Expression analysis of the AtPHO1 gene family in Arabidopsis thaliana and the involvement of AtPHO1 in the regulation of stomatal movements

Résumé Le transfert du phosphate des racines vers les feuilles s'effectue par la voie du xylème. Il a été précédemment démontré que la protéine AtPHO1 était indispensable au transfert du phosphate dans les vaisseaux du xylème des racines chez la plante modèle Arabidopsis thaliana. Le séquençage et l'annotation du génome d'Arabidopsis ont permis d'identifier dix séquences présentant un niveau de similarité significatif avec le gène AtPHO1 et constituant une nouvelle famille de gène appelé la famille de AtPHO1. Basée sur une étude moléculaire et génétique, cette thèse apporte des éléments de réponse pour déterminer le rôle des membres de ia famille de AtPHO1 chez Arabidopsis, inconnue à ce jour. Dans un premier temps, une analyse bioinformatique des séquences protéiques des membres de la famille de AtPHO1 a révélé la présence dans leur région N-terminale d'un domaine nommé SPX. Ce dernier est conservé parmi de nombreuses protéines impliquées dans l'homéostasie du phosphate chez la levure, renforçant ainsi l'hypothèse que les membres de la famille de AtPHO1 auraient comme AtPHO1 un rôle dans l'équilibre du phosphate dans la plante. En parallèle, la localisation tissulaire de l'expression des gènes AtPHO dans Arabidopsis a été identifiée par l'analyse de plantes transgéniques exprimant le gène rapporteur uidA sous le contrôle des promoteurs respectifs des gènes AtPHO. Un profil d'expression de chaque gène AtPHO au cours du développement de la plante a été obtenu. Une expression prédominante au niveau des tissus vasculaires des racines, des feuilles, des tiges et des fleurs a été observée, suggérant que les gènes AtPHO pourraient avoir des fonctions redondantes au niveau du transfert de phosphate dans le cylindre vasculaire de ces différents organes. Toutefois, plusieurs régions promotrices des gènes AtPHO contrôlent également un profil d'expression GUS non-vasculaire, indiquant un rôle putatif des gènes AtPHO dans l'acquisition ou le recyclage de phosphate dans la plante. Dans un deuxième temps, l'analyse de l'expression des gènes AtPHO durant une carence en phosphate a établi que seule l'expression des gènes AtPHO1, AtPHO1; H1 et AtPHO1; H10 est régulée par cette carence. Une étude approfondie de leur expression en réponse à des traitements affectant l'homéostasie du phosphate dans la plante a ensuite démontré leur régulation par différentes voies de signalisation. Ensuite, une analyse détaillée de la régulation de l'expression du gène AtPHO1; H1O dans des feuilles d'Arabidopsis blessées ou déshydratées a révélé que ce gène constitue le premìer gène marqueur d'une nouvelle voie de signalisation induite par l'OPDA, pas par le JA et dépendante de la protéine COI1. Ces résultats démontrent pour la première fois que l'OPDA et le JA peuvent activer différents gènes via des voies de signalisation dépendantes de COI1. Enfin, cette thèse révèle l'identification d'un nouveau rôle de la protéine AtPHO1 dans la régulation de l'action de l'ABA au cours des processus de fermeture stomatique et de germination des graines chez Arabidopsis. Bien que les fonctions exactes des protéines AtPHO restent à être déterminées, ce travail de thèse suggère leur implication dans la propagation de différents signaux dans la plante via la modulation du potentiel membranaire et/ou l'affectation de la composition en ions des cellules comme le font de nombreux transporteurs ou régulateur du transport d'ions. Summary Phosphate is transferred from the roots to the shoot via the xylem. The requirement for AtPHO1 protein to transfer phosphate to the xylem vessels of the root has been previously demonstrated in Arabidopsis thaliana. The sequencing and the annotation of the Arabidopsis genome had allowed the identification of ten sequences that show a significant level of similarity with the AtPHO1 gene. These 10 genes, of unknown functions, constitute a new gene family called the AtPHO1 gene family. Based on a molecular and genetics study, this thesis reveals some information needed to understand the role of the AtPHO1 family members in the plant Arabidopsis. First, a bioinformatics study revealed that the AtPHO sequences contained, in the N-terminal hydrophilic region, a motif called SPX and conserved among multiple proteins involved in phosphate homeostasis in yeast. This finding reinforces the hypothesis that all AtPHO1 family members have, as AtPHO1, a role in phosphate homeostasis. In parallel, we identified the pattern of expression of AtPHO genes in Arabidopsis via analysis of transgenic plants expressing the uidA reporter gene under the control of respective AtPHO promoter regions. The results exhibit a predominant expression of AtPHO genes in vascular tissues of all organs of the plant, implying that these AtPHO genes could have redundant functions in the transfer of phosphate to the vascular cylinder of various organs. The GUS expression pattern for several AtPHO promoter regions was also detected in non-vascular tissue indicating a broad role of AtPHO genes in the acquisition or in the recycling of phosphate in the plant. In a second step, the analysis of the expression of AtPHO genes during phosphate starvation established that only the expression of the AtPHO1, AtPHO1; H1 and AtPHO1; H10 genes were regulated by Pi starvation. Interestingly, different signalling pathways appeared to regulate these three genes during various treatments affecting Pi homeostasis in the plant. The third chapter presents a detailed analysis of the signalling pathways regulating the expression of the AtPHO1; H10 gene in Arabidopsis leaves during wound and dehydrated stresses. Surprisingly, the expression of AtPHO1; H10 was found to be regulated by OPDA (the precursor of JA) but not by JA itself and via the COI1 protein (the central regulator of the JA signalling pathway). These results demonstrated for the first time that OPDA and JA could activate distinct genes via COI1-dependent pathways. Finally, this thesis presents the identification of a novel role of the AtPHO1 protein in the regulation of ABA action in Arabidopsis guard cells and during seed germination. Although the exact role and function of AtPHO1 still need to be determined, these last findings suggest that AtPHO1 and by extension other AtPHO proteins could mediate the propagation of various signals in the plant by modulating the membrane potential and/or by affecting cellular ion composition, as it is the case for many ion transporters or regulators of ion transport.

