infection and may have unearthed a molecular link between inflammation and cancer. The authors used microarrays to monitor expression of a panel of 200 microRNAs in response to polyriboinosinic:polyribocytidylic acid [poly(I:C)], a synthetic double-stranded RNA used to mimic viral infection, or the antiviral cytokine interferon-β (IFN-β). They identified one microRNA, miR-155, that showed increased expression in macrophages in response to both stimuli. Expression of miR-155 was regulated through multiple signaling pathways. The response to poly(I:C) required signaling through Toll receptors and the MyD88 or TRIF adaptor proteins. Interferons, on the other hand, stimulated expression of miR-155 through a slower mechanism that required autocrine signals mediated by tumor necrosis factor α. Both pathways appeared to converge on regulation of an AP-1 transcription factor binding site, and an inhibitor of the JNK mitogen-activated protein kinase inhibited the response to poly(I:C) or interferon. The miR-155 microRNA is also an oncogene, and its overexpression can cause B cell lymphoma. Thus, the authors note that the finding of miR-155 as a common target of pathways activated by viral infection presents a potential link between inflammatory responses and cancer. R. M. O’Connell, K. D. Taganov, M. P. Boldin, G. Cheng, D. Baltimore, MicroRNA-155 is induced during the macrophage inflammatory response. Proc. Natl. Acad. Sci. U.S.A. 104 , 1604-1609 (2007). [Abstract] [Full Text]

An Involucrin Promoter AP1 Transcription Factor Binding Site Is Required for Expression of Involucrin in the Corneal Epithelium In Vivo

Cell division of corneal limbal stem cells gives rise to transient amplifying cells that ultimately differentiate to form the multilayered corneal epithelium. The mechanisms that regulate changes in gene expression during this process are not well understood. In the present study, the involucrin gene was used as a model to study this regulation. Regulation of human involucrin gene expression and promoter activity was assessed using in vivo transgenic mouse models and cultured primary human corneal epithelial cells. Human involucrin (hINV) is a structural protein that is selectively expressed in differentiating corneal epithelial cells. The results reveal that an activator protein one (AP1) DNA-binding site is essential for appropriate basal and stimulus-dependent hINV promoter activity. Mutation of this site, AP1-5, results in a loss of hINV gene expression in the corneal epithelium in vivo and in cultured corneal epithelial cells. A gel mobility supershift analysis revealed interaction of the AP1 factors, Fra-1 and JunB, with this element. Inhibition of AP1 function with a dominant-negative form of AP1 also inhibited expression. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C activator, increased hINV gene expression, a response that correlates with increased AP1 factor (Fra-1 and JunB) binding to the hINV gene AP1-5 response element. These findings point to an essential role for AP1 transcription factors, acting through a distal regulatory region AP1-5 element, in the regulation of involucrin gene expression during corneal epithelial cell differentiation.

Potentially functional polymorphism in the promoter region of prodynorphin gene may be associated with protection against cocaine dependence or abuse.

It has been demonstrated that the opioid peptide dynorphin plays a role in modulating responses to several psychoactive substances including cocaine. Our laboratory and others have found that mRNA levels of dynorphin in the caudate and putamen are elevated after acute or chronic cocaine exposure in rats. Recently, a 68-base pair (bp) repeat polymorphism within the core promoter region of the human prodynorphin gene has been reported to occur in alleles containing one, two, three, or four copies. This repeat contains a putative AP-1 transcription factor binding site; reporter gene constructs with three or four, but not one or two, copies of the tandem repeats were shown to be associated with increases in transcriptional activation in in vitro cellular assays. We hypothesize that this polymorphism may be associated with individual differences in vulnerability to cocaine dependence or abuse. From an ongoing study of the genetics of addiction, 174 subjects were studied, including individuals with a primary diagnosis (DSM-IV criteria) of cocaine dependence (N = 61) or abuse (N = 22), and controls with no history of any substance dependence or abuse (N = 91). We designed primers for polymerase chain reaction (PCR) to amplify sequences of the promoter region of the prodynorphin gene containing the repeat element. The association of alleles containing three or four repeats with cocaine dependence/abuse was examined. With data stratified by ethnic group, pooled relative risk (RR) with Mantel-Haenszel Chi square was calculated: RR = 0.59 (95% confidence interval 0.37-0.95), chi2 (1) = 4.14, P = 0.042. Our results suggest that this allelic variation at the promoter region of the prodynorphin gene (alleles with three or four repeats), which may result in enhanced transcription of the gene, may contribute to relative protection and decrease individual vulnerability to develop cocaine dependence or abuse.

