At the c-fos promoter the presence o f TCF is the determining factor in sensitivity to the actin pathway. TCF binds to SRF and prevents the actin pathway from functioning; MAL22 cannot interact with SRF. On over-expression o f MAL22, the promoter is activated, but not to a large degree. On over-expression o f MAL22 the balance in the competition for SRF between TCF and MAL22 is slightly shifted, but the MAP Kinase activation o f TCF remains the main pathway whereby the c-fos gene is activated.
The vinculin promoter is responsive to the actin pathway. The insertion o f a TCF binding site is not sufficient to restrict the actin pathway. However, over-expression o f NL.Elk in combination with a vinculin promoter with a LexA binding site adjacent to the SRF binding site can inhibit the actin pathway. It is clear that at this promoter it is MAL22 that has the strongest affinity for SRF, the balance at this promoter is shifted towards the actin pathway. Additional proteins/promoter sequence that can interact with MAL22 may affect the balance in pathway sensitivity. The high constitutive activity of SRF in the presence o f myogenic factors, such as MyoD, may reflect interactions between MAL22 and the myogenic proteins, thereby sensitising SRF to basal levels o f active MAL22. We have no evidence that MAL22 interacts with such proteins, however it is reasonable that multiple protein-protein interactions can take place between MAL22 and various transcription factors.
6.5.1 Determination of a promoters sensitivity to the actin pathway
It is of interest to determine what influences the sensitivity o f a promoter to the balance between TCF and factor ‘X ’. Previous studies indicated that the main determinate of sensitivity to the actin pathway is a TCF binding site in the vicinity of the SRE (Murai and Treisman, 2002). Work carried out here suggests that a TCF binding site, albeit necessary to reduce sensitivity to the actin pathway, is not always sufficient. Whether the TCF binding site alone can alter the sensitivity o f a promoter to the actin pathway is promoter specific. It will be important to determine whether
vinculin is a special case, due to the interaction o f multiple promoter elements at this promoter. Or, whether c-fos is the only SRF target gene whose signalling pathway specificity can be altered simply by the change in one transcription factor binding site. Promoter dissection of multiple SRF target genes is needed to address this question, see section 7.1 for possible strategies for future investigation.
It has been established that the SRE o f vinculin is critical for serum induction of the
vinculin gene, however, if s possible that MAL22 has an affinity for other regions of the vinculin promoter. The increased affinity of the vinculin promoter for MAL22 is not great enough to allow serum induced transcription in the absence of SRF, as the SRE.M construct is not induced on serum stimulation, however, it could explain why
vinculin is insensitive to inhibition of the actin pathway by TCF. Likewise, it is known that other elements o f the c-fos promoter are responsive to MAP Kinase signals (De Cesare et al., 1998), which may explain why this promoter is responsive only to the ERK pathway.
6.5.2 Implications of the complexity of the vinculin promoter in understanding signalling specificity at the SRE
It is not always possible to assess the role o f a transcription factor binding site at a promoter simply by removing the site, or adding a new site. It is important to realise that there are multiple interactions between proteins bound to a promoter, both between transcription factors themselves, and between the transcription factors and co activators/the basal transcription machinery. Altering the interaction of one factor with a promoter can effect multiple transcription factor binding elements. We have seen here that particular promoter elements, like the SRE region o f the c-fos gene, can activate transcription strongly in the context o f one promoter, c-fos, but not in the context o f another promoter, vinculin.
Promoters are indeed very complex jigsaw puzzles (Section 1.1) with many interdependent interactions. Removing/adding one factor to the promoter can alter multiple interactions, preventing a step-by-step delineation o f how the promoter functions.
In the case o f the c-fos gene, removing the TCF binding site results in activation via the actin pathway, and therefore leads to important conclusions as to the convergence of signalling pathways at the SRE. It was therefore a reasonable hypothesis to assume the introduction of a TCF binding site into the vinculin promoter would alter sensitivity to the actin pathway.
At the c-fos promoter SRF potentiates the ability o f TCF to mediate transcription, at the vinculin promoter NL.Elk, although able to block the actin pathway, could not be activated on MAP Kinase activation. NL.Elk is able to bind to the hydrophobic groove o f SRF to prevent interaction with MAL22, however, at the c-fos promoter it is also able to mediate transcription induction.
Murai and co-workers predicted that one would be able to design promoters equally sensitive to RhoA-actin and ERK signalling pathways (Murai and Treisman, 2002). However, work carried out here suggests that our knowledge of transcription factor- transcription factor interactions is not sophisticated enough to design artificial promoters capable of responding to specific signalling pathways. We are beginning to understand the interaction of SRF and the MAE proteins (Wang et al., 2001, F.Miralles, A. Zaromytidou, unpublished observations), and we are some way to determining the specific interactions between SRF and TCF (Hassler and Richmond, 2001; Ling et al., 1998). However, the promoter context that these local protein- protein interactions takes place within, is critical in understanding how factors respond to specific signals.
It is possible that an inhibitory domain within the vinculin promoter prevents activation o f Elk-1 at the vinculin promoter, and that MAL22 has an affinity for another element of the vinculin promoter. Such findings would not compromise the ability to predict the signalling sensitivity o f a given promoter, or design an artificial promoter, these observations would simply add another level o f complexity to the interaction between SRF and its co-factors. However, if there are multiple protein- protein interactions at the vinculin/c-fos promoters, and the specific orientations o f specific elements within the vinculin promoter render it refractory to the ERK pathway, then we are far from predicting the sensitivity o f promoters to specific signalling cascades.
The IFN-p promoter involves a complex arrangement of multiple factors, in a specific temporal sequence, to allow induction of this gene in response to virus (Section 1.1.3). It is conceivable that both the vinculin and c-fos promoters use the same level of complexity as the IFN-P promoter to maintain regulation of induction. By altering promoter sequences we are altering a myriad of interactions.