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1.
J Exp Med ; 185(11): 1885-95, 1997 Jun 02.
Article in English | MEDLINE | ID: mdl-9166418

ABSTRACT

Patients with one type of major histocompatibility complex class II combined immunodeficiency have mutations in a gene termed class II transactivator (CIITA), which coordinately controls the transcription of the three major human class II genes, HLA-DR, -DQ, and -DP. However, the experimentally derived B-lymphoblastoid cell line, clone 13, expresses high levels of HLADQ in the absence of HLA-DR and HLA-DP, despite its mapping by complementation analysis to this group. It was possible that one of the clone 13 CIITA alleles bore a mutation that allowed HLA-DQ, but not HLA-DR or -DP transcription. Alternatively, another factor, distinct from CIITA, might control HLA-DQ expression. We report here that ectopic expression of CIITA cDNAs derived by reverse transcriptase polymerase chain reaction from clone 13 do not restore expression of HLA-DQ in another CIITA-deficient cell line, RJ2.2.5. In addition, no CIITA protein is detectable in clone 13 nuclear extracts. In contrast, somatic cell fusion between clone 13 and RJ2.2.5 restored expression of the HLA-DQ haplotype encoded by the RJ2.2.5 DQB gene. Taken together, these data demonstrate the existence of an HLA-DQ isotype-specific trans-acting factor, which functions independently of CIITA.


Subject(s)
Gene Expression Regulation/genetics , Genes, MHC Class II , HLA-DQ Antigens/genetics , Nuclear Proteins , Trans-Activators/genetics , Blotting, Western , DNA Primers , DNA, Complementary/chemistry , Flow Cytometry , HLA-DQ Antigens/immunology , Humans , Hybrid Cells/immunology , In Situ Hybridization, Fluorescence , Lymphocytes , Mutation , Polymerase Chain Reaction , Transfection , Tumor Cells, Cultured
2.
J Immunol ; 158(10): 4741-9, 1997 May 15.
Article in English | MEDLINE | ID: mdl-9144488

ABSTRACT

The MHC class II transactivator gene (CIITA) coordinately controls the expression of the three major human class II genes, HLA-DR, HLA-DQ, and HLA-DP. Indeed, patients with one form of MHC class II immunodeficiency disease, due to defective CIITA genes, lack expression of all three isotypes. Nevertheless, there is substantial evidence that human class II genes are not always coordinately regulated, raising the possibility that CIITA-independent, isotype-specific class II regulatory pathways exist. To address this issue, we have generated a dominant negative mutant of CIITA that lacks the acidic transcription-activating N terminus, but retains the proline/serine/threonine-rich domain. Three newly produced anti-CIITA mAbs revealed that this mutant protein lacked N-terminal epitopes. In this study, we report that this CIITA dominant negative mutant repressed the constitutive expression of all three class II isotypes in human EBV-B cell lines, as well as IFN-gamma-induced class II transcription in HeLa cells. However, in a CIITA-deficient, EBV-transformed B cell line, clone 13, the dominant negative mutant did not alter the endogenous expression of the HLA-DQ gene. Taken together, these data demonstrate the existence of both CIITA-dependent and -independent class II regulatory pathways. Furthermore, our data provide evidence that the latter pathways can be isotype specific.


Subject(s)
Genes, MHC Class II , HLA-D Antigens/genetics , Nuclear Proteins , Trans-Activators/physiology , Gene Expression Regulation , Genes, Dominant , HLA-DQ Antigens/genetics , Humans , Interferon-gamma/pharmacology , Promoter Regions, Genetic , Repressor Proteins/genetics , Sequence Deletion , Structure-Activity Relationship , Transcription, Genetic
3.
J Exp Med ; 183(3): 1063-9, 1996 Mar 01.
Article in English | MEDLINE | ID: mdl-8642248

ABSTRACT

Major histocompatibility complex (MHC) class II combined immunodeficiency (CID), also known as type II bare lymphocyte syndrome, is an autosomal recessive genetic disorder characterized by the complete lack of expression of MHC class II antigens. The defect results from a coordinated lack of transcription of all class II genes. Cell fusion studies using many patient- and experimentally derived class II-negative cell lines have identified four distinct genetic complementation groups. In this report, we present genetic evidence that cell lines derived from two newly described MHC class II-deficient patients, KER and KEN, represent a fifth complementation group. In addition, the KER and KEN cell lines display a unique pattern of dyscoordinate regulation of their MHC class II genes, which is reflected in a new phenotype of in vivo promoter occupancy as revealed by in vivo genomic footprinting. These data point to a new defect that can result in the MHC class II-deficient phenotype.


