SUPPLEMENTARY MATERIAL
 Transcriptional regulatory program in wild-type and retinoblastoma gene deficient mouse embryonic fibroblasts during adipocyte differentiation.
 Robab Hakim-Weber1, Anne-M Krogsdam2, Claus Jørgensen3, Maria Fischer2, Andreas Prokesch4, Juliane G Bogner-Strauss4, Stefan R Bornstein1, Jacob B Hansen5, Lise Madsen6,7, Karsten Kristiansen6, Zlatko Trajanoski2, Hubert Hackl2§
  
 1 Department of Internal Medicine, Technical University Dresden, Germany
 2 Biocenter, Division of Bioinformatics, Innsbruck Medical University, Austria
 3 Cell Communication Team, Section of Cell and Molecular Biology, The Institute of Cancer Research, London, UK
 4 Institute for Genomics and Bioinformatics, Graz University of Technology, Austria
 5 Department of Biomedical Sciences and 6 Department of Biology, University of Copenhagen, Denmark
 7 National Institute of Nutrition and Seafood Research, Bergen, Norway
  
 § Corresponding author: hubert.hackl@i-med.ac.at
  
 Recent studies showed that the retinoblastoma protein (pRb) was expressed in the cell cycle and late cellular differentiation phase during adipogenesis. To investigate this dual role of pRb in the early and late stages of adipogenesis we used microarrays to perform a comprehensive systems-level analysis of the common transcriptional program of the classic 3T3-L1 preadipocyte cell line, wild-type mouse embryonic fibroblasts (MEFs), and retinoblastoma gene-deficient MEFs (Rb-/- MEFs).

Comparative analysis of the expression profiles of 3T3-L1 cells and wild-type MEFs revealed genes involved specifically in early regulation of the adipocyte differentiation as well as secreted factors and signaling molecules regulating the later phase of differentiation. In an attempt to identify transcription factors regulating adipogenesis, bioinformatics analysis of the promoters of coordinately and highly expressed genes was performed. We were able to identify a number of high-confidence target genes for follow-up experimental studies. Additionally, combination of experimental data and computational analyses pinpointed a feedback-loop between Pparg and Foxo1.

To analyze the effects of the retinoblastoma protein at the transcriptional level we chose a perturbated system (Rb-/- MEFs) for comparison to the transcriptional program of wild-type MEFs. Gene ontology analysis of 64 deregulated genes showed that the Rb-/- MEF model exhibits a brown(-like) adipocyte phenotype. Additionally, the analysis results indicate a different or additional role for pRb family member involvement in the lineage commitment.

The data and the analysis provide a starting point for further experimental studies to identify target genes for pharmacological intervention and ultimately remodeling of white adipose tissue into brown adipose tissue.

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