Inr Element Research Articles

Core promoter specificities of the Sp1 and VP16 transcriptional activation domains.

The core promoter compositions of mammalian protein-coding genes are highly variable; some contain TATA boxes, some contain initiator (Inr) elements, and others contain both or neither of these basal elements. The underlying reason for this heterogeneity remains a mystery, as recent studies have suggested that TATA-containing and Inr-containing core promoters direct transcription initiation by similar mechanisms and respond similarly to a wide variety of upstream activators. To analyze in greater detail the influence of core promoter structure on transcriptional activation, we compared activation by GAL4-VP16 and Sp1 through synthetic core promoters containing a TATA box, an Inr, or both TATA and Inr. Striking differences were found between the two activators, most notably in the relative strengths of the TATA/Inr and Inr core promoters: the TATA/Inr promoter was much stronger than the Inr promoter when transcription was activated by GAL4-VP16, but the strengths of the two promoters were more comparable when transcription was activated by Sp1. To define the domains of Sp1 responsible for efficient activation through an Inr, several Sp1 deletion mutants were tested as GAL4 fusion proteins. The results reveal that the glutamine-rich activation domains, which previously were found to interact with Drosophila TAF110, preferentially stimulate Inr-containing core promoters. In contrast, efficient activation through TATA appears to require additional domains of Sp1. These results demonstrate that activation domains differ in their abilities to function with specific core promoters, suggesting that the core promoter structure found in a given gene may reflect a preference of the regulators of that gene. Furthermore, the core promoter preference of an activation domain may be related to a specific mechanism of action, which may provide a functional criterion for grouping activation domains into distinct classes.

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.

Identification of an Upstream Region That Controls the Transcription of the Human Autocrine Motility Factor Receptor

We have isolated from a human placenta cosmid library a 0.7 kb genomic clone that contains the 5′ terminal portion of the autocrine motility factor receptor (hAMFR) coding region. Chloramphenicol Acetyl Transferase (CAT) reporter gene assays have identified this region as the promoter of the hAMFR gene. A single transcription initiation site (+1) has been mapped to 129 bp upstream of the ATG start codon by primer extension. DNA sequence analysis and CAT assay revealed a TATA element at the position -485/-468 which was able to conduct only a marginal transcription (less than 5% of the total activity). The majority of the hAMFR promoter′s activity is contributed by a transcription initiator (Inr) element overlapping the initiation site (+1) which independently controls the transcription of the hAMFR gene.

C-Myc Represses Transiently Transfected HLA Class I Promoter Sequences not Locus-Specifically

Overexpression of the c-myc oncogene is frequently accompanied by downregulation of Major Histocompatibility Complex (MHC, HLA in humans) class I antigens. In human melanoma c-myc overexpression downmodulates HLA-B expression, whereas HLA-A is hardly affected. Repression of HLA-B is mediated through the core promoter, containing a CAAT-box and a non-conventional TATA-box. We show evidence that in transient transfection assays the HLA-A2 and HLA-B7 promoters are repressed by cmyc to the same extent. Therefore, other sequences of the HLA-A and HLA-B genes, possibly intron/exon sequences, should contribute to the locus B-specificity of the downregulation. Furthermore, c-myc does not seem to alter binding of protein complexes to the CAAT- or TATA-box of HLA-B7 or HLA-A2 in gel retardation assays. Comparison of promoters repressed by c-myc reveals a weak consensus sequence of the initiator (Inr) element: TCA(+1)YYYNY. The presence of a TCA sequence in the initiator region of the MHC class I promoter makes downregulation by c-myc through the Inr likely. We speculate that the Inr contributes to MHC class I promoter activity by stimulating recruitment of TFIID to the weak, non-conventional TATA-box, thereby making it susceptible to repression by c-myc through the Inr.