Dynamics of HIV latency and reactivation in a primary CD4+ T cell model.

HIV latency is a major obstacle to curing infection. Current strategies to eradicate HIV aim at increasing transcription of the latent provirus. In the present study we observed that latently infected CD4+ T cells from HIV-infected individuals failed to produce viral particles upon ex vivo exposure to SAHA (vorinostat), despite effective inhibition of histone deacetylases. To identify steps that were not susceptible to the action of SAHA or other latency reverting agents, we used a primary CD4+ T cell model, joint host and viral RNA sequencing, and a viral-encoded reporter. This model served to investigate the characteristics of latently infected cells, the dynamics of HIV latency, and the process of reactivation induced by various stimuli. During latency, we observed persistence of viral transcripts but only limited viral translation. Similarly, the reactivating agents SAHA and disulfiram successfully increased viral transcription, but failed to effectively enhance viral translation, mirroring the ex vivo data. This study highlights the importance of post-transcriptional blocks as one mechanism leading to HIV latency that needs to be relieved in order to purge the viral reservoir.

Superficial and deep changes of cellular mechanical properties following cytoskeleton disassembly.

The cytoskeleton, composed of actin filaments, intermediate filaments, and microtubules, is a highly dynamic supramolecular network actively involved in many essential biological mechanisms such as cellular structure, transport, movements, differentiation, and signaling. As a first step to characterize the biophysical changes associated with cytoskeleton functions, we have developed finite elements models of the organization of the cell that has allowed us to interpret atomic force microscopy (AFM) data at a higher resolution than that in previous work. Thus, by assuming that living cells behave mechanically as multilayered structures, we have been able to identify superficial and deep effects that could be related to actin and microtubule disassembly, respectively. In Cos-7 cells, actin destabilization with Cytochalasin D induced a decrease of the visco-elasticity close to the membrane surface, while destabilizing microtubules with Nocodazole produced a stiffness decrease only in deeper parts of the cell. In both cases, these effects were reversible. Cell softening was measurable with AFM at concentrations of the destabilizing agents that did not induce detectable effects on the cytoskeleton network when viewing the cells with fluorescent confocal microscopy. All experimental results could be simulated by our models. This technology opens the door to the study of the biophysical properties of signaling domains extending from the cell surface to deeper parts of the cell.

TACI, unlike BAFF-R, is solely activated by oligomeric BAFF and APRIL to support survival of activated B cells and plasmablasts.

