title: Yin Yang 1 intronic binding sequences and splicing elicit intron-mediated enhancement of ubiquitin C gene expression.
abstract: In a number of organisms, introns affect expression of the gene in which they are contained. Our previous studies revealed that the 5'-UTR intron of human ubiquitin C (UbC) gene is responsible for the boost of reporter gene expression and is able to bind, in vitro, Yin Yang 1 (YY1) trans-acting factor. In this work, we demonstrate that intact YY1 binding sequences are required for maximal promoter activity and YY1 silencing causes downregulation of luciferase mRNA levels. However, YY1 motifs fail to enhance gene expression when the intron is moved upstream of the proximal promoter, excluding the typical enhancer hypothesis and supporting a context-dependent action, like intron-mediated enhancement (IME). Yet, almost no expression is seen in the construct containing an unspliceable version of UbC intron, indicating that splicing is essential for promoter activity. Moreover, mutagenesis of YY1 binding sites and YY1 knockdown negatively affect UbC intron removal from both endogenous and reporter transcripts. Modulation of splicing efficiency by YY1 cis-elements and protein factor may thus be part of the mechanism(s) by which YY1 controls UbC promoter activity. Our data highlight the first evidence of the involvement of a sequence-specific DNA binding factor in IME.
authors.affiliation: Department of Biomolecular Sciences, Biochemistry and Molecular Biology Section, University of Urbino Carlo Bo, Urbino, Italy. marzia.bianchi@uniurb.it
authors.forename: Marzia
authors.initials: M
authors.lastname: Bianchi
authors.forename: Rita
authors.initials: R
authors.lastname: Crinelli
authors.forename: Elisa
authors.initials: E
authors.lastname: Giacomini
authors.forename: Elisa
authors.initials: E
authors.lastname: Carloni
authors.forename: Lucia
authors.initials: L
authors.lastname: Radici
authors.forename: Mauro
authors.initials: M
authors.lastname: Magnani
mesh_terms: D000704:Analysis of Variance; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D047369:Chromatin Immunoprecipitation; D017931:DNA Primers / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005786:Gene Expression Regulation / Q000235:genetics*; D055785:Gene Knockdown Techniques; D006801:Humans; D047468:Immunoprecipitation; D007438:Introns / Q000235:genetics*; D008156:Luciferases; D016296:Mutagenesis; D011401:Promoter Regions, Genetic / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D025822:Ubiquitin C / Q000235:genetics / Q000378:metabolism*; D050986:YY1 Transcription Factor / Q000235:genetics / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D025822:Ubiquitin C; D050986:YY1 Transcription Factor; D008156:Luciferases