Tetracycline-dependent gene regulation: combinations of transregulators yield a variety of expression windows.

In addition to the originally described Tet transactivator tTA, several variants including transrepressors (tTRs) and reverse transactivators (rtTAs) have been constructed, which we employ here to establish a set of HeLa cell lines carrying different combinations of chromosomally integrated Tet transregulators. We first compare the regulatory properties of these lines using transient transfection of a luciferase reporter gene. Cell lines carrying rtTA-S2 or rtTA-M2 show reduced activity in the absence of dox and higher activation levels in its presence compared to an rtTA line. rtTA-M2 and its synthetic counterpart rtTA2S-M2 show the same regulation pattern. The replacement of the VP16 activation domain in rtTA-S2 or tTA by p65 leads to slightly reduced expression levels. Combination of an rtTA variant with the transrepressor tTR shows active repression of basal expression without affecting the activation level of the transiently transfected reporter gene. However, if the target gene is also chromosomally integrated, then tTR leads to a further reduction of basal expression and also of the maximal expression level. The results demonstrate that different regulatory windows can be achieved using various transregulators or combinations thereof. Thus, the most appropriate combination of regulators can be chosen depending on the application and cell line desired. We suspect that these properties would also allow the construction of transgenic organisms with preselected expression windows.

Tetracycline-inducible systems for Drosophila.

Since their inception, tetracycline (Tet)-inducible systems have become the method of choice for transgenic research. The Tet-Off systems have a number of advantages, including robust target induction using a relatively benign effector molecule. However, use of the Tet-On system has been fraught with difficulties, including high background expression in the absence of effector molecules and inconsistent gene induction. Recently, second generation Tet-On transactivators (TAs) have been described. In HeLa cells, they are far more efficient than the original reverse TA protein, and they exhibit lower background activity in the absence of effectors. Here we examine the most promising TA in transgenic Drosophila and characterize its in vivo properties. We report that low levels of doxycycline, when added to normal fly food, efficiently and rapidly induce target transgenes in adults, larvae, and embryos. This TA is superior to all other Tet-On proteins, and its performance is comparable to that of the widely used Tet-Off TA. In addition, combining the improved Tet-On TA with the Gal4-UAS (upstream-activating sequence) system produces robust, spatially restricted, temporally controlled transgene induction. Because this Tet-On TA is significantly more efficient than previous ones used in Drosophila, it is also possible to modulate gene induction by controlling the dosage of the antibiotic in the food.

Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity.

Regulatory elements that control tetracycline resistance in Escherichia coli were previously converted into highly specific transcription regulation systems that function in a wide variety of eukaryotic cells. One tetracycline repressor (TetR) mutant gave rise to rtTA, a tetracycline-controlled transactivator that requires doxycycline (Dox) for binding to tet operators and thus for the activation of P(tet) promoters. Despite the intriguing properties of rtTA, its use was limited, particularly in transgenic animals, because of its relatively inefficient inducibility by doxycycline in some organs, its instability, and its residual affinity to tetO in absence of Dox, leading to elevated background activities of the target promoter. To remove these limitations, we have mutagenized tTA DNA and selected in Saccharomyces cerevisiae for rtTA mutants with reduced basal activity and increased Dox sensitivity. Five new rtTAs were identified, of which two have greatly improved properties. The most promising new transactivator, rtTA2(S)-M2, functions at a 10-fold lower Dox concentration than rtTA, is more stable in eukaryotic cells, and causes no background expression in the absence of Dox. The coding sequences of the new reverse TetR mutants fused to minimal activation domains were optimized for expression in human cells and synthesized. The resulting transactivators allow stringent regulation of target genes over a range of 4 to 5 orders of magnitude in stably transfected HeLa cells. These rtTA versions combine tightness of expression control with a broad regulatory range, as previously shown for the widely applied tTA.

Gene transfer of human interferon gamma complementary DNA into a renal cell carcinoma line enhances MHC-restricted cytotoxic T lymphocyte recognition but suppresses non-MHC-restricted effector cell activity.

Even though renal cell carcinomas (RCC) are thought to be immunogenic, many tumors express variations in surface molecules and intracellular proteins that hinder induction of optimal antitumor responses. Interferon gamma (IFNgamma) stimulation can correct some of these deficiencies. Therefore, we introduced the complementary DNA (cDNA) encoding human IFNgamma into a well-characterized RCC line that has been selected for development of an allogeneic tumor cell vaccine for treatment of patients with metastatic disease. Studies were performed to determine how endogenous IFNgamma expression influences tumor cell immunogenicity. IFNgamma transductants showed minimal increases in surface expression of MHC class I and adhesion molecules but expression of class II molecules was induced. Proteins of the transporter associated with antigen processing (TAP) and low molecular weight polypeptide (LMP) were constitutively expressed at high levels. The transductants stimulated allospecific cytotoxic T lymphocytes (CTL); however, they were not better than unmodified tumor cells in this capacity. Endogenous IFNgamma expression enhanced tumor cell recognition by MHC-restricted, tumor antigen-specific CTL but suppressed recognition by non-MHC-restricted cytotoxic cells. Thus, the functional consequences of IFNgamma expression varied with respect to the type of effector cell and were not always beneficial for tumor cell recognition.

