La traduction représente un processus central au sein de la cellule, elle assure le transfert de l’information génétique de l’ARNm vers les protéines. De nombreux acteurs y sont impliqués directement ou indirectement et parmi eux, chez les eucaryotes, la petite protéine Trm112. Celle-ci participe à la modification de plusieurs acteurs directs en interagissant et en activant quatre MTases. Le facteur de terminaison eRF1 est méthylé par le complexe Mtq2-Trm112, l’ARNr 18S par Bud23-Trm112 et certains ARNt par les complexes Trm9-Trm112 et Trm11-Trm112. Au cours de ce travail, les structures cristallographiques de Trm9-Trm112 et de Bud23-Trm112 de levure ont été résolues. L’étude comparative structurale de ces complexes et de la structure connue de Mtq2-Trm112, a permis de mettre en évidence que dans un même organisme, les séquences des trois protéines ont évolué de manière à conserver l’interaction avec Trm112. Même si les quatre partenaires présentent moins de 20% d’identité de séquence, les résidus clés pour l’interaction avec la petite protéine activatrice sont conservés ou partagent des caractéristiques identiques. En plus de l’analyse structurale, le complexe Trm9-Trm112 a fait l’objet d’une étude fonctionnelle chez S. cerevisiae ce qui a permis de cartographier le site actif de l’enzyme et de proposer un modèle de mécanisme d’action. Enfin, les premières études in vivo réalisées chez Haloferax volcanii suggèrent que cette plateforme serait également présente chez certains organismes procaryotes. / Protein synthesis is a central process in the cell; it ensures the transfer of genetic information from mRNA in to protein. A lot of actors are involved directly or indirectly in translation. In Eukaryotes, Trm112, a small protein, interacts with and activates four methyltransferases modifying direct actors of translation. The termination factor eRF1 is methylated by the Mtq2-Trm112 complex, the 18S rRNA by Bud23-Trm112 and some tRNA by the Trm9-Trm112 and Trm11-Trm112 complexes. During this work, the crystal structures of Trm9-Trm112 and Bud23-Trm112 complexes from yeast were solved. The comparative analysis of these two new structures with Mtq2-Trm112 structure highlights the structural plasticity allowing Trm112 to interact through a very similar mode with its partners although those share less than 20% sequence identity. In the same organism, the key residues for the interaction with Trm112 are conserved or share similar characteristics. In addition to the structural analysis, the function of the Trm9-Trm112 complex was studied in S. cerevisiae. This analysis allowed to map the active site of the enzyme and to propose a model of its mechanism of action. Finally, the first data obtained in vivo, with the Archaea Haloferax volcanii suggest that the Trm112 platform might also be present in some prokaryotic organisms.

The Aryl Hydrocarbon Receptor (AhR) and the Aryl Hydrocarbon Nuclear Translocator (ARNT) play a role in mediating transcriptional responses to environmental pollutants, including the highly toxic compound 2,3,7,8-tetrachlorodibenzo -p-dioxin (TCDD) but also endogenous physiological ligands. More recent studies have also indicated that the AhR plays a role in the immune system notably in effector Th17 cells where it seems to be critical for the production of the IL-22 cytokine. It is known that AhR ligands such as dioxins can suppress CD8 T cell mediated antiviral immune responses but it is not known whether this reflects a direct role of the AhR in CD8 T cells.Accordingly, one objective of the present study was to explore AhR and ARNT expression in CD8 T cells. The initial strategy was to probe AhR and ARNT expression by western blot analysis. A second approach was to develop a mouse model that would fate mark single lymphocytes that have activated AhR signaling pathways. A third strategy was to examine the impact of deletion of AhR and ARNT on CD8 T-cell function.The data show that AhR and ARNT expression in CD8 T cells is limited to immune activated effector cells and these transcription factors are not expressed in naïve CD8 T cells. There are only low levels of AhR complexes in conventional CD8 positive cytotoxic T cells. To investigate AhR function at the single cell level we developed a mouse model to fate mark cells that have activated AhR signaling. In this model a mouse