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Recent studies on the control of specific metabolic pathways in bacteria have documented the existence of entirely RNA-based mechanisms for controlling gene expression. These mechanisms involve the modulation of translation, transcription termination or RNA self-cleavage through the direct interaction of specific intracellular metabolites and RNA sequences. Here we show that an analogous RNA-based gene regulation system can effectively be designed for mammalian cells via the incorporation of sequences encoding self-cleaving RNA motifs into the transcriptional unit of a gene or vector. When correctly positioned, the sequences lead to potent inhibition of gene or vector expression, owing to the spontaneous cleavage of the RNA transcript. Administration of either oligonucleotides complementary to regions of the self-cleaving motif or a specific small molecule results in the efficient induction of gene expression, owing to inhibition of self-cleavage of the messenger RNA. Efficient regulation of transgene expression is shown in a variety of mammalian cell lines and live animals. In conjunction with other emerging technologies, this methodology may be particularly applicable to the development of gene regulation systems tailored to any small inducer molecule, and provide a novel means of biological sensing in vivo that may have an important application in the regulated delivery of protein therapeutics.

Two promoter elements are important for basal-level transcription, the TATA motif typicaily located 30 nucleotides upsream of the transcription initiation site and the initiator (Inr) MATERIALS AND METHODS Plasmids. pGST-YY1 and pGST-WT1 were constructed by cloning full-length YY1 (6) and WT1 (12) cDNAs into the glutathione S-transferase (GST) expression vector pGEX- 2TK (13). N-and C-terminal deletions ofYY1 were generated by exonuclease III digestion (Erase-A-Base kit, Promega) and fused in frame to GST. pGST-SplQl and pGST-SplZnF were constructed by cloning fragments from Spl cDNA (pSpl-fl, gift of R. Tjian, University of California, Berkeley) in frame into pGEX-2TK. pGST-SplQl and pGST-SplZnF encode aa 1-262 and 620-778 of Spl, respectively. pGAL4-Spl encodes the DNA-binding domain of GAL4 fused at its C terminus to the full-length Spl (gift from G. Gill, University of California, Berkeley). pGAL4E1BCAT contains five GAL4 binding sites inserted 5' of the minimal E1B promoter that is linked to the chloramphenicol acetyltransferase (CAT) gene (gift of A. Levine, Princeton University). Plasmids pCMVYY1/VP16 and various YY1 deletion derivatives were constructed by joining the YY1 fragments in frame with the herpes simplex virus VP16 acidic activation domain (aa 413-490, gift of M. Green, University ofMassachusetts), and the YY1/VP16 fragments were then cloned into a cytomegalovirus (CMV) promoter-driven expression plasmid (6). pCMV-VP16, expressing the activation domain of VP16, was constructed by placing VP16 under the control of the CMV early promoter containing translational initiation consensus sequence and a nuclear localization signal derived from the simian virus 40 large tumor antigen (PKKKRKV; ref. 14). All constructs were verified by sequence analysis.Assay of YY1 and Spl Interaction with GST-YY1 FusionProteins. pGST-YY1 and various C-and N-terminal deletion derivatives were induced with isopropyl f3-D-thiogalactopyranoside to express the fusion proteins, which were bound to glutathione-Sepharose beads and purified as described (13

Previous studies on the marine c-myc promoter demonstrated that a ubiquitously present protein, common factor 1 (CF1), bound at two sites located -260 and -390 bp from the P1 transcription start site. CF1 has been purified to near homogeneity and shown to be identical to the zinc finger protein Yin-yang 1 (YY1) as judged by similarity of molecular weight and other biochemical properties, immunological cross-reactivity, and the ability of recombinant YY1 to bind to CF1 sites. In cotransfection experiments, YY1 is a strong activator of transcription from c-myc promoter-based reporters. Furthermore, in marine erythroleukemia cells, overexpressed YY1 causes increased levels of c-myc mRNA initiated from both major transcription initiation sites of the endogenous c-myc gene.Yin-yang 1 (YY1) is a zinc finger protein cloned by Shi et al. (36) in the course of studies on ElA activation of the adeno-associated virus (AAV) P5 promoter. Recombinant YY1 binds a negative regulatory site at -60 and an initiator site at +1 in the AAV P5 promoter. Cotransfected YY1 functions as a repressor of the AAV P5 promoter, and addition of adenovirus ElA protein relieves YY1-dependent repression. Three other groups also cloned cDNAs encoding the YY1 protein by virtue of its ability to bind functionally important sites in unrelated genes, including the immunoglobulin kappa 3' enhancer and the t.E1 site in the immunoglobulin heavy chain (IgH) enhancer (28), the delta sites of ribosomal proteins L30 and L32 (13), and the long terminal repeat of Moloney murine leukemia virus (8). YY1 has subsequently been shown to compete with serum response factor (SRF) for binding to the c-fos and skeletal a-actin promoters (10, 21). In the Moloney murine leukemia virus long terminal repeat and the 3' kappa enhancer, the YY1 binding sites are negative sites for transcription (8,28). Conversely, the IgH p.E1 site (24,29,39) a third YY1 binding site in the first c-myc exon by virtue of its ability to compete with proteins binding to rpL32 delta sites (1). Thus, we wished to determine the relationship of CF1 to YY1 and to determine how YY1 might affect c-myc transcription.We demonstrate in this paper that YY1 appears to be identical to previously identified CF1, as judged by similarity of mobility in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), binding site specificity, and immunological cross-reactivity. Furthermore, we show that, in a cotransfection assay, recombinant YY1 is a strong activator of a reporter construct dependent on the murine c-myc promoter. The c-myc promoter is the first example of a natural promoter which is activated by cotransfected YY1. Finally, we show that overexpression of exogenous YY1 causes increased mRNAs initiating from both P1 and P2 promoters of the endogenous c-myc gene. These results demonstrate that YY1 binds in the c-myc promoter and activates c-myc transcription. MATERIALS AND METHODSPlasmids and molecular cloning. pGEM-hYY1 contains the human YY1 (hYY1) coding sequence cloned at the EcoRI site...