title: Reconstruction of gene regulatory modules from RNA silencing of IFN-α modulators: experimental set-up and inference method.
abstract: BACKGROUND
  Inference of gene regulation from expression data may help to unravel regulatory mechanisms involved in complex diseases or in the action of specific drugs. A challenging task for many researchers working in the field of systems biology is to build up an experiment with a limited budget and produce a dataset suitable to reconstruct putative regulatory modules worth of biological validation.
  
  
  RESULTS
  Here, we focus on small-scale gene expression screens and we introduce a novel experimental set-up and a customized method of analysis to make inference on regulatory modules starting from genetic perturbation data, e.g. knockdown and overexpression data. To illustrate the utility of our strategy, it was applied to produce and analyze a dataset of quantitative real-time RT-PCR data, in which interferon-α (IFN-α) transcriptional response in endothelial cells is investigated by RNA silencing of two candidate IFN-α modulators, STAT1 and IFIH1. A putative regulatory module was reconstructed by our method, revealing an intriguing feed-forward loop, in which STAT1 regulates IFIH1 and they both negatively regulate IFNAR1. STAT1 regulation on IFNAR1 was object of experimental validation at the protein level.
  
  
  CONCLUSIONS
  Detailed description of the experimental set-up and of the analysis procedure is reported, with the intent to be of inspiration for other scientists who want to realize similar experiments to reconstruct gene regulatory modules starting from perturbations of possible regulators. Application of our approach to the study of IFN-α transcriptional response modulators in endothelial cells has led to many interesting novel findings and new biological hypotheses worth of validation.
authors.affiliation: Department of Surgery, Oncology and Gastroenterology, University of Padova, via Gattamelata 64, 35128, Padova, Italy. angela.grassi@unipd.it.
authors.forename: Angela
authors.initials: A
authors.lastname: Grassi
authors.affiliation: Department of Information Engineering, University of Padova, via Gradenigo 6/B, 35131, Padova, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Di Camillo
authors.affiliation: Istituto Oncologico Veneto - IRCCS, via Gattamelata 64, 35128, Padova, Italy.|Present address: Department of Molecular Medicine, University of Padova, via Gabelli 63, 35121, Padova, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Ciccarese
authors.affiliation: Istituto Oncologico Veneto - IRCCS, via Gattamelata 64, 35128, Padova, Italy.
authors.forename: Valentina
authors.initials: V
authors.lastname: Agnusdei
authors.affiliation: Department of Surgery, Oncology and Gastroenterology, University of Padova, via Gattamelata 64, 35128, Padova, Italy.|Istituto Oncologico Veneto - IRCCS, via Gattamelata 64, 35128, Padova, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Zanovello
authors.affiliation: Department of Surgery, Oncology and Gastroenterology, University of Padova, via Gattamelata 64, 35128, Padova, Italy.|Istituto Oncologico Veneto - IRCCS, via Gattamelata 64, 35128, Padova, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Amadori
authors.affiliation: Institute of Electronics, Computer and Telecommunication Engineering, CNR, via Gradenigo 6/B, 35131, Padova, Italy.
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Finesso
authors.affiliation: Istituto Oncologico Veneto - IRCCS, via Gattamelata 64, 35128, Padova, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Indraccolo
authors.affiliation: Department of Information Engineering, University of Padova, via Gradenigo 6/B, 35131, Padova, Italy.
authors.forename: Gianna Maria
authors.initials: GM
authors.lastname: Toffolo
mesh_terms: D053487:DEAD-box RNA Helicases / Q000235:genetics; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D053263:Gene Regulatory Networks; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D000072640:Interferon-Induced Helicase, IFIH1; D016898:Interferon-alpha / Q000235:genetics*; D008957:Models, Genetic; D034622:RNA Interference; D053633:Receptor, Interferon alpha-beta / Q000235:genetics; D050794:STAT1 Transcription Factor / Q000235:genetics
chemical_list: C507964:IFNAR1 protein, human; D016898:Interferon-alpha; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D053633:Receptor, Interferon alpha-beta; C452815:IFIH1 protein, human; D053487:DEAD-box RNA Helicases; D000072640:Interferon-Induced Helicase, IFIH1
keywords: Experimental set-up; Gene regulatory modules; IFN-α modulators; RNA silencing; Small-scale gene expression screens