Consensus Binding Sites For Sp1 Research Articles

Sp1 is critical for basal and E2-transactivated transcription from the bovine papillomavirus type 1 P89 promoter.

The bovine papillomavirus type 1 (BPV-1) long control region (LCR) contains at least three consensus binding sites for the transcription factor Sp1 at nucleotides (nt) 7800, 7833 and 7854. A high basal-level P89 expression vector consisting of an origin-deleted LCR fused to the chloramphenicol acetyltransferase (CAT) gene was utilized to determine the role of these Sp1 sites in the regulation of transcription from the BPV-1 P89 promoter. The three Sp1 sites were capable of binding Sp1 in vitro. Mutation of these sites in the background of the origin-deleted LCR-CAT or a wild-type LCR-CAT construct resulted in decreased basal expression from P89. In addition, mutation of the Sp1 sites in the wild-type background caused a reduction in E2-transactivation potential. These data illustrate the importance of these Sp1 sites in regulating both basal and E2-transactivated P89 expression.

Isolation and characterization of the promoter of the human 5′-nucleotidase (CD73)-encoding gene

Ecto-5′-nucleotidase (NT, CD73) is a purine salvage-pathway enzyme located on the surface of various cell types, including subsets of human lymphocytes and certain leukemias and lymphomas. In addition to purine salvage, NT has proposed roles in lymphocyte maturation and activation, and its expression has been associated with the resistance of some tumor cell lines to chemotherapeutic agents. To better understand the regulation of NT gene expression in normal lymphocyte development and the elevated expression seen in some drug-resistant tumor cell lines, we isolated NT genomic clones containing the promoter region. The genomic DNA upstream from the NT start codon is high in G+C content, with one cAMP-responsive element and five consensus Sp-1 binding sites, but no TATAA box. RNase protection assays identified a cluster of potential transcription start points ( tsp). One tsp, at −63 bp relative to the start codon, was confirmed as authentic by 5′-RACE (rapid amplification of cDNA ends) cloning. Transient transfection experiments utilizing luc as a reporter gene have demonstrated that a 155-bp NT genomic DNA segment inclusive of the tsp functions as a promoter in both NT + (WI-L2 and MG) and NT − (Jurkat, Hela and Raji) cell lines. The addition of 5′-flanking sequences extending as far as −1.9 kb did not confer cell-type-specific expression to the core promoter. However, nuclear run-on analysis of nascent NT transcripts suggested that differential transcription initiation is at least partially responsible for the regulation of NT expression. Thus, additional information is necessary, either at the chromatin level, or within elements outside of the promoter region, to direct tissue-specific expression of NT.

Identification of three transcriptional regulatory elements in the rat mitochondrial benzodiazepine receptor-encoding gene

The sequence upstream from the first exon in the rat mitochondrial benzodiazepine receptor-encoding gene ( MBR) was analyzed for transcriptional promoter activity by three techniques: promoter deletion analysis in vectors containing the gene cat encoding chloramphenicol acetyltransferase, electrophoretic mobility shift analysis (EMSA) and DNase I protection assay. All three methods are in uniformity with the identification of at least three regulatory elements corresponding to locations -51/-33, -26//-249 and -555/-526. The most distal and proximal domains are positive-acting, whereas the element at -267/-249 acts in a negative manner. The positive-acting -51/-33 element contains the middle of three consensus Sp1-recognition sequences found in this region of the gene. Binding of Y1 cell nuclear protein to a DNA fragment corresponding to this region of the gene is competed by a synthetic oligodeoxyribonucleotide bearing the consensus Sp1-binding site sequence. These studies provide the first reported functional evidence localizing transcriptional elements of MBR.

A negative regulatory element in the promoter region of the rat alpha 2A-adrenergic receptor gene overlaps an SP1 consensus binding site.