expressing Cre recombinase ‘knocked in’ to the CYP1A locus (CYP1A1Cre+/-) was backcrossed to the R26REYFP reporter mouse. In R26REYFP mice, a gene encoding EYFP is knocked into the ubiquitously expressed Rosa26 locus preceded by a loxP flanked stop sequence. CYP1A1 expression is controlled by AhR/ARNT complexes and the concept of our model was that cells that express AhR and ARNT complexes and are triggered with AhR ligands will express Cre recombinase and delete the loxP flanked stop sequence in the R26REYFP reporter locus and hence begin to express YFP.In vitro experiments demonstrated the validity of this AhR reporter model. The in vitro data reveal that expression of functional AhR/ARNT complexes occurs during Th17 and Tc17 cell differentiation but only a very low frequency of cytotoxic T cells activates the AhR. In vivo data found no evidence for AhR activation during T cell development in the thymus but show strong evidence for activation of AhR/ARNT signaling in innate lymphocytes in the gut. The ARNT transcription factor is highly expressed in cytotoxic T cells. These cells do not express functional AhR complexes, yet we considered that ARNT might play a role in CD8 T cell biology because of its ability to dimerise with the transcription factor Hif-1a. Our studies of T cells lacking ARNT expression revealed that in CD4 T cells the ARNT transcription factor regulates IL-17 and IL-22 production. In CD8 T cells we discovered that Hif-1a/ARNT signaling controls glycolysis in immune activated cells by sustaining expression of glucose transporters and multiple rate limiting glycolytic enzymes. ARNT was not required for CD8 T cell proliferation but was required for immune activated CD8 T cells to normally differentiate to express perforin and granzymes and to acquire the migratory program of effector T cells. Importantly, we discovered that Hif-1a/ARNT signaling is regulated by mTOR (mammalian target of rapamycin) thus revealing a fundamental mechanism linking nutrient sensing and transcriptional control of CD8 T-cell differentiation.

Mon étude a porté sur la spécificité de l'interaction entre deux protéines du parasite du paludisme (Plasmodium), tRip (tRNA import protein) et la tyrosyl-ARNt synthétase apicoplastique (api-TyrRS), avec l'ARN de transfert (ARNt). Plasmodium est un parasite intracellulaire qui conserve une organelle vestigiale, l’apicoplaste, qui possède son propre système de traduction. J’ai adapté la séquence de l’ARN messager pour produire l’api-TyrRS in vitro, et j’ai étudié la spécificité de la reconnaissance de l’ARNtTyr apicoplastique, qui évite les interactions erronées plutôt que de favoriser les correctes. La protéine tRip est située à la surface du parasite et est responsable de l’import des ARNt de l’hôte. Mes résultats suggèrent que cet import à lieu pendant la phase sanguine duparasite. Elle ne reconnait pas tous les ARNt de la même façon. Les modifications posttranscriptionnelles modulent l’affinité de tRip, et potentiellement, le taux d’import de cet ARNt. Finalement, j’ai identifié par SELEX une séquence nucléotidique qui se lie spécifiquement à tRip, un début pour la conception d'une molécule qui ciblerait spécifiquement le parasite du paludisme. / My study focused on the specificity of the interaction between two proteins of the malaria parasite (Plasmodium), tRip (tRNA import protein) and the apicoplastic tyrosyl-tRNA synthetase (api-TyrRS), with the transfer RNA (tRNA). Plasmodium is an intracellular parasite with a vestigial organelle, the apicoplast, which has its own translation system. The messenger RNA sequence was adapted to produce api-TyrRS in vitro, and I studied the specificity of apicoplastic tRNATyr recognition, which avoids erroneous interactions rather than favoring the correct ones. The tRip protein is located on the surface of the parasite, and is responsible for importing tRNAs from the host. My results suggest that this import takes place during the blood phase of the parasite. In addition, not all tRNAs are recognized uniformly. The post-transcriptional modifications of the tRNAs define the affinity of tRip, and potentialy, the import rate of this tRNA. Finally, I identified a short nucleotide sequence that binds specifically to tRip. It is a good starting point for designing a molecule that specifically targets the malaria parasite.

