Somatic mutations of immunoglobulin variable genes are restricted to the rearranged V gene.

An important question concerning the mechanism of somatic mutation of immunoglobulin variable (V) genes is whether it involves all of the numerous V genes in a differentiated B cell, independent of location, or if it is restricted to a particular chromosomal site. Comparison of the sequence of two alleles of a given V gene shows that the mutations are limited to the rearranged V gene, while the same V gene on the other chromosome has not undergone mutation. This indicates that a V gene sequence alone is not sufficient for somatic mutation to take place. The mutation is therefore restricted to the rearranged V gene and consequently does not occur before rearrangement.

Differential regulation of gene activity and chromatin structure within the human serpin gene cluster at 14q32.1 in macrophage microcell hybrids.

The human gene encoding alpha1-antitrypsin (alpha1AT, gene symbol PI ) is highly expressed in the liver and in cultured hepatoma cells and, to a lesser extent, in macrophages, where transcription originates from a separate upstream promoter. alpha1AT maps to a region of human chromosome 14q32.1 that includes a related serine protease inhibitor (serpin) gene that encodes corticosteroid-binding globulin (CBG). We recently reported the chromatin organization of this approximately 130 kb region, as defined by DNase I hypersensitive sites (DHSs) and matrix-attachment regions, in expressing and non-expressing cells. Furthermore, we demonstrated that transfer of human chromosome 14 from non-expressing fibroblasts to rat hepatoma cells resulted in activation of both alpha1AT and CBG transcription and gene activation was accompanied by long range chromatin reorganization of the entire region. In this study, we transferred human chromosome 14 from fibroblasts to mouse macrophages and documented activation of alpha1AT but not CBG gene expression. RT-PCR experiments indicated that transcription of the human alpha1AT gene in the microcell hybrids initiated at the macrophage promoter. Furthermore, DHS mapping experiments revealed a distinctive chromatin configuration of the locus that resembled the structure found in human macrophage-like cell lines, with many DHSs around alpha1AT but few in CBG. Thus, mouse macrophage cell lines will provide a useful cell type to study the effects of targeted modifications of the human alpha1AT-CBG locus on the regulation of cell-specific gene activity and chromatin structure.

Stable expression and cell-specific chromatin structure of human alpha1-antitrypsin cosmid transgenes in rat hepatoma cells.

The human gene encoding alpha1-antitrypsin (alpha1AT, gene symbol PI) resides in a cluster of serine protease inhibitor (serpin) genes on chromosome 14q32.1. alpha1AT is highly expressed in the liver and in cultured hepatoma cells. We recently reported the chromatin structure of a >100 kb region around the gene, as defined by DNase I-hypersensitive sites (DHSs) and matrix-attachment regions, in expressing and non-expressing cells. Transfer of human chromosome 14 by microcell fusion from non-expressing fibroblasts to rat hepatoma cells resulted in activation of alpha1AT transcription and chromatin reorganization of the entire region. In the present study, we stably introduced cosmids containing alpha1AT with various amounts of flanking sequence and a linked neo selectable marker into rat hepatoma cells. All single-copy transfectants with >14 kb of 5' flanking sequence expressed wild-type levels of alpha1AT mRNA in a position-independent manner. In contrast, expression of transgenes containing only approximately 1.5-4 kb of flanking sequence was highly variable. Long-term culture of transfectant clones in the absence of selection resulted in gradual loss of neo expression, but expression of the linked alpha1AT gene remained constant. DHS mapping of cosmid transgenes integrated at ectopic sites revealed a hepatoma-specific chromatin structure in each transfectant clone. The implications of these findings are discussed.

Identification and characterization of nuclear matrix-attachment regions in the human serpin gene cluster at 14q32.1.

