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Comparative Study
. 2005 Apr 14:6:8.
doi: 10.1186/1471-2199-6-8.

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

Cécile Delouis  1 , Philippe Prochasson, Madeleine Laithier, Olivier Brison
Affiliations
Comparative Study

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

Cécile Delouis et al. BMC Mol Biol. .

Abstract

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.

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Figures

Figure 1
Figure 1
Effect of E1A-13S or E1A-d30-76 proteins on the activity of the K18 promoter. SW613-3 (T) or SW613-B3 (NT) cells were transfected with plasmid pK18(80)luc (A) or plasmid pK18(41)luc (B) alone (none) or in the presence of plasmid pE1A-13S or pE1A-d30-76, as indicated. Luciferase activities are expressed relative to that obtained with the reporter plasmid alone for each cell clone. Error bars represent the standard deviation of the mean of two experiments. (C) Structure of the promoter of the human K18 gene. The binding site of the Sp1 transcription factor, the initiation site (arrow at position -11; we have shown that the initiation is located 11 bp upstream of previously published +1 position [22]) and the TATA box are indicated. Numbers indicate positions in nucleotides. K18(80) and K18(41), the two forms of the promoter inserted into luciferase expression vectors are shown.
Figure 2
Figure 2
Effect of E1A-13S or E1A-d30-76 proteins on the activity of mutated versions of the K18 promoter. SW613-3 (T) or SW613-B3 (NT) cells were transfected with each of the reporter plasmids carrying the indicated mutated form of the K18(80) promoter, alone or in the presence of plasmid pE1A-13S (A and C) or plasmid pE1A-d30-76 (B and D). Luciferase activities are expressed relative to that obtained with the promoter construct alone in each cell line. Since the activities of all promoter constructs alone were adjusted to 100, they are all represented by a single pair of columns in each panel. The activity of these constructs relative to that of the wild type K18(80) promoter has been reported previously [22]. The structure of the various mutant forms of the K18(80) promoter is schematized in E. Error bars represent the standard deviation of the mean of duplicate experimental points.
Figure 3
Figure 3
Effect of C-terminal and N-terminal deletion mutants of the E1A protein on the activity of the K18 promoter. (A) Schematic representation of the structure of the E1A-13S protein and of the deletion mutants. Know domains of E1A-13S (CR1, CR2, CR3 and CR4 and the N-terminal region) are shown as boxes. Numbers indicate positions in aa. (B and C) SW613-3 (T) or SW613-B3 (NT) cells were transfected with plasmid pK18(80)luc alone (none) or in the presence of the indicated E1A constructs. Luciferase activities are expressed relative to that obtained with the pK18(80)luc plasmid alone. Error bars represent the standard deviation of the mean of at least two experiments.
Figure 4
Figure 4
Expression level of the E1A constructs. SW613-3 cells were transfected with plasmid constructs coding for the indicated wild-type or mutant E1A proteins. Cellular extracts were analyzed by Western blotting with an anti-E1A antibody. (A) N- and C-terminal deletion mutants. The E1A-C29, -C79 and -C109 polypeptides cannot be detected with this antibody. (B) Internal deletion mutants. Note that E1A and some of its mutants are very sensitive to proteolysis (white asterisks) in spite of the presence of protease inhibitors. Similar results were obtained in SW613-B3 cells (not shown).
Figure 5
Figure 5
Effect of internal deletion mutants of the E1A protein on the activity of the K18 promoter. (A) Schematic representation of the structure of the mutants. Symbols are the same as in figure 3. (B) SW613-3 (T) and SW613-B3 (NT) cells were transfected with plasmid pK18(80)luc alone (none) or in the presence of the indicated E1A constructs. Results are presented as in figure 3. Error bars represent standard deviation of the mean of at least two experiments.
Figure 6
Figure 6
