Sp1 binds to the G allele of the-1087 polymorphism in the IL-10 promoter and promotes IL-10 mRNA transcription and protein production.

Interleukin (IL)-10 is an important cytokine in immune regulation and promotes B-cell proliferation and antibody production. High levels of IL-10 were found in subjects with autoimmune diseases. The A to G single nucleotide polymorphism at -1087 of the IL-10 promoter is associated with differences in promoter activity and IL-10 production. The objectives of this study were to analyze differences in the transcription factor binding to the -1087 IL-10 gene polymorphism in B-cells, the influence of the A to G transition on the IL-10 and Sp1 gene expression in B-cells after lipopolysaccharide (LPS) stimulation and the effect of knockdown of Sp1 on IL-10 gene expression. Using B-cell lines obtained from subjects with GG and AA genotypes for the -1087 polymorphism and chromatin immunoprecipitation assay, we showed that the transcription factors PU.1 and Spi-B bound to both G and A alleles, whereas the transcription factor Sp1 only bound to the G allele. LPS stimulation of the B-cells resulted in a larger increase in IL-10 and Sp1 gene expression for GG genotypes than AA genotypes and knockdown of Sp1 gene expression resulted in a decrease in IL-10 mRNA transcription. IL-10 production was higher for the GG genotype than for the AA genotype.

The Sp1 transcription factor binds to the G-allele of the -1087 IL-10 gene polymorphism and enhances transcriptional activation.

The objectives of this study were to evaluate the influence of the -1087 single nucleotide polymorphism (SNP) on the gene expression of interleukin (IL)-10 and to identify transcription factors binding to this site in B cells. Using electrophoretic mobility-shift assays and nuclear extract from the DG75 B-cell line, we demonstrated that the Sp1 transcription factor bound to the -1087 G-allele of the IL-10 promoter and that the transcription factors PU.1 and Spi-B bound to both the G- and A-alleles. Transient transfections showed that lipopolysaccharide stimulation resulted in a 15-fold increase in promoter activity for the G-allele as compared to a 6-fold increase for the A-allele. Co-transfection with Sp1 expression vector in Sp1-deficient SL2 cells leading to Sp1 binding to the G-allele of the -1087 SNP resulted in increased IL-10 promoter activity. The results suggest a role for Sp1 transcription factor in the activation of IL-10 through the G-allele of the -1087 SNP in response to inflammatory signals.

Functional interaction of Oct transcription factors with the family of repeats in Epstein-Barr virus oriP.

The family of repeats (FR) is a major upstream enhancer of the Epstein-Barr virus (EBV) latent C promoter (Cp) that controls transcription of six different latent nuclear proteins following interaction with the EBV nuclear protein EBNA1. Here, it was shown that Cp could also be activated by octamer-binding factor (Oct) proteins. Physical binding to the FR by the cellular transcription factors Oct-1 and Oct-2 was demonstrated by using an electrophoretic mobility-shift assay. Furthermore, Oct-1 in combination with co-regulator Bob.1, or Oct-2 alone, could drive transcription of a heterologous thymidine kinase promoter linked to the FR in both B cells and epithelial cells. Cp controlled by the FR was also activated by binding of Oct-2 to the FR. This may have direct implications for B cell-specific regulation of Cp.

Clinical mass spectrometry in neuroscience. Proteomics and peptidomics.

In this review we discuss the merits and drawbacks with the use of proteomic and peptidomic strategies for identification of proteins and peptides in their multidimensional interactions in complex biological processes. The progress in proteomics and peptidomics during the last years offer us new challenges to study changes in the protein and peptide synthesis. These strategies also offer new tools to follow post-translational modifications and other disturbed chemical processes that may be indicative of pathophysiological alteration(s). Furthermore these techniques can contribute to improvements in the diagnosis and therapy of neurodegenerative diseases, such as Alzheimer's disease, and psychiatric diseases, as depression and post traumatic stress disorders. We also consider different practical aspects of the applications of mass spectrometry in clinical neuroscience, illustrated by example from our laboratories. The new proteomic and peptidomic strategies will further enable the progress for clinical neuroscience research.