Matrix-attachment regions (MARs) are DNA elements that are defined by their abilities to bind to isolated nuclear matrices in vitro. The DNA sequences of different matrix-binding elements vary widely. The locations of some MARs at the ends of chromatin loops suggest that they may represent boundaries of individual chromatin domains. As such, MARs may play important roles in regulating transcription and chromatin structure. As a first step towards assessing the roles of MARs in these processes, we assayed DNA sequences from the human serine protease inhibitor (serpin) gene cluster at 14q32.1 for matrix-binding activity in vitro. This approximately 150 kb region contains the cell-specific genes encoding alpha1-anti-trypsin (alpha1AT) and corticosteroid-binding globulin (CBG), as well as an antitrypsin-related sequence termed ATR. A DNase I-hypersensitive site (DHS) map of the locus has recently been described. We report here that the alpha1AT-ATR-CBG region contains five distinct MARs. There is a strong matrix-binding element approximately 16 kb upstream of alpha1AT; three MARs are between ATR and CBG and one MAR is within the CBG gene itself. These MARs were matrix-associated in all cell types examined. DNA sequencing indicated that the serpin MARs contained predominantly repetitive DNA, although the types of DNA repeats differed among the MARs.

Molecular analysis of the genes for human class II antigens of the major histocompatibility complex.

Different cDNA clones have been isolated that encode each of the three chains of HLA-DR antigens: alpha, intermediate and beta, as well as another beta chain, most likely DC. Whereas the DR alpha and intermediate chains seem encoded by single genes, the DR and DC beta chains are most likely encoded by multiple genes; furthermore, their polymorphism can be readily detected by restriction analysis of cellular DNA. Several genomic DNA clones were isolated for the DR and DC beta chain genes and for the intermediate chain gene. The sum of all distinct cDNA clones and genomic DNA clones for HLA-DR beta chains, isolated from a heterozygous cell line, represent five genes. This implies the existence of at least three nonallelic DR beta chain genes in addition to the DC beta chain genes. The complete sequence of one of the DR beta chains is presented. A genomic DNA clone for a DR beta chain was transferred into mouse L cells and found to be expressed into RNA of the same size as DR beta mRNA. The finding, among the genes for class II antigens, of multiple genes for the beta chain of HLA-DR, distinct from those of other known subregions such as DC, emphasizes the importance of gene transfer experiments, where individual genes can be expressed and tested for their functional role in the immune response.

Structural comparison of the genes of two HLA-DR supertypic groups: the loci encoding DRw52 and DRw53 are not truly allelic.

The organization and sequence of the HLA-DR beta chain genes are compared in the two supertypic groups, DRw52 and DRw53, which together account for more than 80% of HLA-DR alleles. From the structural data, we conclude that these two groups represent distinct lineages which have followed different patterns of evolution. The fine structure of the beta chain locus encoding the DRw53 specificity corresponds most closely to the DR beta II pseudogene in the DRw52 haplotypes. Concomitantly, the DR beta I locus in DRw53 haplotypes is more closely related to both of the two expressed DR beta loci of the DRw52 haplotypes (DR beta I and DR beta III). These two loci are the result of a recent duplication. This leads to the proposal that both expressed DR beta chain genes in the DRw52 haplotypes (DR beta I and DR beta III) are derived from a single precursor locus, while the two loci expressed in the DRw53 haplotypes are derived from distinct ancestral loci. The genes encoding DRw52 and DRw53 are therefore not true alleles of the same original locus. A scheme is proposed that accounts for the evolution of DR specificities within the DRw52 and DRw53 groups of haplotypes. It is evident that the different HLA-DR alleles are not structurally equidistant and that one must take into consideration different degrees of heterozygosity or mismatch among the DR alleles.

Characterization of an HLA-DR beta pseudogene in the DRw52 supertypic group.

The nature of the DR beta II pseudogene in a haplotype of the DRw52 supertypic group was investigated by nucleotide sequence analysis. It revealed several deleterious mutations in the signal sequence and second domain regions in addition to the complete absence of the first domain and adjacent sequences. No expression of DR beta II pseudogene mRNA can be detected. The same DR beta II pseudogene is probably present in other members of the DRw52 supertypic group. The pattern of mutations in this DR beta II pseudogene is different from that observed in the DR beta pseudogene of the DRw53 supertypic group, indicating a distinct evolutionary pathway for these two groups of DR haplotypes.

Molecular linkage of the human alpha 1-antitrypsin and corticosteroid-binding globulin genes on chromosome 14q32.1.