The cytokine BAFF binds to the receptors TACI, BCMA, and BAFF-R on B cells, whereas APRIL binds to TACI and BCMA only. The signaling properties of soluble trimeric BAFF (BAFF 3-mer) were compared with those of higher-order BAFF oligomers. All forms of BAFF bound BAFF-R and TACI, and elicited BAFF-R-dependent signals in primary B cells. In contrast, signaling through TACI in mature B cells or plasmablasts was only achieved by higher-order BAFF and APRIL oligomers, all of which were also po-tent activators of a multimerization-dependent reporter signaling pathway. These results indicate that, although BAFF-R and TACI can provide B cells with similar signals, only BAFF-R, but not TACI, can respond to soluble BAFF 3-mer, which is the main form of BAFF found in circulation. BAFF 60-mer, an efficient TACI agonist, was also detected in plasma of BAFF transgenic and nontransgenic mice and was more than 100-fold more active than BAFF 3-mer for the activation of multimerization-dependent signals. TACI supported survival of activated B cells and plasmablasts in vitro, providing a rational basis to explain the immunoglobulin deficiency reported in TACI-deficient persons.

Stoichiometry of Heteromeric BAFF and APRIL Cytokines Dictates Their Receptor Binding and Signaling Properties.

The closely related TNF family ligands B cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL) serve in the generation and maintenance of mature B-lymphocytes. Both BAFF and APRIL assemble as homotrimers that bind and activate several receptors that they partially share. However, heteromers of BAFF and APRIL that occur in patients with autoimmune diseases are incompletely characterized. The N and C termini of adjacent BAFF or APRIL monomers are spatially close and can be linked to create single-chain homo- or hetero-ligands of defined stoichiometry. Similar to APRIL, heteromers consisting of one BAFF and two APRILs (BAA) bind to the receptors B cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI) but not to the BAFF receptor (BAFFR). Heteromers consisting of one APRIL and two BAFF (ABB) bind to TACI and BCMA and weakly to BAFFR in accordance with the analysis of the receptor interaction sites in the crystallographic structure of ABB. Receptor binding correlated with activity in reporter cell line assays specific for BAFFR, TACI, or BCMA. Single-chain BAFF (BBB) and to a lesser extent single-chain ABB, but not APRIL or single-chain BAA, rescued BAFFR-dependent B cell maturation in BAFF-deficient mice. In conclusion, BAFF-APRIL heteromers of different stoichiometries have distinct receptor-binding properties and activities. Based on the observation that heteromers are less active than BAFF, we speculate that their physiological role might be to down-regulate BAFF activity.

The Elucidation of the involvement of endonuclease DNase y in reducing DNA transfection efficiency in mammalian cells

Gene therapy is predicated upon efficient gene transfer. While viral vectors are the method of choice for transformation efficiency, the immunogenicity and safety concerns remain problematic. Non-viral vectors, on the other hand, have shown high degrees of safety and are mostly non-immunogenic in nature. However, non-viral vectors usually suffer from low levels oftransformation efficiency and transgene expression. Thus, increasing transformation efficiency ofnon-viral vectors, in particular by calcium phosphate co-precipitation technique, is a way of generating a suitable vector for gene therapy and is the aim of this study. It is a long known fact that different cell lines have different transfection efficiencies regardless oftransfection methodology (Lin et a!., 1994). Using commonly available cell lines Madine-Darby Bovine Kidney (MDBK), HeLa and Human Embryonic Kidney (HEK-293), we have shown a decreasing trend ofDNase activity based on a plasmid digestion assay. From densitometry studies, as much as a 40% reduction in DNase activity was observed when comparing HEK-293 (least active) to MDBK (most active). Using various biochemical assays, it was determined that DNase y, in particular, was expressed more highly in MDBK cells than both HeLa and HEK-293. Upon cloning of the bovine DNase y gene, we utilized the sequence information to construct antisense expressing plasmids via both traditional antisense RNA (pASDGneoM) and siRNA (psiRNA-S4, psiRNA-S11 and psiRNA-S16). For the construction ofpASDGneoM, the 3' end of the DNase y was inserted in opposite orientation under a cytomegalovirus (CMV) promoter such that the expression ofRNA complementary to the DNase 2 ymRNA occurred. For siRNA plasmids, the sequence was screened to yield optimal short sequences for siRNA inhibition. The silencing ofbovine DNase y led to an increase in transfection efficiency based on traditional calcium phosphate co-precipitation technique; stable clones of siRNA-producing MDBK cell lines (psiRNA-S4 Bland psiRNA-S4 B4) both demol).strated 4-fold increases in transfection efficiency. Furthermore, serial transfection of antisense DNase y plasmid pASDGneoM and reporter pCMV-~ showed a maximum of 8-fold increase in transfection efficiency when the two separate transfections were carried out 4 hours apart (i.e. transfection ofpASDGneoM, separated by four hours, then transfection ofpCMV-~). Together, these results demonstrate the involvement ofDNase y in reducing transfection efficiency, at least by traditional calcium phosphate technique.