L’import d’ARN dans les mitochondries est un processus ubiquitaire chez les eucaryotes. Dans Saccharomyces cerevisiae un isoaccepteur cytosolique de l’ARNtLys, l’ARNtLys(CUU) (tRK1), est partiellement importé dans les mitochondries. L’adressage vers la surface mitochondriale a été étudié en détail mais l’étape de translocation dans les mitochondries reste toujours à démontrer. L’objectif principal de ce travail de thèse était l’identification et la caractérisation des protéines qui participent dans ce processus. Deux protéines de la membranes externe capables de former des canaux, Tom40 et VDAC1, ont été identifiées et leur rôle dans l’étape de translocation a été évalué in vitro et in vivo en utilisant des souches mutantes appropriées ou des agents capables de bloquer de manière spécifique les canaux formés par les protéines Tom40 et VDAC1. Ainsi il a été démontré que la délétion de VDAC1 ou l’inhibition du canal VDAC1 a conduit à une inhibition importante, néanmoins pas complète, de l’import de tRK1. Le blocage simultané des canaux Tom40 et VDAC1 par contre a causé un arrêt complet de l’import de tRK1 in vitro. Vu ces résultats, nous proposons que la translocation de tRK1 à travers la membrane mitochondriale externe puisse suivre deux chemins alternatifs. / RNA import into mitochondria is a ubiquitous process in eukaryotic cells. In Saccharomyces cerevisiae one cytosolic isoacceptor of tRNALys, tRNALys(CUU) (tRK1), is partially imported into mitochondria. Targeting of tRK1 to the mitochondrial surface is well described but the translocation of tRK1 into mitochondria is still poorly understood. This PhD work aimed to study this translocation step and the main objective was the identification and characterization of mitochondrial membrane proteins participating in this process. The two channel-forming proteins of the mitochondrial outer membrane, Tom40 and VDAC1, were identified and their role in tRK1 translocation further investigated with the help of in vitro and in vivo import studies using appropriate mutant strains or specific agents capable of blocking Tom40 and VDAC1. In this way, it could be demonstrated that deletion of the VDAC1 gene or inhibition of VDAC1 led to an important yet not complete inhibition of tRK1 import into mitochondria. Simultaneous blocking of the two channels formed by Tom40 and VDAC1, however, resulted in a complete inhibition of tRK1 import in vitro. Regarding these results we propose that tRK1 translocation through the mitochondrial outer membrane can use two alternative pathways.

The aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix PER/ARNT/SIM (bHLH-PAS) transcription factor that binds ligands typified by 2,3,7,8-tetracholordibenzo-p-dioxin, translocates to the nucleus, dimerizes with the aryl hydrocarbon nuclear translocator (ARNT) and associates with specific cis xenobiotic response elements to activate transcription of genes involved with xenobiotic metabolism. AHR-mediated signal transduction has been evaluated thoroughly in the C57BL/6J mouse model system. This model system, however, may not be the most accurate model for human comparisons as the AHRb-1 allele carried by C57BL/6J contains a point mutation that prematurely truncates the receptor at 805 amino acids, while the AHRb-2, rat, and human AHR all contain an additional 42-45 amino acids at their carboxy-terminus that have 70% identity. This carboxy-terminal region could be functionally significant and the analysis of AHR-mediated signal transduction in the rat, human, or other mouse strains may better represent the physiology of the AHR pathway. ARNT is another member of the bHLH-PAS family of proteins that is essential in several distinct signal transduction pathways mediated by its dimerization with a variety of bHLH-PAS proteins. Several isoforms of ARNT have been identified in mammalian and aquatic species. While ARNT and ARNT2 exhibit >90% amino acid identity in the bHLH and PAS domains, gene knock-out of either ARNT or ARNT2 results in embryonic/perinatal lethality characterized by distinct phenotypes. This suggests that neither protein can compensate fully for the loss of the other. Since overlapping tissue specific expression of ARNT and ARNT2 does exist, but neither ARNT can compensate fully for loss of the other, this suggests that the two proteins have distinct functions in the presence of various dimerization partners. Thus, the focus of these studies is to examine the discrepancies between the rat, human, or AHRb-2 possessing the extended carboxy-terminal region and that of the AHRb-1 and also to examine the role of both ARNT and ARNT2 during AHR-mediated signal transduction.