The genes encoding alpha 1-antitrypsin (alpha 1AT; gene symbol PI) and corticosteroid-binding globulin (CBG) are part of a cluster of structurally related serine protease inhibitor (serpin) genes on human Chromosome (Chr) 14q32.1. This cluster also includes the genes encoding alpha 1-antichymotrypsin (AACT) and protein C inhibitor (PCI), as well as an alpha 1-antitrypsin-related sequence (ATR; gene symbol PIL). In this report we present a detailed restriction map of a 110-kb region of genomic DNA that includes the alpha 1AT, ATR, and CBG genes. Gene order in this interval is tel-alpha 1AT-ATR-CBG-cen, and all three genes are transcribed in a distal-to-proximal orientation. Within the gene cluster, ATR is approximately 12 kb downstream of alpha 1AT, and CBG is about 57 kb downstream of alpha 1AT. Repetitive DNA sequences have been mapped throughout the interval, and several new restriction site polymorphisms in the region are described.

The HNF-4/HNF-1alpha transactivation cascade regulates gene activity and chromatin structure of the human serine protease inhibitor gene cluster at 14q32.1.

Hepatocyte-specific expression of the alpha1-antitrypsin (alpha1AT) gene requires the activities of two liver-enriched transactivators, hepatocyte nuclear factors 1alpha and 4 (HNF-1alpha and HNF-4). The alpha1AT gene maps to a region of human chromosome 14q32.1 that includes a related serine protease inhibitor (serpin) gene encoding corticosteroid-binding globulin (CBG), and the chromatin organization of this approximately 130-kb region, as defined by DNase I-hypersensitive sites, has been described. Microcell transfer of human chromosome 14 from fibroblasts to rat hepatoma cells results in activation of alpha1AT and CBG transcription and chromatin reorganization of the entire locus. To assess the roles of HNF-1alpha and HNF-4 in gene activation and chromatin remodeling, we transferred human chromosome 14 from fibroblasts to rat hepatoma cell variants that are deficient in expression of HNF-1alpha and HNF-4. The variant cells failed to activate either alpha1AT or CBG transcription, and chromatin remodeling failed to occur. However, alpha1AT and CBG transcription could be rescued by transfecting the cells with expression plasmids encoding HNF-1alpha or HNF-4. In these transfectants, the chromatin structure of the entire alpha1AT/CBG locus was reorganized to an expressing cell-typical state. Thus, HNF-1alpha and HNF-4 control both chromatin structure and gene activity of two cell-specific genes within the serpin gene cluster at 14q32.1.

Long-range chromatin reorganization of the human serpin gene cluster at 14q32.1 accompanies gene activation and extinction in microcell hybrids.

The genes encoding alpha1-antitrypsin (alpha1AT, gene symbol PI) and corticosteroid-binding globulin (CBG) are part of a cluster of six serine protease inhibitor (serpin) genes located on human chromosome 14q32.1. Both genes are actively transcribed in the liver and in human hepatoma cells, but they are not expressed in most other cell types. In this study we mapped DNase I-hypersensitive sites (DHSs) in an approximately 130-kb region of 14q32.1 that includes both genes. The distributions of DHSs in expressing (HepG2) vs nonexpressing (HeLa S3) cells were very different: HepG2 cells displayed 29 DHSs in this interval, but only 7 of those sites were present in HeLa cells. To determine the chromatin organization of activated or extinguished serpin alleles, we transferred human chromosome 14 into rat hepatoma cells or fibroblasts, respectively. Human alpha1AT and CBG gene expression was activated in rat hepatoma microcell hybrids containing human chromosome 14, but extinguished in rat fibroblast hybrids with the same genotype. DHS mapping in these microcell hybrids demonstrated that the chromatin structure of the entire 130-kb region was reorganized in microcell hybrids, and the distributions of DHSs in activated and extinguished alleles recapitulated those of expressing and nonexpressing cells, respectively. Thus, microcell hybrids provide a system in which reproducible changes in gene activity and long-range chromatin organization can be induced experimentally. This provides a basis for studying the effects of targeted modifications of the alpha1AT and CBG loci on the regulation of gene activity and chromatin structure.