Investigations towards the synthesis of isotope labelled analogues of tocopherols and tocotrienols /

Vitamin E is considered as the most effective lipophilic chain breaking antioxidant. a-Tocopherol and its analogues have been studied thoroughly with regards to its biokinetics and bioavailabily. Deuterated tocopherols have been synthesized and utilized in such studies. Tocotrienols are arousing more and more interest because of their high efficiency as antioxidants. However, to date, there is no effective synthetic method reported for deuterated tocotrienols. This thesis is focused on the investigation of the synthetic methods of deuterated tocotrienols and their analogues: 5-trideuteromethyl-a-tocotrienol, 5- trideuteromethyl-p-tocotrienol, tocotrienol acetate, silyl tocotrienol ether, etc. Several synthetic procedures for the preparation of poly-deuterated tocopherols are known. Mainly the deuterium is introduced by use of labelled formaldehyde and deuterated hydrogen chloride under Lewis acid catalysis. However, these methods are not effective in tocotrienols due to exchange of protons for deuterium at other sites under the acidic conditions. We developed several different approaches to generate polydeuterated tocotrienols by using both morpholinomethylation followed by reduction with NaCNBDs as deuterated reducing reagents and transmetalation strategy. The 5-trideuteromethyl-a-tocotrienol was finally obtained in a satisfactory yield of 60%. In addition, this thesis also discussed the study of structural comparison and the chemical property difference of tocopherols and tocotrienols, which provides hints to explain the reactivity difference of them towards oxidation at the C3-C4 positions.Furthermore, the methodology of halogenation and dehydrohalogenation of tocotrienol was explored to prepare a hexaene tocotrienol derivative as a florescent reporter of tocopherol.

Development of packaging cell lines for rescuing BAV2 viral vectors

The construction of adenovirus vectors for cloning and foreign gene expression requires packaging cell lines that can complement missing viral functions caused by sequence deletions and/or replacement with foreign DNA sequences. In this study, packaging cell lines were designed to provide in trans the missing bovine adenovirus functions, so that recombinant viruses could be generated. Fetal bovine kidney and lUng cells, acquired at the trimester term from a pregnant cow, were tranfected with both digested wild type BAV2 genomic DNA and pCMV-EI. The plasmid pCMV-EI was specifically constructed to express El of BAV2 under the control of the cytomegalovirus enhancer/promoter (CMV). Selection for "true" transformants by continuous passaging showed no success in isolating immortalised cells, since the cells underwent crisis resulting in complete cell death. Moreover, selection for G418 resistance, using the same cells, also did not result in the isolation of an immortalised cell line and the same culture-collapse event was observed. The lack of success in establishing an immortalised cell line from fetal tissue prompted us to transfect a pre-established cell line. We began by transfecting MDBK (Mardin-Dardy bovine kidney) cells with pCMV-El-neo, which contain the bacterial selectable marker neo gene. A series of MDBK-derived cell lines, that constitutively express bovine adenoviral (BAV) early region 1 (El), were then isolated. Cells selected for resistance to the drug G418 were isolated collectively for full characterisation to assess their suitability as packaging cell lines. Individual colonies were isolated by limiting dilution and further tested for El expression and efficiency of DNA uptake. Two cell lines, L-23 and L-24, out of 48 generated foci tested positive for £1 expression using Northern Blot analysis. DNA uptake studies, using both lipofectamine and calcium phosphate methods, were performed to compare these cells, their parental MDBK cells, 8 and the unrelated human 293 cells as a benchmark. The results revealed that the new MDBKderived clones were no more efficient than MDBK cells in the transient expression of transfected DNA and that they were inferior to 293 cells, when using lacZ as the reporter gene. In view of the inherently poor transfection efficiency of MDBK cells and their derivatives, a number of other bovine cells were investigated for their potential as packaging cells. The cell line CCL40 was chosen for its high efficiency in DNA uptake and subsequently transfected with the plasmid vector pCMV El-neo. By selection with the drug G418, two cell lines were isolated, ProCell 1 and ProCell 2. These cell lines were tested for El expression, permissivity to BAV2 and DNA uptake efficiency, revealing a DNA uptake efficiency of 37 % , comparable to that of CCL40. Attempts to rescue BAV2 mutants carrying the lacZ gene in place of £1 or £3 were carried out by co-transfecting wild type viral DNA with either the plasmid pdlElE-Z (which contains BAV2 sequences from 0% to 40.4% with the lacZ gene in place of the £1 region from 1.1% to 8.25%) or with the plasmid pdlE3-5-Z (which contains BAV2 sequences from 64.8% to 100% with the lacZ gene in place of the E3 region from 75.8% to 81.4%). These cotransfections did not result in the generation of a viral mutant. The lack of mutant generation was thought to be caused by the relative inefficiency ofDNA uptake. Consequently, cosBAV2, a cosmid vector carrying the BAV2 genome, was modified to carry the neo reporter gene in place of the £3 region from 75.8% to 81.4%. The use of a single cosmid vector earring the whole genome would eliminate the need for homologous recombination in order to generate a viral vector. Unfortunately, the transfection of cosBAV2- neo also did not result in the generation of a viral mutant. This may have been caused by the size of the £3 deletion, where excess sequences that are essential to the virus' survival might have been deleted. As an extension to this study, the spontaneous E3 deletion, accidently discovered in our viral stock, could be used as site of foreign gene insertion.

