Identification des réseaux transcriptionnnels de résistance aux antifongiques chez Candida albicans

Plusieurs souches cliniques de Candida albicans résistantes aux médicaments antifongiques azolés surexpriment des gènes encodant des effecteurs de la résistance appartenant à deux classes fonctionnelles : i) des transporteurs expulsant les azoles, CDR1, CDR2 et MDR1 et ii) la cible des azoles 14-lanostérol déméthylase encodée par ERG11. La surexpression de ces gènes est due à la sélection de mutations activatrices dans des facteurs de transcription à doigts de zinc de la famille zinc cluster (Zn2Cys6) qui contrôlent leur expression : Tac1p (Transcriptional activator of CDR genes 1) contrôlant l’expression de CDR1 et CDR2, Mrr1p (Multidrug resistance regulator 1), régulant celle de MDR1 et Upc2p (Uptake control 2), contrôlant celle d’ERG11. Un autre effecteur de la résistance clinique aux azoles est PDR16, encodant une transférase de phospholipides, dont la surexpression accompagne souvent celle de CDR1 et CDR2, suggérant que les trois gènes appartiennent au même régulon, potentiellement celui de Tac1p. De plus, la régulation transcriptionnelle du gène MDR1 ne dépend pas seulement de Mrr1p, mais aussi du facteur de transcription de la famille basic-leucine zipper Cap1p (Candida activator protein 1), un régulateur majeur de la réponse au stress oxydatif chez C. albicans qui, lorsque muté, induit une surexpression constitutive de MDR1 conférant la résistance aux azoles. Ces observations suggèrent qu’un réseau de régulation transcriptionnelle complexe contrôle le processus de résistance aux antifongiques azolés chez C. albicans. L’objectif de mon projet au doctorat était d’identifier les cibles transcriptionnelles directes des facteurs de transcription Tac1p, Upc2p et Cap1p, en me servant d’approches génétiques et de génomique fonctionnelle, afin de i) caractériser leur réseau transcriptionnel et les modules transcriptionnels qui sont sous leur contrôle direct, et ii) d’inférer leurs fonctions biologiques et ainsi mieux comprendre leur rôle dans la résistance aux azoles. Dans un premier volet, j’ai démontré, par des expériences de génétique, que Tac1p contrôle non seulement la surexpression de CDR1 et CDR2 mais aussi celle de PDR16. Mes résultats ont identifié une nouvelle mutation activatrice de Tac1p (N972D) et ont révélé la participation d’un autre régulateur dans le contrôle transcriptionnel de CDR1 et PDR16 dont l’identité est encore inconnue. Une combinaison d’expériences de transcriptomique et d’immunoprécipitation de la chromatine couplée à l’hybridation sur des biopuces à ADN (ChIP-chip) m’a permis d’identifier plusieurs gènes dont l’expression est contrôlée in vivo et directement par Tac1p (PDR16, CDR1, CDR2, ERG2, autres), Upc2p (ERG11, ERG2, MDR1, CDR1, autres) et Cap1p (MDR1, GCY1, GLR1, autres). Ces expériences ont révélé qu’Upc2p ne contrôle pas seulement l’expression d’ERG11, mais aussi celle de MDR1 et CDR1. Plusieurs nouvelles propriétés fonctionnelles de ces régulateurs ont été caractérisées, notamment la liaison in vivo de Tac1p aux promoteurs de ses cibles de façon constitutive et indépendamment de son état d’activation, et la liaison de Cap1p non seulement à la région du promoteur de ses cibles, mais aussi celle couvrant le cadre de lecture ouvert et le terminateur transcriptionnel putatif, suggérant une interaction physique avec la machinerie de la transcription. La caractérisation du réseau transcriptionnel a révélé une interaction fonctionnnelle entre ces différents facteurs, notamment Cap1p et Mrr1p, et a permis d’inférer des fonctions biologiques potentielles pour Tac1p (trafic et la mobilisation des lipides, réponse au stress oxydatif et osmotique) et confirmer ou proposer d’autres fonctions pour Upc2p (métabolisme des stérols) et Cap1p (réponse au stress oxydatif, métabolisme des sources d’azote, transport des phospholipides). Mes études suggèrent que la résistance aux antifongiques azolés chez C. albicans est intimement liée au métabolisme des lipides membranaires et à la réponse au stress oxydatif.

