PPARγ knockdown by engineered transcription factors: exogenous PPARγ2 but not PPARγ1 reactivates adipogenesis

Abstract

To determine functional differences between the two splice variants of PPAR␥ (␥1 and ␥2), we sought to selectively repress ␥2 expression by targeting engineered zinc finger repressor proteins (ZFPs) to the ␥2-specific promoter, P2. In 3T3-L1 cells, expression of ZFP55 resulted in >50% reduction in ␥2 expression but had no effect on ␥1, whereas adipogenesis was similarly reduced by 50%. However, ZFP54 virtually abolished both ␥2 and ␥1 expression, and completely blocked adipogenesis. Overexpression of exogenous ␥2 in the ZFP54-expressing cells completely restored adipogenesis, whereas overexpression of ␥1 had no effect. This finding clearly identifies a unique role for the PPAR␥2 isoform. The nuclear hormone receptor PPAR␥ is essential for cellular differentiation and lipid accumulation during adipogenesis (Barak et al. 1999;Kubota et al. 1999;Rosen et al. 1999). The adipocyte-specific ␥2 isoform differs from the more widely expressed ␥1 in that it contains additionally 30 amino acid residues at the amino terminus (Kliewer et al. 1994;Tontonoz et al. 1994a;Zhu et al. 1995). Evidence suggests these residues contribute to a constitutive transcription activation function that is 5-10-fold greater than in ␥1 (Werman et al. 1997). PPAR␥2 is selectively expressed in adipose tissue (Fajas et al. 1997) and is strongly up-regulated during adipogenesis (Tontonoz et al. 1994b;Wu et al. 1998), suggesting a specific role for this isoform in fat cell differentiation. Nevertheless, a specific role for ␥2 that could not be substituted by ␥1 has not been clearly determined.The ability to selectively knock out or knock down the expression of a specific gene provides a powerful approach for understanding its biological function. The targeting of individual mRNA splice variants offers an even greater level of selective control and understanding of differential isoform function. Rationally engineered transcription factors potentially provide a powerful tool for targeted regulation of endogenous genes by combining a functional transcription regulatory domain with a customized DNA binding domain that can bind to a specific sequence within the target gene. C2H2 zinc finger proteins (ZFPs) can be engineered to bind with high specificity to wide a diversity of DNA sequences (Desjarlais and Berg 1992;Choo and Klug 1994;Jamieson et al. 1994;Rebar and Pabo 1994;Greisman and Pabo 1997). Previous studies have demonstrated the utility of both engineered activator-and repressor-ZFPs in the regulation of endogenous chromosomal loci (Bartsevich and Juliano 2000;Beerli et al. 2000;Zhang et al. 2000;Liu et al. 2001). Our goal for this study was to selectively inhibit expression of the PPAR␥2 isoform in the adipogenic mouse 3T3-L1 cell line by utilizing engineered zinc finger repressor proteins. Results and DiscussionThe mouse PPAR␥ gene spans >105 kb (Zhu et al. 1995). Coding exons 1 to 6 are conserved between the ␥1 and ␥2 isoforms (Fig. 1A) and transcription of these is driven by an upstream promoter (P1) that also drives expression of two untrans...

Drug-inducible and simultaneous regulation of endogenous genes by single-chain nuclear receptor-based zinc-finger transcription factor gene switches

“…15 Most TF ZF s have been constructed for constitutive regulation of natural promoters driving reporter genes. 11,16,17 TF ZF s have been expressed from constitutive promoters transiently, retrovirally or by generating integrated stable cell lines [18][19][20][21] and have also been expressed in vivo. 22 To effectively study gene function and develop gene therapies, tightly controlled and highly inducible regulation of the target gene is desirable through direct control of the TF ZF with a cell-penetrating drug.…”

PPARs and adipocyte function

“…In order to address the contribution of different PPAR isoforms in adipocyte differentiation, Ren and co-workers silenced expression of PPAR 1 and PPAR 2 in 3T3-L1 cells using zinc finger repressor proteins [30]. By employing this approach it was demonstrated that ectopic expression of PPAR 2 but not PPAR 1 is able to restore adipogenesis.…”

Engineered Zinc Finger Proteins for Controlling Stem Cell Fate

“…ZFP-TFs have been tested in many settings [9][10][11][12][13], and recent work, for example, has shown how they can induce vascular endothelial growth factor A expression and thus stimulate the growth of new blood vessels in mice [14].…”