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  • C07K14/4702—Regulators; Modulating activity

Definitions

• the present invention relates to a method making it possible to identify compounds capable of inhibiting the activation by cytokines, in particular by interferon ⁇ , of the expression of the CIITA gene which itself is involved in the control and regulation of l of genes encoding type II MHC molecules
• MHC major histocompatibility complex
• this class II complex is represented by molecules belonging to the HLA (Human Leukocyte Antigen) system.
• the genes coding for the ⁇ and ⁇ chains constituting the HLA-DR, HLA-DQ and HLA-DP molecules are located in level of region D of chromosome 6
• genes coding for MHC class II molecules are expressed either constitutively or only in a few cell types such that the B lymphocytes, the active T lymphocytes, the macrophages, the cells of the thymic epithelium, the dend ⁇ tic cells such as for example the Langerhans cells, is induced after stimulation, for example by cytokines, and more particularly by Finterferon ⁇ (INF ⁇ ) or interleulcin 4 (IL4), in several other cell types such as, for example, cells belonging to the macrophage or monocyte line, endothehal cells, fibroblasts, muscle cells or cancer cells such as for example melanoma cells
• MHC type II molecules has been shown to be a determining factor in the activation process of T cells. Consequently, it is clear that the molecular mechanisms regulating the expression of these cells genes are a key element in the effectiveness of the immune response. Any defect in this regulatory process can lead to serious immunological disorders, or autoimmune diseases. Thus, in some cases, abnormal expression of MHC class II genes has been observed on the surface of cells which normally would not express such genes. Likewise, an over-expression of these genes can be observed which leads to an aberrant and uncontrolled activation of CD4 + lymphocytes [BOTT ⁇ ZZO et al., 1986, Immunol. Rev., 94, 137-169].
• Such manifestations could be, at least in part, responsible for conditions such as insulin-dependent diabetes, multiple sclerosis, rheumatoid arthritis or lupus erythematosus.
• an immunodeficiency has been demonstrated resulting from a disorder in the expression of MHC class II genes.
• BLS syndrome bare lymphocyte syndrome
• BLS syndrome bare lymphocyte syndrome
• CIITA factor class II transactivator
• WO 9606107 further shows that there are two domains within the CIITA factor more involved in the activation of the transcription of MHC class II genes.
• a sequence capable of expressing transcriptional promoter activity after induction by a cytokine such as for example ⁇ interferon or interleukin 4.
• a cytokine such as for example ⁇ interferon or interleukin 4.
• Such a sequence is represented by the sequence comprising all or part of a sequence identified SEQ ID No. 1, or its complementary sequence. Analysis of this sequence makes it possible to identify several regions corresponding to cis regulation sites of expression, such as in particular the NF-GMa site, the GAS element, the E box or the IRF factor binding site.
• - 1 Mohlethaler-Mottet et al., 1997, EMBO J., 16, 2851 - 2860 and Figure 1.
• This phosphorylation allows, in a third step, the activated STATl factor to migrate into the nucleus where it binds on the GAS box of promoters inducible by cytokines (eg Interferon ⁇ ) thus allowing the activated expression of genes placed under control such promoters.
• cytokines eg Interferon ⁇
• JAK STAT1 activation system in controlling the expression of CIITA genes inducible by interferon ⁇ has been the subject of studies to find that, as with other genes inducible by interferon ⁇ , the expression of the CIITA factor cannot be induced in cell lines deficient for JAK1 (Chang et al., 1994, J. Exp. Med., 180, 1367-1374) Similarly, Meraz et al.
• the factor STAT1 specifically recognizes a particular nucleic acid sequence called “GAS element” (Darnel et al 1997, Science 277, 1630-1635)
• GAS element a particular nucleic acid sequence
• Analysis of the promoter IV sequence of the CIITA gene revealed the presence of such a sequence
• promoter IV also contains the sequence CACGTG (box E, Gregor et al, 1990, Genes Dev, 4, 1730-1740, see Figure 1) This could indicate that a transcription factor belonging to the family of helix loop / hehce / leucine zipper is likely to intervene in the regulation of the expression of genes placed under the control of promoter IV
• the transcription factor USFl is ubiquitously expressed and participates in the regulation of the expression of different genes, some of which are expressed in a "specific tissue” manner or in an inducible manner, such as for example the gene coding for human growth hormone (Pe ⁇ tz et al, 1988,
• the applicant has also shown that the binding of the STATl factor on the GAS site is strongly stabilized by the factor USFl and that these factors bind cooperatively to the binding sites located on the promoter IV, this interaction cooperative playing a decisive role in the control of the specific activation of the IV promoter by cytokines, in particular by interferon ⁇ .
