Vinculin is an immediate early gene. Its promoter contains an SRE, and this promoter element is shown to be critical for serum stimulated induction of transcription. The SRE dependent transactivation is wholly sensitive to inhibitors o f the actin pathway unlike the c-fos gene, where activation is largely sensitive to inhibitors o f the MAP Kinase pathway. The vinculin SRE, unlike that o f c-fos, does not have an adjacent TCF binding site, and it is the TCF binding site that has been demonstrated to be responsible for preventing actin pathway activation o f the Q-fos SRE (Murai and Treisman, 2002). However, here it is shown that introduction o f TCF binding sites into the vinculin promoter is not sufficient to render this promoter insensitive to actin pathway activation. In addition, insertion of the whole c-fos SRE region into the
vinculin promoter, replacing the vinculin SRE region, does not render the promoter insensitive to the actin pathway. The promoter context of the SRE is therefore an important factor in determining whether the SRE can respond to signals downstream of changes in actin dynamics.
2.2 The vinculin promoter
Moiseyeva and co-workers (Moiseyeva et al., 1993) established a 1.1 Kb sequence 5’ to the transcription start site of the human vinculin gene as the vinculin promoter, as described in the introduction (section 1.2.8.3). This promoter region lacks a TATA box, but contains an SRE at position -262, a CCAAT box at position -200, and six Spl binding sites in the same orientation as the direction o f transcription (See Fig 1.9). Moiseyeva and colleagues showed that this 1.1 Kb sequence was responsive to serum in reporter gene assays. In addition, SRF"' embryonic stem cells display a severely reduced induction in vinculin expression (Schratt et al., 2002; Schratt et al., 2001). And, the serum induced expression of vinculin is known to be dependent on changes in actin dynamics (Sotiropoulos et al., 1999), a transcriptional response dependent on the SRE.
At the immediate-early Q-fosand egr-1 promoters SRF forms a ternary complex with TCF. These immediate early genes are not responsive to the actin pathway, induction o f these genes is not inhibited by the actin pathway inhibitor Latrunculin B. SRE containing promoters which lack associated TCF binding sites, such as vinculin, P- actin and s r fpromoters, are responsive to changes in actin dynamics (Gineitis and Treisman, 2001). In addition, the removal of the TCF binding site from the Q-fos promoter renders this promoter sensitive to changes in actin dynamics, induction becomes sensitive to Latrunculin B inhibition (Murai and Treisman, 2002). It has been hypothesised (Gineitis and Treisman, 2001) that the presence o f a TCF binding site renders an SRE unresponsive to the actin pathway, in that the TCF and actin pathways are mutually exclusive. In this chapter I will examine whether the presence o f a TCF binding site, introduced adjacent to the vinculin SRE, can render this promoter insensitive to changes in actin dynamics.
The vinculinpromoter was chosen to identify what makes a promoter sensitive to the actin pathway, because the promoter has a single SRE without characterised transcription binding sites in its vicinity, and hence sequences in the vicinity o f the SRE can be readily modified. In addition, it is known that the promoter is strongly induced by actin stimuli (Gineitis and Treisman, 2001; Sotiropoulos et al., 1999).
2.2.1 Serum stimulation of a vinculin Luciferase reporter gene
The 1.1 kb promoter region o f the human vinculingene (Moiseyeva et al., 1993) was fused to a luciferase reporter gene (pGL3, Promega). Luciferase assays have the advantage of being quick, and many samples can be assayed simultaneously. However, luciferase assays do not measure the induced RNA level on activation of transcription, and they are sensitive to background promoter activity, hence the transience o f induction can be misjudged.
The vinculin luciferase reporter gene was transfected into NIH 3T3 cells, cells were serum starved overnight, and then stimulated with serum for 7 hr, cells were lysed and luciferase activity was measured. As shown in Fig 2.1 the vinculin luciferase construct (wild type) is induced on serum stimulation, albeit to a low level. This induction can be inhibited by a 30 min pre-treatment with Latrunculin B (lanes 3),
illustrating that the induction is entirely due to the actin pathway. Latrunculin B is a drug which sequesters G-actin monomers (Coue et al., 1987), and hence inhibits the actin pathway (see section 1.2.4.3).
