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...

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Transcription factor IIF (TFIIF) cooperates with RNA polymerase II (pol II) during multiple stages of the transcription cycle including preinitiation complex assembly, initiation, elongation, and possibly termination and recycling. Human TFIIF appears to be an ␣ 2 ␤ 2 heterotetramer of RNA polymerase II-associating protein 74-and 30-kDa subunits (RAP74 and RAP30). From inspection of its 517-amino-acid (aa) sequence, the RAP74 subunit appears to comprise separate N-and C-terminal domains connected by a flexible loop. In this study, we present functional data that strongly support this model for RAP74 architecture and further show that the N-and C-terminal domains and the central loop of RAP74 have distinct roles during separate phases of the transcription cycle. The N-terminal domain of RAP74 (minimally aa 1 to 172) is sufficient to deliver pol II into a complex formed on the adenovirus major late promoter with the TATA-binding protein, TFIIB, and RAP30. A more complete N-terminal domain fragment (aa 1 to 217) strongly stimulates both accurate initiation and elongation by pol II. The region of RAP74 between aa 172 and 205 and a subregion between aa 170 and 178 are critical for both accurate initiation and elongation, and mutations in these regions have similar effects on initiation and elongation. Based on these observations, RAP74 appears to have similar functions in initiation and elongation. The central region and the C-terminal domain of RAP74 do not contribute strongly to single-round accurate initiation or elongation stimulation but do stimulate multiple-round transcription in an extract system. RNA polymerase II (pol II) interacts with a number of general and regulatory factors to initiate transcription accurately from a promoter (reviewed in references 34 and 56). In the pathway toward initiation, promoter DNA is bent, and DNA may be wrapped around pol II (24,40). General factors TATA-binding protein (TBP) (or transcription factor IID [TFIID]), TFIIB, TFIIF, and TFIIE cooperate with pol II to strain the DNA helix around the transcriptional start site before ATP-driven helix opening by TFIIH (34,56). After initiation, pol II releases from the promoter (promoter clearance or promoter escape), elongates the RNA chain, terminates transcription, and recycles. TFIIF, made up of RAP30 (RNA polymerase II-associating protein of 30 kDa) and RAP74 (58 kDa) subunits, may participate in each of these stages of the transcription cycle.Inspection of its 517-amino-acid (aa) sequence indicates that human RAP74 can be divided into three regions: (i) a highly basic N-terminal domain with significant globular structure (aa 1 to 217); (ii) an overall acidic, highly charged central region lacking in hydrophobic amino acids but rich in E, D, K, R, S, T, G, and P (aa 218 to 398); and (iii) a very basic C-terminal domain (CTD) with globular structure (aa 399 to 517) (2, 15). The N-terminal domain is important for RAP30 binding (54, 55), preinitiation complex assembly (this report), and elongation stimulation (reference 21 and thi...

Human transcription factor IIF (TFIIF) is an ␣ 2 ␤ 2 heterotetramer of RNA polymerase II-associating 74 (RAP74) and RAP30 subunits. Mutagenic analysis shows that the N-terminal region of RAP74 between L155 (leucine at codon 155) and M177 is important for initiation. Mutants in this region have reduced activity in transcription, but none are inactive. Single amino acid substitutions at hydrophobic residues L155, W164, I176, and M177 have similar activity to RAP74(1-158), from which all but three amino acids of this region are deleted. Residual activity can be explained because each of these mutants forms a complex with RAP30 and recruits RNA polymerase II into the preinitiation complex. Mutants are defective for formation of the first phosphodiester bond from the adenovirus major late promoter but do not appear to have an additional significant defect in promoter escape. Negative DNA supercoiling partially compensates for the defects of TFIIF mutants in initiation, indicating that TFIIF may help to untwist the DNA helix for initiation.Accurate initiation from human pre-mRNA promoters requires the cooperation of general transcription factors and RNA polymerase II (reviewed in references 15, 31, and 38). For promoters containing a TATA box, an ordered in vitro pathway for assembly of an active transcription complex has been defined. TATA-binding protein (TBP) binds to the TAT AAA sequence. Transcription factor IIB (TFIIB) can then associate with TBP and promoter DNA to form a TBP-TFIIBpromoter complex. TFIIF escorts RNA polymerase II to the promoter. TFIIE recruits TFIIH, which is a large protein complex that includes two subunits that are DNA helicases. The helicase activities of TFIIH are believed to open the DNA helix for initiation. In addition to its helicase module, TFIIH also includes a subassembly that contains a kinase-cyclin pair of subunits that phosphorylates the carboxy terminal domain of the largest subunit of RNA polymerase II.Human TFIIF is an ␣ 2 ␤ 2 heterotetramer of RAP74 and RAP30 subunits (RAP for RNA polymerase II-associating protein) (3,8,27,50). Both subunits of TFIIF participate in stable recruitment of RNA polymerase II to the promoter (4, 9, 25), and both are necessary for accurate initiation from linear DNA templates in vitro (24,28,44). From negatively supercoiled DNA templates, the requirement for TFIIH and for ATP hydrolysis can be bypassed for initiation from many promoters (13,33,34,45,46). In these transcription systems, generally, TBP, TFIIB, and RNA polymerase II are minimally required to detect accurate initiation. TFIIE may be dispensable for transcription or it may be stimulatory (19,33,34). TFIIE is thought to have multiple roles in initiation. TFIIE recruits TFIIH, but TFIIE also makes contacts to TFIIF and TBP (30,54), and TFIIE may have a role in DNA untwisting (19). Like TFIIE, TFIIF may be either stimulatory or, in some cases, required for transcription from supercoiled templates. Both TFIIF subunits contribute to transcription from supercoiled templates, although in so...

Transcription factor IIF (TFIIF) is a protein allosteric effector for RNA polymerase II during the initiation and elongation phases of the transcription cycle. In initiation, TFIIF induces promoter DNA to wrap almost a full turn around RNA polymerase II in a complex that includes the general transcription factors TATA-binding protein, TFIIB, and TFIIE. During elongation, TFIIF also supports a more active conformation of RNA polymerase II. This conformational model for elongation is supported by three lines of experimental evidence. First, a region within the RNA polymerase II-associating protein 74 (RAP74) subunit of TFIIF (amino acids T154 to M177), a region that is critical for isomerization of the preinitiation complex, is also critical for elongation stimulation. Amino acid substitutions within this region are shown to have very similar effects on initiation and elongation, and mutagenic analysis indicates that L155, W164, N172, I176, and M177 are the most important residues in this region for transcription. Second, TFIIF is shown to have a higher affinity for rapidly elongating RNA polymerase II than for the stalled elongation complex, indicating that RNA polymerase II alternates between active and inactive states during elongation and that TFIIF stimulates elongation by supporting the active conformational state of RNA polymerase II. The deleterious I176A substitution in the critical region of RAP74 decreases the affinity of TFIIF for the active form of the elongation complex. Third, TFIIF is shown by Arrhenius analysis to stimulate elongation by populating an activated state of RNA polymerase II.

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