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Allele-specific transcriptional regulation of HLA-DQB1 genes
Abstracts
12
042 DOES HLA-Cw7 CONSTITUTE A DISTINCT ALLELIC FAMILY OF THE HLA-Cw LOCUS? Zohreh Tatari*, Jean-Michel Cayuela", CatherineFortier", Colette Raffoux* and DominiqueCharron* * Laboratoire d'Immunologie de d'Histocompatibilite, et Laboratoire d'Hematologie.Hopital Saint Louis. Paris-France. HLA-Cw7 alleles are distinguishedfrom other HLA-Cw alleles by a higher polymorphism and a higher frequency in the Caucasian population. Recentlyit has been describedthat the lower cell surfaceexpressionof HLA-C is due to a negativeregulatoryelement downstreamof the translationstop site and a non-fonctional EnhA which is responsible to prevent the binding of NFkB factors to this region. In order to understand these particularities, we have studied the polymorphism in the 5' upstreamregulatory region of HLACw, We observedthat among of HLA-C alleles, only Cw7 has the conserved sequencesnecessaryfor NaB binding at the Enh A site. The Northern blot analysis shows that HLA-Cw7, on average, is twofold more expressed than the other Cw alleles. The CAT assay tests performed with a fragment from 460 to + I comparingthe transcriptional level of HLA-Cw7 with the other Cw alleles confirmed these results. We are currently quantifying the HLA-Cw alleles includingCw7, using a competitive RT-PCR assay for defining more preciselythe rate of HLA-Cw7transcription in comparaison with other HLAC alleles. These results could be the first to show evidence for a distinct family from a regulatory and polymorphism point of view inside the same locus.
044 MOLECULAR BASIS FOR DIFFERENCES IN THE TRANSCRIPTIONAL ACTIVITIES OF HLA-DRB GENES IN DIFFERENT HAPLOTYPES Xiaohong Qiu, Dharam P. Singal Department of Pathology, McMaster University, Hamilton. ON, Canada In the present study. we examined the effect of polymorphims in the X1 box and the Y box on the transcriptional activity of DRB gene promoters. We utilized normal. chimeric and site-directed mutant promoters and compared their abilities to initiate transcription of the CAT reporter gene. In addition, we examined the effect of these polymorphisms in binding ~-regulatory proteins. The results show that polymorphisms in the X1 box, i.e. substitution of nucleotide C by G at position -188 or of A by G at position -183, significantly reduced (> 70%) the promoter strength. Similarly. nucleotide variations at positions -152, -150 and -149 in the Y box. which affect the inverted CCAAT motif, significantly reduced (>50%) the promoter activity. Polymorphisms in the X1 box affected DNA-protein interactions. However, the role of nuclear proteins that bind to polymorphic X1 box elements on promoter strength is not clear. On the other hand. affinities of NF-Y and NF-YB proteins, which bind to the Y box with an inverted CCAAT and with an imperfect inverted CCAAT motif, respectively, had inverse relationships to the promoter strength. These
results demonstrate that polymorphisms in both X1 and Y box motifs play a dominant role on the promoter strength, and DRB promoters with X1 box (with C at position -188 and A at -183) and Y box
046
043
ALLELE-SPECIFIC TRANSCRIPTIONALREGULATION OF HLA-DQB1 GENES
Gerald T. Nepom, Teresa Sukiennicki, andJon S. Beaty Virginia Mason Research Center, Seattle, WA98101 andthe Department of Immunology, University of Washington School of Medicine, Seattle, WA 98195 USA
DNAsequence polymorphism inthepromoter-proxima! transcriptional regulatory region of the HLA-DQBllocus contributes to variation in locus-andallele-specific expression. A particularly prominent siteof allelic polymorphism consists of a TO dinucleotide present between the Wand XI consensus elements in the BUDQJJl*0302 allele butmissing in the '0301 allele. Using reportergene assays with the cloned promoter elements and RT-peR analysis of endogenous transcripts, we identified two functions of this polymorphic regulatory site: One effect of this polymorphism is to introduce a variation in the spacing between the W and XI elements of these twoalleles, witha negative impact on transcription. A secondary compensatory effect is the binding of a TO-sequence specific nuclear protein complex to the *0302 regulatory regioninunediately 5' of the XI element, witha positive effect on transcription. Screening of a human B-LCL expression library for TO-specific DNA-binding proteins identified a eDNA clone which encodes a ubiquitously expressed zinc-finger transcription factor. This transcription factor binds the HLA-DQBl promoters in an allele-specific manner, depending on the presence of theTOdinucleotide polymorphism. Thepresence of thispolymorphism in multiple HLA-DQBl alleles and in several species suggests selection for two alternative transcriptional regulatory mechanisms influencing expression of alleles of the same HLAlocus. TheTO-binding transcription factor is a likely mediator of thisallele-specific transcriptional regulation.
