A novel short hairpin RNA (shRNA) expression system promotes Sox9-dependent gene silencing

Abstract

Cartilage development and function are dependent on a temporally integrated program of gene expression. With the advent of RNA interference (RNAi), artificial control of these complex programs becomes a possibility, limited only by the ability to regulate and express the RNAi. Using existing methods for production of RNAi’s, we have constructed a plasmid-based short hairpin RNA (shRNA) expression system under control of the human pol III H1 promoter and supplemented this promoter with DNA binding sites for the cartilage-specific transcription factor Sox9. The resulting shRNA expression system displays robust, Sox9-dependent gene silencing. Dependence on Sox9 expression was confirmed by electrophoretic mobility shift assays. The ability of the system to regulate heterologously expressed Sox9 was demonstrated by Western blot, as a function of both Sox9 to shRNA ratio, as well as time from transfection. This novel expression system supports auto-regulatory gene silencing, providing a tissue-specific feedback mechanism for temporal control of gene expression. Its applications for both basic mechanistic studies and therapeutic purposes should facilitate the design and implementation of innovative tissue engineering strategies.

Introduction

Cartilage function and development depends upon a complex pattern of temporally regulated gene expression (Dunn and Kingston, 2007, Lefebvre and Smits, 2005). Cartilaginous differentiation initiates in response to expression of the master transcriptional regulator Sox9, which promotes the transition of mesenchymal stem cells into pre-hypertrophic chondrocytes. Chondrocyte hypertrophy and terminal differentiation, conversely, require the subsequent silencing of Sox9. The need for both events in cartilage development has been demonstrated in gene therapy studies which have shown constitutive over-expression of Sox9 to result in an arrested phenotype at the hypertrophic border. We reasoned that this overexpression and silencing of Sox9 could be achieved through an integrated expression/RNAi system.

RNAi silencing occurs through assembly of the RNA-induced silencing complex (RISC) around a 21–25 base single-stranded guiding RNA (Hamilton and Baulcombe, 1999, Hammond et al., 2000, Zamore et al., 2000), followed by RISC binding its transcript and subsequent degradation by cellular exonucleases (Schwarz et al., 2003). Recognition of the components needed for RISC assembly has resulted in plasmid and virally-based expression systems using short hairpin RNAs (shRNA) (Chang et al., 2006). shRNA expression systems have relied almost exclusively on compact, constitutive pol III promoters derived from human H1 RNA or U6 pol III promoters to drive shRNA expression (Bannister et al., 2007, Cheng and Chang, 2007)). The H1 RNA promoter contains a region (positions −31/−1) adjacent to the TATA box (positions −32/−28) that allows neutral substitution mutations (Myslinski et al., 2001). The promoter includes known upstream transcriptional regulatory sites, including a distal sequence element (DSE), composed of Oct-1 (positions −97/−90) and STAF (positions −88/−69) recognition sequences, and a proximal sequence element (PSE; positions −68/−51) (Hannon et al., 1991, Myslinski et al., 2001). These sequences are thought to play pivotal roles in both basal and inducible gene expression.

Based on this information, regulated pol III expression systems have been designed that usually depend on insertion of the tet or lac operator, where co-expression of the tet or lac repressor suppresses shRNA expression (Higuchi et al., 2004, Hosono et al., 2004, Kappel et al., 2006, Lin et al., 2004, Ohkawa and Taira, 2000). Similarly, use of ecdysone or Cre-lox mediated stuffer reporter deletions has been incorporated into viral gene delivery systems to permit stable, inducible gene silencing (Gupta et al., 2004, Heinonen et al., 2005, Rangasamy et al., 2008, Szulc et al., 2006, Tiscornia et al., 2004, Yu and McMahon, 2006). Although highly effective for in vitro purposes, the in vivo application of these expression systems is limited due to their dependence on heterologous, non-mammalian proteins and due to their requirement for systemic administration of pharmacological inducing agents.

We theorized that we could create a regulated shRNA expression system that was based on the H1 promoter and that could autoregulate Sox9 expression as required in cartilage differentiation and maturation. We therefore constructed an H1-driven shRNA expression plasmid and evaluated the effects of replacement of the STAF binding site with binding sites that recognize the Sox9 transcription factor. Herein, we describe the construction and characterization of such a Sox9-regulated shRNA expression system.