The p65 domain from NF-kappaB is an efficient human activator in the tetracycline-regulatable gene expression system.

The tc-responsive TetR protein allows the investigation of various transcriptional activators in respective fusion proteins. We have fused eight well-known human activator domains to the C-terminus of TetR and determined the properties of the resulting transactivators using a tetracycline-responsive promoter in three human cell lines (HeLa, BJAB, and Jurkat). Several-hundred-fold activation was exclusively obtained with the acidic p65 domain from NF-kappaB and with VP16, which served as a positive control. In contrast, at least 10-fold lower factors of activation were achieved with ITF-1, ITF-2, and MTF-1. The induction properties of the p65 domain are identical to those of VP16 in all three human cell lines and when fused to the reverse TetR. The combination of the novel reverse p65 fusion with the TetR(B/E)-KRAB construct resulted in active silencing and full activation. This is the first report of an expression system with minimal basal activity and high induction levels without viral protein domains.

Tetracycline-inducible expression systems with reduced basal activity in mammalian cells.

We describe a modification of the tetracycline-inducible eukaryotic gene expression system with decreased basal levels of expression in HeLa cells. It employs the tetracycline-inducible transactivator and a tetracycline-regulated repressor fusion acting on the same promoter. To avoid heterodimerization or competition for the same DNA site, each was provided with different DNA recognition and/or protein dimerization specificities. We achieved active silencing in the uninduced state resulting in approximately 6-fold reduced levels of basal transcription and several hundred-fold activation of gene expression upon addition of tetracycline.

Down-regulation of the MHC class I antigen-processing machinery after oncogenic transformation of murine fibroblasts.

Malignant transformation is often associated with genetic alterations providing tumor cells with mechanisms for escape from immune surveillance. Human and murine tumors of various origin as well as in vitro models of viral and oncogenic transformation express reduced levels of major histocompatibility complex (MHC) class I antigens resulting in decreased sensitivity to MHC class I-restricted cytotoxic T lymphocyte (CTL)-mediated lysis. We here investigate whether the suppressed MHC class I surface expression of ras-transformed fibroblasts is due to dysregulation of the genes of the antigen-processing machinery, the peptide transporters TAP-1 and TAP-2 and the proteasome subunits LMP-2 and LMP-7, and whether it can be restored by gene transfer. In comparison to parental NIH3T3 cells, the ras oncogenic transformants revealed reduced TAP and LMP mRNA expression and impaired function of these genes, leading to deficient peptide transport and peptide loading of MHC class I molecules resulting in instable expression of the MHC class I complex on the cell surface. Enhanced H-2 surface expression due to stabilization of the MHC class I complex could be achieved by culturing ras transformants at low, unphysiological temperature (26 degrees C) or by loading these cells with either exogenous human beta2-microglobulin or MHC class I-binding peptide alone or in combination. Furthermore, interferon-gamma treatment was capable to enhance the expression of TAP, LMP and MHC class I molecules in both parental as well as ras-transformed fibroblasts. Stable transfection of the human TAP-1 cDNA into ras transformants caused a partial reconstitution of the peptide transport and an enhancement of the MHC class I surface expression, whereas the level of MHC class I biosynthesis was not affected by TAP-1 overexpression in parental cells. Together these results point to the existence of an association between oncogenic transformation and deficiencies in the MHC class I antigen-restricted immunosurveillance, suggesting intervention strategies involving specific MHC class I-binding peptides or transfection of the LMP and/or TAP genes to overcome the expression of the immune escape phenotype.

Hyperexpression of transporter in antigen processing-1 (TAP-1) in thyroid glands affected by autoimmunity: a contributing factor to the breach of tolerance to thyroid antigens?

