Inhibition of histone deacetylation induces constitutive derepression of the beta interferon promoter and confers antiviral activity.

The induction of alpha/beta interferon (IFN-alpha/beta) genes constitutes one of the first responses of the cell to virus infection. The IFN-beta gene is constitutively repressed in uninfected cells and is transiently activated after virus infection. In this work we demonstrate that histone deacetylation regulates the silent state of the murine IFN-beta gene. Using chromatin immunoprecipitation (ChIP) assays, we show a direct in vivo correlation between the transcriptionally silent state and a state of hypoacetylation of histone H4 on the IFN-beta promoter region. Trichostatin A (TSA), a specific inhibitor of histone deacetylases, induced strong, constitutive derepression of the murine IFN-beta promoter stably integrated into a chromatin context, as well as the hyperacetylation of histone H4, without requiring de novo protein synthesis. We also show in this work that TSA treatment strongly enhances the endogenous IFN level and confers an antiviral state to murine fibroblastic L929 cells. Inhibition of histone deacetylation with TSA protected the cells against the lost of viability induced by vesicular stomatitis virus (VSV) and inhibited VSV multiplication. Using antibodies neutralizing IFN-alpha/beta, we show that the antiviral state induced by TSA is due to TSA-induced IFN production. The demonstration of the predominant role of histone deacetylation during the regulation of the constitutive repressed state of the IFN-beta promoter constitutes an interesting advance on the understanding of the negative regulation of this gene and opens up the possibility of new therapeutic perspectives.

Repression of virus-induced interferon A promoters by homeodomain transcription factor Ptx1.

Interferon A (IFN-A) genes are differentially expressed after virus induction. The differential expression of individual IFN-A genes is modulated by substitutions in the proximal positive virus responsive element A (VRE-A) of their promoters and by the presence or absence of a distal negative regulatory element (DNRE). The functional feature of the DNRE is to specifically act by repression of VRE-A activity. With the use of the yeast one-hybrid system, we describe here the identification of a specific DNRE-binding protein, the pituitary homeobox 1 (Ptx1 or Pitx1). Ptx1 is detectable in different cell types that differentially express IFN-A genes, and the endogenous Ptx1 protein binds specifically to the DNRE. Upon virus induction, Ptx1 negatively regulates the transcription of DNRE-containing IFN-A promoters, and the C-terminal region, as well as the homeodomain of the Ptx1 protein, is required for this repression. After virus induction, the expression of the Ptx1 antisense RNA leads to a significant increase of endogenous IFN-A gene transcription and is able to modify the pattern of differential expression of individual IFN-A genes. These studies suggest that Ptx1 contributes to the differential transcriptional strength of the promoters of different IFN-A genes and that these genes may provide new targets for transcriptional regulation by a homeodomain transcription factor.

Murine interferon-alpha1 gene-transduced ESb tumor cells are rejected by host-mediated mechanisms despite resistance of the parental tumor to interferon-alpha/beta therapy.

The highly metastatic ESb tumor is totally resistant to murine interferon-alpha/beta (IFN-alpha/beta) therapy, regardless of the number of cells injected or the route of inoculation. In contrast, as we show herein, mouse IFN-alpha1-transduced ESb tumor cells were inhibited markedly when injected subcutaneously into immunocompetent mice. IFN-producing ESb tumor rejection was mediated by the immune system, because these tumor cells grew normally in immunosuppressed mice. Tumor regression was accompanied by extensive necrosis and cellular infiltrates in the tumor area. These results further support the use of IFN-alpha in cytokine gene therapy of cancer and suggest the advantage of using gene transfer rather than cytokine administration to enhance an antitumor immune response.

Specific binding of high-mobility-group I (HMGI) protein and histone H1 to the upstream AT-rich region of the murine beta interferon promoter: HMGI protein acts as a potential antirepressor of the promoter.