Increased frequency of a new polymorphism in the cell division cycle 2 (cdc2) gene in patients with Alzheimer's disease and frontotemporal dementia.

Recent studies show linkage between Alzheimer's disease (AD) and two loci on chromosome 10. The cell division cycle 2 (cdc2) gene is located close to one of the chromosome 10 markers, and is a candidate gene for AD since it is involved in the pathogenesis of AD. We sequenced coding exons and flanking intronic sequences and the promoter region on the cdc2 gene and found three new single nucleotide polymorphisms (SNPs). We analyzed 272 Caucasian AD cases, 160 controls and 70 cases with frontotemporal dementia (FTD) for these SNPs. Homozygosity for one of the SNPs (Ex6+7I/D) was more frequent in both AD and FTD cases than in controls. In the combined tauopathy (AD and FTD) group the odds ratio (OR) was 1.77 (95% CI 1.19-2.63) for the Ex6+7II genotype. Our findings suggest that the Ex6+7I allele is associated with tauopathies, both AD and FTD.

The transcobalamin codon 259 polymorphism influences the risk of human spontaneous abortion.

BACKGROUND: The remethylation cycle of methionine is folate and vitamin B(12) (cobalamin) dependent and appears to be crucial for embryonic development, probably through effects on synthesis of DNA, proteins and polyamines. Transcobalamin (TC) transports vitamin B(12) to the tissues. The objective of the present investigation was to explore the putative association between the major TC genetic polymorphism (Pro259Arg) and human spontaneous abortion. METHODS: The prevalence of the TC Pro259Arg polymorphism was determined in DNA samples from embryos that had been spontaneously aborted between the 6th and 20th week after conception, and adult controls using solid-phase minisequencing technique. RESULTS: The 259-Pro allele was significantly less frequent in the spontaneous abortion group than in the control group (42.2 and 57.0% respectively; P = 0.005), while the frequency of 259-Arg was significantly increased. There was a lower prevalence of 259-Pro homozygotes in the spontaneous abortion group compared with the control group (9.1 and 32.2% respectively; P < 0.001). CONCLUSIONS: The 259-Pro allele seems to have beneficial influences during embryogenesis, conceivably through its positive effect on vitamin B(12) intracellular bioavailability. Our results warrant additional investigations addressing the question if vitamin B(12) supplementation in addition to folic acid supplementation may prevent spontaneous abortion in women planning a pregnancy.

Promoter-proximal regulatory elements involved in oriP-EBNA1-independent and -dependent activation of the Epstein-Barr virus C promoter in B-lymphoid cell lines.

The identification of the cellular factors that control the transcription regulatory activity of the Epstein-Barr virus C promoter (Cp) is fundamental to the understanding of the molecular mechanisms that control virus latent gene expression. Using transient transfection of reporter plasmids in group I phenotype B-lymphoid cells, we have previously shown that the -248 to -55 region (-248/-55 region) of Cp contains elements that are essential for oriPI-EBNA1-dependent as well as oriPI-EBNA1-independent activation of the promoter. We now establish the importance of this region by a detailed mutational analysis of reporter plasmids carrying Cp regulatory sequences together with or without oriPI. The reporter plasmids were transfected into group I phenotype Rael cells and group III phenotype cbc-Rael cells, and the Cp activity measured was correlated with the binding of candidate transcription factors in electrophoretic mobility shift assays and further assessed in cotransfection experiments. We show that the NF-Y transcription factor interacts with the previously identified CCAAT box in the -71/-63 Cp region (M. T. Puglielli, M. Woisetschlaeger, and S. H. Speck, J. Virol. 70:5758-5768, 1996). We also show that members of the C/EBP transcription factor family interact with a C/EBP consensus sequence in the -119/-112 region of Cp and that this interaction is important for promoter activity. A central finding is the identification of a GC-rich sequence in the -99/-91 Cp region that is essential for oriPI-EBNA1-independent as well as oriPI-EBNA1-dependent activity of the promoter. This region contains overlapping binding sites for Sp1 and Egr-1, and our results suggest that Sp1 is a positive and Egr-1 is a negative regulator of Cp activity. Furthermore, we demonstrate that a reporter plasmid that in addition to oriPI contains only the -111/+76 region of Cp still retains the ability to be activated by EBNA1.

