Gineitis, Dzugas; (2001) SRF activation in immediate early gene transcription. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The research described in this thesis was focused on the study of signalling pathways that lead to activation of serum response factor (SRF) and its target genes in response to diverse extracellular signals, such as serum, LPA, PDGF or TPA. SRF is able to bind to the serum response element (SRE) which is found in many, but not all immediate early gene promoters. At some promoters, like c-fos and egr-1, in conjunction with SRF, ternary complex factor (TCF) can bind to the SRE. TCF-dependent transcription is regulated by MAP kinases in response to growth factors or stress stimuli, while SRF activity is potentiated by RhoA-dependent signalling pathways in response to serum or LPA. In contrast to transiently transfected templates, an integrated SRF reporter gene is also activated by growth factors or the phorbol ester TPA. Using pharmacological inhibitors it is demonstrated that activation by growth factors and TPA was dependent on PI-3 kinase activity, while activation of SRF by serum and LPA was not. SRF activation by all stimuli was absolutely dependent on RhoA activity and largely independent of MEK activity. Cloning of LIMK, a regulator of the actin treadmilling cycle, as an SRF activator, suggested that actin dynamics are involved in regulating transcriptional activation. Studies with activators and inhibitors of actin polymerisation demonstrated that depletion of the cellular G-actin pool is necessary and sufficient for SRF activation. In contrast, alterations in actin dynamics are neither necessary nor sufficient for activation of TCF. Accordingly, activation of some SRF target genes, such as c-fos and egr-1, was actin and RhoA independent, but MAPK dependent. However, another group of genes, such as srf and vinculin, was actin and RhoA dependent, but largely independent of MEK-ERK signalling. Based on these findings, two classes of SRF target gene can be defined: one regulated by actin dynamics and a second by MEK-ERK signalling. It is proposed that two classes of SRF target gene are regulated in a mutually exclusive manner, and that the presence of TCF may control signalling specificity at SRF target promoters.

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