Functional Characterization of the Promoter Region of the Platelet-activating Factor Receptor Gene.: IDENTIFICATION OF AN INITIATOR ELEMENT ESSENTIAL FOR GENE EXPRESSION IN MYELOID CELLS

To understand the molecular mechanisms that direct the expression of the gene encoding the platelet-activating factor (PAF) receptor, the 5'-flanking region of the human PAF receptor gene was cloned, and its promoter activity in myeloid cell lines was characterized. By the 5'-rapid amplification of cDNA ends method and primer extension, the transcription initiation site was mapped to an adenosine residue 137 bases upstream of the ATG translation initiation codon. The promoter region lacks a typical TATA or CCAAT box. However, the sequence encompassing the transcription initiation site shows high homology to the initiator (Inr) sequence. Transfection of promonocytic U937 cells with recombinant plasmids containing a series of truncated segments of the 5'-flanking region linked to the luciferase reporter gene revealed that the sequence from nucleotides -44 to +27 relative to the transcription initiation site was sufficient to promote a high level of gene expression. The promoter activity was much lower in nonexpressing HeLa cells and promyelocytic HL-60 cells, which express relatively low levels of the PAF receptor. Gel mobility shift analysis demonstrated the binding of nuclear factors extracted from myelocytic cells to the -16/+18 sequence containing the Inr element. No binding activity was detected using the nuclear extracts from the nonmyelocytic HeLa cells. The DNA-protein complexes were sequence-specific since the binding was not significantly affected by the mutated Inr sequences or the Inr sequence of the terminal deoxynucleotidyltransferase gene. Furthermore, point mutations in the Inr element significantly reduced promoter activity in both U937 and THP-1 cell lines. When Me2SO or retinoic acid was used to induce granulocytic differentiation of HL-60 cells, a distinct Inr-protein complex was induced concurrently, but the complex was not observed in 12-O-tetradecanoylphorbol-13-acetate-induced monocytic differentiated HL-60 cells or Me2SO-induced differentiated U937 cells, indicating that the inducible Inr binding activity is granulocyte-specific. These results suggest that distinct nuclear factors interact with the unique Inr element and play a role in the transcriptional regulation of the PAF receptor in various myeloid cells.

Structure and regulation of the luteinizing hormone receptor gene.

Studies of the mechanisms controlling the expression of the rat luteinizing hormone receptor gene were pursued by characterization of the gene structure and identification of regulatory protein binding domains in the 5'-non-coding region of the gene and of 3' non-coding functional domains responsible for generation of the major mRNA forms. The coding region of the rat LHR gene contains 10 introns and 11 exons, of which the first 10 exons comprise the hormone binding extracellular domain and exon 11, the seven transmembrane/G protein coupling module. Several alternative spliced variants of the LHR were identified that conform to deletions of complete and/or partial exons. Within the 6.2 kb of the 3'-non-coding region, two functional LHR pA domains (H1) and (H2) produce two sets of major mRNA transcripts, each coding for both holoreceptor and the form B splice variant. The H1 pA domain is unique to LHR and may represent a recombinant insertion domain. The functional efficiency of each pA domain is related to the specific pA signals, distal downstream elements, and tissue-specific factors. A TATA-less promoter region is present within the 173 bp 5'flanking region of the gene, with Initiator (Inr) elements at transcriptional start sites. Transcription is dependent on the binding of the Sp1 protein at two Sp1 domains that each contribute equally to transcript initiation. Promoter activity is regulated by at least three additional DNA domains, R (-1266 to -1307 bp), C-box (-42 to -73 bp) and M1 (-24 to -42 bp) that bind multiple trans-factors in a tissue-specific manner. Basal promoter activity is enhanced by a functional M1 domain in LHR-expressing mouse Leydig tumor cells (MLTC) but not in non-expressing CHO cells. C-box binding factors either inhibit promoter activity or block inhibition through overlapping but not identical DNA binding domains that carry AP-2 and NF-1 elements. Removal of the AP-2 element within the C-box results in MLTC-specific transcriptional activation that may involve an MTLC M1/C-box interaction. In addition, competition for C-box factors by an upstream regulatory element (R) that is only inhibitory in CHO cells, indicates that both C-box binding factors compete for this upstream (R) domain in a tissue-specific manner. Competition between the inhibitory and neutral DNA binding factors within both upstream (R) and promoter domains (C-box) could provide a mechanism for the control of LH receptor gene expression in gonadal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Cooperation between core promoter elements influences transcriptional activity in vivo.

