Definition of the Ras/ERK signal induced chromatin modification at target gene promoters [RNA-seq & ChIP-seq]. Mus musculus

Ras plays roles across the spectrum of proliferation, invasion and metastasis. The activity of Ras genes, usually K-Ras, is altered by mutations in around 20% of human cancers, and activation of the Ras-ERK pathway is a critical event in melanomas, colorectal and renal cancers but the degree to which Ras- or Raf-driven transformation is dependent on the activity of the SRF network remains largely obscure. Oncogenic active Ras is known to mediate the cellular response to multiple growth factors leading to the activation of immediate-early genes (IE). A key player in IE gene response is the SRF transcriptional network that we identified being crucial to mediate allegedly the entire transcriptional response downstream of Ras/ERK. The Serum response factor (SRF) is a transcription factor with low intrinsic transcriptional activity and requires the recruitment of one of two families of co-activators: the MRTFs (myocardin-related transcription factors) and the TCFs (ternary complex factors, Elk-1, SAP-1, Net). In particular the TCF, member of the wide family of ETS transcription factors, are specifically activated by MAPK signalling downstream of Ras. We characterised the transcriptional program downstream of Ras/ERK signalling using short TPA stimulations (12-O-tetradecanoyl phorbol-13-acetate, compunt known to induced Ras/ERK activation) by RNA-seq. The investigation of the transcriptional response was performed using Mouse Embryonic Fibroblasts (MEFs) derived from different animals: (i) wild type, (ii) TKO (MEFs derived from mice lacking all three TCFs), (iiI) TKO recon pMY Empty (TKO reconstituted with empty vectors by retrovirus infection), (iv) TKO recon Elk-1 wt (TKO reconstituted with retrovirus vectors expressing Elk-1 wild type by retrovirus infection), (v) TKO recon Elk-1 FW (TKO reconstituted with retrovirus vectors expressing Elk-1 mutant lacking the FW residues in its activation domain), (vi) TKO recon Elk-1 nonA (TKO reconstituted with retrovirus vectors expressing Elk-1 mutant where all phosphor-acceptor residues in its activation domain have been mutagenized to alanines). In order to map SRF and Elk-1 binding sites we performed ChIP-seq of SRF in wild type MEFs and TKO and ChIP-seq of Elk-1 in TKO reconstituted cell lines. Overall design: Chromatin immunoprecipitation (ChIP-seq) in Mouse Embryonic Fibroblasts (MEFs) derived from wild type mice (MEF WT) or animals lacking all three TCFs (TCF KO) after TPA (12-O-tetradecanoyl phorbol-13-acetate) using antibodies against SRF (G-20 Lot: D3013 Santa Cruz). In addition the distribution of Elk-1 (in house antibody raised against Elk-1 activation domain able to recognise it regardless of the integrity of the phopsho-acceptor sites) have been assessed in TCF KO cells reconstituted with Elk-1 (one of the member of the TCF factos) variants: (i) TKO recon pMY Empty (TKO reconstituted with empty vectors by retrovirus infection), (ii) TKO recon Elk-1 wt (TKO reconstituted with retrovirus vectors expressing Elk-1 wild type by retrovirus infection), (iii) TKO recon Elk-1 FW (TKO reconstituted with retrovirus vectors expressing Elk-1 mutant lacking the FW residues in its activation domain), (iv) TKO recon Elk-1 nonA (TKO reconstituted with retrovirus vectors expressing Elk-1 mutant where all phosphor-acceptor residues in its activation domain have been mutagenized to alanines). We performed strand specific total-RNA-seq following DSN normalisation and validation by qRT-PCR from the various cellular backgrounds: (i) wild type, (ii) TKO (MEFs derived from mice lacking all three TCFs), (iiI) TKO recon pMY Empty (TKO reconstituted with empty vectors by retrovirus infection), (iv) TKO recon Elk-1 wt (TKO reconstituted with retrovirus vectors expressing Elk-1 wild type by retrovirus infection), (v) TKO recon Elk-1 FW (TKO reconstituted with retrovirus vectors expressing Elk-1 mutant lacking the FW residues in its activation domain), (vi) TKO recon Elk-1 nonA (TKO reconstituted with retrovirus vectors expressing Elk-1 mutant where all phosphor-acceptor residues in its activation domain have been mutagenized to alanines). Each RNA-seq sample has been normalised/standardised to a set of invariant genes across all the condition and background used. The selection of such set was performed using mixed-model distribution of the residuals between samples as genes not changing should present a distribution of the variation approximately normally distributed.