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Descot, Arnaud (2010): Identification and Functional Characterisation of Genes regulated by Monomeric Actin. Dissertation, LMU München: Faculty of Biology
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Abstract

Monomeric actin controls the activity of the transcription factor Serum Response Factor (SRF) via its coactivator MAL/MRTF-A. Upon signal induction, MAL is released from actin, binds SRF and activates target gene expression. In order to characterise the physiological role of this signalling pathway, I screened on a genome wide basis for target genes by transcriptome analysis. A combination of actin binding drugs (Cytochalasin D and Latrunculin B), targeting monomeric actin, was used to specifically and differentially interfere with the complex between MAL and actin. 210 genes primarily controlled by monomeric actin were identified in mouse fibroblasts. Among them more than 30% have been already found in screens for SRF target genes, supporting the validity of the screening approach. As expected, a lot of genes were involved in cytoskeleton organization. However, genes having anti-proliferative or pro-apoptotic features were identified surprisingly to the same extent. Consistently, I could demonstrate an antiproliferative function of MAL. More specifically, several genes interfering with the MAPK pathway were identified. One of them was Mig6/Errfi1, a negative regulator of EGF receptors. Mig6 induction by LPA or FCS revealed to be dependent on MAL, monomeric actin and the small GTPases Rho. Activated forms of MAL or SRF were sufficient to induce Mig6 expression. Subsequently, a Mig6 promoter element was found to be necessary to mediate MAL/SRF induction. Moreover, induction of Mig6 through the Actin-MAL pathway led to the downregulation of the mitogenic EGFR-MAP kinase cascade. For the first time a transcriptional link between G-actin levels sensed by MAL and the regulation of EGFR signalling was established. Furthermore, after having demonstrated that MAL induces apoptosis, I focused on the characterisation of two proapototic targets identified in the screen: Bok and Noxa. Bok and Noxa were induced by activators of the Rho-Actin-Mal-Srf pathway on a MAL dependent manner. The study of the Bok promoter revealed the existence of a response element that was necessary for the induction by MAL-SRF. Interestingly, apoptotic inducers like staurosporine, TNFα, or the DNA damaging agent Doxorubicin triggered MAL-SRF mediated transcription. As SRF controls the expression of the anti-apoptotic genes Bcl2 and Mcl1, the results from this work places thus SRF as a key transcription factor controlling the balance between pro and anti apoptotic genes in response to external cues.