Sequence-specific small interfering RNA (siRNA) duplexes can be used for gene silencing in mammalian cells and as mechanistic probes for determining gene function. Transfection of siRNA for specificity protein 1 (Sp1) in MCF-7 or ZR-75 cells decreased Sp1 protein in nuclear extracts, and immunohistochemical analysis showed that Sp1 protein in transfected MCF-7 cells was barely detectable. Decreased Sp1 protein in MCF-7 was accompanied by a decrease in basal and estrogen-induced transactivation and cell cycle progression. These results clearly demonstrate the key role of Sp1 protein in regulating growth and gene expression of breast cancer cells. The aryl hydrocarbon (AhR) is a ligand-activated nuclear transcription factor. siRNA for the AhR decreased TCDD-induced CYP1A1 protein, CYP1A1dependent activity, and luciferase activity in cells transfected with an Ah-responsive construct. 17β-Estradiol (E2) induces proliferation of MCF-7 cells, and this response is inhibited in cells cotreated with E2 plus TCDD. The effects of TCDD on E2-induced cell cycle progression were partially blocked in MCF-7 cells transfected with siRNA for AhR. The decrease in AhR protein in MCF-7 cells was also accompanied by increased G0/G1 → S phase progression. Surprisingly, TCDD alone induced G0/G1 → S phase progression and exhibited estrogenic activity in MCF-7 cells transfected with siRNA for the AhR. In contrast, degradation of the AhR in HepG2 liver cancer cells resulted in decreased G0/G1 → S phase progression, and this was accompanied by decreased expression of cyclin D1, cyclin E, cdk2 and cdk4. In the absence of ligand, the AhR exhibits growth inhibitory (MCF-7) and growth promoting (HepG2) activity that is cell context-dependent. Sp family proteins play a complex role in regulation of pancreatic cancer cells growth and expression of genes required for growth, angiogenesis and apoptosis. Sp1, Sp3 and Sp4 cooperatively activate VEGF promoter constructs in these cells; however, only Sp3 regulates cell proliferation. siRNA for Sp3 inhibits phosphorylation of retinoblastoma protein, blocks G0/G1 → S phase progression of Panc-1 cells, and upregulates p27 protein/promoter activity. Thus, Sp3 plays a critical role in angiogenesis (VEGF upregulation) and the proliferation of Panc-1 cells by a novel mechanism of Sp3-dependent suppression of the cyclin-dependent kinase inhibitor p27.

Les aminoacil-ARNt sintetases (aaRS) són els enzims encarregats d'aminoacilar els ARN de transferència (ARNt) per tal que puguin ser utilitzats pels ribosomes en el procés de síntesi proteica. La síntesi de les proteïnes codificades en el genoma mitocondrial i constitutives dels complexos de la cadena respiratòria i la fosforilació oxidativa requereixen una maquinària traduccional pròpia de l'orgànul. Per tant, mutacions en els elements que constitueixen l'aparell de traducció genètica mitocondrial poden desencadenar patologies greus en humans. Existeixen malalties mitocondriales humanes causades per mutacions en l'ADN mitocondrial que afecten específicament els ARNt i ARNr i, a més, s'han descrit mutacions en proteïnes mitocondrials codificades en el genoma nuclear, entre les quals es troben mutacions en aaRS mitocondrials. La complexitat de les patologies mitocondrials, degudes a defectes en la síntesi proteica en l'orgànul, justifica la generació de models animals que permetin caracteritzar els efectes de deficiències d'aminoacilació mitocondrial, i el desenvolupament d'estratègies terapèutiques. En base a aquest objectiu, al llarg de la present tesi doctoral hem generat un model de patologia mitocondrial en Drosophila melanogaster amb el propòsit de caracteritzar els efectes d'una deficiència de traducció en l'orgànul com a conseqüència de la manipulació de la seril-ARNt sintetasa mitocondrial (DmSRS2). Els animals sotmesos al silenciament de l'expressió de la DmSRS2, mitjançant l'ARN d'interferència, mostren un descens en els nivells d'ARNtSer mitocondrials aminoacilats. La depleció de la DmSRS2, de manera generalitzada, compromet significativament la viabilitat de l'organisme i, de manera restringida en determinats òrgans i teixits, impedeix el correcte desenvolupament d'aquests. A més, el descens en els nivells de DmSRS2 provoca defectes en la morfologia, en la biogènesi i en la funció mitocondrials, que reprodueixen algunes de les característiques pròpies de malalties mitocondrials humanes. Cal afegir que en el context d'aquest projecte hem descobert una nova proteïna d'insecte homòloga a la seril-ARNt sintetasa mitocondrial (SLIMP), la