SRP066205 PRJNA302207 GSE75002 Definition of the Ras/ERK signal induced chromatin modification at target gene promoters Specific spectra of chromatin modifications are associated with different transcriptional states and gene regulatory elements, but relatively little is known about how such chromatin modifications are established. In particular the relationship of chromatin modifications to the activity of specific transcription factors and the signalling pathways that control them remain largely unclear. We investigated the relationship between chromatin modifications and transcription in response to Ras/ERK activationby stimulating cells with TPA (12-O-tetradecanoyl phorbol-13-acetate) for 30 minutes. 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). In particular the TCF, member of the wide family of ETS transcription factors, are specifically activated by MAPK signalling downstream of Ras. Cells lacking all three TCFs are defective in responding to Ras/MAPK signalling. We analysed changes in 5 main histone modifications (H3K9acS10ph, H4K16ac, H3K27ac, H3K9acK14ac and H3K4me3) associated with active transcription by ChIP-seq in Mouse Embryonic Fibroblasts (MEFs) derived from wild type mice (MEF WT) or animals lacking all three TCFs (TCF KO). In addition the distribution of the 5 modifications have been assessed in TCF KO cells reconstituted with Elk-1 (one of the member of the TCF factos) variants. 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 H3K9acS10ph, H4K16ac, H3K27ac, H3K9acK14ac and H3K4me3 histone modifications. In addition the distribution of the 5 modifications have been assessed in TCF KO cells reconstituted with Elk-1 (one of the member of the TCF factos) variants. Distribution of reads around the TSS of all Ref-seq annotated genes (-2Kb to +1Kb) have been investigated. Each sample has been normalised/standardised to a reference set of TSSs of genes that are constitutively active and invariant across the conditions used. The scaling factors have been compared to a second method of normalisation that involve the identification of those TSSs which frequency distribution of the differences across samples can be approximated to a normal-gaussian distribution. GSE75002