Calcium-dependent Involucrin Expression Is Inversely Regulated by Protein Kinase C (PKC)α and PKCδ

Calcium is an important physiologic regulator of keratinocyte function that may regulate keratinocyte differentiation via modulation of protein kinase C (PKC) activity. PKCalpha and PKCdelta are two PKC isoforms that are expressed at high levels in keratinocytes. In the present study, we examine the effect of PKCdelta and PKCalpha on calcium-dependent keratinocyte differentiation as measured by effects on involucrin (hINV) gene expression. Our studies indicate that calcium increases hINV promoter activity and endogenous hINV gene expression. This response requires PKCdelta, as evidenced by the observation that treatment with dominant-negative PKCdelta inhibits calcium-dependent hINV promoter activity, whereas wild type PKCdelta increases activity. PKCalpha, in contrast, inhibits calcium-dependent hINV promoter activation, a finding that is consistent with the ability of dominant-negative PKCalpha and the PKCalpha inhibitor, Go6976, to increase hINV gene expression. The calcium-dependent regulatory response is mediated by an AP1 transcription factor-binding site located within the hINV promoter distal regulatory region that is also required for PKCdelta-dependent regulation; moreover, both calcium and PKCdelta produce similar, but not identical, changes in AP1 factor expression. A key question is whether calcium directly influences PKC isoform function. Our studies show that calcium does not regulate PKCalpha or delta levels or cause a marked redistribution to membranes. However, tyrosine phosphorylation of PKCdelta is markedly increased following calcium treatment. These findings suggest that PKCalpha and PKCdelta are required for, and modulate, calcium-dependent keratinocyte differentiation in opposing directions.

An allelic variation in the human prodynorphin gene promoter alters stimulus-induced expression.

Prodynorphin, the precursor of the dynorphin opioid peptides, has been shown to play an important role in several aspects of human diseases and complex traits, e.g., drug abuse, epilepsy, and mood disorders. The objective of this study was to identify polymorphisms in the 5' control region of the human prodynorphin gene and to relate these polymorphisms to prodynorphin gene expression. Within the core promoter region, a 68-bp sequence was found to occur as a polymorphic element, either singular or as tandemly repeated element two, three, or four times. This 68-bp repeat element contains an AP-1 transcription factor binding site as demonstrated by electrophoretic mobility shift assay. Reporter gene assays were performed and provided evidence for allele dependent different promoter activity. Dynorphin was found to be involved in many pathophysiological processes so that the described prodynorphin alleles may correlate with the occurrence of several diseases, for example, drug addiction. However, prodynorphin allelic distributions were not significantly different in heroin addicts and control subjects.

Effects of phosphate neutralization on the shape of the AP-1 transcription factor binding site in duplex DNA.

Previous electrophoretic experiments suggest that the AP-1 site in duplex DNA bends in response to the pattern of amino acid charges distal to the basic region in bound bZIP proteins. The extent and direction of apparent DNA bending are consistent with the prediction that DNA will collapse locally upon asymmetric phosphate charge neutralization. To prove that asymmetric phosphate neutralization could produce the observed degree of DNA bending, the present experiments partially substitute anionic phosphate diesters in the AP-1 site with various numbers of neutral methylphosphonate linkages. DNA bending is induced toward the neutralized face of DNA. The degree of DNA bending induced by methylphosphonate substitution (approximately 3.5 degrees per neutralized phosphate) is comparable to that induced by GCN4 variants carrying increasing numbers of additional basic amino acids. It is plausible, therefore, that asymmetric phosphate neutralization is the cause of DNA bending in such complexes.