Core promoters for RNA polymerase II frequently contain either (or both) of two consensus sequence elements, a TATA box and/or an initiator (Inr). Using test promoters consisting of prototypical TATA and/or Inr elements, together with binding sites for sequence-specific activators, we have analyzed the function of TATA and Inr elements in vivo. In the absence of activators, the TATA element was significantly more active than the Inr, and the combination of elements was only slightly more effective than the TATA-only promoter. In the presence of any of several coexpressed activator proteins, the TATA elements was again most active, but here addition of the Inr allowed significant increases in activity, indicating a cooperative interaction between the two elements. An interesting exception was observed with the activator Sp1, which was more effective with the Inr-only promoter, and addition of a TATA box did not enhance activity. Finally, in all cases the TATA plus Inr promoters were found to be partially or completely resistant to the dominant negative effects of a transcription factor TFIIB mutant previously shown to interfere with expression from TATA-only promoters. This result strengthens the conclusion that TATA and Inr elements can cooperate in vivo.

C-Myc represses transcription in vivo by a novel mechanism dependent on the initiator element and Myc box II.

We show that c-Myc, in addition to activating transcription through E-box Myc binding sites (Ems), also represses transcription by a mechanism dependent on initiator (Inr) elements of the basal promoters of susceptible genes. Repression was first observed as a component of c-Myc biphasic regulation of the adenovirus-2 major late promoter (MLP), which contains both Inr and Ems sequences. Two differentiation-specific genes containing Inr, the C/EBP alpha and albumin genes, are repressed through their basal promoters by c-Myc, but are activated by the related B-HLH-LZ factor, USF. Repression requires both the B-HLH-LZ and Myc box II (MBII) domains. Significantly, a MBII deletion mutant which is deficient in repression, but transactivates normally, fails to cooperate with an activated ras gene to transform primary fibroblasts. Thus Myc-dependent transactivation is insufficient for Ras cooperation and the novel transcription repression function is implicated in Ras cooperation as well as the suppression of Inr-dependent genes.

Cloning of a DNA binding protein that is a tyrosine kinase substrate and recognizes an upstream initiator-like sequence in the promoter of the preprodynorphin gene

A 90 bp fragment prepared from the promoter region of the rat preprodynorphin gene formed a complex with rat brain nuclear extracts as assessed by gel mobility shift assays. An 8 base pair sequence, CACTCTCC, termed upstream regulatory element (URE), was identified within this fragment as a binding site by DNase 1 footprint analysis and gel mobility shift assays with synthetic oligonucleotides. The URE is a consensus sequence for a transcription initiator (Inr) element although in the preprodynorphin promoter it is located upstream at -208 and overlaps a region conserved between rat and human promoters. A unique 310 amino acid protein (UreB1) that specifically bound the URE was cloned from a rat brain cDNA library using the URE-containing oligonucleotide. Recombinantly expressed, affinity purified UreB1 protein retains specific binding to the URE oligonucleotide. UreB1 contains a tyrosine kinase phosphorylation consensus and binding is enhanced following phosphorylation with the p43 v-abl tyrosine kinase. The UreB1 tyrosine phosphoprotein increases transcription in vitro, consistent with a positive transcriptional regulatory function. UreB1 transcripts are well expressed in subsets of neurons in multiple brain areas suggesting that, in addition to regulation of the preprodynorphin gene, it may have a more generalized role in gene transcription.

Direct recognition of initiator elements by a component of the transcription factor IID complex.

A core promoter element called an initiator (Inr) overlaps the transcription start site of numerous mammalian protein-coding genes. In promoters that lack a TATA box, the Inr is functionally analogous to TATA, in that it is capable of directing basal transcription by RNA polymerase II and of determining the precise site of transcription initiation. In promoters that contain a TATA box, the Inr can greatly enhance promoter strength. Mammalian Inr consensus sequences have been defined through functional studies and sequence comparisons of the start site regions of protein-coding genes. Here, we show that, in a DNase I footprinting assay with synthetic promoters, the purified TATA-binding protein complex TFIID specifically contacted the Inr. The TFIID-Inr interaction relies on the precise nucleotides needed for Inr function. Detection of the interaction was dependent either on a TATA box or on Sp1 bound to upstream sites. Furthermore, recombinant TFIIB appeared to influence the TFIID-Inr interaction, whereas TFIIA stabilized the TFIID-TATA interaction. These results demonstrate that distinct components of TFIID interact with the TATA boxes and Inr elements of core promoters for RNA polymerase II.