Three subtypes of alpha 2-adrenergic receptors (alpha 2A, alpha 2B and alpha 2C) have been described that differ in their primary sequence and tissue-specific expression and are encoded by three distinct genes. Previous work has shown that the human alpha 2A-adrenergic receptor gene promoter consists of a TATA-box (TATAAA), palindromic sequence (CCCACGTGGG) and GC-box (GGGGCGG) motif. Sequence analysis of the putative promoter region of the rat alpha 2A-adrenergic receptor gene showed that these promoter regions are conserved in their sequence and relative location. We analysed the transcriptional activity of these regions using RINm5F, a rat insulinoma cell line that expresses the endogenous alpha 2A-adrenergic receptor gene. These results showed that the region from -484 to -92 has a negative effect on transcription, as deletion of this region in alpha 2A-adrenergic receptor gene-chloramphenicol acetyltransferase reporter constructs increased reporter gene activity. This region included the GC-box sequence which is a consensus binding site for the nuclear factor SP1, which is a positive activator of transcription. Gel-mobility-shift assays and supershift assays with an antibody that recognizes SP1 showed binding of the SP1 nuclear factor as well as other nuclear factors to this GC-box region. Additional nuclear factors bind to the downstream palindromic region. We suggest that positive- and negative-acting nuclear factors contribute to the activity of the alpha 2-adrenergic receptor promoter.

Transcription factor repression and activation of the human acetylcholinesterase gene.

Acetylcholinesterase in man is encoded by a single gene, ACHE, located on chromosome 7q22. In this study, the transcription start sites and major DNA promoter elements controlling the expression of this gene have been characterized by structural and functional studies. Immediately upstream of the first untranslated exon of the gene are GC-rich sequences containing consensus binding sites for several transcription factors, including Sp1, EGR-1 and AP2. In vitro transcription studies and RNase protection analyses of mRNA isolated from human NT2/D1 teratocarcinoma cells reveal that two closely spaced transcription cap sites are located at a consensus initiator (Inr) element similar to that found in the terminal transferase gene. Transient transfection of mutant genes shows that removal of three bases of this initiator sequence reduces promoter activity by 98% in NT2/D1 cells. In vitro transcription studies and transient transfection of a series of 5' deletion mutants of the ACHE promoter linked to a luciferase reporter show an Sp1 site at -71 to be essential for promoter activity. Purified Sp1 protein protects this site from DNase cleavage during in vitro footprinting experiments. A conserved AP2 consensus binding site, located between the GC box elements and the Inr, is protected by recombinant AP2 protein in DNase footprinting experiments, induces a mobility shift with AP2 protein and AP2-containing cell extracts, and fosters inhibition of transcription by AP2 as measured by transient transfection in mouse and human cell lines and in in vitro transcription reactions. These results indicate that AP2 functions as a repressor of human ACHE and mouse Ache transcription.

Two Sp1-binding Sites Mediate cAMP-induced Transcription of the Bovine CYP11A Gene through the Protein Kinase A Signaling Pathway

Two sequence elements located at -111 to -100 base pairs and -70 to -50 base pairs in the 5'-flanking region of the bovine CYP11A gene and in closely related positions in CYP11A of other species contain G-rich regions that are similar to the consensus Sp1-binding site. These sequences bind the purified transcription factor Sp1 as well as nuclear proteins from mouse Y1 adrenal cells that interact with an antibody specific for Sp1. Both of these CYP11A sequences support basal and cAMP-dependent transcription of reporter gene plasmids transfected into Y1 cells, and mutations within the G-rich -111/-100-base pair sequence that reduce or eliminate the binding of Sp1-related Y1 nuclear proteins also markedly reduce cAMP-induced transcription. cAMP-dependent transcription supported by both CYP11A sequence elements is mediated by protein kinase A at levels comparable to that promoted by different cAMP-response sequences and transcription factors in other genes involved in steroidogenesis. These results indicate that ACTH-dependent regulation of cholesterol side chain cleavage cytochrome P450 levels in the adrenal cortex which is mediated through cAMP involves the ubiquitous transcription factor Sp1.

Regulation of insulin-like growth factor I receptor gene expression by Sp1: physical and functional interactions of Sp1 at GC boxes and at a CT element.

The insulin-like growth factor I (IGF-I) receptor mediates signal transduction by the IGFs and plays a critical role in growth and development. The proximal promoter region of the rat IGF-I receptor gene contains multiple Sp1 consensus-binding sites (GC boxes). Various promoter fragments fused to a luciferase reporter gene were transiently cotransfected together with an Sp1 expression vector into Drosophila Schneider cells, which lack endogenous Sp1. A proximal promoter fragment containing 476 nucleotides of 5'-flanking region and 640 nucleotides of 5'-untranslated region was strongly activated by Sp1 (an average of 116-fold), and progressive 5'-deletions of the promoter that sequentially removed GC boxes reduced Sp1 activation to 15-fold over basal promoter activity. DNase I footprinting studies with purified Sp1 protein revealed four GC boxes in the 5'-flanking region of the promoter and one homopurine/homopyrimidine motif (CT element) in the 5'-untranslated region that bound Sp1. Mutation of the CT element reduced Sp1 activation by 70%. Taken together, these results demonstrate that Sp1 can regulate expression of the IGF-I receptor promoter by acting both on GC boxes in the 5'-flanking region of the promoter and on a CT element in the 5'-untranslated region.