CtBP1 has a role in the stimulatory effect of E1A mutants on the K18 promoter. (A) CtBP1 interacts with E1A-N190 but not with the mutant E1A-N190-mut 281-282. GST pull-down assays were performed on extracts of SW613-3 (3) or SW613-B3 (B3) cells using GST alone (empty vector) or the fusion proteins GST-E1A-N190 or GST-E1A-N190-mut281-282. Bound proteins were analyzed by Western blotting with an anti-CtBP1 antibody. (B) Effect of the E1A-N190 or E1A-N190-mut281-282 proteins on the activity of the K18 promoter. SW613-3 (T) or SW613-B3 (NT) cells were transfected with plasmid pK18(80)luc alone (none) or in the presence of plasmid pE1A-N190 or pE1A-N190-mut281-282 as indicated. Results are presented as in figure 3. Error bars represent standard deviation of the mean of duplicate experimental points. (C) Reversion of the stimulatory effect of E1A-N190 by CtBP1. SW613-3 (T) or SW613-B3 (NT) cells were transfected with plasmid pK18(80)luc alone (none) or in the presence of plasmid pE1A-N190 (2 μg) or pcDNA3-CtBP (24 μg) or with the three plasmids together, as indicated. Results are presented as in figure 3. Error bars represent standard deviation of the mean of triplicate experimental points. (D) The CtBP1 protein has no direct effect on the activity of the K18 promoter. SW613-3 (T) or SW613-B3 (NT) cells were transfected with plasmid pK18(80)luc alone (none) or in the presence of various amounts of plasmid pcDNA3-CtBP, as indicated. Results are presented as in figure 3. Error bars represent standard deviation of the mean of triplicate experimental points.
Figure 7
Figure 7
Expression level of the E1A-N190 and CtBP1 constructs. (A) SW613-B3 or SW613-3 cells were transfected with plasmid pE1A-N190 (wt) or pE1A-N190-mut281-282 (mut). Cellular extracts were analyzed by Western blotting with an anti-E1A antibody. (B) SW613-3 (3) or SW613-B3 (B3) cells were transfected with plasmid pcDNA3-CtBP or untransfected (mock). Cellular extracts were analyzed by Western blotting with an anti-CtBP1 antibody.
Figure 8
Figure 8
The BRCA1 protein has a role in the repression of the K18 promoter in NT cells. (A) SW613-3 (T) or SW613-B3 (NT) cells were transfected with plasmid pK18(80)luc alone (none) or in the presence of 24 μg of plasmids pCBRCA1C (BRCA1wt), pcDNA3-BRCA1(Y1853→STOP) or pcDNA3-BRCT, as indicated. Results are presented as in figure 3. Error bars represent standard deviation of the mean of three experiments. (B) and (C) Expression level of the BRCA1 constructs. SW613-3 (3) or SW613-B3 (B3) cells were untransfected (mock) or transfected with plasmid pCBRCA1C (wt), pcDNA3-BRCA1(Y1853→STOP) (mut) or pcDNA3-BRCT, as indicated. Cellular extracts were analyzed by Western blotting with an anti-HA antibody.
Figure 9
Figure 9
A model for the putative mechanism responsible for the differential activity of the K18 promoter and for the effect of the different E1A mutants. The E1A full-length protein is represented at the top with emphasis on the two domains of interest: aa 12 to 25 and the PLDLS motif. The horizontal line represents the DNA and the initiation site is symbolized by an arrow, whose thickness is an indication of the activity of the promoter. (A) Functioning of the K18 promoter in T and NT cells. The F factor is an as yet unidentified protein which has a HAT/FAT activity and is supposedly more abundant or more active in T cells (bold) than in NT cells. F acetylates (Ac) the substrate protein S, a non-histone protein whose acetylation level controls the activity of the K18 promoter. The acetylation level of S is also under the control of HDAC proteins. The CtBP1 protein interacts with the PLDLS motif of the CtIP protein. CtBP1 associates with some HDAC proteins by a domain that is different from the one recognizing the PLDLS motif [4, 48]. The recruited HDAC proteins deacetylate the S substrate in NT cells where the acetylase activity is already weak and this down-regulates the promoter. CtIP is an interaction partner of BRCA1 which is represented here as part of the preinitiation complex (PIC). (B) Effects of C-terminal deletion mutants of E1A on the activity of the K18 promoter in T and NT cells. The binding of these mutants to the F factor through the 12–25 domain results in a low HAT/FAT activity, deacetylation of S and inhibition of the promoter in T cells. These mutants are expected to have little effect in NT cells since the level of acetylation of S is already low in these cells. (C) Effects of N-terminal deletion mutants of E1A on the activity of the K18 promoter. The C-terminal part of E1A contains the PLDLS motif which permits its interaction with CtBP1. The displacement of CtBP1 from CtIP prevents the recruitment of HDAC proteins to the preinitiation complex. This is of no consequence in T cells where the S protein is maintained in an hyperacetylated state by the high HAT/FAT activity. In contrast, in NT cells removal of HDAC proteins allows the weak HAT/FAT activity to acetylate S to a high level which results in a stimulation of the promoter.
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