Catalase-peroxidase activity is decreased in a Caulobacter crescentus rho mutant

A Caulobacter crescentus rho:Tn5 mutant strain presenting a partially functional transcription termination factor Rho is highly sensitive to hydrogen peroxide in both exponential and stationary phases. The mutant was shown to be permanently under oxidative stress, based on fluorophore oxidation, and also to be sensitive to tert-butyl hydroperoxide and paraquat. However, the results showed that the activities of superoxide dismutases CuZnSOD and FeSOD and the alkylhydroperoxide reductase ahpC mRNA levels in the rho mutant were comparable to the wild-type control in the exponential and stationary phases. In contrast, the KatG catalase activity of the rho mutant strain was drastically decreased and did not show the expected increase in the stationary phase compared with the exponential phase. Transcription of the katG gene was increased in the rho mutant and the levels of the immunoreactive KatG protein do not differ considerably compared with the wild type in the stationary phase, suggesting that KatG activity is affected in a translational or a post-translational step.

Characterization of hLodestar: A CDC5L interacting protein

Lodestar, a Drosophila maternal-effect gene, is essential for proper chromosome segregation during embryonic mitosis. Mutations in lodestar cause chromatin bridging in anaphase, preventing the sister chromatids from fully separating and leaving chromatin tangled at the metaphase plate. Drosophila lodestar protein was originally identified, in purified fractions of Drosophila Kc cell nuclear extracts, by its ability to suppress the generation of long RNA polymerase II transcripts. The human homolog of this protein (hLodestar) was cloned and studied in comparison to the Drosophila lodestar activities. The results of these studies show, similar to the Drosophila protein, hLodestar has dsDNA-dependent ATPase and transcription termination activity in vitro. hLodestar has also been shown to release RNA polymerase I and II stalled at a cyclobutane thymine dimer. Lodestar belongs to the SNF2 family of proteins, which are members of the DExH/D helicase super-family. The SNF2 family of proteins are believed to play a critical role in altering protein-DNA interactions in a variety of cellular contexts. We have recently isolated a human cDNA (hLodestar) that shares significant homology to the Drosophila lodestar gene. The 4.6 kb clone contains an open reading frame of 1162 amino acids, and shares 55% similarity and 46% identity to the Drosophila Lodestar protein sequence. Our studies looking for hLodestar interacting proteins revealed an association with CDC5L in the yeast two-hybrid system and co-immunoprecipitation experiments. CDC5L has been well documented to be a component of the spliceosome. Our data suggests hLodestar is involved in splicing through in vitro assembly and splicing reactions, in addition to its association with spliceosomes purified from HeLa nuclear extract. Although many other members of the DExH/D helicase super-family have been linked to splicing, this is the first SNF2 family member to be implicated in the splicing reaction. ^

Stability and potential application of spliced nuclear pre -mRNA introns

A major portion of this thesis work was dedicated to study the nature and significance of spliced introns. The initial work was focused on studying the IVS1$\sb{\rm C\beta 1}$ intron from a T-cell receptor (TCR)-$\beta$ gene. Compared to an intron lariat control from adenovirus pre-mRNA that was spliced in vitro, IVS1$\sb{\rm C\beta 1}$ was debranched less efficiently by HeLa S100 extracts, although IVS1$\sb{\rm C\beta 1}$ also used the consensus branchpoint in vivo. Subcellular-fractionation analysis showed that most IVS1$\sb{\rm C\beta 1}$ lariats cofractionated with pre-mRNA in the nucleus, consistent with the possibility that intron degradation releases splicing factors which will be available for further rounds of splicing. The half-life of IVS1$\sb{\rm C\beta 1}$ from the endogenous TCR-$\beta$ gene was measured using the general transcription inhibitor actinomycin D to be about $\sim$15 min, which was similar to that of unstable mRNAs such as c-myc mRNA.^ The general transcription inhibitor DRB was also used for intron stability analysis. Unexpectedly, DRB decreased intron and pre-mRNA levels only initially, it later increased the levels of intron-containing RNAs. Inhibition of transcription initiation appeared to be the major early effect (the reduction phase); whereas enhanced premature transcription termination was dominant later (the induction phase).^ Having established the procedures for studying in vivo spliced introns, this approach was applied to study the mechanism of nonsense-mediated downregulation (NMD), a phenomena in which premature termination codons (PTCs) decrease the levels of mRNAs. In this study, the novel intron-oriented approach was applied to study the mechanism of NMD. The levels of spliced introns immediately upstream and downstream of a PTC-bearing exon in a TCR-$\beta$ gene were identified and analyzed along with their pre-mRNA. Although PTC reduced the mRNA levels by 4 to 9 fold, the steady-state levels of spliced introns and the pre-mRNA-to-intron ratios were not significantly altered, indicating that the PTC did not significantly inhibit TCR-$\beta$ RNA splicing. Consistent with this conclusion, the half-lives of the PTC$\sp+$ and PTC$\sp-$ pre-mRNA were similar. The protein synthesis inhibitor cyclohexmide (CHX) upregulated the levels of the PTC$\sp+$ mRNA over 10 fold without affecting the levels of the spliced introns, suggesting that the reversal effect of CHX was through stabilization, not production. These results indicated that inhibition of splicing could not be the major mechanism for the NMD pathway of the TCR-$\beta$ gene, instead, suggesting that mRNA destabilization may be more important. (Abstract shortened by UMI.) ^

