In this study, we have examined the mechanisms involved in E2-ERα mediated transcriptional repression of early primary target genes, and we describe a new mechanism for ER-mediated transcriptional repression that involves the recruitment of a p300-dependent CtBP1 corepressor complex following ERα failure to activate gene transcription. We demonstrate that ERα can be recruited directly, albeit transiently and less strongly, to DNA elements close to E2-repressed genes where ERα recruits p300 and is able to transiently increase the transcriptional output; however, ERα and p300 are unable to become a nucleation site for p160 coactivators and to sustain positive transcriptional regulation. This leads to recruitment of the corepressor CtBP1, via p300, with RNA polymerase II eviction and histone deacetylation that result in transcriptional repression. Of note, CtBP1 was a crucial factor for gene repression, whereas it was irrelevant for gene stimulation, by estrogen. Furthermore, the important effects of CtBP1 in E2-ERα-mediated gene repression were unique to CtBP1 and were not reproduced by the related CtBP2 protein.
Modulation of gene transcription by ERα: stimulation versus repression.
It is now well accepted that the ER is a master regulator of gene transcription, as demonstrated by numerous studies using both cell culture models and whole-animal target tissues (11
). From these studies it is evident that, upon E2 treatment, gene transcription is widely impacted, creating highly complex regulatory networks whose ultimate goal is the stimulation or suppression of specific biological processes. In fact, in MCF-7 breast cancer cells, the expression of more genes is repressed than stimulated by the E2-occupied ERα (14
). Hence, it was of interest to understand how the ER, being a strong transcriptional activator, can also behave as a transcriptional repressor.
Thus far, several mechanisms have been hypothesized for E2-mediated gene repression, including physiological squelching of cofactors (e.g., p160s and CBP), direct action of corepressors (NCoR, SMRT, and NRIP1) accompanied by histone deacetylation, and participation of elements of the basal transcriptional machinery (e.g., TAFII30). Most of these studies, though, employed exogenous reporter systems or overexpression of selected factors and/or considered events mostly at late time points of E2 treatment (8 to 24 h). What we addressed in this study is the analysis of early, primary ERα-repressed target genes and the mechanisms that occur at their ERα binding sites in the endogenous cell chromatin setting.
From genome-wide studies of ERα binding sites, it appears that ca. 60% of E2-repressed genes at early time points (1 to 4 h) possess at least one ERα binding site in their proximity, indicating that direct effects of ERα are likely and that they may account for at least a portion of the repressive events. In this study, we characterized a group of primary E2-repressed target genes and document that ERα is recruited to these binding sites by E2 treatment but, interestingly, in a manner that is different from recruitment to the enhancer of the TFF1/pS2 gene, which is strongly E2 stimulated. At the ERα binding sites that we studied, ERα occupancy increased comparatively similarly to that at the TFF1/pS2 enhancer in the first 5 to 15 min of E2 treatment, after which ERα occupancy decreased or remained constant at approximately 10 to 15% of the level of TFF1/pS2 enhancer occupancy. This indicates that ERα may interact less efficiently and more transiently with these sites and also points to the fact that the number of binding sites close to E2-repressed genes may have been underestimated by these genome-wide techniques, because only the strongest interactions would be detected and also because, based on our study, a different result might be obtained by using earlier time points (i.e., 15 min) of E2 treatment versus the typical 45-min time point most commonly examined (5
). From our bioinformatic analysis of the composition of the ERα binding sites, there appears not to be any preferential factor linked to E2-repressed versus E2-stimulated genes (F. Stossi, unpublished observation), suggesting that it may be difficult to isolate specific transcription factors that are associated only with E2-mediated transcriptional repression.
p300 plays a central role in both gene activation and repression.
Nuclear receptor coregulators encompass a large family of proteins with multiple enzymatic activities that appear to be essential in performing and fine-tuning the actions of the ER at the chromatin level (29
). Several studies (16
) have highlighted a very dynamic picture of multiple coactivating and corepressing complexes exchanging during the transcriptional process, adding an important level of complexity and control in the regulation and direction (up/down) of the transcriptional output.
Using time-course ChIP assays, we could establish that p300 was the only cofactor that appeared to be recruited at all the sites analyzed, while other factors, like CBP, NRIP1, and p160s, might play more gene-specific roles. Also of note, an important finding in our study was the essential role of p300 in gene repression as well as gene stimulation. p300 was found to be recruited to the binding sites of both E2-stimulated and E2-repressed genes, and p300 knockdown fully prevented E2-mediated gene repression and also markedly reduced E2-mediated gene stimulation.
The role of p300 in ERα-mediated gene stimulation has been extensively characterized where it plays a central role in transcriptional initiation, but not reinitiation (27
), and is normally seen before recruitment of SRCs to the TFF1 gene (31
). There is evidence that ERα and p300 interact directly (12
), although some have suggested that this may involve mediating proteins (e.g., SRC-3).
p300 is also known to elicit negative roles in transcription, as recently shown in a completely purified in vitro transcription system (36
) where p300 acted as a negative cofactor whose repressive activity was reversed by the addition of acetyl-coenzyme A. Moreover, p300 contains a strong repressive domain (cell cycle regulatory domain 1, amino acids 1017 to 1029) that functions independently from the HAT domain via sumoylation and HDAC6 recruitment (17
Since a role for p300 in transcriptional initiation has been extensively characterized, we hypothesized that the p300-ERα-containing complex might be trying to stimulate gene transcription also at E2-repressed targets but ultimately fails to continue the process. To investigate this possibility, we first performed nuclear run-on assays that clearly demonstrated that, at early time points, ERα can transiently stimulate the transcription of E2-repressed genes. Second, after we cleared the coding sequences of the genes from transcribing RNA polymerase II, E2 treatment resulted in the reloading of RNA polymerase II at both E2-repressed and E2-stimulated genes, indicating that ERα is capable of driving positive transcription from binding sites close to E2-repressed genes. The results of these two experiments also lead us to speculate that elements in the basal transcription machinery and/or in the elongation complexes might be important in choosing the direction of regulation of transcription by ERα after this initial phase of stimulation at both types of genes.