• bind in a cooperative manner is meant to indicate that there is an interaction between the protein factors in question, which can take place before or after their fixation on their respective site, which makes it possible in particular to define sites specific interaction between said protein factors and which results in a cooperative effect.
• This cooperative effect is understood as a result which can only be observed when the interaction in question takes place, for example this cooperative effect will consist of a stabilization of the binding of at least one of the protein factors on its site ( being understood, thanks to the interaction existing between protein factors).
• the cooperative effect will be a synergistic effect characterized in that the effect observed by the use of protein factors will be greater than the expected effect corresponding to the sum of the individual effects observed for each of the factors .
• This mechanism of induction of the expression of a gene placed under the control of the promoter IV differs from the other systems previously described for the genes inducible by interferon ⁇ in that the latter requires the factor USFl as a partner. essential for fixation and therefore for the activity of the STATl factor.
• the discovery of this mechanism involved in particular in the activation by interferon ⁇ of the expression of the CIITA gene, and consequently in the induction by interferon ⁇ of MHC class II molecules, led the depositor to develop a new method of identifying molecules capable of inhibiting expression genes placed under the control of a promoter whose activity is induced by the cooperative fixation of STAT l and USF l, and more particularly of all or part of promoter IV.
• said gene is the gene coding for CIITA
• nucleic acid sequence coding for the CIITA polypeptide comprises all or part of a nucleic acid sequence corresponding to mRNAs originating from different tissues or cell lines expressing CIITA activity of constitutively or after induction II can therefore be sequences that are at least partially coding as well as sequences intervening in the control of 1 expression, in particular sequences having transc ⁇ ptional promoter activity
• nucleic acid sequence is meant a fragment of DNA and / or RNA, double strand or single strand, isolated natural or synthetic, designating a precise sequence of nucleotides, modified or not, making it possible to define a fragment or a region of a nucleic acid
• polypeptide is meant to designate a precise sequence of amino acids, regardless of its size or of its function, natural isolated or synthetic, modifies or not
• nucleic acid sequence having transc ⁇ ptional promoter activity is meant a nucleic acid sequence making it possible to control, ie to initiate and / or modulate, the transcription of at least one gene, homologous or heterologous, located downstream of said sequence Likewise, we will speak of the promot ⁇ ce function of said sequences or of promoter
• reporter gene any nucleic acid sequence located downstream of a second nucleic acid sequence, making it possible to analyze 1 activity of transc ⁇ ptional promoter of said second sequence.
• transc ⁇ ption of this reporter gene is translated by 1 appa ⁇ tion of a product (RNA or polypeptide) easily detectable according to conventional techniques well known
• transcription factor or STATl polypeptide is meant to designate the transcription factor STATl which is capable of binding to the level of the GAS element of the promoters inducible by the interferon ⁇ (Damell, 1997, Science 277, 1630-1635)
• transcription factor or polypeptide USF 1 is intended to denote the transcription factor USF 1 which is capable of binding at the level of the E box of promoters such as for example the promoter of the gene coding for human growth hormone ( Pe ⁇ tz et al, 1988, J Biol Chem, 263, 5005-5007, of the gene coding for the ⁇ 2 chain of immunoglobulins (Chang et al, 1992, Nucleic Acid Res, 20, 287-293) or of the gene coding for p53 ( Reisman and Rotter, 1993, Nucleic Acid Res, 21, 345-350).
• promoter of the gene coding for human growth hormone Pe ⁇ tz et al, 1988, J Biol Chem, 263, 5005-5007
• the gene coding for the ⁇ 2 chain of immunoglobulins (Chang et al, 1992, Nucleic Acid Res, 20, 287-293) or of the gene coding for p53 ( Reisman and Rotter, 1993, Nucleic Acid Res, 21, 345-
• said factors STAT 1 and USF1 can be of recombinant or natural origin, and more particularly consist of factors available in cellular extracts, in particular nuclear, prepared from cell line, possibly stimulated by a cytokine, in particular by interferon ⁇ , expressing said factors.