Subject(s)
Gene Expression Regulation , Genes, MHC Class II , HLA-D Antigens/genetics , Lymphocytes/immunology , Severe Combined Immunodeficiency/genetics , Severe Combined Immunodeficiency/immunology , Cell Fusion , Cell Line , Chromosome Mapping , DNA Footprinting , Genes, Recessive , HLA-D Antigens/biosynthesis , HLA-DR Antigens/biosynthesis , Humans , Immunophenotyping , Macromolecular Substances , Transcription, Genetic
4.
Int Immunol ; 8(2): 255-65, 1996 Feb.
Article in English | MEDLINE | ID: mdl-8671611

ABSTRACT

We have examined the expression and regulation of the MHC class II E beta d gene in both cell lines and in transgenic mice. In transient transfection assays, as little as 192 bp of the E beta d proximal promoter was sufficient to direct constitutive expression of a reporter gene in a B cell line and to confer inducibility by IFN-gamma in a macrophage cell line. To determine if the same E beta d promoter sequences were also sufficient to direct correct expression in vivo, E beta d transgenes bearing either 4.1 or 0.2 kb of upstream sequence were introduced into an inbred mouse strain with a non-expressed endogenous E beta gene. Expression of both transgenes mirrored the expression of the endogenous I-A protein in thymus, B cells and macrophages with regard to both constitutive and cytokine-inducible expression. These results indicate that for the E beta gene only 200 bp of proximal promoter sequence are required to achieve tissue-specific and cytokine-inducible expression. This is in striking contrast to the E alpha gene, the only other murine class II gene whose promoter has been analyzed in vivo, which has been shown to require 2.0 kb of upstream sequence for appropriate expression. These data demonstrate, therefore, that the location of critical regulatory elements for the E beta and E alpha genes may differ.


Subject(s)
Gene Expression Regulation/immunology , Genes, MHC Class II/immunology , Histocompatibility Antigens Class II/genetics , Animals , B-Lymphocytes/immunology , Cell Line , Crosses, Genetic , Cytokines/pharmacology , Epitopes/genetics , Gene Expression Regulation/drug effects , Genes, Reporter/immunology , Macrophages/immunology , Mice , Mice, Transgenic , Organ Specificity/genetics , Organ Specificity/immunology , Plasmids/immunology , T-Lymphocytes/immunology , Transfection/immunology , Transgenes/drug effects , Transgenes/immunology
5.
J Exp Med ; 180(4): 1559-64, 1994 Oct 01.
Article in English | MEDLINE | ID: mdl-7931088

ABSTRACT

Collagen-induced arthritis (CIA) is an animal model of autoimmune inflammatory polyarthritis that has features similar to rheumatoid arthritis (RA). Much like RA, susceptibility to mouse CIA is influenced by the major histocompatibility complex (MHC), H-2, and restricted to the H-2q and H-2r haplotypes. Whereas the role of the H-2A molecule in susceptibility to CIA is well established, little is known about the role of H-2E molecule in the disease. In this study, we analyzed the effect of a transgenic E beta d molecule on CIA susceptibility in a recombinant mouse B10.RQB3, which expresses the CIA susceptible Aq genes and an Eak gene, but does not produce an E molecule since Ebq is nonfunctional. In the presence of an Ebd transgene, a viable E molecule is generated. Whereas B10.RQB3 were susceptible to CIA, B10.RQB3-E beta d+ showed a dramatic reduction in the incidence of arthritis as well as a decrease in the level of anti-mouse and anti-bovine CII antibodies in their serum. No clear cut differences in the expression of T cell receptor (TCR) V beta was observed between E beta d+ and E beta d- transgenic mice. Mechanisms underlying the protective effect of E beta d transgenic molecule on CIA may shed light on how HLA-DR molecules influence human RA.


Subject(s)
Arthritis/prevention & control , Collagen/immunology , H-2 Antigens/physiology , Animals , Genes, MHC Class II , H-2 Antigens/genetics , HLA-DR Antigens/physiology , Mice , Mice, Inbred BALB C , Mice, Transgenic
6.
EMBO J ; 9(5): 1543-9, 1990 May.
Article in English | MEDLINE | ID: mdl-2328725

ABSTRACT

The class II major histocompatibility complex (MHC, Ia) antigens are a family of membrane proteins whose expression is strictly regulated. They have a limited tissue distribution and their expression is regulated both developmentally and in response to external stimuli. Here we report the identification of a DNA binding protein complex (termed complex A) within the murine E beta MHC gene, which is restricted to cells that express Ia antigens. Complex A binding activity is developmentally regulated in cells of the B lineage in accordance with class II expression and is responsive to two different Ia-inducing lymphokines, interferon-gamma in macrophages and interleukin-4 in pre-B cells. The DNA target sequence in complex A includes three previously defined transcriptional motifs W, X and Y, and acts as a cis-acting transcription element. Complex A is present both in cells that are constitutive for class II MHC expression and in cells that have been induced for class II MHC expression. These results suggest that complex A may play a critical role in the regulation of class II MHC gene expression.