Quantitative analysis of specific mRNA species in minute cell samples by RT-PCR and flow cytometry.

A method for the quantitative determination of specific mRNAs in small numbers of cells, freshly isolated from tissues or early cell cultures, was developed by combining quantitative reverse transcription-polymerase chain reaction (RT-PCR) and quantitative flow cytometry. Freshly isolated umbilical vein endothelial cells were sorted by flow cytometry and then lysed. The number of cells in the lysate was determined by counting of nuclei after propidium iodide staining using flow cytometry. The number of plasminogen activator inhibitor-1 (PAI-1) mRNA copies per cell was determined by quantitative RT-PCR using point-mutated PAI-1 cRNA as an internal standard. The cells were shown to contain 400-900 copies of PAI-1 mRNA molecules per cell which confirms that endothelial cells in vivo express PAI-1. PAI-1 mRNA expression was also analyzed in small numbers of endothelial cells in primary culture in basal conditions and after incubation with different interleukins. The method allowed reliable and reproducible estimation of the number of mRNA copies per cell from original cell samples containing less than 1000 cells. This method can be used for the quantitative determination of various mRNA species in specified cell populations from small tissue samples or cultured cells.

No association between the alpha2-macroglobulin (A2M) deletion and Alzheimer's disease, and no change in A2M mRNA, protein, or protein expression.

A polymorphism consisting of a deletion near the 5' splice site of exon 18 on the alpha2-macroglobulin (A2M) gene (A2M-2) has been suggested to be associated with Alzheimer's disease (AD) in family-based studies. We studied the A2M-2 allele together with the ApoE alleles in a large series on patients with AD (n = 449) and age-matched controls (n = 349). Neuropathologically confirmed diagnoses were available in 199 cases (94 AD and 107 control cases). We found no increase in A2M-2 genotype or allele frequencies in AD (27.5% and 14.6%) versus controls (26.4% and 14.9%). In contrast, a marked increase (p < 0.0001) in ApoE epsilon4 genotype or allele frequencies was found in AD (66.6% and 41.2%) as compared with controls (29.8% and 16.5%), suggesting sufficient statistical power in our sample. No relation was found between the A2M-2 and the ApoE epsilon4 allele. No change in A2M exon 17-18 mRNA size or sequence or A2M protein size was found in cases carrying the A2M-2 deletion, suggesting that there is no biological consequences of the A2M intronic deletion. No change in A2M protein level in cerebrospinal fluid was found in AD, suggesting that the A2M-2 allele does not effect the A2M protein expression in the brain. The lack of an association between the A2M-2 allele and AD in the present study, and the lack of abnormalities in the A2M mRNA or protein suggest that the A2M-2 allele is not associated with AD.

Angiotensin-converting enzyme gene I/D polymorphism in malignant hypertension.