Identification of an AP-1 transcription factor binding site within the human cholecystokinin (CCK) promoter

Neuropeptide gene expression, i.e. proenkephalin A, may be modulated by activating protein (AP-1) transcription factor complexes which associate with specific DNA sequence motifs, known as the AP-1 binding sites. Gel mobility shift assays revealed that nuclear extracts prepared from a human neuroblastoma cell-line, SK-N-MC, bind to the 5'-CTGCGTCAGCG-3' motif, present within the human cholecystokinin (CCK) promoter. In contrast, the mutated 5'-CTGCAACAGCG-3' motif did not bind any specific protein complex. In vitro run-off transcription and Western blot analysis revealed the expression of the protooncogenes Fos and Jun. This suggests that Fos/Jun homo- or heterodimeric complexes may interact with the 5'-CTGCGTCAGCG-3' motif, present within the human CCK promoter.

Neocarzinostatin-mediated DNA damage in a model AGT.ACT site: mechanistic studies of thiol-sensitive partitioning of C4' DNA damage products.

Double-strand (DS) DNA damage caused by neocarzinostatin (NCS) has been studied in the trinucleotide AGT-ACT sequence in an AP-1 transcription factor binding site. There are strong similarities between bistranded lesions produced at AGT.ACT and AGC-GCT, including the fact that DS lesions outnumber SS lesions on the AGT and AGC strands, while SS exceed DS on the ACT and GCT strands. Structure-function studies revealed that a variety of different thiols produced bistranded lesions in this model by predominantly C4'-hydrogen atom abstraction (84-93%) at the T of AGT and C5'-hydrogen atom abstraction (87-91%) at the T of ACT. Single-strand (SS) lesions were found to represent a variable mixture of C4' and C5' chemistry. The C4'-hydroxylated abasic site occurred in both SS and DS lesions at both sites and accounted for most of the DS damage at AGT (60-83%); the remaining damage consisted of 3'-phosphoglycolate- and 3'-phosphate-ended fragments. The nature of the thiol was found to affect the partitioning of the breakdown products arising from C4' and, to a lesser extent, C5' hydrogen atom abstraction. Production of 3'-phosphoglycolate residues, restricted mainly to the T of AGT in bistranded lesions, correlated with the incidence of direct DS breaks in the AGT.ACT model and in plasmid DNA and appeared to be influenced by the reducing power of the thiol activator. Furthermore, hydrazine and sodium borohydride both inhibited the formation of glycolate, an effect that was exploited to determine the rate constant for 3'-phosphoglycolate formation: 0.06 min-1 at 0 degree C, pH 7.4. Under anaerobic conditions, the nitroaromatic radiation sensitizer misonidazole caused a large increase in glycolate production in both SS and DS lesions formed by NCS, which suggests that the formation of 3'-phosphoglycolate, like 3'-formylphosphate generated by C5' chemistry, involves an oxyradical intermediate. The pathways for DNA damage involving C4' and C5' hydrogen atom abstraction thus share many common features, several of which are consistent with a mechanism for the production of NCS-mediated bistranded lesions at AGT.ACT that involves a tetraoxide bridge joining the lesions on opposite strands of DNA.

The AP-1 Site and the cAMP- and Serum Response Elements of the c-fosGene Are Constitutively OccupiedIn Vivo

The c-fos proto-oncogene is inducible by cAMP, phorbol esters, serum, and growth factors. The induction by cAMP is mediated by the conserved cAMP response element (CRE), while induction by phorbol esters, serum, and growth factors requires a distal element called the serum response element (SRE). In addition to these elements, a consensus AP-1 transcription factor binding site is located next to SRE. Upstream regions of the mouse and human c-fos genes were footprinted in vivo by the ligation-mediated polymerase chain (PCR). Our results show that all three elements are constitutively protected in mouse liver and lung and in cultured human A431 cells. No major change in the protection profile was detected in A431 cells following stimulation with epidermal growth factor or in mice at birth, when c-fos is known to be induced. These results suggest that the inducible cis elements of the c-fos gene are poised, ready to respond immediately to external signals.