045 RFX BINDS TO S BOX IN ORA PROMOTER Nabila Jabrane-Ferrat, JosephD. Fontes and R Matija Peterlin Howard Hughes Medical Institute and Departments of Medicine, Microbiology and Immunology, University of California, San francisco, San Francisco, California 94143 The S box (also known at the H, W or the Z box) is the 5' most element of the conserved upstream sequences in MHC class :n: promoters. It is important for their IS cell-specific and j-interferon inducible expression. Previously we reported that the S box is i\ partial duplication of the downstream X box. In this study we. demonstrate that RFX from nuclear extracts and affinity purification binds to the 5 box and its S' flanking sequence. The.. spacing between S and X boxes must be concerved for the. expression of the DRA promoter suggesting that RFX protein bound to these sites must interact with each other. Using an RFX5 VP16 fusion protein, we demonstrate this binding interaction ill vivo. We conclude that RFX binds to both the S and X boxes and functions as the regulator of the B cell-specific and interferon '6" inducible expression of MHC class II promoters.
047
GENE THERAPY IN CIITA DEFICIENT BARE LYMPHOCYTE SYNDROME
INDUCTION OF CIITA AND MODIFICATIONS OF IN VIVO HLA-DR PROMOTER OCCUPANCY IN NORMAL THYMIC EPITHELiAl CELLS TREATED WITH IFN-r
JanetS. Lee, M.BrigidBradley, Grace Ungers, Paul Szabolcs', James Young', and Richard O'Reilly. Immunology Program,Memorial SloanKetteringCancerCenter, New York, NY 10021,and 'Rockefeller University, New York, NY 10021.
Rigaud Gildas", De Lenna Barbaro Andrea', Ramani Dunia', Riviera Anna Pia', Accolla RobertO', 'Instttute of Immunology and Infectious Diseases, Verona and 'Advanced Biotechnology Center, Genova, Raly. 'InstitutPasteur,Paris,France.
One group HLA class II negative immunodeficiency or Bare Lymphocyte Syndrome (BLS), is due to a defect in the transactivator, CIITA. Using recombinant CIITA retroviruses,we have transduced B cell lines and primary cellsfromBLS patientsand stained the cells with antibodiesagainst HLA-DR. Irradiating virus producer cells and then co-cultivating with target cells achieved efficiencies of20 to 50"10 72 hr post infection. We have also observed that allclassII negative celllinesthat we have infected with this vector, can be inducedto express HLA class II. C034+ cells were obtained and cocultured withthe producersin the presenceofFCS andcytokines. Expandingcells were harvested and cultured with e-kit ligand, GM-CSF, and TNFa. The myeloid progenywere successfully transduced, based on HLA-OR expressionby 29"10 of the bulk population. Moreover,after one week of expansion,37% of COIa positive dendritioLangerhans cellsexpressedHLA-DR. Cells from the patient have been restored to functional competence in their abilities to stimulate in MLR, present superantigens, and specific antigens.
In this study, the INF-r induction of MHC class II gene expression in primary cuhures of thymic epithelial cells (TEC) was analyzed. This cellular system offers the advantagethat MHC class II induction is studied in a 'phy$iologlc' celllineage that, as a resuR of this expression within the thymus, is thought to partecipate to the seleclion and maturation of the T cells. Rwas found that the MHe class II gene expression was associated with the de 110110 transcription of the gene encoding the CIITA lrans-activator, a crucial MHC class II gene regulatory factor. Furthermore, the anatomy of interaction between the MHC class II ORA promoter and corresponding binding factors was analyzed by in vivo DNAse I footpl1nt. It wasfound that treatmentwith INF-r Induces changes In the occupancy of the DRA gene regulatory sequences by nuclearfaclors. The resuhlng occupancy displays strong similarities with the one observed in the MHC class II-constitutiVe Beells,repnlS8I1ted by both the Bur1d!t lymphoma line Raji and normal tonsIklerived B cells. However some peculiar differences were observed between the TEC, either INF-r induced or not, and the constttutive B cells. These results Indicate that both common mechanisms, such as the one mediated by the CIITA lrans-activator, and distinct tissue ,specific constraints contrillUte to the transcriptional control of constitutiVe and IFN--y induced MHeclass II gene expression.