caracterització de la qual s'ha convertit en un objectiu més d'aquesta tesi doctoral. La llarga història evolutiva de les aaRS explica l'àmplia varietat de funcions associades amb elles, i de proteïnes homòlogues, independents del paper convencional que desenvolupen en la traducció genètica. SLIMP representa una d'aquestes proteïnes, que probablement va sorgir de la duplicació d'una SRS mitocondrial a la base de l'evolució dels metazous, i s'ha mantingut en tots els insectes, en una espècie d'aràcnid i en una d'eriçó de mar. Tot i que SLIMP no desenvolupa una funció com aaRS, reté algunes de les característiques típiques de les SRS mitocondrials, com ho són la conformació dimèrica i la capacitat d'unir específicament els ARNtSer mitocondrials. SLIMP té un paper essencial en D. melanogaster atès que la reducció constitutiva i ubiqua de la seva expressió disminueix la viabilitat, i la depleció restringida en teixits concrets afecta el desenvolupament d'aquests. Proposem que la funció de SLIMP pot estar relacionada amb el metabolisme mitocondrial, ja que la seva depleció produeix greus anormalitats estructurals en l'orgànul, un increment en la biogènesi i una reducció de la capacitat respiratòria mitocondrial. Addicionalment, una dieta enriquida en molècules antioxidants produeix un efecte pal·liatiu en la viabilitat de les mosques sotmeses al silenciament global de SLIMP. / Aminoacyl-tRNA synthetases (aaRS) aminoacylate transfer RNAs (tRNAs) for their use by the ribosome during protein synthesis. The synthesis of mitochondrially encoded proteins is performed by a translation apparatus specific for the organelle, and mutations in elements that constitute this apparatus can trigger severe pathologies in humans. Mutations in the mitochondrial DNA that specifically affect tRNA and rRNA, as well as mutations in nuclear encoded mitochondrial proteins, for instance, those affecting mitochondrial aaRS, are related to disease. The complexity of mitochondrial diseases caused by defects in protein synthesis justifies the generation of animal models which permit the characterization of the pathogenic mechanisms and the development of therapeutic strategies. In the present doctoral thesis we use Drosophila melanogaster to construct a model of a mitochondrial disease caused by a deficiency in mitochondrial translation, by means of RNAi-mediated silencing of mitochondrial seryl-tRNA synthetase (DmSRS2). Silencing of DmSRS2 causes a decrease in mitochondrial seryl-tRNASer levels. Global DmSRS2 depletion compromises viability, and when restricted to particular organs, it prevents their proper development. Moreover, DmSRS2 reduction affects mitochondrial morphology, biogenesis, and function.Within the context of this project we discovered a new insect protein, called SLIMP, homologous to mitochondrial seryl-tRNA synthetase, whose characterization became an additional objective of this thesis. Owing to their long evolutionary history, aaRS and their homologues can perform a wide variety of non-canonical functions. SLIMP is likely the result of a duplication of a mitochondrial SRS gene that occurred early in the evolution of metazoans and was maintained in insects. SLIMP is a dimeric protein and is able to bind mitochondrial tRNASer, properties it has retained from the ancestral mitochondrial SRSs. Although SLIMP does not function as an aaRS, it carries out an essential role in D. melanogaster mitochondria, as its depletion by RNAi decreases the viability of the organism and affects mitochondrial structure, biogenesis, and respiratory capacity.

Ma thèse porte sur l'étude fonctionnelle et structurale des partenaires de la réaction d'aminoacylation spécifique de la tyrosine dans la mitochondrie humaine. Contrairement à ce qui a été observé pour les autres systèmes d'aminoacylation, les réactions de charges croisées entre bactéries et archaea/eucaryotes sont impossibles suite à la nature différente de la première paire de bases de l'ARNtTyr. La tyrosyl-ARNt synthétase (TyrRS) mitochondriale humaine est la première TyrRS connue à ce jour qui s'affranchisse de la barrière d'espèces et qui ne discrimine pas les ARNtTyr en fonction de la nature de leur première paire de bases. La TyrRS mitochondriale est un homodimère de forme allongée susceptible de fixer l'ARNtTyr à cheval sur ses deux monomères. Elle se distingue des autres TyrRS par la présence de deux insertions à sa surface, l'une potentiellement impliquée dans la reconnaissance de l'ARNtTyr et l'autre qui pourrait constituer une zone d'interaction avec un cofacteur.