2.2.2 An intact SRE is essential for serum stimulated transcription of vinculin
To verify that the SRE is responsible for induction o f the Vinculin gene by actin pathway stimuli the SRE of the vinculin gene was mutated to the binding site of M CM l. The sequence of the mutant SRE (SRE.M) is identical to the sequence introduced into the Q-fos promoter, which was shown to be incapable of binding SRF by Hill and co-workers (Hill et al., 1993). Vinculin luciferase with the SRE.M mutation was expressed in NIH 3T3 cells, cells were serum stimulated and induction of the gene was monitored. No induction in transcription following serum stimulation could be observed (Fig 2.1, lanes 4-6). The SRE o f the vinculin promoter is therefore essential for serum stimulation of this gene. The necessity of the SRE for serum stimulated transcription o f the vinculin gene as shown here, is comparable to the effect of knocking out SRF, where serum induction of vinculin is similarly abolished (Schratt et al., 2002; Schratt et al., 2001). Basal transcription levels o f this reporter gene are affected by the mutation o f the SRF binding site (Fig 2.1), luciferase activity levels are approximately half that driven by the wild type construct. This suggests the SRE has a role in maintaining basal transcription levels of the Vinculin gene.
2.2.3 Expression of SRF VP 16 in combination with the vinculin reporter genes To verify that SRF binds to the vinculin promoter in the absence of extracellular stimuli, similarly to the binding of SRF to the Q-fospromoter (Herrera et al., 1989), a constitutively active SRF molecule was expressed in NIH 3T3 cells. SRF fused to the VP 16 transcription activation domain was expressed in combination with the vinculin
luciferase reporter gene. Cells were incubated for 24 hr prior to lysis. The level of luciferase activity was vastly increased in cells expressing the SRF VP 16 fusion protein. At low levels of SRF VP 16 expression vector luciferase activity was higher on serum stimulation, this is due to the endogenous SRF bound to a proportion of
vinculin reporter gene promoters. At higher SRF VP 16 expression levels there was little induction seen on serum stimulation (Fig 2.2a), suggesting that SRF can bind to the vinculin promoter in the absence o f extracellular signals. In comparison the SRE.M vinculin construct was not induced to very high levels by the expression o f this
constitutive active SRF (Fig 2.2a, lanes 9-16), this was predicted as this reporter gene does not have a high affinity SRF binding site.
A comparison was made as to the level o f transcription o f several SRE driven reporter genes on expression of 0.1 pg SRF VP 16. This allows one to determine whether the mutations introduced into the vinculin promoter have an effect on the affinity o f SRF for the CArG box o f the promoter. However, it is not possible to compare binding affinity o f SRF VP 16 for different types of promoter (e.g. the vinculin promoter compared to the Q-fos promoter) solely on luciferase activity, due to fundamental differences in promoter structure, such as the TATA box and the presence o f other transcription factor binding sites. Again SRE.M vinculin luciferase was not expressed to levels comparable to the wild type vinculin gene (Fig 2.2b), as SRF cannot bind efficiently to this promoter. High levels of transcription are seen from the c-fos
luciferase and SDA.luciferase (a reporter gene consisting of 3 SRF binding sites without adjacent TCF binding sites in the context o f a minimal P-actin promoter, see section 8.12.1) constructs compared to the vinculin promoter driven luciferase reporter genes. This may be a property of the interactions of the basal transcription machinery with the promoter, and in keeping with this hypothesis vinculin does not contain a canonical TATA box. Alternatively, the vinculin promoter may contain elements inhibitory to SRF mediated transactivation. However, this experiment shows that SRF VP 16 binds at comparable levels to both the wild type and 5T3A (section 2.3.1)
vinculin promoters, and binding is slightly reduced at 5TCF and 3'TCF vinculin
promoters (section 2.3) compared to the wild type promoter.