12
042 DOES HLA-Cw7 CONSTITUTE A DISTINCT ALLELIC FAMILY OF THE HLA-Cw LOCUS? Zohreh Tatari*, Jean-Michel Cayuela", CatherineFortier", Colette Raffoux* and DominiqueCharron* * Laboratoire d'Immunologie de d'Histocompatibilite, et Laboratoire d'Hematologie.Hopital Saint Louis. Paris-France. HLA-Cw7 alleles are distinguishedfrom other HLA-Cw alleles by a higher polymorphism and a higher frequency in the Caucasian population. Recentlyit has been describedthat the lower cell surfaceexpressionof HLA-C is due to a negativeregulatoryelement downstreamof the translationstop site and a non-fonctional EnhA which is responsible to prevent the binding of NFkB factors to this region. In order to understand these particularities, we have studied the polymorphism in the 5' upstreamregulatory region of HLACw, We observedthat among of HLA-C alleles, only Cw7 has the conserved sequencesnecessaryfor NaB binding at the Enh A site. The Northern blot analysis shows that HLA-Cw7, on average, is twofold more expressed than the other Cw alleles. The CAT assay tests performed with a fragment from 460 to + I comparingthe transcriptional level of HLA-Cw7 with the other Cw alleles confirmed these results. We are currently quantifying the HLA-Cw alleles includingCw7, using a competitive RT-PCR assay for defining more preciselythe rate of HLA-Cw7transcription in comparaison with other HLAC alleles. These results could be the first to show evidence for a distinct family from a regulatory and polymorphism point of view inside the same locus.
044 MOLECULAR BASIS FOR DIFFERENCES IN THE TRANSCRIPTIONAL ACTIVITIES OF HLA-DRB GENES IN DIFFERENT HAPLOTYPES Xiaohong Qiu, Dharam P. Singal Department of Pathology, McMaster University, Hamilton. ON, Canada In the present study. we examined the effect of polymorphims in the X1 box and the Y box on the transcriptional activity of DRB gene promoters. We utilized normal. chimeric and site-directed mutant promoters and compared their abilities to initiate transcription of the CAT reporter gene. In addition, we examined the effect of these polymorphisms in binding ~-regulatory proteins. The results show that polymorphisms in the X1 box, i.e. substitution of nucleotide C by G at position -188 or of A by G at position -183, significantly reduced (> 70%) the promoter strength. Similarly. nucleotide variations at positions -152, -150 and -149 in the Y box. which affect the inverted CCAAT motif, significantly reduced (>50%) the promoter activity. Polymorphisms in the X1 box affected DNA-protein interactions. However, the role of nuclear proteins that bind to polymorphic X1 box elements on promoter strength is not clear. On the other hand. affinities of NF-Y and NF-YB proteins, which bind to the Y box with an inverted CCAAT and with an imperfect inverted CCAAT motif, respectively, had inverse relationships to the promoter strength. These
results demonstrate that polymorphisms in both X1 and Y box motifs play a dominant role on the promoter strength, and DRB promoters with X1 box (with C at position -188 and A at -183) and Y box
046
043
ALLELE-SPECIFIC TRANSCRIPTIONALREGULATION OF HLA-DQB1 GENES
Gerald T. Nepom, Teresa Sukiennicki, andJon S. Beaty Virginia Mason Research Center, Seattle, WA98101 andthe Department of Immunology, University of Washington School of Medicine, Seattle, WA 98195 USA
DNAsequence polymorphism inthepromoter-proxima! transcriptional regulatory region of the HLA-DQBllocus contributes to variation in locus-andallele-specific expression. A particularly prominent siteof allelic polymorphism consists of a TO dinucleotide present between the Wand XI consensus elements in the BUDQJJl*0302 allele butmissing in the '0301 allele. Using reportergene assays with the cloned promoter elements and RT-peR analysis of endogenous transcripts, we identified two functions of this polymorphic regulatory site: One effect of this polymorphism is to introduce a variation in the spacing between the W and XI elements of these twoalleles, witha negative impact on transcription. A secondary compensatory effect is the binding of a TO-sequence specific nuclear protein complex to the *0302 regulatory regioninunediately 5' of the XI element, witha positive effect on transcription. Screening of a human B-LCL expression library for TO-specific DNA-binding proteins identified a eDNA clone which encodes a ubiquitously expressed zinc-finger transcription factor. This transcription factor binds the HLA-DQBl promoters in an allele-specific manner, depending on the presence of theTOdinucleotide polymorphism. Thepresence of thispolymorphism in multiple HLA-DQBl alleles and in several species suggests selection for two alternative transcriptional regulatory mechanisms influencing expression of alleles of the same HLAlocus. TheTO-binding transcription factor is a likely mediator of thisallele-specific transcriptional regulation.