The high-mobility-group I (HMGI) protein is a nonhistone component of active chromatin. In this work, we demonstrate that HMGI protein specifically binds to the AT-rich region of the murine beta interferon (IFN-beta) promoter localized upstream of the murine virus-responsive element (VRE). Contrary to what has been described for the human promoter, HMGI protein did not specifically bind to the VRE of the murine IFN-beta promoter. Stably transfected promoters carrying mutations on this HMGI binding site displayed delayed virus-induced kinetics of transcription. When integrated into chromatin, the mutated promoter remained repressed and never reached normal transcriptional activity. Such a phenomenon was not observed with transiently transfected promoters upon which chromatin was only partially reconstituted. Using UV footprinting, we show that the upstream AT-rich sequences of the murine IFN-beta promoter constitute a preferential binding region for histone H1. Transfection with a plasmid carrying scaffold attachment regions as well as incubation with distamycin led to the derepression of the IFN-beta promoter stably integrated into chromatin. In vitro, HMGI protein was able to displace histone H1 from the upstream AT-rich region of the wild-type promoter but not from the promoter carrying mutations on the upstream high-affinity HMGI binding site. Our results suggest that the binding of histone H1 to the upstream AT-rich region of the promoter might be partly responsible for the constitutive repression of the promoter. The displacement by HMGI protein of histone H1 could help to convert the IFN-beta promoter from a repressed to an active state.

Silencer activity in the interferon-A gene promoters.

Interferon-A (IFN-A) differential gene expression is modulated by a complex interplay between cis-acting DNA elements and the corresponding specific trans-regulating factors. Substitutions in the proximal virus-responsive element of the interferon-A (IFN-A) promoters contribute to their differential gene expression. The 5' distal silencing region in the weakly virus-inducible murine IFN-A11 gene has been previously delimited. DNase I footprinting experiments and transient gene expression assays demonstrate identical silencing activity in equivalent regions of the genes for IFN-A11 and IFN-A4 promoters. A minimal 20-mer distal negative regulatory element (DNRE) in both promoters is necessary and sufficient for the silencing and a region in the highly inducible IFN-A4 promoter located between the silencer and the virus-responsive element overrides the silencer activity. Mutations in the central region of the DNRE, causing derepression, also altered the formation of one of the two major DNA-protein complexes. One of these contains a protein related to or identical to the high mobility group I(Y) proteins, while the other complex contains a major protein present in uninduced and virus-induced cells with a molecular mass of 38 kDa, which may be related to the silencer activity. Similar DNREs are present in other virus-uninducible IFN-A promoters, and these data suggest that a common silencer may mediate the transcriptional repression in different genes of this family.

Synergism between multiple virus-induced factor-binding elements involved in the differential expression of interferon A genes.

Comparative transfection analysis of murine interferon A4 and interferon A11 promoter constructs transiently transfected in mouse L929 and human HeLa S3 cells infected with Newcastle disease virus showed that the second positive regulatory domain I-like domain (D motif), located between nucleotides -57 and -46 upstream of the transcription start site, contributes to the activation of virus-induced transcription of the interferon (IFN)-A4 gene promoter by cooperating with the positive regulatory domain I-like and TG-like domains previously described. Electrophoretic mobility shift assay performed with the virus-inducible fragments containing these motifs indicated that the binding activity that we have denoted as virus-induced factor (Génin, P., Bragança, J., Darracq, N., Doly, J., and Civas, A. (1995) Nucleic Acids Res. 23, 5055-5063) is different from interferon-stimulated gene factor 3. It binds to the D motif but not to the virus-unresponsive form of the D motif disrupted by a G-57 --> C substitution. We show that the low levels of IFN-A11 gene expression are caused essentially by the lack of two inducible enhancer domains disrupted by the A-78 --> G and the G-57 --> C substitutions. These data suggest a model taking account of the differential regulation of IFN-A gene family members. They also suggest that virus-induced factor may correspond to the primary transcription factor directly activated by virus that is involved in the initiation of IFN-A gene transcription.

Identification of distal silencing elements in the murine interferon-A11 gene promoter.

