Transcription factor binding sites in the pol gene intragenic regulatory region of HIV-1 are important for virus infectivity

We have previously identified in the pol gene of human immunodeficiency virus type 1 (HIV-1) a new positive transcriptional regulatory element (nt 4481–4982) containing recognition sites for nuclear proteins (sites B, C, D and a GC-box) [C. Van Lint, J. Ghysdael, P. Paras, Jr, A. Burny and E. Verdin (1994) J. Virol. 68, 2632–2648]. In this study, we have further physically characterized each binding site and have shown that the transcription factors Oct-1, Oct-2, PU.1, Sp1 and Sp3 interact in vitro with the pol region. Chromatin immunoprecipitation assays using HIV-infected cell lines demonstrated in the context of chromatin that Sp1, Sp3, Oct-1 and PU.1 are recruited to the HS7 region in vivo. For each site, we have identified mutations abolishing factor binding to their cognate DNA sequences without altering the underlying amino acid sequence of the integrase. By transient transfection assays, we have demonstrated the involvement of the pol binding sites in the transcriptional enhancing activity of the intragenic region. Our functional results with multimerized wild-type and mutated pol binding sites separately (i.e. in the absence of the other sites) have demonstrated that the PU.1, Sp1, Sp3 and Oct-1 transcription factors regulate the transcriptional activity of a heterologous promoter through their respective HS7 binding sites. Finally, we have investigated the physiological role of the HS7 binding sites in HIV-1 replication and have shown that these sites are important for viral infectivity

The Mediator complex subunit MED25 is targeted by the N-terminal transactivation domain of the PEA3 group members

International audiencePEA3, ERM and ER81 belong to the PEA3 subfamily of Ets transcription factors and play important roles in a number of tissue-specific processes. Transcriptional activation by PEA3 subfamily factors requires their characteristic amino-terminal acidic transactivation domain (TAD). However, the cellular targets of this domain remain largely unknown. Using ERM as a prototype, we show that the minimal N-terminal TAD activates transcription by contacting the activator interacting domain (ACID)/Prostate tumor overexpressed protein 1 (PTOV) domain of the Mediator complex subunit MED25. We further show that depletion of MED25 disrupts the association of ERM with the Mediator in vitro . Small interfering RNA-mediated knockdown of MED25 as well as the overexpression of MED25-ACID and MED25-VWA domains efficiently inhibit the transcriptional activity of ERM. Moreover, mutations of amino acid residues that prevent binding of MED25 to ERM strongly reduce transactivation by ERM. Finally we show that siRNA depletion of MED25 diminishes PEA3-driven expression of MMP-1 and Mediator recruitment. In conclusion, this study identifies the PEA3 group members as the first human transcriptional factors that interact with the MED25 ACID/PTOV domain and establishes MED25 as a crucial transducer of their transactivation potential

Regulation of ploidy and senescence by the AMPK-related kinase NUAK1.

Senescence is an irreversible cell-cycle arrest that is elicited by a wide range of factors, including replicative exhaustion. Emerging evidences suggest that cellular senescence contributes to ageing and acts as a tumour suppressor mechanism. To identify novel genes regulating senescence, we performed a loss-of-function screen on normal human diploid fibroblasts. We show that downregulation of the AMPK-related protein kinase 5 (ARK5 or NUAK1) results in extension of the cellular replicative lifespan. Interestingly, the levels of NUAK1 are upregulated during senescence whereas its ectopic expression triggers a premature senescence. Cells that constitutively express NUAK1 suffer gross aneuploidies and show diminished expression of the genomic stability regulator LATS1, whereas depletion of NUAK1 with shRNA exerts opposite effects. Interestingly, a dominant-negative form of LATS1 phenocopies NUAK1 effects. Moreover, we show that NUAK1 phosphorylates LATS1 at S464 and this has a role in controlling its stability. In summary, our work highlights a novel role for NUAK1 in the control of cellular senescence and cellular ploidy.We thank the members of the Laboratory for helpful discussions. We also thank Virginie Glippa and Julie Bertout for technical assistance. We thank H Esumi for the NUAK1 cDNA, E Hara and H Saya for the LATS1‐encoding vector. This work was carried out with the support of the ‘Association pour la Recherche sur le Cancer’, the ‘Fondation pour la Recherche Médicale Nord Pas de Calais’, the ‘Comité du Pas de Calais de la Ligue Nationale contre le Cancer’, the RTRS Fondation Synergie Lyon Cancer, and the Medical Research Council, UK.S

Regulation of DNA Methylation Patterns by CK2-Mediated Phosphorylation of Dnmt3a

DNA methylation is a central epigenetic modification that is established by de novo DNA methyltransferases. The mechanisms underlying the generation of genomic methylation patterns are still poorly understood. Using mass spectrometry and a phosphospecific Dnmt3a antibody, we demonstrate that CK2 phosphorylates endogenous Dnmt3a at two key residues located near its PWWP domain, thereby downregulating the ability of Dnmt3a to methylate DNA. Genome-wide DNA methylation analysis shows that CK2 primarily modulates CpG methylation of several repeats, most notably of Alu SINEs. This modulation can be directly attributed to CK2-mediated phosphorylation of Dnmt3a. We also find that CK2-mediated phosphorylation is required for localization of Dnmt3a to heterochromatin. By revealing phosphorylation as a mode of regulation of de novo DNA methyltransferase function and by uncovering a mechanism for the regulation of methylation at repetitive elements, our results shed light on the origin of DNA methylation patterns

Influence of L-thyroxine, L-triiodothyronine, thyroid stimulating hormone, or estradiol on the cell kinetics of cultured mammary cancer cells.