Interaction between the phage HK022 Nun protein and the nut RNA of phage lambda.

The nun gene product of prophage HK022 excludes phage lambda infection by blocking the expression of genes downstream from the lambda nut sequence. The Nun protein functions both by competing with lambda N transcription-antitermination protein and by actively inducing transcription termination on the lambda chromosome. We demonstrate that Nun binds directly to a stem-loop structure within nut RNA, boxB, which is also the target for the N antiterminator. The two proteins show comparable affinities for boxB and they compete with each other. Their interactions with boxB are similar, as shown by RNase protection experiments, NMR spectroscopy, and analysis of boxB mutants. Each protein binds the 5' strand of the boxB stem and the adjacent loop. The stem does not melt upon the binding of Nun or N, as the 3' strand remains sensitive to a double-strand-specific RNase. The binding of RNA partially protects Nun from proteolysis and changes its NMR spectra. Evidently, although Nun and N bind to the same surface of boxB RNA, their respective complexes interact differently with RNA polymerase, inducing transcription termination or antitermination, respectively.

NusG is required to overcome a kinetic limitation to Rho function at an intragenic terminator.

Rho-dependent transcription termination at certain terminators in Escherichia coli also depends on the presence of NusG [Sullivan, S. L. & Gottesman, M. E. (1992) Cell 68, 989-994]. We have found that termination at the first intragenic terminator in lacZ (tiZ1) is strongly dependent on NusG when transcription is done in vitro with the concentrations of NTPs found in vivo. With a lower level of NTPs, and consequently a slower rate of RNA-chain growth, Rho causes some termination by itself that is enhanced with NusG. These results suggest that NusG serves to overcome a kinetic limitation of Rho to function at certain terminators. At a second intragenic terminator within the lacZ reading frame (tiZ2) the efficiency of Rho-mediated termination was unaffected by either NusG or by RNA polymerase elongation kinetics. Thus, using purified components and intracellular levels of NTPs, we have confirmed the in vivo finding that certain Rho-dependent terminators also depend on NusG, whereas others do not.

Reduced transcription of TCOF1 in adult cells of Treacher Collins syndrome patients

Background: Treacher Collins syndrome (TCS) is an autosomal dominant craniofacial disorder caused by frameshift deletions or duplications in the TCOF1 gene. These mutations cause premature termination codons, which are predicted to lead to mRNA degradation by nonsense mediated mRNA decay (NMD). Haploinsufficiency of the gene product (treacle) during embryonic development is the proposed molecular mechanism underlying TCS. However, it is still unknown if TCOF1 expression levels are decreased in postembryonic human cells. Methods: We have estimated TCOF1 transcript levels through real time PCR in mRNA obtained from leucocytes and mesenchymal cells of TCS patients (n = 23) and controls (n = 18). Mutational screening and analysis of NMD were performed by direct sequencing of gDNA and cDNA, respectively. Results: All the 23 patients had typical clinical features of the syndrome and pathogenic mutations were detected in 19 of them. We demonstrated that the expression level of TCOF1 is 18-31% lower in patients than in controls (p < 0.05), even if we exclude the patients in whom we did not detect the pathogenic mutation. We also observed that the mutant allele is usually less abundant than the wild type one in mesenchymal cells. Conclusions: This is the first study to report decreased expression levels of TCOF1 in TCS adult human cells, but it is still unknown if this finding is associated to any phenotype in adulthood. In addition, as we demonstrated that alleles harboring the pathogenic mutations have lower expression, we herein corroborate the current hypothesis of NMD of the mutant transcript as the explanation for diminished levels of TCOF1 expression. Further, considering that TCOF1 deficiency in adult cells could be associated to pathologic clinical findings, it will be important to verify if TCS patients have an impairment in adult stem cell properties, as this can reduce the efficiency of plastic surgery results during rehabilitation of these patients.

