Comment[ArrayExpressAccession]	E-GEOD-53098
MAGE-TAB Version	1.1		
Public Release Date	2014-03-06
Investigation Title	Gene expression changes associated with 3D growth of TAF4 knock out fibroblasts		
Comment[Submitted Name]	Gene expression changes associated with 3D growth of 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. 6 samples corresponding to non-confluent cells, confluent cells and cells growing as fibrospheres were analyzed. Each growing condition 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	Martianov	Davidson
Person First Name	Igor	Igor	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-GSE53098-2	P-GSE53098-1	
Protocol Description	Illumina Casava1.8 software was used for image analysis and basecalling. Sequence reads mapped to reference genome mm9/NCBI37 using Tophat (Trapnell et al., 2009) v1.2.0 with bowtie v0.12.7. Genome_build: mm9 Supplementary_files_format_and_content: The ReadCountPerGene.tsv file is a tab separated text file containing the raw read count and the normalized number of reads for each Ensembl Gene (Ensembl v61).	Libraries for high throughput DNA sequencing were created using M-bM-^@M-^\TruSeqM-bM-^DM-" RNA Sample Preparation v2 KitM-bM-^@M-^] (Illumina). Briefly, mRNA was purified from 2 M-5g total RNA using poly-T oligo-attached magnetic beads and fragmented using divalent cations at 94M-0C for 8 minutes. The cleaved mRNA fragments were reverse transcribed to cDNA using random primers then the second strand of the cDNA was synthesized using DNA Polymerase I and RNase H. The double stranded cDNA fragments were blunted using T4 DNA polymerase, Klenow DNA polymerase and T4 PNK. A single M-bM-^@M-^XAM-bM-^@M-^Y nucleotide was added to the 3M-bM-^@M-^Y ends of the blunt DNA fragments using a Klenow fragment (3' to 5'exo minus) enzyme. The cDNA fragments were ligated to double stranded adapters using T4 DNA Ligase. The ligated products were enriched by PCR amplification (30 sec at 98M-0C; [10 sec at 98M-0C, 30 sec at 60M-0C, 30 sec at 72M-0C] x 12 cycles; 5 min at 72M-0C). Then surplus PCR primers were removed by purification using AMPure XP beads (Agencourt Biosciences Corporation). DNA libraries were checked for quality and quantified using 2100 Bioanalyzer (Agilent). The libraries were loaded in the flow cell at 8pM concentration and clusters were generated in the Cbot and sequenced in the Illumina Hiseq 2500 as single-end 50 base reads. FASTQ files were generated with CASAVA v1.8.2.	
Protocol Type	normalization data transformation protocol	nucleic acid library construction protocol	
Experimental Factor Name	TREATMENT		
Experimental Factor Type	treatment		
Comment[SecondaryAccession]	GSE53098		
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]	GSE53098_ReadCountPerGene.tsv		
Comment[SecondaryAccession]	SRP033571		
Comment[SequenceDataURI]	http://www.ebi.ac.uk/ena/data/view/SRR1044935-SRR1044940		
SDRF File	E-GEOD-53098.sdrf.txt