A functional RNAi screen for regulators of the serum response factor (SRF).
Doctoral thesis, UCL (University College London).
Serum response factor (SRF) controls transcription of numerous immediate early and muscle-specific genes by its association with diverse co-activators. Amongst the SRF binding regulators are the TCF family proteins, including Elk1, SAP1 and SAP2 and the myocardin-related transcription factor (MRTF) family proteins including MAL and MAL16. Both types of transcriptional coactivators form a complex with SRF, which binds to serum response elements (SREs) upon serum activation by different upstream signal transduction pathways. While SRF activation by TCFs occur through the MAP kinase pathway, MRTFs regulate SRF specifically through the RhoA pathway. MAL, a G-actin binding protein, continuously shuttles between cytoplasm and nucleus in many unstimulated cells and appears cytoplasmic. Upon RhoA-induced actin polymerization, MAL nuclear export is blocked due to disruption of the MAL-actin interaction. MAL accumulates in the nucleus where it binds to SRF, which results in target gene expression. Although many of the pathway components are already known, it is probable that there are still uncharacterized factors that regulate SRF. Therefore this thesis addresses the identification of new regulators of SRF that are specifically dependent on the RhoA pathway. Since the interaction of MAL with SRF and actin represents an evolutionarily conserved regulatory mechanism between Drosophila and human and the Drosophila genome lacks much of the genetic redundancy observed in human, a genome-wide RNA interference (RNAi) screen in cultured Drosophila cells was performed to identify key regulators of SRF. Using RNAi, 295 proteins in Drosophila cells were identified, of which 50 were further validated in human cells. Amongst the final confirmed hits, eukaryotic elongation translation factor (eEF1A) was a promising regulator for RhoA induced SRF activation. eEF1A2 was shown to interact with MAL and depletion of eEF1A2 in unstimulated cells led to nuclear accumulation of MAL, suggesting its role as a MAL regulator. Potential roles of the remaining hits confirmed in the rescreen are also discussed in this thesis.
|Title:||A functional RNAi screen for regulators of the serum response factor (SRF)|
|Additional information:||Authorisation for digitisation not received|
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