Expression of the carboxypeptidase E gene: Characterization of the initiator-binding proteins

Several of the genes for enzymes involved in peptide hormone processing, such as carboxypeptidase E (CPE), do not contain a TATA box. The region surrounding the major transcription initiation site of the CPE gene has sequence homology with the ‘initiator’ (Inr) elements of the TATA-less terminal deoxynucleotidyltransferase (TdT) gene, and the adenovirus major late (AdML) and other promoters. To investigate the promoter region of the CPE gene, GH 4C 1 cells were transiently transfected with constructs containing the luciferase reporter gene attached to various portions of the rat CPE gene (−395 to +45). Positive regulator elements were detected in positions −84 to −12 and +30 to +47. However, the Inr-like element of the CPE gene (−12 to +20) produced detectable luciferase activity in the absence of upstream and downstream sequences. This region of the CPE gene was much more active when expressed in the normal (sense) orientation than when expressed in the antisense orientation. A mutation within the consensus sequence between CPE and other Inr elements was much less active than the wild-type sequence. Interestingly, deletion of the Inr and surrounding sequences produced a large increase in the transcription from upstream sites, suggesting that proteins which bind at, or near, the Inr sequence suppress transcription from other sites. To characterize GH 4C 1 nuclear proteins which bind to the CPE gene, Southwestern blotting, UV cross-linking, and gel shift analyses were performed. The Southwestern analysis showed that the CPE and AdML Inr sequences labeled several proteins of similar sizes which are distinct from the transcription factor USF; this factor has been previously reported to bind to the AdML Inr sequence. A CPE Inr-binding protein co-purifies with an AdML Inr-binding protein on a CPE Inr affinity column. Gel shift assays showed that with some binding conditions, the proteins that bind to the CPE sequence also bind to the TdT and AdML Inr elements. Taken together, these results indicate that the −12 to +20 region of the CPE gene has the properties of an Inr element which binds some, but not all, of the factors which bind to other Inr elements.

DNA sequence requirements for transcriptional initiator activity in mammalian cells

A transcriptional initiator (Inr) for mammalian RNA polymerase II can be defined as a DNA sequence element that overlaps a transcription start site and is sufficient for (i) determining the start site location in a promoter that lacks a TATA box and (ii) enhancing the strength of a promoter that contains a TATA box. We have prepared synthetic promoters containing random nucleotides downstream of Sp1 binding sites to determine the range of DNA sequences that convey Inr activity. Numerous sequences behaved as functional Inrs in an in vitro transcription assay, but the Inr activities varied dramatically. An examination of the functional elements revealed loose but consistent sequence requirements, with the approximate consensus sequence Py Py A+1 N T/A Py Py. Most importantly, almost every functional Inr that has been described fits into the consensus sequence that we have defined. Although several proteins have been reported to bind to specific Inrs, manipulation of those elements failed to correlate protein binding with Inr activity. The simplest model to explain these results is that all or most Inrs are recognized by a universal binding protein, similar to the functional recognition of all TATA sequences by the same TATA-binding protein. The previously reported proteins that bind near specific Inr elements may augment the strength of an Inr or may impart transcriptional regulation through an Inr.

DNA sequence requirements for transcriptional initiator activity in mammalian cells.

A transcriptional initiator (Inr) for mammalian RNA polymerase II can be defined as a DNA sequence element that overlaps a transcription start site and is sufficient for (i) determining the start site location in a promoter that lacks a TATA box and (ii) enhancing the strength of a promoter that contains a TATA box. We have prepared synthetic promoters containing random nucleotides downstream of Sp1 binding sites to determine the range of DNA sequences that convey Inr activity. Numerous sequences behaved as functional Inrs in an in vitro transcription assay, but the Inr activities varied dramatically. An examination of the functional elements revealed loose but consistent sequence requirements, with the approximate consensus sequence Py Py A+1 N T/A Py Py. Most importantly, almost every functional Inr that has been described fits into the consensus sequence that we have defined. Although several proteins have been reported to bind to specific Inrs, manipulation of those elements failed to correlate protein binding with Inr activity. The simplest model to explain these results is that all or most Inrs are recognized by a universal binding protein, similar to the functional recognition of all TATA sequences by the same TATA-binding protein. The previously reported proteins that bind near specific Inr elements may augment the strength of an Inr or may impart transcriptional regulation through an Inr.

