Yin Yang 1 Intronic Binding Sequences and Splicing Elicit Intron-Mediated Enhancement of Ubiquitin C Gene Expression

Marzia Bianchi,Elisa Carloni,Elisa Giacomini,Lucia Radici,Rita Crinelli,Mauro Magnani,Emanuele Buratti

doi:10.1371/journal.pone.0065932

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.

Highlights

Ubiquitin (Ub), in the context of the ubiquitin proteasome system (UPS) is responsible for much of the regulated proteolysis in the cell, but has non-degradative functions as well: it is involved in the control of many cellular activities, ranging from signal transduction to transcription, from endocytosis to protein trafficking, from DNA repair to cell survival and proliferation [1]
We previously cloned the upstream sequence of the human ubiquitin C gene and found that the maximal promoter activity is achieved when to the proximal promoter (PP) region of 371 nt, is added the unique 59 untranslated region (59-UTR) intron, which is crucial for basal transcriptional activity (Figure 1) [19]
Electrophoretic mobility shift assay revealed the presence of multiple cis-acting elements for Sp1 and Yin Yang 1 (YY1) transcription factors, within specific intron sequences, which we previously termed ODN 1, ODN 3, ODN 5, probe II and probe IV [19]

Summary

Introduction

Ubiquitin (Ub), in the context of the ubiquitin proteasome system (UPS) is responsible for much of the regulated proteolysis in the cell, but has non-degradative functions as well: it is involved in the control of many cellular activities, ranging from signal transduction to transcription, from endocytosis to protein trafficking, from DNA repair to cell survival and proliferation [1]. The peculiar features of ubiquitin at the function level are displayed at the gene level. The UbC gene, coding for polyubiquitin, is the most studied and characterized because of its inducibility in response to various cell challenges [7]. The UbC promoter is a widely used regulatory sequence to drive a high and sustained level of transgene expression [8,9,10]

Methods

Results

Conclusion