The aryl hydrocarbon receptor (Ahr) and the aryl hydrocarbon receptor nuclear translocator (Arnt) are well characterized bHLH-PAS transcription factors shown to regulate expression of xenobiotic metabolism genes. Extensive study has shown that upon treatment with certain aromatic hydrocarbons, mammalian cells rapidly activate the Ahr signaling pathway in order to stimulate gene expression and attempt to metabolize the xenobiotic compounds. It has been shown that after DNA-binding, the Ahr but not the Arnt protein, is quickly eliminated from the nuclear compartment thereby attenuating the dose of gene regulation administered by the Ahr*Arnt transcription factor complex. Previous studies have implicated involvement of the 26S proteasome complex in the degradation process, but the exact identity of the intermediary proteins and/or ligases remains to be defined. Identification and characterization of the protein(s) involved in degrading the receptor is essential for understanding the signaling pathway in its entirety including the mechanism for regulating the genetic response to Ahr ligands. The model organism, Saccharomyces cerevisiae, was used in order to characterize the Ahr signaling pathway and degradation mechanism in a more simplified cellular setting in which the major processes required for growth and development are conserved. First, the AHR and ARNT cDNAs were stably inserted into the yeast genome such that protein expression was inducible. A time course of induction demonstrated detectable levels of Ahr and Arnt proteins via western blotting while protein function was confirmed by detection of ligand-dependent reporter activity in an expressor strain carrying the pLXRE5-Z beta-galactosidase reporter plasmid. Additionally, a rapid reduction in protein levels was observed upon turning off the inducible GAL1 promoter located upstream of both AHR and ARNT cDNAs. Studies in mammalian cell culture have demonstrated that disrupting receptor chaperoning results in rapid Ahr protein turnover, as demonstrated by treatment with Hsp90 inhibitors. In order to determine if reduced Ahr protein expression in the yeast system was attributed to improper chaperoning of the exogenous protein; human heat shock proteins were constitutively expressed from yeast expression vectors in the Ahr and Arnt expressing strains, but did not confer any effect on Ahr stability when protein levels were evaluated by western blotting. Additionally, a strain of yeast was constructed such that the gene encoding the cell-wall protein, ERG6, was deleted from the yeast genome to allow for permeation of proteasome inhibitors. Treatment of this strain with proteasome inhibitors blocked the receptor degradation, therefore implicating the 26S proteasome in Ahr degradation when expressed exogenously in yeast.

Sequence-specific small interfering RNA (siRNA) duplexes can be used for gene silencing in mammalian cells and as mechanistic probes for determining gene function. Transfection of siRNA for specificity protein 1 (Sp1) in MCF-7 or ZR-75 cells decreased Sp1 protein in nuclear extracts, and immunohistochemical analysis showed that Sp1 protein in transfected MCF-7 cells was barely detectable. Decreased Sp1 protein in MCF-7 was accompanied by a decrease in basal and estrogen-induced transactivation and cell cycle progression. These results clearly demonstrate the key role of Sp1 protein in regulating growth and gene expression of breast cancer cells. The aryl hydrocarbon (AhR) is a ligand-activated nuclear transcription factor. siRNA for the AhR decreased TCDD-induced CYP1A1 protein, CYP1A1dependent activity, and luciferase activity in cells transfected with an Ah-responsive construct. 17β-Estradiol (E2) induces proliferation of MCF-7 cells, and this response is inhibited in cells cotreated with E2 plus TCDD. The effects of TCDD on E2-induced cell cycle progression were partially blocked in MCF-7 cells transfected with siRNA for AhR. The decrease in AhR protein in MCF-7 cells was also accompanied by increased G0/G1 → S phase progression. Surprisingly, TCDD alone induced G0/G1 → S phase progression and exhibited estrogenic activity in MCF-7 cells transfected with siRNA for the AhR. In contrast, degradation of the AhR in HepG2 liver cancer cells resulted in decreased G0/G1 → S phase progression, and this was accompanied by decreased expression of cyclin D1, cyclin E, cdk2 and cdk4. In the absence of ligand, the AhR exhibits growth inhibitory (MCF-7) and growth promoting (HepG2) activity that is cell context-dependent. Sp family proteins play a complex role in regulation of pancreatic cancer cells growth and expression of genes required for growth, angiogenesis and apoptosis. Sp1, Sp3 and Sp4 cooperatively activate VEGF promoter constructs in these cells; however, only Sp3 regulates cell proliferation. siRNA for Sp3 inhibits phosphorylation of retinoblastoma protein, blocks G0/G1 → S phase progression of Panc-1 cells, and upregulates p27 protein/promoter activity. Thus, Sp3 plays a critical role in angiogenesis (VEGF upregulation) and the proliferation of Panc-1 cells by a novel mechanism of Sp3-dependent suppression of the cyclin-dependent kinase inhibitor p27.