Structure and Chromosomal Localization of the Mouse Neurocan Gene

Cosmid clones containing the mouse neurocan gene were isolated from a genomic library using rat neurocan cDNA fragments as probe. The murine gene has a size of approximately 25 kb and contains the coding sequence for the mRNA on 15 exons. The exon-intron structure reflected the structural organization of neurocan, which is a multidomain protein belonging to the aggrecan/versican preteoglycan family. All introns between conserved modular protein domains are phase I introns. Primer extension experiments indicate a transcriptional start point 28 bases downstream of a consensus TATA sequence. Further analysis of 1 kb of 5′ flanking sequence revealed in addition to AP1, AP2, and SP1 consensus binding sites multiple E-box elements and a glucocorticoid responsive element. Single-strand conformation polymorphism was used to map neurocan to chromosome 8 between the microsatellite markers D8Mit29 and D8Mit78. Among mouse mutants that have been mapped around this region are the three allelic neurological diseases tottering, leaner, and rolling. The multidomain structure and the preferential expression of neurocan in the brain suggest a potential involvement in these diseases.

Inverse PCR-mediated cloning of the promoter for the rat vasopressin V2 receptor gene

The adenylyl cyclase-coupled vasopressin V2 receptor has been cloned recently and shown, in rats, to be produced from the predominant form of two alternate spliced variants. To begin to unravel the transcriptional regulation of this receptor, we have isolated the 5' flanking region of the rat vasopressin V2 receptor gene and characterized its promoter sequence. The method of inverse polymerase chain reaction (PCR), which allows the amplification of DNA fragments adjacent to a segment of known sequence, was used as an alternative approach to genomic DNA library screening. Using a probe encompassing part of the coding region, first we identified by Southern blot analysis, a single BstX I hybridizing fragment of 2.3 kilobases (kb). This size predicted a BstX I restriction site 1.5 kb upstream to the gene coding region. Cloning of this fragment was accomplished through circularization of BstX I restriction digests and inverse PCR-mediated amplification. Sequence analysis of the gene 5' flanking domain enabled the design of oligonucleotide primers with the usual forward/reverse orientation, and additional clones were generated from native genomic DNA using a high fidelity thermoresistant DNA polymerase. Reverse transcription-PCR (RT-PCR) and primer extension analysis mapped the major transcription start site 422 nucleotides upstream to the translation initiation codon. The promoter region lacks a TATA box but contains a CAAT box and a consensus binding site for transcription factor Sp1. Multiple potential binding sites for the transcription factor PEA3 are clustered in two DNA portions located 0.6 kb and 1 kb upstream to the coding region. In addition, sequences homologous to glucocorticoid response elements are present and might be responsible for the regulation by adrenal steroids of vasopressin-dependent adenylyl cyclase activity in the kidney.

Characterization of a GC-rich Region Containing Sp1 Binding Site(s) as a Constitutive Responsive Element of the α2(I) Collagen Gene in Human Fibroblasts

To analyze regulatory elements in the human alpha 2(I) collagen gene (COL1A2) promoter, a series of deletion mutants from -323 to -186 base pairs was tested in transient transfection assays in human fibroblasts. A strong positive responsive element was mapped to a GC-rich region located between base pairs -303 and -271. This region contains three binding sites (GC-boxes) resembling recognition sites for the transcription factor Sp1. Substitution mutations in the GC-boxes abolished binding to the GC-rich region in gel shift analyses and resulted in 90% reduction of promoter activity in transient transfection assays. We demonstrated that transcription factor Sp1 is essential for binding based on the following observations. 1) Sp1 consensus binding site alone competes by binding to the GC-rich region in the DNase I protection assay; 2) both Sp1 consensus binding site and Sp1 antibodies prevent the formation of a DNA-protein complex in the mobility shift assay; 3) anti-Sp1 antibodies recognize a component of the complex competed for by Sp1 consensus binding site.