Direct role for Myc in transcription initiation mediated by interactions with TFII-I.

The nuclear proto-oncoprotein Myc has been implicated in the control of cell proliferation and differentiation. Myc participates in transcription and belongs to the basic-helix-loop-helix (bHLH) family of regulatory proteins. Here we show that Myc interacts with TFII-I, a transcription initiation factor that activates core promoters through an initiator element (Inr). As previously observed for the bHLH activator USF, Myc was found to interact cooperatively with TFII-I at both Inr and upstream E-box promoter elements. However, in this case Myc interactions with TFII-I at the Inr lead to an inhibition of transcription initiation. This inhibition is selective for a TFII-I-dependent (as opposed to TFIIA-dependent) initiation pathway and correlates with the prevention of complex formation between the TATA-binding protein TBP (TFIID tau), TFII-I and the promoter. TBP probably interacts with Myc, but only slowly. These observations indicate that Myc has the potential to interact physically and functionally with components of the general transcription machinery.

Evidence for physical interaction between the zinc-finger transcription factors YY1 and Sp1.

Two promoter elements are important for basal-level transcription, the TATA motif typically located 30 nucleotides upstream of the transcription initiation site and the initiator (Inr) element encompassing the start site. The mechanism of how Inr elements work is poorly understood, partly because very few proteins that bind to Inr elements have been identified and isolated. The recently cloned YY1 is such an Inr-binding protein. YY1 is able to direct transcription upon binding to its recognition sequence in vitro. The ability of YY1 to initiate transcription is augmented by the presence of a TATA motif or binding sites for transcription factor Sp1. To study the mechanism underlying the apparent functional cooperation between YY1 and Sp1, we explored the possibility of protein-protein interactions between these two transcription factors. We found that YY1 and Sp1 can form a physical complex. In addition, we identified domains within YY1 and Sp1 that mediate their interactions with each other. The physical interaction between YY1 and Sp1 may thus form the basis for the functional interplay observed previously.

Temporal Embryonic Expression of the Sea Urchin Early H1 Gene Is Controlled by Sequences Immediately Upstream and Downstream of the TATA Element

The sea urchin early histone H1 gene is expressed only during a very short interval in the life cycle of the organism. In addition to a pool of stored maternal transcripts, the gene is active from the 16-cell stage to the early blastula stage of development, at which time the gene is transcriptionally repressed. We have defined the minimal sequences required for the proper temporal expression of this gene during early embryogenesis. These sequences from -65 to +39 include the TATA element, a conserved sequence CCCACGTACGCAA (part of USE 0) just upstream of the TATA element, an Inr element at the transcription start site, and an internal sequence in the leader region of the H1 transcript. Surprisingly, we could find no role for two highly conserved elements, a GC box (USE I) and an H1-specific element (USE II), that are important in the expression of vertebrate H1 genes. The internal element functions as a positive regulator when three copies are ectopically placed upstream of an early histone H3 TATA box. A temporal regulator is localized within the USE 0 sequence CCCACGTACGCAA, and it apparently plays a role in activation and probably repression of the gene.

Human transcription factor USF stimulates transcription through the initiator elements of the HIV-1 and the Ad-ML promoters.

