title: Identification of novel Sp1 targets involved in proliferation and cancer by functional genomics.
abstract: Sp1 is a transcription factor regulating many genes through its DNA binding domain, containing three zinc fingers. We were interested in identifying target genes regulated by Sp1, particularly those involved in proliferation and cancer. Our approach was to treat HeLa cells with a siRNA directed against Sp1 mRNA to decrease the expression of Sp1 and, in turn, the genes activated by this transcription factor. Sp1-siRNA treatment led to a great number of differentially expressed genes as determined by whole genome cDNA microarray analysis. Underexpressed genes were selected since they represent putative genes activated by Sp1 and classified in six Gene Onthology categories, namely proliferation and cancer, mRNA processing, lipid metabolism, glucidic metabolism, transcription and translation. Putative Sp1 binding sites were found in the promoters of the selected genes using the Match™ software. After literature mining, 11 genes were selected for further validation. Underexpression by qRT-PCR was confirmed for the 11 genes plus Sp1 in HeLa cells after Sp1-siRNA treatment. EMSA and ChIP assays were performed to test for binding of Sp1 to the promoters of these genes. We observed binding of Sp1 to the promoters of RAB20, FGF21, IHPK2, ARHGAP18, NPM3, SRSF7, CALM3, PGD and Sp1 itself. Furthermore, the mRNA levels of RAB20, FGF21 and IHPK2 and luciferase activity for these three genes related to proliferation and cancer, were determined after overexpression of Sp1 in HeLa cells, to confirm their regulation by Sp1. Involvement of these three genes in proliferation was validated by gene silencing using polypurine reverse hoogsteen hairpins.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Barcelona, Barcelona, Spain. coleaga@ub.edu
authors.forename: Carlota
authors.initials: C
authors.lastname: Oleaga
authors.forename: Sabine
authors.initials: S
authors.lastname: Welten
authors.forename: Audrey
authors.initials: A
authors.lastname: Belloc
authors.forename: Anna
authors.initials: A
authors.lastname: Solé
authors.forename: Laura
authors.initials: L
authors.lastname: Rodriguez
authors.forename: Núria
authors.initials: N
authors.lastname: Mencia
authors.forename: Elisabet
authors.initials: E
authors.lastname: Selga
authors.forename: Alicia
authors.initials: A
authors.lastname: Tapias
authors.forename: Veronique
authors.initials: V
authors.lastname: Noé
authors.forename: Carlos J
authors.initials: CJ
authors.lastname: Ciudad
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D049109:Cell Proliferation; D047369:Chromatin Immunoprecipitation; D017931:DNA Primers; D023281:Genomics; D006367:HeLa Cells; D006801:Humans; D009369:Neoplasms / Q000473:pathology*; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D060888:Real-Time Polymerase Chain Reaction; D016329:Sp1 Transcription Factor / Q000235:genetics* / Q000378:metabolism
chemical_list: D017931:DNA Primers; D016329:Sp1 Transcription Factor