Richard Treisman

Growth factor-induced transcriptional regulation

Growth factor-induced transcriptional regulation through alterations in actin dynamics and MAP kinase signalling

We study the molecular mechanisms by which signalling pathways regulate gene transcription. Our principal focus is on Serum Response Factor (SRF), which controls growth factor-regulated genes such as c-fos, egr-1, vinculin and actin, and plays a major role in maintenance of the actin cytoskeleton ¹. SRF activity is controlled through its differential association with two families of signal-regulated cofactors, the MRTFs (Myocardin-Related Transcription Factors) and the TCFs (Ternary Complex Factors), which compete for a common surface on its DNA-binding domain ^{2, 7}.

Two MRTFs, MAL/MKL1 and MAL16/MKL2, serve to link SRF activity to Rho GTPase signalling ⁴. These actin-binding proteins are predominantly cytoplasmic, in unstimulated cells, and Rho-induced depletion of the G-actin pool is sufficient to cause their nuclear accumulation and activation ^{3, 6}. Our recent studies have demonstrated MRTFs continuously shuttle through the nucleus, and that actin controls their activity at multiple levels ⁸.

We have also defined the actin binding elements within the regulatory domain ¹⁰. The three TCFs, SAP-1, Elk-1 and Net, control SRF through classical MAP kinase signalling, via phosphorylation of their C-terminal regulatory domains ¹, and we have shown that the SAP-1 TCF plays an important role in T cell development ^{5, 9}.

Our aim is to understand the function of the SRF network, and signalling mechanisms controlling MRTF and TCF activity, in molecular detail. Our current approaches include mammalian cell transfection, yeast two-hybrid and siRNA screening, FACS analysis, immunofluorescence microscopy and FRET, protein-DNA interaction analysis and structural studies.

Regulatory pathways controlling MRTF activity

Current work on regulation of the MRTF proteins aims to understand the nature of the regulatory pathways involved. Actin binding is of critical importance controlling both import, export, and activity within the nucleus, and structural studies are in progress to elucidate the structure and significance of actin-MAL complexes ⁸.

We are carrying out functional screens using RNAi libraries to identify new components of the MAL pathway. The focus is on factors regulating nuclear export and import, and transcriptional activation and repression. The molecular mechanisms of regulation will be defined using molecular cell biology, biochemistry, and biophysics approaches such as FRET/FLIM imaging. The functional significance of pathway components will be evaluated using standard gene regulation assays, ChIP analysis, and their effects on cellular processes controlled by SRF including cell adhesion, cell transformation and tumorigenesis.

Function of the SRF network in the mouse

We inactivated the SAP-1 TCF in the mouse, demonstrating that SAP-1 is not required for viability but is required for thymocyte development, acting redundantly with other TCF family members ^{5, 9}. The immune system provides an attractive and readily manipulated system for the study of the relationship between signalling to SRF and cellular behaviour in vivo. Conditional gene inactivation, transgene and siRNA expression will be used to ablate or alter the activity of SRF signalling system in both thymocytes and peripheral T cells.

Mouse tumour models will be used to evaluate the significance of the SRF network for tumorigenesis in vivo, and reporter genes will be developed to measure MRTF pathway activity and actin-binding status of MRTF proteins in situ.

References

1. Treisman R. Journey to the surface of the cell: Fos regulation and the SRE. EMBO J 1995; 14: 4905-4913.
2. Gineitis D and Treisman R. Differential usage of signal transduction pathways defines two types of SRF target gene. J Biol Chem 2001; 276: 24531-24539.
3. Posern G, Sotiropoulos A and Treisman R. Mutant cytoskeletal actins reveal a role for unpolymerised actin in control of transcription by Serum Response Factor. Mol Biol Cell 2002; 13: 4167-4178.
4. Miralles F, Posern G, Zaromytidou A-I and Treisman R. Actin dynamics control SRF activity by regulation of its coactivator MAL. Cell 2003; 113: 329-342.
5. Costello PS, Nicolas RH, Watanabe Y, Rosewell I and Treisman R. Ternary Complex Factor SAP-1 is required for ERK-mediated thymocyte positive selection. Nature Immunology 2004; 5: 289-298.
6. Posern G, Miralles F, Güttler S and Treisman R. Mutant actins that stabilise F-actin use distinct mechanisms to activate the SRF coactivator MAL. EMBO J 2004; 14: 3973-3983.
7. Zaromytidou A-I, Miralles F and Treisman R. MAL and TCF use different mechanisms to contact a common surface on the SRF DNA-binding domain. Mol and Cell Biol 2006; 26: 4134-4148.
8. Vartiainen MK, Güttler S, Larijani B and Treisman R. Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL. Science 2007; 317: 1749-1752.
9. Willoughby JE, Costello PS, Nicholas RH, Robinson J, Stamp, G, Powrie F and Treisman R. ERK signalling but not SAP-1 activation is involved in regulatory T cell development. J Immun 2007; in press.
10. Güttler S, Vartiainen MK, Miralles F, Larijani B and Treisman R. RPEL motifs link the SRF cofactor MAL but not myocardin to Rho signalling via actin binding; submitted August 2007.