The region of mouse mammary tumor virus DNA containing the long terminal repeat includes a long coding sequence and signals for hormonally regulated transcription.

Starting from a biologically active recombinant DNA clone of exogenous unintegrated GR mouse mammary tumor virus, we have generated three subclones of PstI fragments of 1.45, 1.1, and 2.0 kb in the plasmid vector PBR322. The nucleotide sequence has been determined for the clone of 1.45 kb which includes almost the complete region of the long terminal repeat (LTR) plus an adjacent stretch of unique sequence DNA. A short region of the 2.0 kb clone, containing the beginning of the LTR, has also been sequenced. Starting with the A of an initiation codon outside the LTR, we detected an open reading frame of 960 nucleotides, potentially coding for a protein of 320 amino acids (36K). Two hundred nucleotides downstream from the termination codon, and approximately 25 nucleotides upstream from the presumptive initiation site of viral RNA synthesis, we found a promoter-like sequence. The sequence AGTAAA was detected approximately 15-20 nucleotides upstream from the 3' end of virion RNA and probably serves as a polyadenylation signal. The 1.45 kb PstI fragment has been transfected into Ltk- cells together with a plasmid containing the thymidine kinase gene of herpes simplex virus. The virus-specific RNA synthesis detected in a Tk+ cell clone was strongly stimulated by the addition of dexamethasone.

STRUCTURAL ANALYSIS OF THE TRANSCRIPTION PRODUCTS OF A TEMPERATURE-SENSITIVE PHENOTYPIC MUTANT OF MOLONEY SARCOMA VIRUS--MUSV TS110

Cells infected with a temperature sensitive phenotypic mutant of Moloney sarcoma virus (MuSVts110) exhibit a transformed phenotype at 33('(DEGREES)) and synthesize two virus specific proteins, p85('gag-mos), a gag-mos fusion protein and p58('gag), a truncated gag precursor protein (the gag gene codes for viral structural proteins and mos is the MuSV transforming gene). At 39('(DEGREES)) only p58('gag) is synthesized and the morphology of the cells is similar to uninfected NRK parental cells. Two MuSVts110 specific RNAs are made in MuSVts110-infected cells, one of 4.0 kb in length, the other of 3.5 kb. Previous work indicated that each of these RNAs arose by a single central deletion of parental MuSV genetic material, and that p58('gag) was made by the 4.0 kb RNA and p85('gag-mos) from the 3.5 kb RNA. The objective of my dissertation research was to map precisely the deletion boundaries of both of the MuSVts110 RNAs, and to determine the proper reading frame across both deletion borders. This work succeeded in arriving at the following conclusions: (a) Using S-1 nuclease analysis and primer extension sequencing, it was found that the 4.0 kb MuSVts110 RNA arose by a 1488 base deletion of 5.2 kb parental MuSV genomic RNA. This deletion resulted in an out of frame fusion of the gag and mos genes that resulted in the formation of a "stop" codon which causes termination of translation just beyond the c-terminus of the gag region. Thus, this RNA can only be translated into the truncated gag protein p58('gag). (b) S-1 analysis of RNA from cells cultivated at different temperatures demonstrated that the 4.0 kb RNA was synthesized at all temperatures but that synthesis of the 3.5 kb RNA was temperature sensitive. These observations supported the data derived from blot hybridization experiments the interpretation of which argued for the existence of a single provirus in MuSVts110 infected cells, and hence only a single primary transcript (the 4.0 kb RNA). (c) Analyses similar to those described in (a) above showed that the 3.5 kb RNA was derived from the 4.0 kb MuSVts110 RNA by a further deletion of 431 bases, fusing the gag and mos genes into a continuous reading frame capable of directing synthesis of the p85('gag-mos) protein. These sequence data and the presence of only one MuSVts110-specific provirus, indicate that a splice mechanism is employed to generate the 3.5 kb RNA since the gag and mos genes are observed to be fused in frame in this RNA. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