Organization of the Gene Encoding the Human Kell Blood Group Protein

Kell is one of the major blood group systems in human erythrocytes. It is a complex system containing a large number of different antigens. Previously we cloned the Kell cDNA, which was predicted to encode an integral membrane protein with 731 amino acids. Now we have isolated overlapping genomic clones and determined the exon-intron structure of the KEL gene; it spans approximately 21.5 kb with its coding sequence being organized in 19 exons that range in size from 63 bp to 288 bp. The size of introns ranges from 93 bp to approximately 6 kb. The donor and acceptor splice sites all conform to the consensus splicing sequences. Exon 1 encodes only the initiation amino acid, methionine, and contains a consensus Sp1 binding site. The single membrane spanning region of Kell protein is encoded in exon 3 and the putative zinc endopeptidase active site is in exon 16. The amino acids encoded by the 19 exons are identical to those of a person with a common Kell phenotype, as determined by RNA polymerase chain reaction of peripheral blood. Amplification of cDNA 5' ends, derived from human fetal liver, indicated three transcription initiation sites located 30, 81, and 120 bp upstream of the initiation codon. The 5' flanking region of KEL from -176 does not contain a TATA sequence, but has possible GATA-1 binding sites and has significant promoter activity when determined by chloramphenicol acetyltransferase activity in K562 cells.

Clustering of Sp1 sites near the promoter region of ICP34.5 in herpes simplex virus type 1.

We report that a host nuclear protein of approximately 100 kDa binds to the tandemly reiterated DR2 sequence of herpes simplex virus type 1 (HSV-1). The DR2 sequence is a repeated component in the "a" sequence, which defines the signals for cleavage and encapsidation of viral DNA; the "a" sequence also contains the promoter regulatory signals for the gene encoding the viral neurovirulence factor, ICP34.5. Characterization of the host binding protein by means of gel shifts and DNase I footprinting revealed this protein is the eukaryotic transcription factor, Sp1. Furthermore, as judged from the sequence homology, the DR2 region contains clustered matches to the consensus binding site for Sp1. Comparison of the host factor and purified Sp1 (by means of gel shifts and footprinting) confirmed these findings. Since clustered DNA recognition elements represent unusually high affinity binding sites, these repeated Sp1 motifs proximal to the ICP34.5 gene suggest that this region may be a major Sp1 binding site in the viral genome.

Analysis of the rat lactate dehydrogenase A subunit gene promoter/regulatory region.

The rat lactate dehydrogenase (LDH) A subunit gene promoter contains a putative AP-1 binding site at -295/-289 bp, two consensus Sp1 binding sites at -141/-136 bp and -103/-98 bp, and a single copy of a consensus cyclic AMP-responsive element (CRE) at -48 to -41 bp upstream of the transcription initiation site. Additionally, an as yet unidentified silencer element is located within the -1173/-830 bp 5'-flanking region. Transient transfection analyses of a -1173/+25 bp LDH A-chLoramphenicol acetyltransferase fusion gene has indicated a complete inability of the promoter fragment to direct basal or forskolin-induced transcription. Deletion of the -1173/-830 bp sequence restored basal and cyclic AMP (cAMP)-inducible activity. Point mutations in the Sp1 binding sites of a -830/+25 bp promoter fragment reduced basal but not the relative degree of cAMP-inducible activity. cAMP-regulated transcriptional activity was dependent upon an 8 bp CRE, -TGACGTCA-, located at the -48/-41 bp upstream region. Mutations in the CRE abolished cAMP-mediated induction and reduced basal activity by about 65%. The CRE binds a 47 kDa protein which has previously been identified as CRE binding protein (CREB)-327, an isoform of the activating transcription factor/CREB transcription factor gene family. Co-transfection of a vector that expresses the catalytic subunit of cAMP-dependent protein kinase stimulates LDH A subunit promoter activity suggesting that cAMP induces LDH A subunit gene expression through phosphorylative modification of CREB-327. This study emphasizes a fundamental role of several modules including Sp1 and CREB binding sites in regulating basal and cAMP-mediated transcriptional activity of the LDH A gene.

Structure of the human APO-1 gene.

