Two types of SRF target gene

The use o f pharmacological inhibitors allowed the relative importance o f the TCF dependent, and TCF-independent pathways, to be evaluated on several SRF target genes (Gineitis and Treisman, 2001). Latrunculun B, which sequesters G-actin monomers and inactivates the Rho-actin pathway, was found to inhibit activation of

vinculin and s r f genes. This inhibitor did not significantly inhibit the induction of c-

fo s and egr-1 genes. In contrast, U0126, which inhibits TCF phosphorylation by inhibiting MEK (Duncia et al., 1998), severely inhibited transcriptional activation following serum stimulation o f c-fos and egr-1 genes, yet had little effect on vinculin

and s r f genes.

The promoters o f vinculin and s r f do not have well defined TCF binding sites adjacent to the SRF binding site (Moiseyeva et al., 1993; Spencer and Misra, 1996),

and hence it is reasonable that the serum induction of these genes is not dependent on activation o f the MAP kinase pathway. However, that c-fos and egr-1 are not sensitive to Latrunculin B inhibition suggest that the actin pathway doesn’t function at these promoters, a result supported by the lack o f induction o f these genes by the actin pathway activating drug, Jasplakinolide (Sotiropoulos et al., 1999). There is a direct correlation between the presence o f a TCF binding site adjacent to the SRE, and the lack of sensitivity to the actin pathway. The recruitment o f the effector o f the actin pathway to SRF may therefore be sterically hindered by TCF. This would have interesting implications on the signalling specificity of transcriptional activation by SRF, and defines two sets o f SRF target genes (Gineitis and Treisman, 2001).

Hill and co-workers, first established that the actin pathway activator maybe blocked from binding SRF by TCF using SRE reporter genes. An SRF reporter gene was established that bound SRF poorly, and therefore required over-expression of SRF for efficient transcriptional activation. Over-expression o f a TCF derivative that could bind to this reporter gene potentiated serum induced activation. However, over-expression of a TCF derivative deleted in C box residues important for transactivation resulted in the abolition of TCF independent transactivation. The over-expression of a TCF molecule, itself unable to activate transcription, abolished the ability o f SRF to activate transcription independently o f TCF (Hill et al., 1994).

1.2.5.1 Alteration of the signalling specificity at the SRE

Murai and Treisman tested the model that a TCF binding site adjacent to the SRE renders a promoter refractive to the actin pathway. Murai and Treisman removed the TCF binding site o f the c-fos SRE, and determined what effect this had on the signalling specificity (Murai and Treisman, 2002). It was found that this RhoA-actin pathway unresponsive gene became sensitive to inhibitors of this pathway, such as the G-actin sequestering drug Latrunculin B, on removal o f the TCF binding site. Furthermore, it was possible to negate the sensitivity to Latrunculin B, by expressing a derivative o f the TCF Elk-1, which could bind to the mutated c-fos promoter.

The experiments carried out by Murai and Treisman suggest that TCF does indeed bind to SRF in such a way as to block the actin pathway. A TCF derivative with

point mutations in the B-box, L158P or Y159A, which abolish the interaction between SRF and TCF, was not able to change the sensitivity of the Q-fos mutant promoter to the actin pathway (Murai and Treisman, 2002). It is therefore assumed that TCF and the downstream activator o f the actin pathway compete for common surfaces on SRF, and hence their interaction with SRF is mutually exclusive.

The use o f the NL.Elk TCF derivative established that TCF inhibits the sensitivity to the actin pathway, however the NL.Elk construct is dependent on SRF for recruitment to the promoter, and hence one cannot decipher whether physical contact between Elk-1 and SRF is required for inhibition. Murai and Treisman established that SRF-TCF interaction is required to inhibit the actin pathway by the use of Gal4- TCF fusion proteins that are able to bind to a derivative o f the Q-fos promoter independently o f SRF. Gal4-TCF fusions with an intact B-box inhibited the actin pathway, however Gal4-TCF fusion proteins with mutations in the B-box critical for SRF interaction failed to inhibit the actin pathway.

The hydrophobic groove o f SRF (section 1.2.2.2 and 1.2.3) appears to play a significant role in interaction with multiple factors. The interaction between the B- box o f TCF and SRF recruits TCF to a promoter, however this SRF-TCF interaction prevents the interaction of SRF with the co-activator of the actin pathway. It is reasonable that the binding o f one factor can restrict the activation of the promoter to another pathway that targets the same region of SRF.

In addition, removal of the API binding site found 3' to the Q-fos SRF binding site renders the serum induction of this gene partially sensitive to the actin pathway inhibitor, Latrunculin B (Murai and Treisman, 2002). However, the effect o f mutating the TCF binding site has a more striking effect. It therefore seems that removal o f the steric constraints from SRF bound at the Q-fospromoter can render the gene responsive to the actin pathway. However, the B box interaction o f TCF with SRF has the greatest effect on inhibition o f the actin pathway, and the effect of the API site may be purely steric, a hindrance of access o f the actin pathway SRF co-factor to SRF.

Work in this thesis describes the introduction o f a TCF binding site into the actin pathway responsive gene, vinculin. Vinculin is an immediate early gene, whose induction on serum stimulation is abolished in the absence o f SRF (Schratt et al., 2002; Schratt et al., 2001). The promoter is described further in section 1.2.8.3. It is found that a TCF binding site alone is not sufficient to render the promoter insensitive to the actin pathway (Chapter 2), however targeting an altered specificity Elk-1 derivative to the promoter can inhibit serum induction o f this gene (Chapter 3). It appears that promoter context plays an important role in the interaction between SRF and its cofactors, and that the interaction between multiple promoter elements determine the sensitivity o f a promoter to the actin pathway. That the removal o f the API binding site 3’ to the Q-fos SRE rendered the promoter more sensitive to the actin pathway (Murai and Treisman, 2002) supports the conclusion that other promoter elements, in addition to a TCF binding site, alter the sensitivity of an SRE to the actin pathway.