BACKGROUND: The mechanism of the rapid transition of a stable benign hypertensive disease to a severe and devastating malignant hypertension is not fully understood. However, the renin angiotensin system, which is highly activated in malignant hypertension, is established as an important pathogenetic factor in different cardiovascular and renal diseases. Over the last decade, a polymorphism in genes regulating this system has been found. This includes the 287 bp sequence deletion (D)/insertion (I) polymorphism in the angiotensin-converting enzyme (ACE) gene and the methionine (M) to threonine (T) point mutation polymorphism in the angiotensinogen (AGT) gene. These gene polymorphisms have been associated with various cardiovascular and renal diseases and the aim of this study was to investigate whether they were linked to malignant hypertension. METHODS: Forty-two patients with malignant hypertension (mean age 55 years), 42 patients with non-malignant hypertension (mean age 57 years) and 85 normotensive control subjects (mean age 42 years) were investigated with respect to ACE I/D and AGT M/T genotypes. DNA was prepared by standard methods from isolated white blood cells and analysed by the PCR technique. The PCR reaction used in the detection of the ACE I/D polymorphism was optimized for an equal amplification of the I and D alleles. RESULTS: The frequency of the DD genotype was significantly increased in patients with malignant hypertension (43%) compared with patients with non-malignant hypertension (14%) and normotensive control subjects (18%) (p <0.01) for both. The frequency distribution of AGT M/T genotype did not differ between patients with malignant and non-malignant hypertension. CONCLUSION: The DD genotype of the ACE gene occurred more than twice as often in malignant hypertension than in non-malignant hypertension and indicates that ACE gene polymorphism is a significant risk factor for initiation of malignant hypertension.

Angiotensin I-converting enzyme gene polymorphism in non-diabetic renal disease.

BACKGROUND: The insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene determines the concentration of ACE in serum and local tissues. The role of this polymorphism in progressive chronic renal disease is still not fully clear. METHODS: We analysed the impact of the D/D polymorphism on the rate of decline in renal function in patients with non-diabetic, chronic progressive renal insufficiency. Seventy non-diabetic patients, aged 21-69 years at baseline, with moderately advanced renal insufficiency due to primary chronic renal disease were followed for an average of 3 years with repeated measurements of their glomerular filtration rate (GFR). Their mean GFR at baseline was 41 ml/min/1.73 m(2) body surface area (BSA). The polymerase chain reaction (PCR) amplification method was used to detect the I/D polymorphism of the ACE gene. GFR was measured as the clearance of (51)Cr-EDTA and the individual rate of progression was calculated using linear regression. RESULTS: The distributions of the genotypes were: D/D 30%, I/D 49%, and I/I 21%. The rates of progression in the three ACE genotype groups were an annual decline in renal function of -4.2 (SD 4.6) ml/minx1.73 m(2) BSA in the D/D group, -2.7 (SD 3. 4) in the I/D group and -1.7 (SD 3.4) in the I/I group (ANOVA P=0. 12). In patients with proteinuria below 3.5 g/24 h, the D/D group had a significantly higher rate of progression than patients with the I allele. The same was found in a separate analysis when only patients with normal apoliprotein B (below 155 mg/dl) levels were analysed. Furthermore, the D/D genotype was a significant predictor of a more rapid decline in renal function in male, but not female, patients. CONCLUSION: The results in this study in non-diabetic patients with chronic renal disease indicate that the presence of the D allele in the ACE genotype may be of particular importance as a predictor of a high rate of progression in male patients who otherwise do not have a major burden of documented and important prognostic factors for progressive renal insufficiency.

Relative levels of EBNA1 gene transcripts from the C/W, F and Q promoters in Epstein-Barr virus-transformed lymphoid cells in latent and lytic stages of infection.

