Comment[ArrayExpressAccession] E-GEOD-53097 MAGE-TAB Version 1.1 Public Release Date 2014-03-06 Investigation Title Retinoic acid induced changes in gene expression in TAF4 knock out fibroblasts Comment[Submitted Name] Retinoic acid induced changes in gene expression in TAF4 knock out fibroblasts Experiment Description Collagen 6A3 (Col6a3), a component of extracellular matrix, is often up-regulated in tumours and is believed to play a pro-oncogenic role. However the mechanisms of its tumorigenic activity are poorly understood. We show here that Col6a3 is highly expressed in densely growing mouse embryonic fibroblasts (MEFs). In MEFs where the TAF4 subunit of general transcription factor IID (TFIID) has been inactivated, elevated Col6a3 expression prevents contact inhibition promoting their 3 dimensional growth as foci and fibrospheres. Analyses of gene expression in densely growing Taf4-/- MEFs revealed repression of the Hippo pathway and activation of Wnt signalling. The Hippo activator Kibra/Wwc1 is repressed under dense conditions in Taf4-/- MEFs, leading to nuclear accumulation of the proliferation factor YAP1 in the cells forming 3D foci. At the same time, Wnt9a is activated and the Sfrp2 antagonist of Wnt signalling is repressed. Surprisingly, treatment of Taf4-/- MEFs with all-trans retinoic acid (ATRA) restores contact inhibition suppressing 3D growth. ATRA represses Col6a3 expression independently of TAF4 expression and Col6a3 silencing is sufficient to restore contact inhibition in Taf4-/- MEFs and to suppress 3D growth by reactivating Kibra expression to induce Hippo signalling and by inducing Sfrp2 expression to antagonize Wnt signalling. All together, these results reveal a critical role for Col6a3 in regulating both Hippo and Wnt signalling to promote 3D growth, and show that the TFIID subunit TAF4 is essential to restrain the growth promoting properties of Col6a3. Our data provide new insight into the role of extra cellular matrix components in regulating cell growth. 7 samples corresponding to non-treated cells, 12 hours RA treated cells and72 hours RA treated cells were analyzed. First two conditions were done in triplicate. 72 hs point was done in duplicate. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Martianov Cler Davidson Person First Name Igor Emilie Irwin Person Email martiano@igbmc.fr Person Affiliation IGBMC Person Phone 0033388653440 Person Address IGBMC, 1 rue Laurent Fries, Illkirch, France Person Roles submitter Protocol Name P-GSE53097-2 P-GSE53097-1 Protocol Description Illumina Pipeline v1.4 software was used for image analysis and basecalling. The 16-base long reads (excluding the 4-base DpnII recognition site) were aligned to DpnII tag tables generated by Stowers Institute http://research.stowers-institute.org/microarray/tag_tables/index.html using Eland. Genome_build: mm9 Supplementary_files_format_and_content: The NormalizedTagCountPerGenes.tsv file is a tab separated text file containing the normalized number of reads for each Ensembl Gene (Annotations from Ensembl Core v53_37f) . Sequencing libraries were prepared from 1 M-5g of total RNA using reagents from the DpnII Digital Gene Expression Tag Profiling kit (Part # 1003803 Rev. B protocol, Illumina). mRNA was captured on magnetic oligo(dT) beads and reverse transcribed into single-strand cDNA and double-stranded cDNA was then synthesized. The cDNA attached to the beads was digested using the restriction enzyme DpnII. A DpnII adapter was ligated to the DpnII cleavage sites. The adapter-ligated cDNA was then digested with MmeI to release 17bp fragments of cDNA from the magnetic bead. The fragments were dephosphorylated and purified by phenol-chloroform. A second adapter was ligated at the MmeI cleavage sites. The adapter-ligated cDNA fragments were amplified by PCR (30 sec at 98M-0C; [10 sec at 98M-0C, 30 sec at 60M-0C, 15 sec at 72M-0C] x 15 cycles; 10 min at 72M-0C) and the PCR products were purified on a 6% TBE PAGE gel. The 85 bp PCR products were excised from the gel and eluted overnight, followed by ethanol precipitation. DNA libraries were checked for quality and quantified using 2100 Bioanalyzer (Agilent).The libraries were loaded in the flowcell at 8pM concentration and clusters were generated following IlluminaM-bM-^@M-^Ys instructions. Libraries were sequenced on the Illumina Genome Analyzer II as single read 18 bases reads, following the manufacturer's protocols. Protocol Type normalization data transformation protocol nucleic acid library construction protocol Experimental Factor Name TREATMENT TIME Experimental Factor Type treatment time Comment[SecondaryAccession] GSE53097 Comment[GEOReleaseDate] 2014-03-06 Comment[ArrayExpressSubmissionDate] 2013-12-06 Comment[GEOLastUpdateDate] 2014-03-12 Comment[AEExperimentType] RNA-seq of coding RNA Comment[AdditionalFile:Data1] GSE53097_NormalizedTagCountPerGenes.tsv Comment[SecondaryAccession] SRP033570 Comment[SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/SRR1044927-SRR1044934 SDRF File E-GEOD-53097.sdrf.txt