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

Histoire des pratiques et des représentations politiques à l’époque contemporaine

Stéphane Audoin-Rouzeau, Christophe Prochasson, directeurs d’étudesAnne Rasmussen, maître de conférences à l’Université Strasbourg-I Atelier d’historiographie : comment on écrit l’histoire de la Grande Guerre Comme chaque année, le séminaire s’est voulu le lieu de comptes rendus et de discussions d’expériences intellectuelles centrées sur l’étude socioculturelle de la Grande Guerre. La première séance a permis à deux jeunes historiens de venir faire état du travail accompli lors d’expositions..

The Snf2 Homolog Fun30 acts as a homodimeric ATP-dependent chromatin-remodeling enzyme

The Saccharomyces cerevisiae Fun30 (Function unknown now 30) protein shares homology with an extended family of Snf2-related ATPases. Here we report the purification of Fun30 principally as a homodimer with a molecular mass of about 250 kDa. Biochemical characterization of this complex reveals that it has ATPase activity stimulated by both DNA and chromatin. Consistent with this, it also binds to both DNA and chromatin. The Fun30 complex also exhibits activity in ATP-dependent chromatin remodeling assays. Interestingly, its activity in histone dimer exchange is high relative to the ability to reposition nucleosomes. Fun30 also possesses a weakly conserved CUE motif suggesting that it may interact specifically with ubiquitinylated proteins. However, in vitro Fun30 was found to have no specificity in its interaction with ubiquitinylated histones

The Saccharomyces cerevisiae Histone Chaperone Rtt106 Mediates the Cell Cycle Recruitment of SWI/SNF and RSC to the HIR-Dependent Histone Genes

In Saccharomyces cerevisiae, three out of the four histone gene pairs (HTA1-HTB1, HHT1-HHF1, and HHT2-HHF2) are regulated by the HIR co-repressor complex. The histone chaperone Rtt106 has recently been shown to be present at these histone gene loci throughout the cell cycle in a HIR- and Asf1-dependent manner and involved in their transcriptional repression. The SWI/SNF and RSC chromatin remodeling complexes are both recruited to the HIR-dependent histone genes; SWI/SNF is required for their activation in S phase, whereas RSC is implicated in their repression outside of S phase. Even though their presence at the histone genes is dependent on the HIR complex, their specific recruitment has not been well characterized. In this study we focused on characterizing the role played by the histone chaperone Rtt106 in the cell cycle-dependent recruitment of SWI/SNF and RSC complexes to the histone genes.Using GST pull-down and co-immunoprecipitation assays, we showed that Rtt106 physically interacts with both the SWI/SNF and RSC complexes in vitro and in vivo. We then investigated the function of this interaction with respect to the recruitment of these complexes to HIR-dependent histone genes. Using chromatin immunoprecipitation assays (ChIP), we found that Rtt106 is important for the recruitment of both SWI/SNF and RSC complexes to the HIR-dependent histone genes. Furthermore, using synchronized cell cultures, we showed by ChIP assays that the Rtt106-dependent SWI/SNF recruitment to these histone gene loci is cell cycle regulated and restricted to late G1 phase just before the peak of histone gene expression in S phase.Overall, these data strongly suggest that the interaction between the histone chaperone Rtt106 and both the SWI/SNF and RSC chromatin remodeling complexes is important for the cell cycle regulated recruitment of these two complexes to the HIR-dependent histone genes

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

SWI/SNF Displaces SAGA-Acetylated Nucleosomes

SWI/SNF is a well-characterized chromatin remodeling complex that remodels chromatin by sliding nucleosomes in cis and/or displacing nucleosomes in trans. The latter mechanism has the potential to remove promoter nucleosomes, allowing access to transcription factors and RNA polymerase. In vivo, histone acetylation often precedes apparent nucleosome loss; therefore, we sought to determine whether nucleosomes containing acetylated histones could be displaced by the SWI/SNF chromatin remodeling complex. We found that SAGA-acetylated histones were lost from an immobilized nucleosome array when treated with the SWI/SNF complex. When the nucleosome array was acetylated by SAGA in the presence of bound transcription activators, it generated a peak of acetylation surrounding the activator binding sites. Subsequent SWI/SNF treatment suppressed this acetylation peak. Immunoblots indicated that SWI/SNF preferentially displaced acetylated histones from the array relative to total histones. Moreover, the Swi2/Snf2 bromodomain, an acetyl-lysine binding domain, played a role in the displacement of acetylated histones. These data indicate that targeted histone acetylation by the SAGA complex predisposes promoter nucleosomes for displacement by the SWI/SNF complex

Rtt106 is essential for recruitment of RSC to the HIR-dependent histone genes.

<p>(A) Chromatin immunoprecipitation of the RSC complex were carried out as described in Material and Methods. An antibody directed against the TAP tag of the Rsc8-TAP subunit in wild-type and <i>rtt106Δ</i> strains was used. Presence of the indicated histone gene loci promoter sequence was monitored by qPCR. A non-transcribed region of chromosome V (InterV) was used as an internal background control. Error bars show the range between biological duplicates. (B) 25 µg of whole cell extract prepared from the <i>RSC8-TAP</i> (wild-type background) and <i>RSC8-TAP rtt106Δ</i> strains were loaded per lane. Protein levels of two representative RSC subunits, Rsc8-TAP and Sth1, were compared between the <i>RSC8-TAP</i> (lane 1) and <i>RSC8-TAP rtt106Δ</i> strain (lane 2) by western blots using an antibody targeting the TAP-tag in Rsc8-TAP, or an antibody against endogenous Sth1, which is the catalytic subunit of the RSC complex. Actin was used as a loading control. (C) ChIP assays were performed as in A to monitor the presence of the Rsc8-TAP tagged subunit at the <i>HTA1-HTB1</i> promoter in wild-type (WT) strain and strains deleted of <i>RTT106 (rtt106Δ)</i>, <i>ASF1 (asf1Δ)</i>, or <i>HPC2 (hpc2Δ)</i>. Enrichment of <i>HTA1-HTB1</i> promoter sequence in immunoprecipitated material was monitored by qPCR and normalized to the control region, InterV. The graph shows the average of duplicate IP reactions, and the error bars show the range between individual IPs.</p

Rtt106 is required for SWI/SNF localization to <i>HTA1-HTB1</i> loci.

<p>(A) Chromatin immunoprecipitation was carried out as described in Material and Methods. Antibodies specific for the N-terminus or C-terminus of the endogenous Swi2/Snf2 subunit were used for immunoprecipitation of the SWI/SNF complex in cross-linked chromatin extracts from the wild-type strain and strains deleted of <i>RTT106, ASF1</i>, or <i>HPC2</i>. The amount of <i>HTA1-HTB1</i> promoter (black bars) precipitated was monitored by qPCR and inter V region (grey bars) was used as an internal background control from a non-transcribed region on chromosome V. Data is expressed as percentage of ChIP/Input and error bars show the range of repeated IPs. (B) 25 µg of whole cell extracts prepared from the untagged wild-type and <i>rtt106Δ</i> strains were loaded per lane. Protein levels of four SWI/SNF subunits, Swi2/Snf2, Swi1, Snf5 and Swi3, were compared between the wild-type strain (lane 1) and the <i>rtt106Δ</i> strain (lane 2) by western blot analysis using specific antibodies against each individual protein. Actin was included as a loading control.</p