045 RFX BINDS TO S BOX IN ORA PROMOTER Nabila Jabrane-Ferrat, JosephD. Fontes and R Matija Peterlin Howard Hughes Medical Institute and Departments of Medicine, Microbiology and Immunology, University of California, San francisco, San Francisco, California 94143 The S box (also known at the H, W or the Z box) is the 5' most element of the conserved upstream sequences in MHC class :n: promoters. It is important for their IS cell-specific and j-interferon inducible expression. Previously we reported that the S box is i\ partial duplication of the downstream X box. In this study we. demonstrate that RFX from nuclear extracts and affinity purification binds to the 5 box and its S' flanking sequence. The.. spacing between S and X boxes must be concerved for the. expression of the DRA promoter suggesting that RFX protein bound to these sites must interact with each other. Using an RFX5 VP16 fusion protein, we demonstrate this binding interaction ill vivo. We conclude that RFX binds to both the S and X boxes and functions as the regulator of the B cell-specific and interferon '6" inducible expression of MHC class II promoters.
047
GENE THERAPY IN CIITA DEFICIENT BARE LYMPHOCYTE SYNDROME
INDUCTION OF CIITA AND MODIFICATIONS OF IN VIVO HLA-DR PROMOTER OCCUPANCY IN NORMAL THYMIC EPITHELiAl CELLS TREATED WITH IFN-r
JanetS. Lee, M.BrigidBradley, Grace Ungers, Paul Szabolcs', James Young', and Richard O'Reilly. Immunology Program,Memorial SloanKetteringCancerCenter, New York, NY 10021,and 'Rockefeller University, New York, NY 10021.
Rigaud Gildas", De Lenna Barbaro Andrea', Ramani Dunia', Riviera Anna Pia', Accolla RobertO', 'Instttute of Immunology and Infectious Diseases, Verona and 'Advanced Biotechnology Center, Genova, Raly. 'InstitutPasteur,Paris,France.
One group HLA class II negative immunodeficiency or Bare Lymphocyte Syndrome (BLS), is due to a defect in the transactivator, CIITA. Using recombinant CIITA retroviruses,we have transduced B cell lines and primary cellsfromBLS patientsand stained the cells with antibodiesagainst HLA-DR. Irradiating virus producer cells and then co-cultivating with target cells achieved efficiencies of20 to 50"10 72 hr post infection. We have also observed that allclassII negative celllinesthat we have infected with this vector, can be inducedto express HLA class II. C034+ cells were obtained and cocultured withthe producersin the presenceofFCS andcytokines. Expandingcells were harvested and cultured with e-kit ligand, GM-CSF, and TNFa. The myeloid progenywere successfully transduced, based on HLA-OR expressionby 29"10 of the bulk population. Moreover,after one week of expansion,37% of COIa positive dendritioLangerhans cellsexpressedHLA-DR. Cells from the patient have been restored to functional competence in their abilities to stimulate in MLR, present superantigens, and specific antigens.
In this study, the INF-r induction of MHC class II gene expression in primary cuhures of thymic epithelial cells (TEC) was analyzed. This cellular system offers the advantagethat MHC class II induction is studied in a 'phy$iologlc' celllineage that, as a resuR of this expression within the thymus, is thought to partecipate to the seleclion and maturation of the T cells. Rwas found that the MHe class II gene expression was associated with the de 110110 transcription of the gene encoding the CIITA lrans-activator, a crucial MHC class II gene regulatory factor. Furthermore, the anatomy of interaction between the MHC class II ORA promoter and corresponding binding factors was analyzed by in vivo DNAse I footpl1nt. It wasfound that treatmentwith INF-r Induces changes In the occupancy of the DRA gene regulatory sequences by nuclearfaclors. The resuhlng occupancy displays strong similarities with the one observed in the MHC class II-constitutiVe Beells,repnlS8I1ted by both the Bur1d!t lymphoma line Raji and normal tonsIklerived B cells. However some peculiar differences were observed between the TEC, either INF-r induced or not, and the constttutive B cells. These results Indicate that both common mechanisms, such as the one mediated by the CIITA lrans-activator, and distinct tissue ,specific constraints contrillUte to the transcriptional control of constitutiVe and IFN--y induced MHeclass II gene expression.