Nomenclature for factors of the HLA system, 1996.
Explore the source record for details and available documents.
Alleles↗
Biomedical subjects
B Mach
Publications and source records attributed to B Mach.
[Genetic genius and medicine].
Explore the source record for details and available documents.
Genetic Diseases, Inborn↗
Locations of human and mouse genes encoding the RFX1 and RFX2 transcription factor proteins.
RFX transcription factors constitute a highly conserved family of site-specific DNA binding proteins involved in the expression of a variety of cellular and viral genes, including major histocompatibility complex class II genes and genes in human hepatitis B virus. Five members of the RFX gene family have been isolated from human and mouse, and all share a highly characteristic DNA binding domain that is distinct from other known DNA binding motifs. The human RFX1 and RFX2 genes have been assigned by in situ hybridization to chromosome 19p13.1 and 19p13.3, respectively. In this paper, we present data that localize RFX1 and RFX2 precisely within the detailed physical map of human chromosome 19 and genetic data that assign Rfx1 and Rfx2 to homologous regions of mouse chromosomes 8 and 17, respectively. These data define the established relationships between these homologous mouse and human regions in further detail and provide new tools for linking cloned genes to phenotypes in both species.
Animals↗
RFX proteins, a novel family of DNA binding proteins conserved in the eukaryotic kingdom.
Until recently, the RFX family of DNA binding proteins consisted exclusively of four mammalian members (RFX1-RFX4) characterized by a novel highly conserved DNA binding domain. Strong conservation of this DNA binding domain precluded a precise definition of the motif required for DNA binding. In addition, the biological systems in which these RFX proteins are implicated remained obscure. The recent identification of four new RFX genes has now shed light on the evolutionary conservation of the RFX family, contributed greatly to a detailed characterization of the RFX DNA binding motif, and provided clear evidence for the function of some of the RFX proteins. RFX proteins have been conserved throughout evolution in a wide variety of species, including Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, mouse and man. The characteristic RFX DNA binding motif has been recruited into otherwise very divergent regulatory factors functioning in a diverse spectrum of unrelated systems, including regulation of the mitotic cell cycle in fission yeast, the control of the immune response in mammals, and infection by human hepatitis B virus.
Amino Acid Sequence↗
Major histocompatibility complex class II deficiency: a disease of gene regulation.
Explore the source record for details and available documents.
Gene Expression Regulation↗
Genetic complexity of the human hsp 60 gene.
Hsp 60 is a chaperonin protein, homologous to GroEL of Escherichia coli and highly conserved across species. Immune response induced by the hsp 60 equivalent of numerous microorganisms elicits in animals and man a dominant cross-reactive T lymphocyte response. Hsp 60 has been strongly implicated as an example of molecular mimicry in the pathogenicity of autoimmune diseases and, more recently, in T cell-mediated protection. Curiously, in spite of this interest, the gene encoding HSP 60 has not yet been cloned. Sequencing of numerous PCR-derived HSP 60 clones. obtained following amplification of genomic DNA revealed multiple distinct but highly related sequences. These were all different from the sequence encoding the expressed protein and all had interrupted reading frames. PCR amplification from mRNA, however, yielded only the sequence expected for the expressed hsp 60 protein. This apparent paradox was resolved by cloning and sequencing HSP 60-specific genomic clones: the majority of these clones corresponded to intronless genes having the characteristics of retro-pseudogenes and were flanked by unrelated DNA sequences. In addition, several genomic clones were isolated that corresponded to a unique functional HSP 60 gene. This gene is composed of multiple exons, some very short. The transcription start site was identified and 750 bp of 5' flanking sequence were determined. The human HSP 60 gene is induced by heat. We conclude that hsp 60 is encoded by a single highly fragmented gene, that co-exists with multiple HSP 60 retro-pseudogenes, normally not expressed.
Amino Acid Sequence↗
Highly efficient peptide binding and T cell activation by MHC class II molecules of CIITA-transfected cells.
Expression of MHC class II, DM and Ii genes is controlled by the transactivator CIITA, a mediator of the activation of these genes by IFN-gamma. Surprisingly, MHC class II molecules expressed on CIITA transfectants behave very differently from those expressed at the same level on IFN-gamma-induced cells in terms of peptide binding and peptide-specific T cell activation. MHC class II-positive CIITA transfectants exhibit an unusually high capacity for binding exogenous peptides, with a higher percentage of DR molecules occupied by a given peptide and are much more efficient at peptide-specific, HLA-DR-restricted activation of T lymphocytes. This unexpected phenotype reflects the antigen processing defect observed in CIITA transfectants. It suggests novel strategies for the use of CIITA-transformed cells in peptide-based immunization.
Antigen Presentation↗
A consensus motif in the RFX DNA binding domain and binding domain mutants with altered specificity.