Mechanisms of transcription inhibition by 8-amino-adenosine

Nucleoside analogs are a class of chemotherapeutic agents with tremendous utility in treating viral infections and cancers. Traditional nucleoside analogs are DNA-directed. However, there is a new group of nucleoside analogs that induce cell death by a direct effect on RNA synthesis. The adenosine analog, 8-chloroadenosine, is incorporated into RNA and is currently in clinical trials. Another congener, 8-amino-adenosine has demonstrated toxicity in multiple myeloma cell lines. Like other nucleoside analogs, 8-amino-adenosine must be metabolized to its triphosphate to elicit a cytotoxic effect. Furthermore, 8-amino-adenosine causes a decline of the intracellular ATP pool and inhibits mRNA poly(A) adenylation. ^ Because of the previously known adenosine analog mechanism as well as the scope of the RNA directed nucleoside analog field, I hypothesized there are multiple mechanisms of transcription inhibition mediating 8-amino-adenosine-induced cell death. Prior to investigating these mechanisms, cell death by 8-amino-adenosine was characterized. 8-Amino-adenosine activates PARP cleavage and induces the caspase cascade. 8-Amino-adenosine increases Annexin V binding and the mitochondrial membrane permeability in wild-type MEF cells. In BAX/BAK deficient MEF cells, 8-amino-adenosine decreases the mitochondrial membrane permeability and induces autophagy. ^ Once cell death was characterized, the mechanisms of 8-amino-adenosine transcription inhibition were assessed. It was established that 8-aminoadenosine treatment causes 8-amino-ATP accumulation and decreases the intracellular ATP concentration, resulting in RNA synthesis inhibition. Several other mechanisms are identified. First, a relationship between ATP decline by 8-amino-adenosine or other known ATP synthesis inhibitors and RNA synthesis is established indicating that effects on cellular bioenergy, regardless of the mechanism of ATP decline, can decrease RNA synthesis. Second, 8-aminoadenosine treatment decreases the phosphorylation of serine residues on the RNA polymerase II C-terminal domain which regulates transcription initiation and elongation. Third, evidence is provided to demonstrate 8-amino-ATP is a substrate for RNA synthesis. Fourth, 8-amino-ATP is incorporated at the 3'-terminal position leading to chain termination. Finally, in vitro transcription assays show that 8-amino-ATP may compete with ATP to decrease de novo mRNA synthesis. Overall, this work demonstrates 8-amino-adenosine is a cytotoxic nucleoside analog that functions to inhibit RNA transcription through multiple mechanisms. ^

Escherichia coli RNA polymerase terminates transcription efficiently at rho-independent terminators on single-stranded DNA templates

Several models have been proposed for the mechanism of transcript termination by Escherichia coli RNA polymerase at rho-independent terminators. Yager and von Hippel (Yager, T. D. & von Hippel, P. H. (1991) Biochemistry 30, 1097–118) postulated that the transcription complex is stabilized by enzyme–nucleic acid interactions and the favorable free energy of a 12-bp RNA–DNA hybrid but is destabilized by the free energy required to maintain an extended transcription bubble. Termination, by their model, is viewed simply as displacement of the RNA transcript from the hybrid helix by reformation of the DNA helix. We have proposed an alternative model where the RNA transcript is stably bound to RNA polymerase primarily through interactions with two single-strand specific RNA-binding sites; termination is triggered by formation of an RNA hairpin that reduces binding of the RNA to one RNA-binding site and, ultimately, leads to its ejection from the complex. To distinguish between these models, we have tested whether E. coli RNA polymerase can terminate transcription at rho-independent terminators on single-stranded DNA. RNA polymerase cannot form a transcription bubble on these templates; thus, the Yager–von Hippel model predicts that intrinsic termination will not occur. We find that transcript elongation on single-stranded DNA templates is hindered somewhat by DNA secondary structure. However, E. coli RNA polymerase efficiently terminates and releases transcripts at several rho-independent terminators on such templates at the same positions as termination occurs on duplex DNAs. Therefore, neither the nontranscribed DNA strand nor the transcription bubble is essential for rho-independent termination by E. coli RNA polymerase.

Intron insertion facilitates amplification of cloned virus cDNA in Escherichia coli while biological activity is reestablished after transcription in vivo.