APO-1/Fas (CD95) is a type 1 transmembrane protein that belongs to the tumor necrosis factor/nerve growth factor receptor family characterized by cysteine-rich extracellular domains. Cross-linking of APO-1 mediates apoptosis in a variety of cells. In the present study we report the isolation and characterization of the human APO-1 gene spanning approximately 25 kb of human chromosome 10. The gene consists of nine exons (25 bp to > 1.44 kb) separated by eight introns (152 bp to approximately 12 kb). The boundaries of exon 2 to 5 encoding the extracellular region do not match the boundaries of the three APO-1 protein subdomains. Exon structure and functional protein domains correspond for exon 6 encoding the transmembrane region and for exon 9 encoding the "death domain". By a polymerase chain reaction-based approach we localized major transcriptional start sites in human spleen cells 77 and 73 nucleotides upstream of the translation initiation codon of the human APO-1 gene. Minor initiation sites were found at positions -128, -111, -91, and -74. The 5' flanking sequence of the human APO-1 gene is GC rich, contains a high number of CpG dinucleotides and lacks a consensus TATA box. Consensus binding sites for the transcription factors Sp1, AP-1, AP-2, GAF, NF-kappa B, and NF-AT were found. The elucidation of the human APO-1 gene structure will facilitate the study of its involvement in various diseases such as in autoimmunity.

Platelet-derived growth factor A-chain gene transcription is mediated by positive and negative regulatory regions in the promoter.

Platelet-derived growth factor (PDGF) is a disulphide-linked heterodimer of two polypeptide chains, the A and B chains, which are encoded by genes on separate chromosomes. The A-chain gene is transcribed in a number of transformed and non-transformed cell lines and is inducible by a wide variety of growth factors, cytokines and other mitogenic agonists. To localize DNA elements that mediate basal transcription in the promoter regulatory region of the A-chain gene, we have employed 5'-endpoint deletion mutagenesis and transient expression analysis in the renal epithelial cell line BSC-1 (African green monkey). Studies conducted in this cell line, which expresses high concentrations of PDGF A-chain mRNA, reveal a positive regulatory element (PRE) in a GC-rich stretch of the A-chain promoter between -82 and -40, relative to the transcription start site. Two discrete regions of the promoter were identified as negative regulatory elements (NREs), located between -1029 and -880 (NRE1) and between -1800 and -1029 (NRE2). The -1800 to -812 region, which contains both NREs, functions as a potent NRE when relocated in either orientation adjacent to the herpes simplex virus thymidine kinase promoter, reducing transcription activity by 60% in the positive orientation and 85% in the negative orientation. Comparison of BSC-1 cells and Saos-2 cells (human osteogenic sarcoma), which do not express significant quantities of PDGF A-chain mRNA or protein, indicates that basal transcription of the gene is determined by enhancer activity mediated by the GC-rich region rather than through de-repression of the upstream NREs. Electrophoretic gel mobility shift assays reveal a complex pattern of nuclear protein binding to the GC-rich PRE (-73 to -46). Competition studies conducted with mutant oligonucleotides that alternately disrupt consensus binding sites for Sp-1 or Egr-1 demonstrate a requirement for the presence of an Sp1-like core sequence (GGCGGG) but not Egr-1/Krox-24 [GCG(G/T)-GGGCG] for the formation of specific DNA-protein complexes. Our observations suggest that basal transcription of the A-chain gene in renal epithelial cells is achieved through active enhancement, mediated by a GC-rich PRE and nuclear proteins that bind to Sp-1-like consensus DNA sequences.

Activation of a heterologous promoter by human immunodeficiency virus type 1 Tat requires Sp1 and is distinct from the mode of activation by acidic transcriptional activators.

We have previously shown that the Tat protein of the human immunodeficiency virus type 1 (HIV-1) is a modular transcriptional activator that can be targeted upstream of either a synthetic promoter or the intact HIV promoter to activate transcription. This activation was shown to be largely dependent on the presence of consensus binding sites for the cellular transcription factor Sp1. Since the use of heterologous promoters may provide further insight into Tat-mediated transactivation, we have analyzed the transactivation of the thymidine kinase promoter of herpes simplex virus by Tat and by the acidic transcriptional transactivator VP16. The effects of mutations of defined upstream promoter elements show that Tat transactivation is dependent on Sp1 binding sites in a site-specific manner. In contrast, transactivation by the acidic transactivator VP16 is completely independent of any of the defined promoter elements upstream of the TATA box. These results suggest that Tat and the classically defined modular acidic transcriptional activators have different modes of transactivation. In addition, the substitution of the HIV-1 TATA box for the thymidine kinase TATA box substantially increases Tat transactivation, indicating that Tat transactivation may also ultimately involve TATA box-associated cellular transcription factors.

The heat stable antigen (mouse CD24) gene is differentially regulated but has a housekeeping promoter.