Four promoters, Cp, Wp, Fp and Qp, are known to participate in transcription of the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) gene in EBV-infected cell lines. The promoters are used differentially during the different phases of infection and establishment of the stages of latency. This has raised questions about the regulation of the promoters and the molecular mechanisms underlying the switches between them. To obtain a measure of the activity of the different EBNA1 transcription units in EBV-transformed cell lines of different phenotypes, RNA probes were constructed that allowed the detection and relative quantification, by RNase protection analysis, of EBNA1 transcripts initiated at Fp and Qp and, in an indirect manner, Cp/Wp. RNase protection and PCR assays were performed with cytoplasmic RNA from B-lymphoid cell lines in latency stages I, II-III and III and after induction of the virus lytic cycle. The experiments demonstrated that, in addition to previously identified EBNA1 transcripts, cell lines of all latency types also contained different mRNAs that carried sequences from the EBNA1-encoding K exon. Induction of the virus lytic cycle resulted in low levels of an FpQ/U/K-spliced transcript. However, there was a large increase of FpQ- and FpQ/U-spliced transcripts with unknown 3' sequences. Furthermore, a new transcript, initiated at an unidentified site 5' of the BamHI f/K cleavage site and continuing through BamHI K into the EBNA1-encoding K exon without interruption, was produced in substantial amounts in the lytic cycle.

Silencing of the Epstein-Barr virus latent membrane protein 1 gene by the Max-Mad1-mSin3A modulator of chromatin structure.

The tumor-associated latent membrane protein 1 (LMP1) gene in the Epstein-Barr virus (EBV) genome is activated by EBV-encoded proteins and cellular factors that are part of general signal transduction pathways. As previously demonstrated, the proximal region of the LMP1 promoter regulatory sequence (LRS) contains a negative cis element with a major role in EBNA2-mediated regulation of LMP1 gene expression in B cells. Here, we show that this silencing activity overlaps with a transcriptional enhancer in an LRS sequence that contains an E-box-homologous motif. Mutation of the putative repressor binding site relieved the repression both in a promoter-proximal context and in a complete LRS context, indicating a functional role of the repressor. Gel retardation assays showed that members of the basic helix-loop-helix transcription factor family, including Max, Mad1, USF, E12, and E47, and the corepressor mSin3A bound to the E-box-containing sequence. The enhancer activity correlated with the binding of USF. Moreover, the activity of the LMP1 promoter in reporter constructs was upregulated by overexpression of USF1 and USF2a, and the transactivation was inhibited by the concurrent expression of Max and Mad1. This suggests that Max-Mad1-mediated anchorage of a multiprotein complex including mSin3A and histone deacetylases to the E-box site constitutes the basis for the repression. Removal of acetyl moieties from histones H3 and H4 should result in a chromatin structure that is inaccessible to transcription factors. Accordingly, inhibition of deacetylase activity with trichostatin A induced expression of the endogenous LMP1 gene in EBV-transformed cells.

Upregulation of Epstein-Barr virus-encoded latent membrane protein by human herpesvirus 6 superinfection of EBV-carrying Burkitt lymphoma cells.

The effect of HHV-6 strain A infection on the expression of Epstein-Barr virus- (EBV-) encoded growth transformation-associated genes in two EBV-positive Burkitt lymphoma cell lines, Akata and P3HR-3, was investigated. The results indicate that HHV-6A upregulates the expression of the latent membrane protein LMP-1 in both cell lines. Expression of EBNA-2 was also upregulated in Akata cells following HHV-6A infection. Transfection of reporter constructs carrying the LMP-1 regulatory sequences (LRS; -634/+40) or its 5' deleted derivatives in Akata and in a T-lymphoblastoid cell line, J-Jhan, confirmed the presence of positive and negative regulatory elements responsive to HHV-6A infection in LMP-1 regulatory sequence (LRS). The majority of LRS constructs were under the influence of dominant negative factors. HHV-6A was able to override the effect of such factors acting on reporter plasmids containing the -634/-54, -324/-54, -214/-54, and -106/-54 parts of LRS. The plasmid that carried only the -54/+40 LRS region was constitutively active in both Akata and J-Jhan cells; in Akata, its activity was influenced by HHV-6A. The finding that HHV-6A infection may activate LMP-1 and EBNA-2 expression, which is essential for the immortalization of B-lymphocytes by EBV, shows a novel aspect of the interaction between these two herpesviruses.

