Descripción de los subprocesos correspondientes al proceso de Facturación

The role o f RhoA mediated activation of SRF, independent o f TCF activation, was established by Hill and co-workers (Hill and Treisman, 1995; Hill et al., 1995; Hill et al., 1994, and section 1.2.4). Studies have shown that the activation of SRF by this pathway is dependent on a decrease in G-actin levels (Sotiropoulos et al., 1999). This thesis describes a 1-hybrid screen to identify the factor responsible for mediating the actin pathway through interactions with SRF. However, during this investigation Olson and co-workers identified an unknown cardiac specific gene, Myocardin, whose protein product was found to induce transcription mediated by SRF to a very high level (Wang et al., 2001). Ubiquitously expressed homologues o f this cardiac specific factor, the MAL proteins, are also able to mediate transcription in an SRF dependent manner (F.Miralles, personal communication). A discussion o f the interaction of MAL containing proteins with SRF can be found in Chapter 1 (Section 1.2.6).

Over-expression o f MAL22 in NIH 3T3 cells leads to a striking increase in transcription of SRF dependent genes. In this thesis the effect o f increasing doses of MAL22 on transcription from the vinculin promoter was monitored (Chapter 4). It is found that the level o f transcription induced by MAL22 is far greater than serum stimulated increases in transcription of the vinculin promoter. The effect of MAL22 over-expression is far less striking in the case o f the c-fos promoter, where induced levels of transcription are no greater than double that induced by serum, and likewise the basal levels of transcription of this gene increases, but only 2-fold. The actin pathway cannot normally function at the c-fos promoter as it is inhibited by TCF, as previously described. MAL22 over-expression can induce a marginal increase in transcription of c-fos, however this induction is likely to be inhibited by over-

expression o f TCF. The hydrophobic groove o f SRF interacts with MAL22 and TCF in a mutually exclusive manner (Murai and Treisman, 2002).

Transcriptional induction of vinculin is significantly increased on over-expression of MAL22 (Fig 4.1). As the actin pathway is solely responsible for the serum induction of the vinculin gene, it is unsurprising that MAL22 is able to activate this promoter. However, the induction by MAL22 far exceeds the induction seen on serum stimulation. The SRE L2 luciferase is responsive to the actin pathway, yet induction by

MAL22 as assayed by luciferase activity shows an increase in transcription levels 2- fold that seen on serum stimulation (Fig 4.2). The basal level o f transcription is induced to levels seen on serum stimulation, as with transcription from the vinculin

promoter, but this is unsurprising considering the factor responsive for activation is present in the nucleus in the absence o f serum stimulation (F.Miralles, personal communication).

A comparison o f transcriptional induction on over-expressing a limiting downstream factor o f a pathway, and stimulating cells with an extracellular stimuli, is clearly difficult. However, one cannot exclude the possibility that activation o f transcription from the vinculin promoter on serum stimulation is partially inhibited, and over­ expression o f MAL22 is sufficient to over-come this inhibition. Further analysis of the promoter would be needed to determine whether elements o f the vinculin promoter are inhibitory to serum mediated transactivation.

An inhibitory domain o f the vinculin promoter may explain why NL.Elk is unable to mediate a TCF dependent response at this promoter. This could work in multiple ways. An inhibitor may prevent interaction between elements bound to the promoter and the transcription machinery. Or, an inhibitor may mediate an inhibitory chromatin structure at the promoter, even in the context of a transiently transfected reporter gene. One would be able to distinguish between these possibilities if a region of the vinculin

promoter was found that was inhibitory to transcription activation. However, Wang and co-workers saw a similarly large induction o f muscle specific SRF target genes on expression of Myocardin. Both, SMC22 and ANF were induced to very high levels on over-expression o f Myocardin (Wang et al., 2001). A similar mechanism may act at these promoters to prevent high levels of induction on serum stimulation.

Alternatively, we are simply observing the induction level possible on over-expression o f a limiting factor directly involved in transactivation.

6.4.1 The necessity of the SRE in MAL22 mediated induction of the vinculin

gene

An explanation for the difference in sensitivity o f the SRE o f the vinculin promoter to the actin pathway compared to SRF target genes like c-fos, maybe an increased affinity o f the vinculin promoter for the SRF co-activator o f the actin pathway. It is possible that this factor ‘X ’ is able to interact with the vinculin promoter at an additional element to the SRE. To test this hypothesis MAL22 was over-expressed in the presence o f a vinculin reporter gene with a mutated SRE, SRE.M, which is severely compromised in ability to bind SRF (Fig 2.12). MAL22 is able to induce transcription fi*om this vinculin promoter (Fig 4.1).

The vinculin promoter without a wild type SRF binding site is induced to ten fold higher levels than the basal level on expression of MAL22. The same increase in induction is not seen fi*om the c-fos promoter with a similar mutation at the SRE. It is therefore possible that MAL22 does indeed interact with the vinculin promoter at an additional element to the SRE. This is an interesting observation, and it will be important to decipher which region o f the vinculin promoter is required for this transcription induction. However, it is also possible that over-expression o f MAL22 is sufficient to recruit a MAL22-SRF complex to a promoter with only a very weak affinity for SRF, as Fig 2.2a shows that the SRE.M sequence may have a weak affinity for SRF. It remains possible however, that MAL22 activates transcription fi*om promoters that don’t contain SRE sequences, in an SRF independent manner. Determining whether there are regions of the vinculin promoter which allow SRF independent transcription, would allow identification o f a MAL22 binding sequence, and allow analysis o f whether this element is found at other promoters.

The binding o f MAL22, at cellular concentrations, to an additional element in the

vinculin promoter is not sufficient to mediate transcription induction, as a vinculin

promoter without a functional SRF binding site is not induced on serum stimulation (Fig 2.6).

6.5

Competition model; The hydrophobic groove on SRF is the