The corepressor CtBP1 is utilized by ERα, via p300, to repress gene transcription.
In addressing how ERα and p300 elicit transcriptional repression, we focused on the corepressor CtBP1. Although CtBP1 had not previously been directly linked to ERα action, it had been shown to interact directly with p300 and inhibit the HAT activity of p300 via interaction with its bromodomain, thus impeding p300's recognition and acetylation of histone tails (24
). Moreover, CtBP1-containing complexes have been characterized as containing numerous enzymatic activities, including histone deacetylation via multiple HDACs (i.e., HDAC1 and HDAC2). Although the relationships between histone posttranslational modifications and positive or negative gene activities are known to be very complex (9
), it was striking that robust H3K14 and H3K9 deacetylation accompanied the gene repression by E2 and that these were prevented by depletion of CtBP1.
We demonstrated that CtBP1, in complex with p300, is recruited to E2-repressed genes and is essential for the repressive process and histone tail deacetylation events, highlighting a central role for this factor in ERα-mediated transcriptional repression. In addition, p300 recruitment appeared to be a prerequisite for CtBP1 recruitment, although there may also be additional mechanisms, because CtBP1 has been shown to interact with the corepressors NRIP1/RIP140 and LCoR, which can directly interact with ERα (13
A model for E2-mediated gene repression of early target genes.
Based on our observations, we present a model for E2-mediated transcriptional repression of early target genes (Fig. ). In this scenario, ERα would interact either directly, indirectly, or cooperatively with DNA elements in a manner that is comparable for stimulated and repressed target genes in the first 5 to 15 min after E2 treatment. During this first phase, p300 and, in a gene-specific fashion (i.e., MMD), other cofactors (i.e., SRC-3) are being recruited by ERα, causing a spike in transcriptional activation, possibly due to the RNA polymerase II already cycling at these genes. After this first phase, ERα occupancy starts to decrease or remains constant, and this is followed by a loss of capacity for sustaining a steady increase in transcription that causes RNA polymerase II loss, recruitment of CtBP1-containing complexes, and histone deacetylation at early repressed genes.
FIG. 7. Proposed model for ERα-mediated repression of early target genes. Following E2 treatment, ERα is initially recruited to ERα binding sites of both E2-stimulated and E2-repressed target genes, either via direct DNA binding or tethering (more ...)
It is notable that even at E2-repressed genes, one sees some molecular attributes of gene stimulation, albeit transiently: recruitment of ERα, p300, and to some genes, CBP, and a transient increase in nuclear run-on and RNA polymerase II recruitment on α-amanitin-cleared genes. While it might seem paradoxical to see such changes at genes that show a net reduction of RNA levels, it is clear that elevated RNA production from repressed genes is, at most, brief. Also, at the E2-repressed genes, there is a net dismissal of RNA polymerase II upon E2 treatment, RNA polymerase II recruitment by E2 being evident only on the artificially lowered background following α-amanitin treatment.
The most significant and durable differences between the E2-repressed genes and E2-stimulated genes we have studied appear to be the relative instability of the recruitment of ERα and the clear differential recruitment of certain coregulator complexes, e.g., the corepressor CtBP1 to repressed genes versus the coactivator SRC-3 to stimulated genes. It is the consequences of the known differential chromatin-modifying activities of these coregulators that appear to be responsible for the ultimate differential effects on the production of RNA from the repressed versus the stimulated genes.
Our work raises two interesting questions. First, what is responsible for the fact that the E2-ERα-p300 complex, which forms at both stimulated and repressed genes, recruits CtBP1 only to E2-repressed genes? It is possible that differential posttranslational modifications of p300 or other coregulators by specific enzymatic complexes will determine the choice of protein partners for p300. It is indeed known that a “posttranslational code” exists for coactivators like SRC-3 (29
), and p300 is known to possess multiple sites of posttranslational modification that influence its activity (17
). Second, if E2 treatment results in dismissal of the CtBP1 corepressor system from E2-stimulated genes, by what mechanism is CtBP1 present in the absence of hormone, ERα, and p300? Presumably, in the absence of hormone, CtBP1 is held at stimulated genes through other transcription factors via different coregulatory proteins, like TBL1 (35
). It would be interesting to investigate this system, because such transcription factor-CtBP1 corepressor complexes might be responsible for maintaining the low basal activity expected of genes poised to be stimulated by estrogens acting through ERα.
Our studies highlight a new mechanism utilized by the ER to elicit transcriptional repression of target genes. This mechanism includes a new role for p300 as a bridging factor between ERα and coregulator complexes that appears to be crucial in deciding the direction of transcription after ERα activation and binding to DNA. Moreover, we demonstrate for the first time the involvement of the corepressor CtBP1 in estrogen-mediated gene repression. Thus, the cooperation between CtBP1 and p300 appears to be central in discriminating nuclear receptor repression versus stimulation of genes at early times after hormone exposure.