Transfection of newt blastema mesenchyme using the techniques of lipofection and direct gene transfer

The regenerating amphibian limb provides a useful system for studying genes involved in the establishment of positional information. While a number of candidate genes that may playa role in pattern formation have been identified, their function in vivo is unknown in this system. To better ascertain the role of these genes, it would be useful to be able to alter their normal patterns of expression in vivo and to assess the effects of this misexpression on limb pattern. In order to achieve this, a method of introducing a plasmid containing the eDNA of a gene of interest into a newt blastema (a growth zone of mesenchymal progenitor cells) is needed. Unfortunately, most commonly used transfection techniques cannot be used with newt blastema cells. In this study, I have used the techniques of lipofection and direct gene transfer to introduce plasmid DNA containing reporter genes into the cells of a regenerating newt limb. The technique of lipofection was most effective when the blastema cells were transfected in vitro. The optimal ratio for transfection was shown to be 1:3 DNA:Lipofectin (W/w) , and an increase in the amount of DNA present in the mixture (1:3 ratio maintained) resulted in a corresponding increase in gene expression. The technique of direct gene transfer was used to transfect newt blastema cells with and without prior complex formation with Lipofectin. Injection of plasmid DNA alone provided the most 3 promising results. It was possible to introduce plasmid DNA containing the reporter gene ~-galactosidase and achieve significant gene expression in cells associated with the injection site. In the future, it would be interesting to use this technique to inject plasmid DNA containing a gene which may have a role in pattern formation into specific areas of the newt blastema and to analyze the resulting limb pattern that emerges.

Adenovirus-based exogenous gene expression in mammalian cells

Thesis (Ph.D.)--Brock University, 2010.

Investigating the role of apoptosis regulator EndoG on exogenous DNA uptake, stability, replication and recombination