Insertion of introns into cloned cDNA of two isolates of the plant potyvirus pea seedborne mosaic virus facilitated plasmid amplification in Escherichia coli. Multiple stop codons in the inserted introns interrupted the open reading frame of the virus cDNA, thereby terminating undesired translation of virus proteins in E. coli. Plasmids containing the full-length virus sequences, placed under control of the cauliflower mosaic virus 35S promoter and the nopaline synthase termination signal, were stable and easy to amplify in E. coli if one or more introns were inserted into the virus sequence. These plasmids were infectious when inoculated mechanically onto Pisum sativum leaves. Examination of the cDNA-derived viruses confirmed that intron splicing of in vivo transcribed pre-mRNA had occurred as predicted, reestablishing the virus genome sequences. Symptom development and virus accumulation of the cDNA derived viruses and parental viruses were identical. It is proposed that intron insertion can be used to facilitate manipulation and amplification of cloned DNA fragments that are unstable in, or toxic to, E. coli. When transcribed in vivo in eukaryotic cells, the introns will be eliminated from the sequence and will not interfere with further analysis of protein expression or virus infection.

Bipartite function of a small RNA hairpin in transcription antitermination in bacteriophage lambda.

Transcription of downstream genes in the early operons of phage lambda requires a promoter-proximal element known as nut. This site acts in cis in the form of RNA to assemble a transcription antitermination complex which is composed of lambda N protein and at least four host factors. The nut-site RNA contains a small stem-loop structure called boxB. Here, we show that boxB RNA binds to N protein with high affinity and specificity. While N binding is confined to the 5' subdomain of the stem-loop, specific N recognition relies on both an intact stem-loop structure and two critical nucleotides in the pentamer loop. Substitutions of these nucleotides affect both N binding and antitermination. Remarkably, substitutions of other loop nucleotides also diminish antitermination in vivo, yet they have no detectable effect on N binding in vitro. These 3' loop mutants fail to support antitermination in a minimal system with RNA polymerase (RNAP), N, and the host factor NusA. Furthermore, the ability of NusA to stimulate the formation of the RNAP-boxB-N complex is diminished with these mutants. Hence, we suggest that boxB RNA performs two critical functions in antitermination. First, boxB binds to N and secures it near RNAP to enhance their interaction, presumably by increasing the local concentration of N. Second, boxB cooperates with NusA, most likely to bring N and RNAP in close contact and transform RNAP to the termination-resistant state.

Impact of alternative initiation, splicing, and termination on the diversity of the mRNA transcripts encoded by the mouse transcriptome

We analyzed the FANTOM2 clone set of 60,770 RIKEN full-length mouse cDNA sequences and 44,122 public mRNA sequences. We developed a new computational procedure to identify and classify the forms of splice variation evident in this data set and organized the results into a publicly accessible database that can be used for future expression array construction, structural genomics, and analyses of the mechanism and regulation of alternative splicing. Statistical analysis shows that at least 41% and possibly as much as 60% of multiexon genes in mouse have multiple splice forms. Of the transcription units with multiple splice forms, 49% contain transcripts in which the apparent use of an alternative transcription start (stop) is accompanied by alternative splicing of the initial (terminal) exon. This implies that alternative transcription may frequently induce alternative splicing. The fact that 73% of all exons with splice variation fall within the annotated coding region indicates that most splice variation is likely to affect the protein form. Finally, we compared the set of constitutive (present in all transcripts) exons with the set of cryptic (present only in some transcripts) exons and found statistically significant differences in their length distributions, the nucleoticle distributions around their splice junctions, and the frequencies of occurrence of several short sequence motifs.

Essential regulatory elements for NHE3 gene transcription in renal proximal tubule cells

The objectives of the present study were to identify the cis-elements of the promoter absolutely required for the efficient rat NHE3 gene transcription and to locate positive and negative regulatory elements in the 5’-flanking sequence (5’FS), which might modulate the gene expression in proximal tubules, and to compare this result to those reported for intestinal cell lines. We analyzed the promoter activity of different 5’FS segments of the rat NHE3 gene, in the OKP renal proximal tubule cell line by measuring the activity of the reporter gene luciferase. Because the segment spanning the first 157 bp of 5’FS was the most active it was studied in more detail by sequential deletions, point mutations, and gel shift assays. The essential elements for gene transcription are in the region -85 to -33, where we can identify consensual binding sites for Sp1 and EGR-1, which are relevant to NHE3 gene basal transcription. Although a low level of transcription is still possible when the first 25 bp of the 5’FS are used as promoter, efficient transcription only occurs with 44 bp of 5’FS. There are negative regulatory elements in the segments spanning -1196 to -889 and -467 to -152, and positive enhancers between -889 and -479 bp of 5’FS. Transcription factors in the OKP cell nuclear extract efficiently bound to DNA elements of rat NHE3 promoter as demonstrated by gel shift assays, suggesting a high level of similarity between transcription factors of both species, including Sp1 and EGR-1.