• the factor STAT l this can be either in an non-activated form (non-phosphorilated) or in an activated form (phosphorilee, in particular by the action of the enzyme JAK1).
• the activated form of ST AT 1 will preferably be chosen.
• the present invention relates firstly to a method for determining whether a candidate compound is capable of inhibiting the binding between the STAT1 and USF1 polypeptides comprising the steps following:
• the STAT l and USF1 polypeptides used in the context of the present invention can either be natural polypeptides, extracted for example from cell lines expressing the corresponding genes, such as for example the line
• the STATl and USFl polypeptides used in the present invention may or may not retain their activat ⁇ ce function of other genes for which the cooperative effect of the two polypeptides is not observed (the polypeptides then act separately) More particularly it is possible to '' use only part of said STATl and / or USFl polypeptides provided that they have retained their prop ⁇ etes to bind to each other, and possibly the property of binding to their respective sites
• the STATl polypeptide is capable of binding to the GAS element (5'-TTCTGATAAA-3 ') because it is in its activated form (phosphorilated)
• This phospho ⁇ lation of the STATl polypeptide can in particular be obtained a) naturally, in a cell expressing said polypeptide induced by a cytokine, preferably by interferon ⁇ , b) by the action of a kinase, such as for example JAK1 or c) chemically, by synthesis
• step (d) consists of an indirect measurement that is to say that in this particular case the formation of complexes comprising STATl is determined.
• step (d) consists of an indirect measurement that is to say that in this particular case the formation of complexes comprising STATl is determined.
• USFl and a double b ⁇ n nucleic acid sequence comprising the element GAS (5'-TTCTGATAAA-3 ') and the box E (5'-CACGTG-3')
• GAS 5'-TTCTGATAAA-3 '
• E 5'-CACGTG-3'
• step (d) consists of another indirect measurement, that is to say that in this case, the expression of a nucleic acid sequence coding for all or part of a polypeptide is measured, said expression being placed under the control of a promoter containing at least 1 GAS element (5'-TTCTGATAAA-3 ') and box E (5'-CACGTG-3'), or its complementary sequence
• said promot ⁇ ce sequence is selected from the sequences which contain all or part of promoter IV (SEQ ID No. 1), or of its complementary sequence
• said acid sequences nucleic acid whose expression is measured in step (d) can 1) be reporter genes! such as for example the gene for rabbit ⁇ globin, luciferase or ⁇ lactamase or 2) to code for all or part of polypeptides having the amino acid sequence ri a CIITA factor as described in the French patent application N ° 97 04954 and more particularly as defined by SEQ ID N ° 2 According to this latter case, it will then be said that they code for all or part of the CIITA polypeptide
• Measuring the expression of the nucleic acid sequence may in particular consist of a) measuring the specific messenger RNAs expressed from said nucleic acid sequence or b) measuring the expressed polypeptide
• Examples of such methods are widely developed in the literature and their implementation is within the scope of those skilled in the art as A examples include techniques such as those based on the hyb ⁇ dation of oligonucléiques labeled probes whose sequence is specific for RNA that is to be detected, 1 amplification of Dar by PCR using primers whose sequence is specific for said RNA, the technique of protection against degradation by
• the step of measuring the expression of the sequence of nucleic acid is carried out under conditions allowing the induction of said expression by a cytokine, and more particularly by interferon v
• step (d) consists in a direct measurement of the formation of complexes between the STATl and USFl polypeptides, for example by the use of antibodies specific for said complexes or any other suitable means.