Endonuclease G (EndoG) is a well conserved mitochondrial nuclease with dual lethal and vital roles in the cell. It non-specifically cleaves endogenous DNA following apoptosis induction, but is also active in non-apoptotic cells for mitochondrial DNA (mtDNA) replication and may also be important for replication, repair and recombination of genomic DNA. The aim of our study was to examine whether EndoG exerts similar activities on exogenous DNA substrates such as plasmid DNA (pDNA) and viral DNA vectors, considering their importance in gene therapy applications. The effects of EndoG knockdown on pDNA stability and levels of encoded reporter gene expression were evaluated in the cervical carcinoma HeLa cells. Transfection of pDNA vectors encoding short-hairpin RNAs (shRNAs) reduced levels of EndoG mRNA and nuclease activity in HeLa cells. In physiological circumstances, EndoG knockdown did not have an effect on the stability of pDNA or the levels of encoded transgene expression as measured over a four day time-course. However, when endogenous expression of EndoG was induced by an extrinsic stimulus (a cationic liposome transfection reagent), targeting of EndoG by shRNA improved the perceived stability and transgene expression of pDNA vectors. Therefore, EndoG is not a mediator of exogenous DNA clearance, but in non-physiological circumstances it may non-specifically cleave intracellular DNA regardless of its origin. To investigate possible effects of EndoG on viral DNA vectors, we constructed and evaluated AdsiEndoG, a first generation adenovirus (Ad5 ΔE1) vector encoding a shRNA directed against EndoG mRNA, along with appropriate Ad5 ΔE1 controls. Infection of HeLa cells with AdsiEndoG at a multiplicity of infection (MOI) of 10 p.f.u./cell resulted in an early cell proliferation defect, absent from cells infected at equivalent MOI with control Ad5 ΔE1 vectors. Replication of Ad5 ΔE1 DNA was detected for all vectors, but AdsiEndoG DNA accumulated to levels that were 50 fold higher than initially, four days after infection, compared to 14 fold for the next highest control Ad5 ΔE1 vector. Deregulation of the cell cycle by EndoG depletion, which is characterized by an accumulation of cells in the G2/M transition, is the most likely reason for the observed cell proliferation defect. The enhanced replication of AdsiEndoG is consistent with this conclusion, as Ad5 ΔE1 DNA replication is intimately related to cell cycling and prolongation or delay in G2/M greatly enhances this process. Furthermore, infection of HeLa with AdsiEndoG at MOI of 50 p.f.u./cell resulted in an almost complete disappearance of viable, adherent tumour cells from culture, whereas almost a third of the cells were still adherent after infection with control Ad5 ΔE1 vectors, relative to the non-infected control. Therefore, targeting of EndoG by RNAi is a viable strategy for improving the oncolytic properties of first generation adenovirus vectors. In addition, AdsiEndoG-mediated knockdown of EndoG reduced homologous recombination between pDNA substrates in HeLa cells. The effect was modest but, nevertheless demonstrated that the proposed role of EndoG in homologous recombination of cellular DNA also extends to exogenous DNA substrates.

Ann Eliza Hepburne Rooth fonds, 1837-1897

Ann Eliza Hepburne was born in Chippawa, Ontario, in 1821, to William Hepburne and Susan Shannon. In 1842, she married William Anthony Rooth in St. James Cathedral in Toronto. They continued to live in different parts of the Niagara region, including Drummondville, Welland and Port Colborne. William was the editor and proprietor of the Drummondville Reporter, as well as an accountant and insurance agent, and later worked for the Customs Service in Port Colborne. He died in 1878, and Eliza in 1899. Both are buried in Drummond Hill Cemetery in Niagara Falls, Ontario.

Association between disruption of CD4 receptor dimerization and increased human immunodeficiency virus type 1 entry

Affiliation: Département de microbiologie et immunologie, Faculté de médecine, Université de Montréal & Institut de Recherches Cliniques de Montréal

Genome-wide location analysis and expression studies reveal a role for p110 CUX1 in the activation of DNA replication genes.

Proteolytic processing of the CUX1 transcription factor generates an isoform, p110 that accelerates entry into S phase. To identify targets of p110 CUX1 that are involved in cell cycle progression, we performed genome-wide location analysis using a promoter microarray. Since there are no antibodies that specifically recognize p110, but not the full-length protein, we expressed physiological levels of a p110 isoform with two tags and purified chromatin by tandem affinity purification (ChAP). Conventional ChIP performed on synchronized populations of cells confirmed that p110 CUX1 is recruited to the promoter of cell cycle-related targets preferentially during S phase. Multiple approaches including silencing RNA (siRNA), transient infection with retroviral vectors, constitutive expression and reporter assays demonstrated that most cell cycle targets are activated whereas a few are repressed or not affected by p110 CUX1. Functional classes that were over-represented among targets included DNA replication initiation. Consistent with this finding, constitutive expression of p110 CUX1 led to a premature and more robust induction of replication genes during cell cycle progression, and stimulated the long-term replication of a plasmid bearing the oriP replicator of Epstein Barr virus (EBV).