The locus control region activates serpin gene expression through recruitment of liver-specific transcription factors and RNA polymerase II.

The human serine protease inhibitor (serpin) gene cluster at 14q32.1 comprises 11 serpin genes, many of which are expressed specifically in hepatic cells. Previous studies identified a locus control region (LCR) upstream of the human alpha1-antitrypsin (alpha1AT) gene that is required for gene activation, chromatin remodeling, and histone acetylation throughout the proximal serpin subcluster. Here we show that the LCR interacts with multiple liver-specific transcription factors, including hepatocyte nuclear factor 3beta (HNF-3beta), HNF-6alpha, CCAAT/enhancer binding protein alpha (C/EBPalpha), and C/EBPbeta. RNA polymerase II is also recruited to the locus through the LCR. Nongenic transcription at both the LCR and an upstream regulatory region was detected, but the deletion of the LCR abolished transcription at both sites. The deletion of HNF-3 and HNF-6 binding sites within the LCR reduced histone acetylation at both the LCR and the upstream regulatory region and decreased the transcription of the alpha1AT, corticosteroid binding globulin, and protein Z-dependent protease inhibitor genes. These results suggest that the LCR activates genes in the proximal serpin subcluster by recruiting liver-specific transcription factors and components of the general transcription machinery to regulatory regions upstream of the alpha1AT gene.

Topoisomerase II cleavage activity within the human D11Z1 and DXZ1 alpha-satellite arrays.

Topoisomerase II (Topo II) is a major component of mitotic chromosomes and its unique decatenating activity has been implicated in many aspects of chromosome dynamics including DNA replication, transcription, recombination, chromosome condensation and segregation. Of these, chromosome segregation is the most seriously affected by loss of Topo II, most probably because of residual catenations between sister chromatids. At metaphase, vertebrate chromatids are attached principally through their centromeric regions. Intriguingly, evidence has recently been presented for Topo II cleavage activity within the centromeric alpha-satellite DNA arrays of the human X and Y chromosomes. In this report we extend these observations by mapping distinct sites of Topo II cleavage activity within the alpha-satellite array of human chromosome 11. A single major site of cleavage has been assigned within the centromeric DNA of each of three independently derived, and active, 11 centromeres. Unlike the X and Y centromeres, where cleavage sites mapped close to (within 150 kb of) the short arm edge of the arrays, on chromosome 11, the cleavage sites lie many hundreds of kilobases into each alpha-satellite array. We also demonstrate that catalytically active Topo II is concentrated within the centromere domain through an extended period of G2 and M, with levels declining in G1 and S.

Formation of a large, complex domain of histone hyperacetylation at human 14q32.1 requires the serpin locus control region.

The human serine protease inhibitor (serpin) gene cluster at 14q32.1 is a useful model system to study cell-type-specific gene expression and chromatin structure. Activation of the serpin locus can be induced in vitro by transferring human chromosome 14 from non-expressing to expressing cells. Serpin gene activation in expressing cells is correlated with locus-wide alterations in chromatin structure, including the de novo formation of 17 expression-associated DNase I-hypersensitive sites (DHSs). In this study, we investigated histone acetylation throughout the proximal serpin subcluster. We report that gene activation is correlated with high levels of histone H3 and H4 acetylation at serpin gene promoters and other regulatory regions. However, the locus is not uniformly hyperacetylated, as there are regions of hypoacetylation between genes. Furthermore, genetic tests indicate that locus-wide controls regulate both gene expression and chromatin structure. For example, deletion of a previously identified serpin locus control region (LCR) upstream of the proximal subcluster reduces both gene expression and histone acetylation throughout the approximately 130 kb region. A similar down regulation phenotype is displayed by transactivator-deficient cell variants, but this phenotype can be rescued by transfecting the cells with expression cassettes encoding hepatocyte nuclear factor-1alpha (HNF-1alpha) or HNF-4. Taken together, these results suggest that histone acetylation depends on interactions between the HNF-1alpha/HNF-4 signaling cascade and the serpin LCR.

Organization and expression of the human serpin gene cluster at 14q32.1.

The serpins are a superfamily of gene sequences that have been conserved through evolution. These genes encode protein products that perform a variety of functions in vivo, and their regulation differs among different cell types. About one-third of the serpin genes in the human genome are located at 14q32.1, and the serpin genes in this ~370 kb region are organized into discrete proximal, central, and distal subclusters of four, three, and four genes each. In this report we discuss the genomic organization of the 14q32.1 serpin gene cluster, and we summarize what is known about the regulation of each serpin gene in this region. An approach for studying locus-wide regulation of chromosomal serpin genes in situ is also described. Using this approach, specific mutations in the proximal serpin subcluster were prepared by homologous recombination. These mutant alleles define a serpin locus control region that regulates gene activity and chromatin structure of the entire proximal subcluster. Prospects for further analyses of this complex genomic domain are discussed.

Human matrix attachment regions are necessary for the establishment but not the maintenance of transgene insulation in Drosophila melanogaster.

Human matrix attachment regions (MARs) can insulate transgene expression from chromosomal position effects in Drosophila melanogaster. To gain insight into the mechanism(s) by which chromosomal insulation occurs, we studied the expression phenotypes of Drosophila transformants expressing mini-white transgenes in which MAR sequences from the human apoB gene were arranged in a variety of ways. In agreement with previous reports, we found that a single copy of the insulating element was not sufficient for position-independent transgene expression; rather, two copies were required. However, the arrangement of the two elements within the transgene was unimportant, since chromosomal insulation was equally apparent when both copies of the insulator were upstream of the mini-white reporter as when the transcription unit was flanked by insulator elements. Moreover, experiments in which apoB 3' MAR sequences were removed from integrated transgenes in vivo by site-specific recombination demonstrated that MAR sequences were required for the establishment but not for the maintenance of chromosomal insulation. These observations are not compatible with the chromosomal loop model in its simplest form. Alternate mechanisms for MAR function in this system are proposed.