The murine interferon-A11 (Mu IFN-A11) gene is a member of the IFN-A multigenic family. In mouse L929 cells, the weak response of the gene's promoter to viral induction is due to a combination of both a point mutation in the virus responsive element (VRE) and the presence of negatively regulating sequences surrounding the VRE. In the distal part of the promoter, the negatively acting E1E2 sequence was delimited. This sequence displays an inhibitory effect in either orientation or position on the inducibility of a virus-responsive heterologous promoter. It selectively represses VRE-dependent transcription but is not able to reduce the transcriptional activity of a VRE-lacking promoter. In a transient transfection assay, an E1E2-containing DNA competitor was able to derepress the native Mu IFN-A11 promoter. Specific nuclear factors bind to this sequence; thus the binding of trans-regulators participates in the repression of the Mu IFN-A11 gene. The E1E2 sequence contains an IFN regulatory factor (IRF)-binding site. Recombinant IRF2 binds this sequence and anti-IRF2 antibodies supershift a major complex formed with nuclear extracts. The protein composing the complex is 50 kDa in size, indicating the presence of IRF2 or antigenically related proteins in the complex. The Mu IFN-A11 gene is the first example within the murine IFN-A family, in which a distal promoter element has been identified that can negatively modulate the transcriptional response to viral induction.

The essential role of endogenous IFN alpha/beta in the anti-metastatic action of sensitized T lymphocytes in mice injected with Friend erythroleukemia cells.

Adoptive transfer of splenic T lymphocytes from DBA/2 mice immunized against Friend erythroleukemia cells (FLC) inhibited the development of visceral metastases and increased the survival time of DBA/2 mice challenged i.v. with parental FLC 24 hr to 2 months later. Immune spleen cells were ineffective in mice pre-treated with potent neutralizing antibody to mouse IFN alpha/beta (but not to IFN gamma), demonstrating the essential participation of endogenous IFN alpha/beta in the inhibitory action of immune T lymphocytes against FLC metastases. These findings suggest that the reported inability of immune T lymphocytes to exert an anti-FLC effect in immunodeficient DBA/2 mutant beige (bg/bg) mice (unless these mice had also been treated with IFN alpha/beta), may have been due to lower levels of endogenous IFN alpha/beta in DBA/2 bg/bg mice than in normal DBA/2+/bg mice. Experimental results in support of this hypothesis are presented.

IFN-alpha 1 gene transfection completely abolishes the tumorigenicity of murine B16 melanoma cells in allogeneic DBA/2 mice and decreases their tumorigenicity in syngeneic C57BL/6 mice.

The murine B16 melanoma (H-2b) was transfected with a retroviral vector containing the mouse IFN-alpha 1 gene. IFN-alpha 1-transfected cells produced IFN-alpha in vitro and exhibited an altered phenotype characterized by a decreased rate of multiplication, enhanced expression of H-2 antigens, an antiviral state to VSV, and decreased pigmentation. Control and IFN-alpha 1-transfected cells were tested for their ability to grow in syngeneic (H-2b) C57Bl/6 and allogeneic (H-2d) DBA/2 mice. IFN-alpha 1-producing B16 clones were less tumorigenic after s.c., i.p., and i.v. routes of injection than IFN-non-producer B16 clones in syngeneic C57Bl/6 mice. IFN-alpha 1-producing B16 cells were, however, totally rejected by allogeneic DBA/2 mice regardless of the routes and inocula tested, while control B16 cells grew in and killed DBA/2 mice. The total rejection of IFN-alpha 1-transfected B16 cells in allogeneic mice appeared to be dependent on T cells as these cells grew in DBA/2 nude mice. Incubation of IFN-alpha-producing clones with anti-mouse IFN-alpha/beta prior to injection into C57Bl/6 mice did not enhance their tumorigenicity. Likewise, injection of C57Bl/6 and DBA/2 mice with antibody to IFN-alpha/beta did not enhance the tumorigenicity of IFN-alpha 1-transfected cells. C57Bl/6 mice immunized with irradiated IFN-alpha 1 cells were only slightly protected against a subsequent challenge with parental B16 cells. In contrast, DBA/2 mice immunized with irradiated IFN-alpha 1 cells exhibited tumor-specific, long-lasting immunity to subsequent challenge with parental B16 cells.