A 370-kb cosmid contig of the serpin gene cluster on human chromosome 14q32.1: molecular linkage of the genes encoding alpha 1-antichymotrypsin, protein C inhibitor, kallistatin, alpha 1-antitrypsin, and corticosteroid-binding globulin.

The human genes encoding alpha 1-antitrypsin (alpha 1AT, gene symbol PI), corticosteroid-binding globulin (CBG), alpha 1-antichymotrypsin (AACT), and protein C inhibitor (PCI) are related by descent, and they all map to human chromosome 14q32.1. This serine protease inhibitor (serpin) gene cluster also contains an antitrypsin-related sequence (ATR, gene symbol PIL), but the precise molecular organization of this region has not been defined. In this report we describe the generation and characterization of an approximately 370-kb cosmid contig that includes all five serpin genes. Moreover, a newly described serpin, kallistatin (KAL, gene symbol PI4), was also mapped within the region. Gene order within this interval is cen-CBG-ATR-alpha 1 AT-KAL-PCI-AACT-tel. The genes occupy approximately 320 kb of genomic DNA, and they are organized into two discrete subclusters of three genes each that are separated by approximately 170 kb. The distal subcluster includes KAL, PCI, and AACT; it occupies approximately 63 kb of DNA, and all three genes are transcribed in a proximal-to-distal orientation. Within the subcluster, there is approximately 12 kb of intergenic DNA between KAL and PCI and approximately 19 kb between PCI and AACT. The proximal subcluster includes alpha 1AT, ATR, and CBG; it occupies approximately 90 kb of genomic DNA, with approximately 12 kb of DNA between alpha 1AT and ATR and approximately 40 kb between ATR and CBG. These genes are all transcribed in a distal-to-proximal orientation. This represents the first detailed physical map of the serpin gene cluster on 14q32.1.

Linkage map of three HLA-DR beta-chain genes: evidence for a recent duplication event.

The predominant class II, or Ia, antigen of the human major histocompatibility complex is HLA-DR. It consists of an alpha and a beta chain, the latter being responsible for the remarkable polymorphism of these Ia antigens. Studies with cloned genes had shown the existence of more than one DR beta-chain locus. We have isolated about 100 kilobases of the HLA-DR beta-chain gene region from a cosmid library generated from a consanguineous homozygous B-cell line of the DR3 haplotype. Three HLA-DR beta-chain genes have been characterized. They are arranged in a head-to-tail orientation. One of the genes lacks the region encoding the first domain of the DR beta chain. The two other genes are transcribed, as shown by RNA blot hybridization analysis. A striking restriction site homology has been found within the DR beta-chain gene cluster, suggesting a recent duplication event involving at least 25 kilobases of DNA. Moreover, the molecular map of DR beta chain genes cloned from B-cell lines of two other HLA-DR haplotypes shows extensive homology between alleles of a given DR beta-chain locus.

Expression pattern of mouse mammary tumor virus in transgenic mice carrying exogenous proviruses of different origins.

To study the tissue specificity of mouse mammary tumor virus (MMTV) gene expression, we developed two series of transgenic mice, containing the MMTV proviral DNA of mammary (GR) and kidney (C3H-K) origin. The expression pattern in the MMTV(GR) transgenic mice is very similar to that observed in infected animals, e.g., a strong preference for viral expression in the lactating mammary glands and lower levels of expression in salivary glands, lymphoid tissues, and male reproductive organs. One line of transgenic mice carrying the C3H-K provirus has a similar expression pattern, indicating that MMTV(C3H-K), despite a striking alteration in the U3 region of its long terminal repeat, can be expressed in the same tissues as the wild-type MMTV.

Molecular organization of the HLA-SB region of the human major histocompatibility complex and evidence for two SB beta-chain genes.