An ATF/CRE element mediates both EBNA2-dependent and EBNA2-independent activation of the Epstein-Barr virus LMP1 gene promoter.

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is a viral oncogene whose expression is regulated by both viral and cellular factors. EBV nuclear antigen 2 (EBNA2) is a potent transactivator of LMP1 expression in human B cells, and several EBNA2 response elements have been identified in the promoter regulatory sequence (LRS). We have previously shown that an activating transcription factor/cyclic AMP response element (ATF/CRE) site in LRS is involved in EBNA2 responsiveness. We now establish the importance of the ATF/CRE element by mutational analysis and show that both EBNA2-dependent activation and EBNA2-independent activation of the promoter occur via this site but are mediated by separate sets of factors. An electrophoretic mobility shift assay (EMSA) with specific antibodies showed that the ATF-1, CREB-1, ATF-2 and c-Jun factors bind to the site as ATF-1/CREB-1 and ATF-2/c-Jun heterodimers whereas the Sp1 and Sp3 factors bind to an adjacent Sp site. Overexpression of ATF-1 and CREB-1 in the cells by expression vectors demonstrated that homodimeric as well as heterodimeric forms of the factors transactivate the LMP1 promoter in an EBNA2-independent manner. The homodimers of ATF-2 and c-Jun did not significantly stimulate promoter activity. In contrast, the ATF-2/c-Jun heterodimer had only a minor stimulatory effect in the absence of EBNA2 but induced a strong transactivation of the LMP1 promoter when coexpressed with this protein. Evidence for a direct interaction between the ATF-2/c-Jun heterodimeric complex and EBNA2 was obtained by EMSA and coimmunoprecipitation experiments. Thus, our results suggest that EBNA2-induced transactivation via the ATF/CRE site occurs through a direct contact between EBNA2 and an ATF-2/c-Jun heterodimer. EBNA2-independent promoter activation via this site, on the other hand, is mediated by a heterodimeric complex between the ATF-1 and CREB-1 factors.

Human 4-hydroxyphenylpyruvate dioxygenase gene (HPD).

Overlapping DNA fragments spanning approximately 21 kb of genomic DNA and encompassing the human 4-hydroxyphenylpyruvate dioxygenase gene (HPD) have been cloned by screening a human leukocyte genomic library and by PCR amplification of human fibroblastic DNA. A continuous gene sequence of 20,890 nucleotides was established, including 1957 bp of the 5'-flanking region. The 4-hydroxyphenylpyruvate dioxygenase gene is composed of 14 exons interrupted by 13 introns, all exhibiting conventional vertebrate splicing. Computer analysis of the DNA sequence revealed 12 complete repetitive Alu elements, 1 in the 5'-flanking region and 11 in the intervening segments of the gene. The transcriptional initiation site was mapped to a position 35 nt upstream of the translational start point. The computer analysis also identified several potential transcription regulatory elements, including one CRE site, two AP-2 sites, and two Sp1 sites, in the sequence upstream of the transcription initiation site. Functional analysis of promoter activity by transient transfection of chloramphenicolacetyl transferase reporter plasmids revealed a possible involvement of cyclic adenosine monophosphate in the regulation of transcription. The highest level of expression of 4-hydroxyphenylpyruvate dioxygenase was found in human liver tissue as demonstrated by Northern blot analysis.

B cell-specific activation of the Epstein-Barr virus-encoded C promoter compared with the wide-range activation of the W promoter.

We have studied the activity of reporter plasmids, carrying the Epstein-Barr virus (EBV) nuclear antigen (EBNA) promoters Wp and Cp, in a group of somatic cell hybrids obtained by fusing EBV-positive lymphoblastoid cell lines or group II/III Burkitt's lymphoma cell lines with non-B cell lines. In B/non-B cell hybrids of this type, B cell markers are extinguished as a rule, in parallel with the inactivation of Cp or Wp and down-regulation of EBNA-2-6 expression. A Wp-carrying reporter construct was active in non-B cell lines. Only cells with a B cell phenotype could support the activity of Cp-carrying plasmids. EBNA-2 transactivated Cp only in B cells. Our data suggest that while Wp can be used for EBNA transcription in B and non-B cells, Cp activity is restricted to B cells. The inability of EBNA-2 to transactivate Cp in non-B cells indicates that other factors present in B cells might be involved in Cp transactivation.