Domains of interaction between alpha interferon and its receptor components.

We describe how constraints on the binding of human interferons (IFNs), alpha1 and alpha2 and alpha8 on mouse cells are partially relieved by the expression of the bovine (Bo) or human (Hu) IFN alpha/beta receptor (IFNAR) component in these cells. We show that, while the binding of all three is substantially increased by the transfection of Bo IFNAR, it is accompanied by an increase in activity only in the case of alpha2 and alpha8 (IFNs that otherwise have little activity on mouse cells). IFN alpha1, which shows some partial activity on mouse cells, responds to the presence of Bo IFNAR by acting, at low concentrations, as a competitive antagonist to IFNs alpha2 and alpha8. A review of published results on IFN hybrid scanning and on the effects of expressing Bo IFNAR in human cells led us to propose that an N-terminal segment of the IFN molecule interacts directly with IFNAR. Applying site-directed mutagenesis to an IFN hybrid; alpha8[60]alpha1[92]alpha8, we show that the point mutations K84 to E84 and Y90 to D90 act synergistically to cause the hybrid to behave as the parental IFN alpha8, switching the preference from Mu to Hu IFNAR in transfected mouse cells. The published structural models for IFN reveal that positions 84 and 90 span the exposed residues of the alpha-helix C of the IFN molecule. We derive a model of IFN-receptor interaction in which the A helix and the C helix of IFN interact with IFNAR and in which a binding phase can be distinguished from a binding/activity phase. We propose that the so-called "hot" domains of the IFN molecule (the AB loop and the D helix) are presented by IFNAR to interact with an additional component of the functional receptor.

Behavior of a cloned murine interferon alpha/beta receptor expressed in homospecific or heterospecific background.

A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes.

Murine tumor cells expressing the gene for the human interferon alpha beta receptor elicit antibodies in syngeneic mice to the active form of the receptor.

The cellular receptor for the human alpha and beta interferons (IFN) was expressed, by gene transfer, in a murine hepatoma-derived cell line, BTG 9A. Injected subcutaneously into the syngeneic mouse (C57BL/6), the parental and the transfected cells grew and formed tumors which later regressed. More than half the mice bearing tumors derived from cells expressing the receptor, developed IgG antibodies capable of blocking the activity, on human cells, of human recombinant IFN-alpha B, -alpha A, -alpha D and of natural human IFN-beta, but not of recombinant IFN-gamma. Cross-reactivity of human IFN-alpha on murine and bovine cells was unaffected by these antibodies. The binding of human IFN-alpha to solubilized receptors from human lymphoid cell lines was also blocked and complexes of radiolabeled recombinant IFN-alpha A or IFN-alpha B, chemically cross-linked to their human receptor could be immunoprecipitated by the antisera. IFN alpha beta receptor protein, purified by electrophoresis in sodium dodecylsulfate, was not recognized. We conclude that the antibodies are directed against the forms of the IFN alpha beta receptor actually expressed on the membrane.

Importance of interferon alpha in the resistance of allogeneic C57B1/6 mice to the multiplication of Friend erythroleukemia cells in the liver.

Friend erythroleukemia cells (FLC) (H-2d) injected intravenously multiply extensively in the livers of syngeneic DBA/2 mice and not at all in the livers of allogeneic C57B1/6 mice. Our results indicate that interferon alpha (IFN-alpha) is an important factor in the resistance of allogeneic mice to the multiplication of FLC in the liver. (a) After i.v. inoculation of FLC there was an inverse correlation between the presence of IFN-alpha in the serum and the capacity of FLC to multiply in the liver. Thus, all 44 FLC-injected adult C57B1/6 mice had circulating IFN-alpha and FLC did not multiply in the liver of any of the mice. Interferon was not detected in the serum of 83% of 41 FLC-injected DBA/2 mice (and was found only at a low titer in 17% of the mice) and FLC multiplied in the liver of all mice. (b) FLC did multiply in the livers of newborn C57B1/6 mice and in the livers of irradiated adult C57B1/6 mice, and IFN-alpha was not detected in their sera. In contrast, after i.v. inoculation of FLC, IFN-alpha was detected in the sera of 3-week-old and athymic nu/nu adult C57B1/6 mice while FLC failed to multiply in the liver. (c) FLC also induced IFN-alpha in congenic B10.D2 (H-2d) mice and FLC did not multiply in the liver. We suggest that, depending on the site of tumor implantation, different host mechanisms have various degrees of importance in controlling the growth and/or rejection of allogeneic tumor cells, and that IFN-alpha is particularly important when FLC are injected i.v.