• the invention also relates to a method for determining whether a candidate compound is capable of inhibiting the expression of a nucleic acid sequence encoding all or part of a polypeptide, preferably all or part of the CIITA polypeptide (SEQ LD N ° 2) or all or part of a reporter gene placed under the control of all or part d 'a promoter containing at least the GAS element (5'-TTCTGATAAA-3') and box E (5'-CACGTG-3 '), and preferably a promoter IV (SEQ LD No. 1), or their respective complementary sequence comprising the following steps
• (c) have a nucleic acid sequence encoding all or part of a polypeptide preferably for all or part of the CIITA polypeptide (SEQ ID N ° 2) or for all or part of a reporter gene, the expression of which is placed under the control of all or part of a promoter containing at least the GAS element (5'- TTCTGATAAA-3 ') and the box E (5'-CACGTG-3 ') ; and preferably a promoter IV (SEQ ID No. 1), (d) bringing said polypeptides as defined in (a) and (b) into contact, a said nucleic acid sequence as defined in (c ) and said candidate,
• said candidate compound is capable of inhibiting the expression of a nucleic acid sequence coding for all or part of a polypeptide, in particular for all or part of the CIITA polypeptide or for all or part of a reporter gene placed under the control of all or part of a promoter containing at least the GAS element (5'-TTCTGATAAA-3 ') and the E box
• polypeptide in particular for all or part of the CIITA polypeptide (SEQ ID No. 2) or for all or part of a reporter gene, placed under the control of all or part of a promoter containing at least the GAS element (5 ′ -TTCTGATAAA-3 ') and the E-box (5'-CACGTG-3'), and most preferably a promoter IV (SEQ ID N ° l), (c) bringing said cells into contact with a said candidate compound or transfect the cells with an expression vector allowing the expression of a said compound inside said cells, under conditions allowing activation of the expression of the nucleic acid sequence by a cytokine, preferably by l ' ⁇ interferon (d) measuring the expression of said one sequence of nucleic acid, and (e) comparing this measurement with the measurement of the expression of said nucleic acid sequence observed under the same experimental conditions, in particular of activation of expression, in the absence of said candidate compound, a decrease said expression allowing the conclusion that said candidate compound is capable of inhibiting activ
• An expression vector comprising at least one nucleic acid sequence coding for all or part of a polypeptide placed under the control of all or part of the promoter IV containing the element GAS (5'-TTCTGATAAA-3 ') and the box E (5'-CACGTG-3 ') may in particular consist of a vector as described in French patent application No. 97 04954, the content of which forms part of the present application
• the invention also relates to methods as defined above making it possible to identify candidate compounds capable of inhibiting the expression of genes coding for MHC class II molecules when the latter is desired, in particular under conditions for which it is desired to act after induction by a cytokine, and more particularly by the interferon ⁇ .
• Figure 1 shows the different elements involved in the regulation of expression induced by cytokines of genes placed under the control of promoter IV
• Me67 8 (melanoma) and THP1 (monocyte) cell lines are cultured in RPMI-1640 medium.
• the 2FTGH (fibrosarcoma) and U3A lines (2FTGH line not expressing STATl) are cultured on modified Dulbecco medium. .
• the media are supplemented with 10% fetal calf serum, 10 U / ml of penicillin, 10 mg / ml of streptomycin and 2 mM glutamine. Incubations are carried out at 37 ° C under 5% CO2
• the expression of the reporter genes is measured by quantitative RT-PCR as described in Sperisen et al, 1992, PCR Meth Appli. 1, 164-170 Les transfections, RNA preparation and RT-PCR analyzes are carried out as previously described (Muhlethaler-Mottet et al, 1997, EMBO J, 16, 2851 - 2860)
• the plasmid PIV-308 contains the fragment -308 to -75 of the flanking region of the promoter IV of the CIITA gene under a clone downstream of the gene coding for the rabbit beta globuhne of the plasmid pG ⁇ G (+)
• the activity of the promoter is measured by Phosphoimager
• the cells used are or are not stimulated by interferon ⁇ (500U / ml) for 30 mm before the preparation of the nuclear extracts according to the method described by Harroch et al, 1994, EMBO J, 13, 1942-1949
• oligonucleotides radiolabelled at one of their ends by the addition of [ ⁇ -32P] ATPs are hybridized to their complementary sequence and purified by electrophoresis on polyacrylamide gel so as to obtain probes labeled with double stranded DNA corresponding to all or part of CIITA promoter IV
• the mixture is incubated for 30 minutes at 20 ° C.

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• 1998
  • 1998-02-19 FR FR9802025A patent/FR2775003A1/fr active Pending
• 1999
  • 1999-02-19 AU AU24312/99A patent/AU2431299A/en not_active Abandoned
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