Additional SRF interacting factors; Clone

A 1-hybrid screen to identify factors that interact with SRF and mediate the actin pathway was conducted (Chapter 5). This screen identified SRF, which would be expected as the reporter genes used in this screen were driven by an SRE, and the expressed cDNA library had a constitutively active transcription domain at their N- terminus. However, TCF was not identified. This could because TCF is a large protein with a long 3’UTR, a TCF clone with the SRF interacting B-box may not therefore be present in this cDNA library which was made using primers complementary to the polyA tail, however this was not investigated. The finding that SRF was included in positives identified from the screen was promising, and indicated the screen was carried out in a manner which would allow identification of interesting proteins. SRF was also identified in the mammalian 1-hybrid screen conducted by Sotiropoulos and co-workers which identified LIM Kinase, and it was this screen that led to the identification o f the role of actin-remodelling in the activation o f SRF by the RhoA pathway (Sotiropoulos et al., 1999). That TCF, and other factors known to interact with SRF, were not identified was disappointing.

A clone that interacted with SRF at the hydrophobic groove, known to be important in mediating the Rho-actin pathway, was termed clone 208 (c208). Clone 208 has proteins identified as homologues in S.cerevisiae, C.elegans^ D.melanogaster^ and is known to be ubiquitously expressed in mouse (Unigene database). Comparison o f the homologous proteins shows a region of strongest homology at their N-terminus (Fig 5.11). This region o f homology is a predicted methyltransferase domain (Niewmierztcka and Clarke, 1999). Deletion of a motif of the predicted methyltransferase domain is sufficient to render the protein incapable o f interacting with SRF in the 1-hybrid system. However, verification o f the interaction between

c208 and SRF through alternative methodology, such as immuno-precipitation was not conducted in this study.

Clone 208 activated both the His and LacZ reporters in the strain o f yeast used for this 1-hybrid screen, strain S62/His3. However, expression of c208, with a VP 16 activation domain, in NIH 3T3 cells elicited only a moderate increase in reporter gene activity, and furthermore, this induction was only seen at those SRF reporter genes whose SRE was found adjacent to a TCF binding site. Induction of transcription by this clone was most striking when basal levels of transcription were assayed by RNase protection (Fig 5.15). It is not clear as to why this assay was most sensitive for measuring an induction in transcription. It is possible that the association of c208 with SRF is o f a very transient nature, and hence transcription levels measured over longer time periods, as with luciferase assays, are less sensitive in recording such a short association. However, a clear difference can be seen on basal levels o f transcription from the Q-fos promoter, and for this measurement there is no signal to induce association between c208 and SRF; the cells are incubated for 24 hours post­ transfection before RNA is extracted.

Transcription, induced on over-expression of clone 208 fused to the constitutively active transcription activation domain VP 16, is not seen when a motif o f the putative methyltransferase domain is deleted (Fig 5.14). There is therefore a direct correlation between the effects o f this protein expressed in NIH 3T3 cells, and the effects seen in the 1-hybrid screen. However, the protein with a deleted motif II is expressed at lower levels than the wild type protein (Fig 5.14b), and it is surprising that the induction levels seen with wild type c208 in mammalian cells is so low.

6.6.1 Promoter Specificity in the interaction between clone 208 and SRF

It is of interest that an induction in transcription by c208 fused to a VP 16 activation domain can only be seen on those promoters with TCF binding sites adjacent to the SRE. An induction in transcription is seen both with the SREi and c-fos promoter driven luciferase reporter genes, and also seen in an RNase protection assay using a probe against the c-fos promoter. Induction is not seen when assayed on SRE Lz and

To explain the interaction o f c208 with SRF at those promoters with TCF binding sites, one could speculate that the DNA sequence, and hence structure surrounding the SRE is critical in mediating a conformation of SRF that can interact with clone 208. One has to invoke a model by which sequence itself is important, and not the presence of TCF, as S.cerevisiae do not have a TCF homologue, and therefore in the screen c208 was able to interact with SRF in the absence o f such an SRF co-factor. This would not be the only explanation for the restriction in clone 208s interaction with SRF. It is also conceivable that in the absence o f TCF a factor binds to SRF, for example MAL22, and this factor prevents the interaction of c208 with SRF. However, at promoters with TCF binding sites, MAL22 does not bind, and hence c208 is able to interact with SRF.

It is also possible that c208 is able to bind to SRF at promoters that do not contain TCF binding sites. However, at these promoters the transcriptional activation domain, VP 16, is not in the correct orientation to interact with the basal machinery, and hence we cannot assay its presence by transcription activation methods. This scenario is unlikely, in that VP 16 is a potent transcriptional activator whose ability to ‘squelch’ the basal machinery is well documented. Chromatin immuno-precipitation experiments would allow further analysis as to at what promoters c208 can interact with SRF, however if the nature of the interaction between c208 and SRF is highly transient then these assays may be difficult to perform.

Further studies are required to probe the interaction between c208 and SRF. It is clear that c208 is not the downstream activator o f the actin pathway, as this clone is not a potent activator of SRF, and its activity is not restricted to those promoters known to be controlled by the actin pathway. However, this factor could be important in restricting the signalling specificity to SRF at given promoters. Further work is needed to define clearly the interaction of clone 208 with SRF, and the functional role of this interaction.