Interferon removes its own receptors as it blocks the division of Daudi cells.

The Burkitt-derived line, Daudi, whose proliferation is inhibited by human alpha-interferon (IFN-alpha), was treated with 125I-labelled recombinant human IFN-alpha A. After separation from unbound ligand, cell-bound IFN was extracted with the detergent digitonin yielding soluble and insoluble complexes of IFN and receptor, together with a certain amount of uncomplexed IFN. 1. Soluble complexes were stable enough to be separated from uncomplexed IFN by permeation chromatography. Treatment of soluble complexes with the bifunctional reagent, disuccinimidyl suberate, yielded a radioactive product separating with an Mr of 130,000 on electrophoresis in sodium dodecyl sulphate. Similar complexes could be recovered with sodium dodecyl sulphate from the digitonin-insoluble residue, treated with the bi-functional reagent. 2. The total (soluble and insoluble) of complexed IFN obtained after digitonin extraction was a constant fraction (0.62) of the total cell-bound radioactivity, being independent of the concentration of IFN added to the cells (less than pM to greater than nM), and of the time of incubation (1 min to 20 h). However, between 30 min and 3 h of incubation, the insoluble complex increased, at the expense of the soluble complex, and there appeared a cellular pool of degraded ligand. From 3 h to 20 h the distribution of ligand-derived radioactivity remained constant while the total amount decreased to less than 10% of its value at 30 min. This decrease in binding was matched by the appearance of an equivalent quantity of radiolabelled fragments in the culture medium. 3. The inhibition of cellular division due to IFN was shown to be coincident with the disappearance of cellular binding and with the cell-mediated degradation of receptor-complexed IFN. We propose that IFN removes its own receptor and, in doing so, blocks a linked function necessary for the stimulated growth of Daudi cells.

Electrostatic interactions in the cellular dynamics of the interferon-receptor complex.

Using membrane preparations of the interferon receptor, prepared from cells of the Burkitt line, Daudi, we have examined the binding of three human recombinant alpha-interferons. 1. We discovered a binding titration of the interferons IFN-alpha A and IFN-alpha D in the pH range 6-9. Receptor binding, negligible at pH 6, rises to a maximum close to pH 9. We have shown that binding of IFN-alpha A at basic pH is to the same receptors as at neutrality and that IFN-receptor complexes extracted with digitonin are more stable at basic pH than they are at neutrality. 2. The recombinant interferon, IFN-alpha B, shows little change of binding in the pH range 6-9. At its basic optimum the binding of IFN-alpha A approaches that of IFN-alpha B, while at neutral pH the binding of IFN-alpha A is 3-4 times less. This difference at neutral pH is seen on intact cells as well as on membrane preparations. The specific activity of IFN-alpha B is close to that of IFN-alpha A, both of which are 10-20 times more active than IFN-alpha D; and the binding titration is, therefore, independent of the initial binding affinities. 3. Using hybrid IFNs constructed from the DNA sequences of alpha D and alpha B, we have isolated the sequence responsible for the binding titration to the segment comprising amino acids 61-92. Examination of these sequences reveals that Lys-84 is present in all the IFN-alpha except IFN-alpha B where it is replaced by Glu; and Tyr-90, present in most of the common IFN-alpha including alpha A and alpha D, is replaced by Asp in IFN-alpha B. Lys and Tyr would normally titrate in the pH range 6-9. We conclude that the binding titration is due to an electrostatic interaction and we propose that the interaction is between IFN-receptor complexes. The role of the interaction in the binding losses that accompany the antiproliferative effects of IFN is discussed.

