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...
The development of leukemias in several children with severe combined immunodeficiency disease who were transplanted with retroviral vector-transduced bone marrow cells has renewed concerns about the risks associated with the random integration of proviral sequences into chromosomal DNA. One theoretical way to reduce the risks of insertional mutagenesis would be to employ transduction/transplantation protocols that minimize the total number of genetically modified cells and associated proviral integration "events" introduced into recipients. Toward this end, we have developed a transduction protocol that involves the short-term incubation of highly purified murine stem cells with high-titer recombinant lentivirus vectors in the presence of serum-free medium and the cytokines SCF and TPO. Competitive repopulation studies showed that stem cells transduced in this way possessed the same reconstitutive ability as fresh, unmanipulated cells. Animals transplanted with only 200-2000 transduced cells were efficiently reconstituted with the genetically modified cells, and most hematopoietic cells in the recipients expressed the transgene. In contrast, the use of high-titer oncoretroviral vectors in conjunction with the same transduction/transplantation protocol resulted in only low levels of gene marking in vivo. The use of a similar transduction/transplantation strategy in future clinical studies may offer distinct advantages over current protocols.
Vascular endothelial growth factor (VEGF) exerts crucial functions during pathological angiogenesis and normal physiology. We observed increased hematocrit (60-75%) after high-grade inhibition of VEGF by diverse methods, including adenoviral expression of soluble VEGF receptor (VEGFR) ectodomains, recombinant VEGF Trap protein and the VEGFR2-selective antibody DC101. Increased production of red blood cells (erythrocytosis) occurred in both mouse and primate models, and was associated with near-complete neutralization of VEGF corneal micropocket angiogenesis. High-grade inhibition of VEGF induced hepatic synthesis of erythropoietin (Epo, encoded by Epo) >40-fold through a HIF-1alpha-independent mechanism, in parallel with suppression of renal Epo mRNA. Studies using hepatocyte-specific deletion of the Vegfa gene and hepatocyte-endothelial cell cocultures indicated that blockade of VEGF induced hepatic Epo by interfering with homeostatic VEGFR2-dependent paracrine signaling involving interactions between hepatocytes and endothelial cells. These data indicate that VEGF is a previously unsuspected negative regulator of hepatic Epo synthesis and erythropoiesis and suggest that levels of Epo and erythrocytosis could represent noninvasive surrogate markers for stringent blockade of VEGF in vivo.
YY1 is a C2H2-type zinc finger transcription factor that is a member of the human GLl-Kruppel family of proteins. YY1 represses transcription when bound upstream of transcription initiation sites. The repression can be relieved by adenovirus E1A and activation of target genes occurs. We have mapped the repression domain of YY1 to the C-terminal region, overlapping its DNA binding domain. We have also identified an activation domain within the first 69 amino acids of YY1. The YY1 C-terminal region is involved in physical interactions with E1A and is functionally necessary for YY1 to respond to E1A. This suggests that relief of YY1 repression by E1A involves YY1-E1A physical interactions. Although not involved in interactions with E1A, the N-terminal activation domain is also necessary for YY1 to respond to E1A. Presumably, under repressing conditions, the activation domain is masked by the conformation of YY1, but is released upon binding of E1A and is required to subsequently activate transcription. Consistent with this hypothesis, an ATF-2-YY1 chimeric protein containing the activation domain of ATF-2 and the C-terminal two-thirds of YY1 is still a potent repressor. Unlike the mutant YY1 lacking its own N-terminal activation domain, the chimeric protein is fully responsive to E1A.
Background. Patients with advanced hematologic malignancies remain at risk for relapse following reducedintensity conditioning (RIC) allogeneic hematopoietic stem cell transplantation (allo-HSCT). We conducted a prospective clinical trial to test whether vaccination with whole leukemia cells early after transplantation facilitates the expansion of leukemia-reactive T cells and thereby enhances antitumor immunity. Methods. We enrolled 22 patients with advanced chronic lymphocytic leukemia (CLL), 18 of whom received up to 6 vaccines initiated between days 30 and 45 after transplantation. Each vaccine consisted of irradiated autologous tumor cells admixed with GM-CSF-secreting bystander cells. Serial patient PBMC samples following transplantation were collected, and the impact of vaccination on T cell activity was evaluated.Results. At a median follow-up of 2.9 (range, 1-4) years, the estimated 2-year progression-free and overall survival rates of vaccinated subjects were 82% (95% CI, 54%-94%) and 88% (95% CI, 59%-97%), respectively. Although vaccination only had a modest impact on recovering T cell numbers, CD8 + T cells from vaccinated patients consistently reacted against autologous tumor, but not alloantigen-bearing recipient cells with increased secretion of the effector cytokine IFN-γ, unlike T cells from nonvaccinated CLL patients undergoing allo-HSCT. Further analysis confirmed that 17% (range, 13%-33%) of CD8 + T cell clones isolated from 4 vaccinated patients by limiting dilution of bulk tumor-reactive T cells solely reacted against CLL-associated antigens. Conclusion.Our studies suggest that autologous tumor cell vaccination is an effective strategy to advance longterm leukemia control following allo-HSCT.Trial registration. Clinicaltrials.gov NCT00442130.
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