We describe the in vitro influence of 3,5,3'-triiodo-L-thyronine (T3), L-thyroxine (T4), a thyroid-stimulating hormone (TSH), and/or estradiol (E2: chosen as the control of the methodology) on the cell kinetics (cell distribution in the S + G2 + M phases) of mouse MXT and human MCF-7 mammary cancer cells. Experiments were performed by means of a cell image processor, analyzing MCF-7 or MXT cells that had been grown on glass cover slips and whose nuclei had been stained by the Feulgen reaction, which is selective and quantitative (stoichiometric) with respect to DNA. We show that T3, T4, and TSH at 0.01 microM dramatically stimulate the cell kinetics of the MXT mouse and the MCF-7 human mammary cancer cell lines. Indeed, the three hormones bring about a significant transient increase in the S + G2 + M fraction as does E2. Furthermore, our data indicate that E2 and TSH are antagonistic with regards to MXT or MCF-7 cell kinetics.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Etude in vivo des facteurs de transcription du groupe PEA3 appartenant à la famille Ets dans la glande mammaire normale cancéreuse

Les membres du groupe PEA3 (Pea3, Er81 et Erm) sont des facteurs de transcription appartenant à la famille Ets. Il existe des arguments en faveur d'un rôle de ces facteurs dans le développement de cancers mammaires et surtout dans la formation de métastases. En effet, ces gènes sont fortement exprimés dans des lignées cancéreuses mammaires humaines qui possèdent un fort pouvoir métastatique. De plus, des gènes associés à la progression tumorale sont les cibles potentielles des protéines du groupe PEA3; il s'agit de ceux codant certaines enzymes de dégradation de la matrice extra-cellulaire. Nous avons établi un modèle in vivo d'étude de ces facteurs de transcription. Pour cela, nous avons réalisé des souris transgéniques pour er81 sous le contrôle d'un promoteur spécifique de la glande mammaire et induit lors de la lactation: le long terminal repeat (LTR) du rétrovirus MMTV. Ces souris exprimant er81 dans la glande mammaire ont été suivies pendant plus d'un an et aucune n'a développé de tumeur ce qui suggère que Er81 n'est pas capable d'induire seul la formation de tumeurs mammaires. Une étude des gènes cibles a permis de détecter dans les glandes mammaires exprimant er81 une sur-expression de deux gènes. Il s'agit de la métalloprotéase stromélysine-1 (MMP-3) et de l'urokinase plasminogen activator (uPA) dont les produits sont impliqués dans la dégradation de la matrice extracellulaire associée à l'invasion tumorale. Nous avons ensuite analysé l'expression des membres du groupe PEA3 dans des tumeurs mammaires issues de modèles établis de souris transgénique. Une forte expression des membres du groupe PEA3 a été détectée dans les tumeurs des souris MMTV-neu et MMTV-ras. Ces deux gènes impliqués dans la formation de tumeurs du sein chez la femme sont responsables chez la souris de tumeurs invasives capables de métastaser [...]LILLE1-BU (590092102) / SudocSudocFranceF

In vivo influence of androgens on the cell kinetics and chromatin pattern of the MXT mouse mammary tumor treated or not by aminoglutethimide.

We characterized the influence of androstenedione, 5-alpha-dihydrotestosterone and 17-beta-estradiol on the chromatin organization and the cell kinetics (distribution of the cells into the G0-G1, S and G2+ M fractions) of MXT mouse mammary tumors grafted onto animals that were left intact or that were oophorectomized and/or treated with aminoglutethimide. The cell kinetics and chromatin pattern were monitored by analyzing Feulgen-stained MXT imprint smears through a cell image processor, the SAMBA 200 system. We have also assayed the estrogen and progesterone receptors of these MXT tumors, which appeared to possess both of these biological markers. Our results show that ovariectomy or aminoglutethimide treatment slowed down the MXT tumor growth without any additive effect between them. Androstenedione exerted a stimulating influence on the cell kinetics, which were very similar to those observed after estradiol administration; the treatment of castrated animals with aminoglutethimide completely abolished this androstenedione-induced stimulating influence, however. This androgen lacked any apparent efficiency to transform cell nuclei from a state of castration-induced chromatin condensation into a thinly textured and decondensed state, as did estradiol. Dihydrotestosterone was able to activate the cell kinetics of MXT tumor grafted onto castrated animals as well as those of MXT neoplasms grafted on oophorectomized mice treated with aminoglutethimide. Dihydrotestosterone also possesses the property to transform condensed chromatin into decondensed chromatin. It thus appeared that androstenedione and dihydrotestosterone could activate MXT cell proliferation, as did estradiol, although it appeared that their mechanisms of action were quite distinct from each other.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

In vivo relationship between morphonuclear parameters and hormone sensitivity of the murine MXT mammary tumor.

The initially hormonosensitive (HS) MXT mouse mammary tumor spontaneously evolved to hormonoin-dependence (HI). Using a SAMBA 200 cell image processor, we compared the DNA content and the chromatin structure of HS and HI tumor cells squashed onto histologic slides; the nuclei were colored by the Feulgen reaction. We compared HI and HS nuclei by a discriminant analysis using the 15 parameters obtained on each nucleus. We show that the percentage of well-classified granulocytes (2n DNA content control) versus HS or HI nuclei exceeded 99. On the other hand, this percentage did not reach 70 when we compared HS and HI. The cell cycle analysis revealed that the percentage of cells in S and G2 + M phases were significantly higher in HI tumors than in the HS. Hence, HI and HS MXT tumor nuclei seem to be morphologically identical, but are significantly different if we refer to cell proliferation rates.Comparative StudyJournal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Is melatonin really an in vitro inhibitor of human breast cancer cell proliferation? [8]

SCOPUS: le.jinfo:eu-repo/semantics/publishe