The RFX DNA binding domain is a novel motif that has been conserved in a growing number of dimeric DNA-binding proteins, having diverse regulatory functions, in eukaryotic organisms ranging from yeasts to humans. To characterize this novel motif, we have performed a detailed dissection of the site-specific DNA binding activity of RFX1, a prototypical member of the RFX family. First, we have performed a site selection procedure to define the consensus binding site of RFX1. Second, we have developed a new mutagenesis-selection procedure to derive a precise consensus motif, and to test the accuracy of a secondary structure prediction, for the RFX domain. Third, a modification of this procedure has allowed us to isolate altered-specificity RFX1 mutants. These results should facilitate the identification both of additional candidate genes controlled by RFX1 and of new members of the RFX family. Moreover, the altered-specificity RFX1 mutants represent valuable tools that will permit the function of RFX1 to be analyzed in vivo without interference from the ubiquitously expressed endogenous protein. Finally, the simplicity, efficiency, and versatility of the selection procedure we have developed make it of general value for the determination of consensus motifs, and for the isolation of mutants exhibiting altered functional properties, for large protein domains involved in protein-DNA as well as protein-protein interactions.
Amino Acid Sequence↗
Regulation of MHC class II genes: lessons from a disease.
Precise regulation of major histocompatibility complex class II (MHC-II) gene expression plays a crucial role in the control of the immune response. A major breakthrough in the elucidation of the molecular mechanisms involved in MHC-II regulation has recently come from the study of patients that suffer from a primary immunodeficiency resulting from regulatory defects in MHC-II expression. A genetic complementation cloning approach has led to the isolation of CIITA and RFX5, two essential MHC-II gene transactivators. CIITA and RFX5 are mutated in these patients, and the wild-type genes are capable of correcting their defect in MHC-II expression. The identification of these regulatory factors has furthered our understanding of the molecular mechanisms that regulate MHC-II genes. CIITA was found to be a non-DNA binding transactivator that functions as a molecular switch controlling both constitutive and inducible MHC-II expression. The finding that RFX5 is a subunit of the nuclear RFX-complex has confirmed that a deficiency in the binding of this complex is indeed the molecular basis for MHC-II deficiency in the majority of patients. Furthermore, the study of RFX has demonstrated that MHC-II promoter activity is dependent on the binding of higher-order complexes that are formed by highly specific cooperative binding interactions between certain MHC-II promoter-binding proteins. Two of these proteins belong to families of which the other members, although capable of binding to the same DNA motifs, are probably not directly involved in the control of MHC-II expression. Finally, the facts that CIITA and RFX5 are both essential and highly specific for MHC-II genes make possible novel strategies designed to achieve immunomodulation via transcriptional intervention.
Animals↗
A novel antigen-processing-defective phenotype in major histocompatibility complex class II-positive CIITA transfectants is corrected by interferon-gamma.
Presentation of exogenous protein antigens to T lymphocytes is based on the intersection of two complex pathways: (a) synthesis, assembly, and transport of major histocompatibility complex (MHC) class II-invariant chain complexes from the endoplasmic reticulum to a specialized endosomal compartment, and (b) endocytosis, denaturation, and proteolysis of antigens followed by loading of antigenic peptides onto newly synthesized MHC class II molecules. It is believed that expression of MHC class II heterodimers, invariant chain and human leukocyte antigen-DM is both necessary and sufficient to reconstitute a functional MHC class II loading compartment in antigen-presenting cells. Expression of each of these essential molecules is under the control of the MHC class II transactivator CIITA. Unexpectedly, however, whereas interferon gamma stimulation does confer effective antigen-processing function to nonprofessional antigen presenting cells, such as melanoma cells, expression of the CIITA transactivator alone is not sufficient. Activation of antigen-specific T cells thus requires additional CIITA-independent factor(s), and such factor(s) can be induced by interferon gamma.
Antigen-Presenting Cells↗
Purified X2 binding protein (X2BP) cooperatively binds the class II MHC X box region in the presence of purified RFX, the X box factor deficient in the bare lymphocyte syndrome.
The conserved X2 box sequence of MHC class II promoters is homologous to TRE/CRE elements, and is required for B cell expression and IFN-gamma induction of MHC class II genes. The X2 binding protein (X2BP) was initially identified as a DNA-binding activity that specifically interacts with the conserved X2 box sequence in both the MHC HLA-DRA and HLA-DRB promoters. To begin to demonstrate that X2BP is the X2 box factor responsible for class II expression in B cells, we have purified X2BP to homogeneity from B cell nuclear extracts using DNA-affinity chromatography. X-box DNA-affinity purification indicates that X2BP is most likely composed of two polypeptides of 120 kDa and 46 kDa. The 120-kDa protein was specifically cross-linked to an X-box probe by exposure to UV irradiation. The 46-kDa subunit of X2BP cross-reacted with anti-rat CREB polyclonal Abs but not to anti-human CREB Abs in Western analysis and supershift assays, indicating that it may be a novel member of the ATF/CREB family. Purified X2BP interacted with purified RFX, a factor that binds to the adjacent X1 box and is absent in some cell lines that are mutant for MHC class II transcription. This interaction increases the DNA-binding half-life of RFX from 5 to at least 60 min, suggesting that X2BP functions in class II MHC gene expression by forming a stable complex with RFX.
Base Sequence↗
RB and a novel E2F-1 binding protein in MHC class II deficient B-cell lines and normal IFN-gamma induction of the class IL transactivator CIITA in class II non-inducible RB-defective tumor lines.