Subject(s)
DNA-Binding Proteins/physiology , Histocompatibility Antigens Class II/genetics , Animals , B-Lymphocytes/physiology , Base Sequence , Gene Expression Regulation/genetics , Mice , Molecular Sequence Data , Regulatory Sequences, Nucleic Acid , Transcription, Genetic/genetics , Transfection
7.
Proc Natl Acad Sci U S A ; 87(3): 914-8, 1990 Feb.
Article in English | MEDLINE | ID: mdl-2105504

ABSTRACT

Interferon gamma (IFN-gamma) is a potent inducer of major histocompatibility complex (MHC) antigens during normal immune responses and in abnormal responses in autoimmune disease. In this report we identify two nuclear factors whose binding to the murine E beta class II MHC beta-chain gene is regulated by this cytokine. IFN-gamma stimulation of murine macrophages results in the appearance of increased binding of one protein complex, complex A, and decreased binding of a second, faster migrating protein complex, complex B. Although the contact residues for both of these proteins lie within the highly conserved Y-box transcriptional element, their binding specificity differs. The protein in complex B is a CCAAT-box-binding protein that may be similar or identical to NF-Y or YB1, previously identified class II Y-box-binding proteins. The DNA sequence requirements for the binding of the slower migrating complex, complex A, are not limited to CCAAT-box sequences but include sequences upstream of the Y box. These upstream sequences are required both for IFN-gamma-induced gene transcription and for IFN-gamma-induced modulation of binding activity. These data suggest a model in which upstream sequences contribute to formation of a lymphokine-regulated complex downstream. The IFN-gamma-induced binding protein described as complex A in this report differs from the IFN-gamma, -alpha, or -beta-induced nuclear factors previously identified.


Subject(s)
DNA-Binding Proteins/metabolism , Genes, MHC Class II/drug effects , Interferon-gamma/pharmacology , Macrophages/metabolism , Nuclear Proteins/metabolism , Animals , Base Sequence , Cell Line , Histocompatibility Antigens Class II/genetics , Macromolecular Substances , Macrophages/drug effects , Mice , Molecular Sequence Data , Oligonucleotide Probes , Plasmids , Promoter Regions, Genetic , Protein Binding , Recombinant Proteins , Transfection
8.
Cell ; 47(5): 807-16, 1986 Dec 05.
Article in English | MEDLINE | ID: mdl-2946418

ABSTRACT

Although lambda repressor and lambda Cro bind to the same six operators on the phage chromosome, the fine specificities of the two proteins differ: repressor binds more tightly to OR1 than to OR3, and vice versa for Cro. In this paper, we change base pairs in the operators and amino acids in the proteins to analyze the basis for these preferences. We find that these preferences are determined by residues 5 and 6 of the recognition helices of the two proteins and by the amino-terminal arm, in the case of repressor. We also find that the most important base pairs in the operator which enable repressor and Cro to discriminate between OR1 and OR3 are position 3 (for Cro) and positions 5 and 8 (for repressor). These and previous results show how repressor and Cro recognize and distinguish between two related operator sequences.


Subject(s)
Bacteriophage lambda/genetics , Genes, Regulator , Genes, Viral , Repressor Proteins/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Base Sequence , Binding Sites , DNA-Binding Proteins/metabolism , Nucleic Acid Conformation , Protein Binding , Protein Conformation , Repressor Proteins/genetics , Viral Proteins , Viral Regulatory and Accessory Proteins
9.
Proc Natl Acad Sci U S A ; 77(9): 5230-3, 1980 Sep.
Article in English | MEDLINE | ID: mdl-6159642

ABSTRACT

We applied the method of Guarente et al. [Guarente, L., Lauer, G., Roberts, T.M. & Ptashne, M. (1980) Cell 20, 543-553] to construct plasmids that direct expression in Escherichia coli of the human fibroblast interferon (F-IF) gene. Two plasmids were recovered. One directs efficient synthesis of a protein whose primary sequence is that of pre-F-IF and the other, that of mature F-IF. Extracts of bacteria synthesizing mature F-IF display antiviral activity characteristic of human F-IF. This activity is lower than that expected from the differential rate of synthesis of the protein. We have detected no such activity in extracts of bacteria synthesizing pre-F-IF.


Subject(s)
DNA, Recombinant , Escherichia coli/genetics , Interferons/genetics , Base Sequence , Cloning, Molecular , Fibroblasts , Gene Expression Regulation , Genetic Vectors , Humans , Interferons/biosynthesis , Methods , Operon , Plasmids , Vesicular stomatitis Indiana virus/drug effects
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