Use of adenoviral E1A protein to analyze K18 promoter deregulation in colon carcinoma cells discloses a role for CtBP1 and BRCA1

BACKGROUND: The promoter of the keratin 18 (K18) gene is 5- to 10-fold more active in tumorigenic (T-type) cell clones derived from the SW613-S human colon carcinoma cell line than in non-tumorigenic (NT-type) clones. We have reported previously that the mechanism responsible for this differential activity is acting on the minimal K18 promoter (TATA box and initiation site). This mechanism does not require the binding of a factor to a specific site on the DNA but involves the acetylation of a non-histone substrate. To get further insight into this mechanism, we investigated the effect of the adenovirus E1A protein on the activity of the K18 promoter, both in T and NT cells. RESULTS: Wild type adenovirus E1A protein and C-terminal deletion mutants inhibit the K18 promoter, specifically in T-type cells. The domain responsible for this inhibitory effect is located in the 12–25 region of the viral protein. E1A mutants that have lost this region but retain the PLDLS motif (the C-terminal binding site for CtBP1) stimulate the K18 promoter, specifically in NT cells. The inhibitory or stimulatory effects of the different E1A mutants are not dependent on a particular sequence of the promoter. An E1A N-terminal deletion mutant carrying point mutations in the PLDLS motif cannot stimulate the K18 promoter. CtBP1 interacts with CtIP, which is a known partner of BRCA1, itself a component of the RNA polymerase II holoenzyme. The stimulatory effect of two BRCA1 mutants, specifically in NT cells, implicates a tripartite BRCA1-CtIP-CtBP1 complex in the regulation of the K18 promoter. CONCLUSION: Since we have shown previously that the K18 promoter is stimulated by deacetylase inhibitors, specifically in NT cells, we conclude that the activity of the promoter is repressed in NT cells by a mechanism involving the recruitment, by a BRCA1/CtIP complex, of CtBP1 and associated deacetylases to the preinitiation complex. We propose a model depicting the mechanism responsible for the differential activity of the K18 promoter between T and NT cells of the SW613-S cell line

Updating the mechanisms of common fragile site instability: how to reconcile the different views?

Common fragile sites (CFSs) are large chromosomal regions long identified by conventional cytogenetics as sequences prone to breakage in cells subjected to replication stress. The interest in CFSs came from their key role in the formation of DNA damage, resulting in chromosomal rearrangements. The instability of CFSs was notably correlated with the appearance of genome instability in precancerous lesions and during tumor progression. Identification of the molecular mechanisms responsible for their instability therefore represents a major challenge. A number of data show that breaks result from mitotic entry before replication completion but the mechanisms responsible for such delayed replication of CFSs and relaxed checkpoint surveillance are still debated. In addition, clues to the molecular events leading to breakage just start to emerge. We present here the results of recent reports addressing these questions

High frequency trans-splicing in a cell line producing spliced and polyadenylated RNA polymerase I transcripts from an rDNA-myc chimeric gene

The 2G1MycP2Tu1 cell line was obtained following transfection of human colon carcinoma cells from the SW613-S cell line with a plasmid carrying a genomic copy of the human MYC gene. 2G1MycP2Tu1 cells produce MYC mRNAs and proteins of abnormal size. In order to analyze the structure of these abnormal products, a cDNA library constructed using RNA isolated from these cells was screened with a MYC probe. Fifty clones were studied by DNA sequencing. The results indicated that a truncated copy of the MYC gene had integrated into an rDNA transcription unit in 2G1MycP2Tu1 cells. This was confirmed by northern blot analysis, PCR amplification on genomic DNA and fluorescent in situ hybridization (FISH) experiments on metaphase chromosomes. 2G1MycP2Tu1 cells produce hybrid rRNA-MYC RNA molecules that are polyadenylated and processed by splicing reactions involving natural and cryptic splice sites. These transcripts are synthesized by RNA polymerase I, as confirmed by actinomycin D sensitivity experiments, suggesting that 3′ end processing and splicing are uncoupled from transcription in this case. 2G1MycP2Tu1 cells also produce another type of chimeric mRNAs consisting of correctly spliced exons 2 and 3 of the MYC gene fused to one or more extraneous 5′ exons by proper splicing to the acceptor sites of MYC exon 2. These foreign exons belong to 33 different genes, which are located on 14 different chromosomes. These observations and the results of FISH and Southern blotting experiments lead us to conclude that trans-splicing events occur at high frequency in 2G1MycP2Tu1 cells

MECANISMES TRANSCRIPTIONNELS DE LA SUREXPRESSION DU GENE DE LA KERATINE 18 DANS DES CELLULES DE CARCINOME DU COLON HUMAIN

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocPARIS-BIUM (751062103) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Mécanismes impliqués dans le contrôle de l'initiation de la réplication chez les mammifères

La réplication est une étape clef du cycle cellulaire au cours de laquelle l'ADN est dupliqué une fois et une seule. Chez les mammifères, de nombreuses origines potentielles sont présentes le long du génome mais les mécanismes qui contrôlent leur localisation et leur efficacité sont mal compris. Durant ma thèse, j'ai tout d'abord montré que la vitesse de progression des fourches de réplication contrôle la densité en origines de réplication actives et leur sélection parmi l'ensemble des origines possibles. La sélection de l'origine majeure passe par une localisation préférentielle a la matrice nucléaire durant la phase G1. J'ai également montré que le niveau d'acétylation des histones n'est associé ni à la localisation, ni à l'efficacité des origines de réplication situées dans des régions riches en gènes. En revanche, l'inhibition des desacétylases par des drogues, comme la trichostatine A conduit à une modification de la dynamique de réplication. L'hyperacétylation provoquée entraine une diminution de l'expression de deux enzymes impliquées dans les voies de biosynthèse des dNTP, la thymidylate synthase et la CTP synthetase. Cette diminution d'expression induit une diminution de la quantité de dCTP et dTTP disponible dans la cellule, une diminution de la vitesse de réplication et une augmentation du nombre d'origines actives. Ces résultats réconcilient des données contradictoires présentes dans la littérature et pose la question de l'existence d'une nouvelle voie de contrôle de la dynamique de réplication par l'acétylationPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Role de l'amplification de l'oncogenèse MYC dans des cellules de carcinome du colon humain

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Characterization at nucleotide resolution of the homogeneously staining region sites of insertion in two cancer cell lines

International audienceThe mechanisms of formation of intrachromosomal amplifications in tumours are still poorly understood. By using quantitative polymerase chain reaction, DNA sequencing, chromosome walking, in situ hybridiza-tion on metaphase chromosomes and whole-genome analysis, we studied two cancer cell lines containing an MYC oncogene amplification with acquired copies ectopically inserted in rearranged chromosomes 17. These intrachromosomal amplifications result from the integration of extrachromosomal DNA molecules. Replication stress could explain the formation of the double-strand breaks involved in their insertion and in the rearrangements of the targeted chromosomes. The sequences of the junctions indicate that homolo-gous recombination was not involved in their formation and support a non-homologous end-joining process. The replication stress-inducible common fragile sites present in the amplicons may have driven the intrachromosomal amplifications. Mechanisms associating break-fusion-bridge cycles and/ or chromosome fragmentation may have led to the formation of the uncovered complex structures. To our knowledge, this is the first characterization of an intrachromosomal amplification site at nucleotide resolution