The major histocompatibility (MHC) class II genes encode cell surface proteins that bind antigenic peptide for presentation to T-cells. The class II proteins are expressed constitutively on B-cells and EBV-transformed B-cells, and are inducible by IFN-gamma on a wide variety of cell types. Retinoblastoma protein (RB) is a tumor suppressor and functions as a transcriptional repressor by binding and inactivating the transactivator E2F-I. RB-defective tumor lines are non-inducible for MHC class II by IFN-gamma, or very weakly inducible, but transfection of 2 different lines with RB expression vectors re-establishes or substantially enhances class II inducibility. Therefore, we examined the RB status of a series of B-cell mutants that are defective in class II expression, generated either in vitro or derived from Bare Lymphocyte Syndrome (BLS) patients. Nuclear matrix-bound RB was detectable in all cases, indicating that loss of RB is not responsible for decreased class II expression in these lines. A second E2F-I binding protein, most likely DP-I, was also apparently normal in both class II-positive and -negative B-cell lines. We also examined the IFN-gamma induction of CIITA in RB-defective lines. CIITA is a class II gene transactivator known to be defective in one form of BLS and to be required for the induction of MHC class II by IFN-gamma. CIITA mRNA is normally inducible by IFN-gamma in class II non-inducible, RB-defective lines, and in one line, re-expression of RB has no effect on CIITA mRNA induction levels. Thus, the block in MHC class II inducibility in RB-defective cells is not due to a block in CIITA inducibility.
Antigens, Nuclear↗
A novel DNA-binding regulatory factor is mutated in primary MHC class II deficiency (bare lymphocyte syndrome).
Regulation of MHC class II gene expression is an essential aspect of the control of the immune response. Primary MHC class II deficiency is a genetically heterogeneous disease of gene regulation that offers the unique opportunity of a genetic approach for the identification of the functionally relevant regulatory genes and factors. Most patients exhibit a characteristic defect in the binding of a nuclear complex, RFX, to the X box motif of MHC class II promoters. Genetic complementation of a B-lymphocyte cell line from such a patient with a cDNA expression library has allowed us to isolate RFX5, the regulatory gene responsible for the MHC class II deficiency. This gene encodes a novel DNA-binding protein that is indeed a subunit of the RFX complex. Mutations in the RFX5 gene have been characterized in two patients. Transfection of the patient's cells with the RFX5 cDNA repairs the binding defect and fully restores expression of all the endogenous MHC class II genes in vivo.
Amino Acid Sequence↗
Identification of cytosolic proteins that bind to the Fanconi anemia complementation group C polypeptide in vitro. Evidence for a multimeric complex.
The oligomeric structure of Fanconi anemia complementation group C (FACC) was investigated in mammalian cell lysates. Using an affinity-purified polyclonal antibody, FACC was immunoprecipitated from radiolabeled cell lysates and shown to form monomers of 63 kDa. Association of FACC with heterologous proteins was investigated by co-precipitation of radiolabeled proteins with a recombinant chimeric FACC molecule fused to the constant portion of the human IgG1 heavy chain (FACC gamma 1). Expression of FACC gamma 1 in FACC-deficient Fanconi anemia (FA) lymphoblasts corrected the hypersensitivity of these cells to mitomycin C. Binding of FACC gamma 1 to protein A-agarose and incubation with radiolabeled cell lysates identified three polypeptides with molecular masses of 65, 50, and 35 kDa that were also detected on immunoblots probed with the purified FACC gamma 1 polypeptide. FACC, as well as the three FACC-binding polypeptides, co-fractionated with cytosolic and membrane extracts. Binding was specific for the FACC moiety of FACC gamma 1 and was detected in cytosolic extracts of a number of FA and non-FA mammalian cells. These results demonstrate that FACC binds directly to a family of ubiquitous cytosolic proteins and is conserved in a wide range of mammalian cells.
Amino Acid Sequence↗
MHC class II regulation--lessons from a disease.
Explore the source record for details and available documents.
Antibody Formation↗
Molecular defects in the bare lymphocyte syndrome and regulation of MHC class II genes.
The complex pattern of expression of major histocompatibility complex (MHC) class II molecules plays an essential role in the control of the immune response. Our understanding of the molecular mechanisms controlling this expression has benefited greatly from the identification of the regulatory factors defective in two forms of a hereditary disease of MHC class II regulation: bare lymphocyte syndrome. This has also provided new tools for the experimental modulation of MHC class II expression.
Gene Expression Regulation↗
Nomenclature for factors of the HLA system, 1995.
Explore the source record for details and available documents.
Alleles↗
Complementation cloning of mammalian transcriptional regulators: the example of MHC class II gene regulators.
Cloning by complementation of mutant cell lines is a powerful way in which to identify and isolate important regulatory genes on the basis of functional assays. The recent cloning of two essential regulators of major histocompatibility complex (MHC) class II gene expression has not only advanced our understanding of the complex mechanisms controlling these genes, but also helps to illustrate the feasibility of this approach for the study of mammalian gene regulation.
Animals↗