Identification and Functional Characterisation of Genes regulated by Monomeric Actin

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.