Expression of Epstein-Barr nuclear antigen 2 in kidney tubule cells induce tumors in transgenic mice.

The effect of EBNA2 in normal cells in vivo has not as yet been explored. The experiments described here were initiated to follow the consequences of the expression of EBNA2 in different tissues in transgenic mice. EBNA2 transgenic strains were generated using a vector containing EBNA2 encoding sequences under the control of the simian virus 40 (SV 40) early enhancer/promoter fused to the endogenous EBNA2 Wp promoter. Control mice carrying a transgene with the same sequence but lacking the EBV DNA part remained healthy during observation periods of up to 15 months. The SV-EBNA2 transgenic animals, however, over time developed abdominal masses that on necropsy showed to be due to kidney tumors. Histological examination revealed the presence of tumors with the morphology of kidney adenocarcinoma with a solid growth pattern. At the age of 20 weeks the kidneys of all animals investigated showed disseminated islands of tubular hyperplasia but no true malignant neoplasms. At about 50 weeks of age multiple foci of microscopic tubular adenocarcinomas were found in both kidneys. Eventually, tumors could be diagnosed in about 90% of the SV-EBNA2 transgenic mice. EBNA2-encoding RNA was expressed in both non-malignant kidney tissue and in tumors as shown by cDNA/PCR analysis. Immunoprecipitation and immunoblot analysis showed that the tumor cells contained a polypeptide of the same size as EBNA2 in B95-8 cells that reacted with a monoclonal anti-EBNA2 antibody. Immunohistochemistry demonstrated nuclear expression of EBNA2 in hyperplastic tubules and in tumor tissue.

PU box-binding transcription factors and a POU domain protein cooperate in the Epstein-Barr virus (EBV) nuclear antigen 2-induced transactivation of the EBV latent membrane protein 1 promoter.

Expression of the Epstein-Barr virus (EBV) latent membrane protein (LMP1) is regulated by virus- and host cell-specific factors. The EBV nuclear antigen 2 (EBNA2) has been shown to transactivate a number of viral and cellular gene promoters including the promoter for the LMP1 gene. EBNA2 is targeted to at least some of these promoters by interacting with a cellular DNA binding protein, RBP-J kappa. In the present report we confirm and extend our previous observation that the LMP1 promoter can be activated by EBNA2 in the absence of the RBP-J kappa-binding sequence in the LMP1 promoter regulatory region (LRS). We show that two distinct LRS regions, -106 to +40 and -176 to -136, contribute to EBNA2 responsiveness. Site-directed mutagenesis analysis of the upstream -176/-136 EBNA2 responsive element revealed that two critical cis-acting elements are required for full promoter function. These same elements analysed by electrophoretic mobility shift assays define two binding sites recognized by nuclear factors derived from B cells. An octamer-like sequence (-147 to -139) contained overlapping binding sites for an unidentified transcriptional repressor on the one hand and a factor(s) belonging to the POU domain family but distinct from Oct-1 and Oct-2 on the other. An adjacent purine tract (-171 to -155) held a PU.1 binding site, which was also recognized by a related factor. The results suggest that the POU domain protein and either of two PU box-binding factors bind simultaneously to LRS, creating a ternary complex that might be in part responsible for mediating the transactivation of the LMP1 promoter by EBNA2. There were no qualitative differences between EBV-negative and EBV-positive cells with regard to transcription factor binding to the octamer-like sequence and the PU.1 recognition site, as revealed by electrophoretic mobility shift assays.