Receptor mediated pathways for interferon action: in vivo implications.

High affinity receptors for human interferon (IFN) are a common surface component of human nucleated cells. A model culture system has been used to mark the activation of surface receptors by IFN as the first step in several, possibly separate, pathways of action, some of which depend on the continued presence of IFN. Two types of equilibrium binding can be seen: an initial one of short duration corresponding to receptor activation and a second of longer duration corresponding to activation of a second site by IFN. Various different responses to IFN are seen to be determined by one or other of these equilibria. In vivo, implications are discussed in terms of the pharmacokinetic distribution of IFN, with particular reference to intravenous injection. Desensitization of target cells with respect to late equilibrium effects is seen as a possible consequence of receptor activation against the background of changing IFN concentration in vivo. A modulating effect of combined IFN species is proposed.

Inhibitory effect of mouse interferon on the growth of an embryonal carcinoma in mice.

Embryonal carcinoma cells (F9) were resistant to the inhibitory effects of interferon (IFN) on viral multiplication and cell division in vitro. Nevertheless, daily administration of IFN resulted in a marked inhibition of the growth of this carcinoma injected intraperitoneally (ip) and increased mouse survival time. An incidental finding was that daily IFN treatment increased the serum level of alpha-fetoprotein in normal 129 mice.

Kinetic evidence for an activation step following binding of human interferon alpha 2 to the membrane receptors of Daudi cells.

A single species of human interferon alpha (IFN alpha) was labelled with 125I to high incorporation for binding studies on the B-lymphoblastoid cell line, Daudi, whose growth is inhibited by low doses of IFN, the effect being saturated at about 100 U/ml (25 pM). The radiolabelled IFN was shown to be fully active and the binding affinity to cellular sites was shown to be unchanged by iodination. Experimental conditions were standardized such that binding and cell growth experiments could be performed on the same initial culture of cells. 125I-labelled IFN alpha 2 (IFN alpha prepared from Escherichia coli carrying human alpha 2 gene) was added to exponentially growing cultures (mean specific growth rate 0.77 +/- 0.07 days-1) at a mean concentration of 235000 +/- 20000 cells ml-1. Two types of binding could be discerned on growing cultures: the first with a transient peak followed by a loss or discharge of available sites, the second reaching equilibrium some 3 h after the addition of IFN. Large differences in the apparent dissociation constants were evident. The affinity of binding at the 'steady-state', appeared to be much higher. An analysis of the displacement rates for bound IFN suggested that the two reactions were occurring consecutively over the whole of the dose range studied (1-100 U/ml; 0.25-25 pM IFN). In this dose range we found that Daudi cells would eventually stop growing at all doses and that the rates of deceleration of cellular growth were linearly proportional to the dose of IFN in a double-reciprocal plot (i.e. in analogy to Michaelis-Menten kinetics). A good congruence was found between the equilibrium constants for binding and for growth inhibition (2.65 pM and 2.39 pM, respectively). The amount of IFN bound at steady state thus determines the rate at which growth is inhibited. We propose that the first reaction represents binding of IFN to surface receptors, and the second transfer of IFN to an activation complex on the cell membrane. Appropriate models and their general applicability to IFN action are discussed.

Indomethacin and aspirin do not inhibit the antiviral or anti-proliferative actions of interferon.

Neither indomethacin nor aspirin, at concentrations which inhibited the formation of prostaglandins and prevented the interferon-induced increase in the intracellular concentration of cyclic GMP, had any significant effect on the development of the interferon-induced antiviral state either in mouse L1210 cells challenged with vesicular stomatitis virus or in mice infected with encephalomyocarditis virus. Furthermore, neither drug had any significant effect on the interferon-induced inhibition of cell multiplication in cultures of mouse leukaemia L1210 cells. The differences in the effects of these cyclo-oxygenase inhibitors on different interferon effects may provide some insight into the different pathways of interferon action.