The class II products of the major histocompatibility complex, also called Ia antigens, are composed of two polypeptide chains, the alpha and beta chains, both encoded within the major histocompatibility complex. In man, the class II antigens can be divided into three biochemically distinct groups called HLA-DR, HLA-DC, and HLA-SB. Our isolation of cDNA clones for the polymorphic beta chain of HLA-DR and HLA-DC has allowed us to study the organization of the class II genes. Here we identify the HLA-SB beta-chain gene in recombinant clones from a cosmid library generated from a consanguineous homozygous B-cell line. The SB beta-chain gene is linked to the SB alpha-chain gene and the two genes are in opposite orientation. A second SB beta-chain gene, corresponding to a new SB beta II locus, has also been identified and cloned. The SB beta-chain genes show much less allelic restriction site polymorphism than the genes for the beta chains of HLA-DR or HLA-DC.

Partial activation of gene activity and chromatin remodeling of the human 14q32.1 serpin gene cluster by HNF-1 alpha and HNF-4 in fibroblast microcell hybrids.

The genes encoding alpha 1-antitrypsin (alpha 1AT, gene symbol P I) and corticosteroid-binding globulin (CBG) are part of a cluster of serine protease inhibitor (serpin) genes on human chromosome 14q32.1. Both genes are highly expressed in the liver and in cultured hepatoma cells, and the approximately 100-kb region around these genes contains an extensive array of expression-associated DNase I-hypersensitive sites (DHSs). Activation of human alpha 1AT and CBG transcription occurred when human chromosome 14 was transferred from nonexpressing cells to rat hepatoma cells. This activation event was accompanied by long-range chromatin reorganization of the entire region and the de novo formation of 17 expression-associated DHSs. Both gene activation and chromatin remodeling in hepatic cells required the liver-enriched transactivators hepatocyte nuclear factors-1 alpha and -4 (HNF-1 alpha and HNF-4). In this study, we tested whether ectopic expression of HNF-1 alpha and HNF-4 in nonexpressing cells could activate alpha 1AT and/or CBG transcription, and we monitored the chromatin structure of the locus in stably transfected fibroblasts. We report that both alpha 1AT and CBG mRNAs were expressed in fibroblast transfectants that stably expressed HNF-1 alpha and HNF-4, but expression was only approximately 1-10% of that observed in hepatic cells. Gene activation in these cells was accompanied by partial chromatin remodeling, as 6 of 17 expression-associated DHSs were formed. The potential implications of these results are discussed.

Remarkable sequence conservation of the HLA-DQB2 locus (DX beta) within the highly polymorphic DQ subregion of the human MHC.

The HLA-D region of the major histocompatibility complex (MHC) is characterized by a remarkable diversity. Most of the HLA class II genes are highly polymorphic, and in addition, the number and organization of individual loci in that region varies in different haplotypes. This extensive allelic polymorphism of immune response genes has well-known functional implications. Within the HLA-D region, two loci, DQA2 and DQB2 (formerly called DX alpha and DX beta), represent a very special case: the detailed structure of these two genes is entirely compatible with expression, yet their expression has never been demonstrated in any tissue. Consequently, there exists no known corresponding protein product. Pseudogenes are known to accumulate mutations, as observed for instance in the case of HLA-DPA2,-DPB2, or -DRB2 genes. We have therefore investigated the extent of DQ2 genes' polymorphism by DNA sequence comparison and by oligonucleotide hybridization across a large number of different haplotypes, and compared it with other genes in the HLA-D region. We show here that, contrary to the adjacent DQ1 genes, DQ2 genes exhibit little and possibly no polymorphism. This conservation of DQ2 genes in many haplotypes indicates that the DQ1-DQ2 duplication event must have preceeded the extensive diversification of DQ1 genes and raises the puzzling question of why DQ2 genes have remained nonpolymorphic. This suggests that either these genes correspond to an unusually invariant region of the MHC or they are under a strong selective pressure for the conservation of the amino acid sequence of a putative DQ2 gene product. The latter would imply that the HLA-DQ2 genes are expressed into a protein product endowed with essential functional properties.

Clonal deletion of V beta 14-bearing T cells in mice transgenic for mammary tumour virus.

Autoreactive T lymphocytes are clonally deleted during maturation in the thymus. Deletion of T cells expressing particular receptor V beta elements is controlled by poorly defined autosomal dominant genes. A gene has now been identified by expression of transgenes in mice which causes deletion of V beta 14+ T cells. The gene lies in the open reading frame of the long terminal repeat of the mouse mammary tumour virus.