Earlier in vitro studies identified USF as a cellular factor which activates the adenovirus major late (Ad-ML) promoter by binding to an E-box motif located at position -60 with respect to the cap site. Purified USF contains 44 and 43 kDa polypeptides, and the latter was found (by cDNA cloning) to be a helix-loop-helix protein. In this report, we demonstrate a 25-to 30-fold stimulation of transcription via an upstream binding site by ectopic expression of the 43 kDa form of USF (USF43) in transient transfection assays. More recent data have also revealed alternate interactions of USF43 at pyrimidine-rich (consensus YYAYTCYY) initiator (Inr) elements present in a variety of core promoters. In agreement with this observation, we show here that USF43 can recognize the initiator elements of the HIV-1 promoter, as well as those in the Ad-ML promoter, and that ectopic expression of USF43 can stimulate markedly the corresponding core promoters (TATA and initiator elements) when analyzed in transient co-transfection assays. Mutations in either Inr 1 or Inr 2 reduced the USF43-dependent transcription activity in vivo. In addition, in vitro transcription assays showed that mutations in either or both of the Inr 1 and Inr 2 sequences of the HIV-1 and Ad-ML promoters could affect transcription efficiency, but not the position of the transcriptional start site. These results indicate that USF43 can stimulate transcription through initiator elements in two viral promoters, although the exact mechanism and physiological significance of this effect remain unclear.

Transcription by RNA polymerase II: initiator‐directed formation of transcription‐competent complexes 1

Studies of transcription by RNA polymerase II have revealed two promoter elements, the TATA motif and the initiator (Inr), capable of directing specific transcription initiation. Although binding to the TATA motif by one of the components of the transcription machinery has been shown to be the initial recognition step in transcription complex formation, many promoters that lack a traditional TATA motif have recently been described. In such TATA-less promoters, the Inr element is critical in positioning RNA polymerase II. Various Inr elements have been described and classified according to sequence homology. These Inr elements are recognized specifically by Inr-binding proteins. Interaction between these Inr-binding proteins and components of the basal transcription machinery provides a means through which a transcription competent complex can be formed.

Role of sequences within the first intron in the regulation of expression of eukaryotic initiation factor 2 alpha.

Resting human peripheral blood T cells synthesize proteins at very low rates and contain very low levels of eukaryotic initiation factor (eIF) 2 alpha mRNA. During mitogenic activation, the level of eIF-2 alpha mRNA increases at least 50-fold, an effect thought to be due primarily to intranuclear stabilization of the primary transcript (Cohen, R. B., Boal, T. R., and Safer, B. (1990) EMBO J. 9, 3831-3837). Analysis of sequences within the first intron revealed a region with homology to the "initiator" (Inr) sequence first described by Smale and Baltimore (Smale, S. T., and Baltimore, D. (1989) Cell 57, 103-113). This Inr element is positioned 450 bases downstream of the eIF-2 alpha promoter and is oriented to generate an overlapping antisense transcript. Deletion or mutation of the Inr element results in a reproducible 5-8-fold increase in the activity of an eIF-2 alpha promoter-driven CAT reporter gene and a corresponding 2.5-fold decrease in activity of an antisense driven luciferase reporter gene in vivo in 293 cells. In vitro transcription analysis also reveals antisense transcripts which depend on an intact Inr element and whose 5' ends map to sequences surrounding the Inr consensus sequence. A potential role for double-stranded RNA generated by these overlapping divergent transcription units in the regulation of eIF-2 alpha gene expression in T cells is suggested.

Identification of regulatory elements within the minimal promoter region of the human endogenous ERV9 proviruses: accurate transcription initiation is controlled by an Inr-like element.

ERV9 is a low repeated family of human endogenous retroviral elements whose expression is mainly detectable in undifferentiated embryonal carcinoma NT2/D1 cells. In this report we have analyzed the minimal promoter region located within the ERV9 LTR. Using the transient CAT expression assay we have identified the minimal promoter region, which includes sequences spanning from -70 to +6 relative to the major transcription start site. Deletion analysis, primer extension mapping of the transcription start sites and DNA-protein interactions assays have allowed us to define two important regions within the ERV9 minimal promoter. One region located between -70 to -39 acts as a transcriptional activating sequence and contains an Sp 1 binding site. The second region from -7 to +6, which resembles an initiator element (Inr), was necessary for the correct transcription start site utilization, and binds to a regulatory protein. Cross-competition experiments using various Inr elements have indicated that the protein that binds to the ERV9 Inr element can be competed by the HIV-1 and TdT Inr sequences.