According to the 'aberrant HLA expression' hypothesis, endocrine autoimmunity is driven by presentation of self antigens by target cells over-expressing HLA molecules. In autoimmune thyroid diseases (AITD), thyroid follicular cells (thyrocytes) over-express HLA class I and HLA class II molecules. Since efficient presentation of endogenous peptides via class I requires transporters that translocate endogenous peptides from the cytoplasm to the endoplasmic reticulum, i.e. transporters associated with antigen processing (TAP) -1 and -2, the capability of thyrocytes to express TAP and whether TAP is hyperexpressed in AITD glands are issues relevant to the above hypothesis. Results from immunofluorescence and Northern blotting studies on primary thyrocyte cultures and on a thyroid cell line demonstrate that thyrocytes express constitutively TAP-1 at a low level, and that this expression is readily induced by interferon-gamma (IFN-gamma) and to a lesser extent by IFN-alpha. In AITD, but not in non-autoimmune glands, thyrocytes hyperexpress TAP-1, as demonstrated by both immunohistopathology and flow cytometry. The cytokine pattern does not bear, as assessed by reverse transcriptase-polymerase chain reaction (RT-PCR), a clear relationship with TAP-1 expression. These results have broad implications and suggest that the core concept of the 'aberrant HLA expression' hypothesis of endocrine autoimmunity could be incorporated in the currently prevailing view of 'autoimmunity by breach of peripheral tolerance'.

Intracellular location, complex formation, and function of the transporter associated with antigen processing in yeast.

Peptide transport across the membrane of the endoplasmic reticulum (ER) gains increasing importance in view of its potential function in selective protein degradation and antigen processing. An example for peptide transport in the ER is the transporter associated with antigen processing (TAP), which supplies peptides for the formation of major-histocompatibility-complex class-I complexes. Here, we have expressed human TAP1 and TAP2 in the yeast Saccharomyces cerevisiae. Expression of both genes resulted in the formation of a stable TAP heterodimer that was localized mainly in the ER. Although a minor fraction of TAP is found in the plasma membrane, TAP is unable to restore a-factor secretion in a mutant cell line that lacks the yeast mating-factor transporter Ste6. Nevertheless, in vitro studies with microsomal vesicles demonstrated that the TAP complex is fully functional in the ER membrane in terms of selective peptide binding, ATP-dependent transport, and specific inhibition by the viral protein of herpes simplex virus ICP47. This offers opportunities for topological, structural and mechanistic studies as well as genetic screenings for TAP functionality.

Interferon-gamma rescues HLA class Ia cell surface expression in term villous trophoblast cells by inducing synthesis of TAP proteins.

Human placental trophoblast cells that constitute the materno-fetal interface during pregnancy escape maternal alloimmune attack. The different trophoblast cell subpopulations have developed efficient regulatory mechanisms to prevent expression of beta2-microglobulin-associated HLA class Ia molecules at their cell surface. We previously reported the presence of HLA class Ia messages in villous cytotrophoblast cells and in the syncytiotrophoblast differentiated in vitro purified from term placenta. In this study, we found that these transcripts are translated in heavy chain proteins that are endoglycosidase H sensitive and thus retained in the endoplasmic reticulum or cis-Golgi. Moreover, these class Ia heavy chains can be co-immunoprecipitated with the chaperone protein calnexin resident in the endoplasmic reticulum. When these trophoblast cells are treated with interferon (IFN)-gamma, part of the class Ia heavy chains become endoglycosidase H resistant, demonstrating that they have left the endoplasmic reticulum. Furthermore, after such a treatment, these heavy chains are detectable at the cell surface of these trophoblast cells, as assessed by two-color flow cytometry analysis and immunoprecipitation of cell surface biotinylated proteins using the W6/32 anti-HLA class I monoclonal antibody (mAb). IFN-gamma treatment induces a significant enhancement of the transcription of transporters associated with antigen processing (TAP1 and TAP2) rather than an increase of HLA class I or beta2-microglobulin messages. Finally, we demonstrate that an anti-TAP1 mAb co-immunoprecipitates TAP1 proteins and HLA class Ia heavy chains in these IFN-gamma-treated trophoblast cells. Thus, the constitutive absence of HLA class Ia cell surface expression in term villous cytotrophoblast and syncytiotrophoblast is likely to be due to a lack of transporter proteins that participate in the proper assembly of these molecules in the endoplasmic reticulum. Such a defect can be modified upon IFN-gamma treatment.

The activity of the gluconate-H+ symporter of Schizosaccharomyces pombe cells is down-regulated by D-glucose and exogenous cAMP.

Schizosaccharomyces pombe cells take up D-gluconate, as an alternative carbon source for growth, during glucose starvation or when cultured on glycerol-containing medium. Gluconate uptake is not detectable while cells are growing logarithmically on glucose. The addition of D-glucose as well as its non-metabolizable analogues to glycerol-grown cells causes an immediate loss of gluconate transport within 1 min. The reversible down-regulation of the gluconate carrier occurs after glucose has been internalized. This regulation is triggered not only by D-glucose but also by extracellular cAMP even in the absence of the cAMP-dependent protein kinase (PKA1).