Expression of the mouse heat stable antigen (HSA or mouse CD24) shows tissue-specific as well as developmental regulation. During the maturation of several hematopoietic lineages, HSA expression is generally high in immature precursor cells and low or absent in terminally differentiated cells. We present evidence suggesting that this regulation of the HSA gene (Cd24a) occurs at the transcriptional level. In addition, sequence and methylation analysis of the Cd24a promoter revealed characteristics of both "housekeeping" and tissue-specific promoters, including a methylation-free, HpaII tiny fragment (HTF) island, multiple putative SP1 and AP-2 consensus binding sites, and a TATA box. Functional analysis of a 0.6-kilobase DNA fragment containing these elements fused to the CAT reporter gene in transient transfection experiments showed activity in both HSA expressing and non-expressing cell lines with a strength similar to that of the herpes-simplex virus-thymidine kinase promoter. Large fragments from the flanking region of the Cd24a promoter did not influence the ubiquitous nature of this promoter. Finally, we mapped the Cd24a, Cd24b, and Cd24c genes to mouse chromosomes 10, 8, and 14 respectively.

Determinants of the brain-specific expression of the rat aldolase C gene: ex vivo and in vivo analysis.

A 115-bp promoter fragment of the aldolase C gene is sufficient for conferring neural cell specificity on a reporter gene, in cultured PC12 cells and in transgenic mice. In vitro DNase I protection experiments detected two footprints on the promoter, termed boxes A/A', and B. The 5' A/A' box contains overlapping Sp1 and Krox20/Krox24 binding sites; it binds Sp1 in fibroblasts (box A') and a different complex in brain (box A). Any deletion or mutation of this box that impairs protein recognition also suppresses promoter activity. The replacement of box A/A' by a Sp1 consensus binding site results in the loss of the brain specificity of expression in transgenic mice. Further 3', box B is composed of a 5' direct repeat and a 3' GC box consisting of overlapping Sp1 and Krox20/Krox24 binding sites. Mutation of the direct repeat subregion appears to be more deleterious for the promoter activity than mutation of the G+C-rich subregion.

Cloning and Functional Analysis of the Mouse c-kit Promoter

The c-kit protooncogene encodes a tyrosine kinase receptor expressed during ontogeny and adult life by several important and developmentally distinct cell lineages. Mice carrying germ line c-kit mutations exhibit deficiencies in most of these lineages, demonstrating that c-kit function is necessary for their normal development. To facilitate the identification of cis-acting elements which regulate tissue-specific c-kit expression, we cloned and characterized a mouse c-kit promoter which is functional in different cell types. A major c-kit transcription initiation site (TIS), located 58 bp upstream from the translation initiation codon, is utilized in mouse mast cells and in c-kit-positive cells in the mouse cerebellum. The effects of deletions in the 5′ flanking region on reporter gene activity identify three short regulatory regions which function in both mouse and human c-kit positive cell lines. The nucleotide sequence of this region does not include CCAAT or TATA boxes but contains consensus binding sites for Sp1, Ap-2 and several short GA-rich elements which resemble binding sites for the ETS-domain proteins.

Multiple in vivo footprints are specific to the active allele of the X-linked human hypoxanthine phosphoribosyltransferase gene 5' region: implications for X chromosome inactivation.

Dosage compensation of X-linked genes in male and female mammals is accomplished by random inactivation of one X chromosome in each female somatic cell. As a result, a transcriptionally active allele and a transcriptionally inactive allele of most X-linked genes reside within each female nucleus. To examine the mechanism responsible for maintaining this unique system of differential gene expression, we have analyzed the differential binding of regulatory proteins to the 5' region of the human hypoxanthine phosphoribosyltransferase (HPRT) gene on the active and inactive X chromosomes. Studies of DNA-protein interactions associated with the transcriptionally active and inactive HPRT alleles were carried out in intact cultured cells by in vivo footprinting by using ligation-mediated polymerase chain reaction and dimethyl sulfate. Analysis of the active allele demonstrates at least six footprinted regions, whereas no footprints were detected on the inactive allele. Of the footprints on the active allele, at least four occur over canonical GC boxes or Sp1 consensus binding sites, one is associated with a potential AP-2 binding site, and another is associated with a DNA sequence not previously reported to interact with a sequence-specific DNA-binding factor. While no footprints were observed for the HPRT gene on the inactive X chromosome, reactivation of the inactive allele with 5-azacytidine treatment restored the in vivo footprint pattern found on the active allele. Results of these experiments, in conjunction with recent studies on the X-linked human PGK-1 gene, bear implications for models of X chromosome inactivation.