Sequence organization and matrix attachment regions of the human serine protease inhibitor gene cluster at 14q32.1.

The human serine protease inhibitor (serpin) gene cluster at 14q32.1 is a useful model system for studying the regulation of gene activity and chromatin structure. We demonstrated previously that the six known serpin genes in this region were organized into two subclusters of three genes each that occupied approximately 370 kb of DNA. To more fully understand the genomic organization of this region, we annotated a 1-Mb sequence contig from data from the Genoscope sequencing consortium (http://www.genoscope.cns.fr/ ). We report that 11 different serpin genes reside within the 14q32.1 cluster, including two novel alpha1-antiproteinase-like gene sequences, a kallistatin-like sequence, and two recently identified serpins that had not been mapped previously to 14q32.1. The genomic regions proximal and distal to the serpin cluster contain a variety of unrelated gene sequences of diverse function. To gain insight into the chromatin organization of the region, sequences with putative nuclear matrix-binding potential were identified by using the MAR-Wiz algorithm, and these MAR-Wiz candidate sequences were tested for nuclear matrix-binding activity in vitro. Several differences between the MAR-Wiz predictions and the results of biochemical tests were observed. The genomic organization of the serpin gene cluster is discussed.

Chromosomal elements regulate gene activity and chromatin structure of the human serpin gene cluster at 14q32.1.

The human serine protease inhibitor (serpin) gene cluster at 14q32.1 contains a number of genes that are specifically expressed in hepatic cells. Cell-specific enhancers have been identified in several of these genes, but elements involved in locus-wide gene and chromatin control have yet to be defined. To identify regulatory elements in this region, we prepared a series of mutant chromosomal alleles by homologous recombination and transferred the specifically modified human chromosomes to hepatic cells for functional tests. We report that deletion of an 8-kb DNA segment upstream of the human alpha1-antitrypsin gene yields a mutant serpin allele that fails to be activated in hepatic cells. Within this region, a 2.3-kb DNA segment between kb -8.1 and -5.8 contains a previously unrecognized control region that is required not only for serpin gene activation but also for chromatin remodeling of the entire locus.

The 5' boundary of the human apolipoprotein B chromatin domain in intestinal cells.

The 5' boundary of the chromosomal domain of the human apolipoprotein B (apoB) gene in intestinal cells has been localized and characterized. It is composed of two kinds of boundary elements; the first, functional boundary is an insulator activity exhibited by a 1.8 kb DNA fragment located between -58 and -56 kb upstream of the human apoB promoter. In this region, an enhancer-blocking activity has been mapped to a CTCF binding site that is located upstream of two apoB intestinal enhancers (IEs), the 315 IE and the 485 IE. The CTCF site represents a boundary between two types of chromatin structure: an open, DNaseI-sensitive region 3' of the CTCF site containing the intestinal regulatory elements and a closed, DNaseI-resistant region 5' of the CTCF site. The 1.8 kb fragment harboring the CTCF site also insulated mini-white transgenes against position effects in Drosophila melanogaster. The second, structural boundary is represented by a nuclear matrix attachment region (MAR), situated about 3 kb 5' of the CTCF site. This MAR may represent the 5' anchorage site for a chromosomal loop that functions to bring the intestinal regulatory elements closer to the apoB promoter.

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.

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.

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.

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.

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.

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.

Human matrix attachment regions insulate transgene expression from chromosomal position effects in Drosophila melanogaster.

Germ line transformation of white- Drosophila embryos with P-element vectors containing white expression cassettes results in flies with different eye color phenotypes due to position effects at the sites of transgene insertion. These position effects can be cured by specific DNA elements, such as the Drosophila scs and scs' elements, that have insulator activity in vivo. We have used this system to determine whether human matrix attachment regions (MARs) can function as insulator elements in vivo. Two different human MARs, from the apolipoprotein B and alpha1-antitrypsin loci, insulated white transgene expression from position effects in Drosophila melanogaster. Both elements reduced variability in transgene expression without enhancing levels of white gene expression. In contrast, expression of white transgenes containing human DNA segments without matrix-binding activity was highly variable in Drosophila transformants. These data indicate that human MARs can function as insulator elements in vivo.

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.

Construction of a panel of canine-rodent hybrid cell lines for use in partitioning of the canine genome.

We have constructed a collection of canine-rodent microcell hybrid cell lines by fusion of canine fibroblast microcell donors with immortalized rodent recipient cells. Characterization of the hybrid cell lines using a combination of fluorescence in situ hybridization and PCR analysis of canine microsatellite repeat sequences allowed selection of a panel of hybrids in which most canine chromosomes are represented. Approximately 90% of genetic markers and genes that were tested could be assigned to 1 of 31 anonymous canine chromosome groups, based on common patterns of retention in the hybrid set. Many of these putative chromosome groups have now been validated by linkage analysis. This panel of cell lines provides a tool for development of genetic, physical, and comparative maps of the canine genome.

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.

Isolation and characterization of human hepatoma cells with targeted insertions of a gpt selectable marker in the alpha 1-antitrypsin locus.

The bacterial xanthine-guanine phosphoribosyl transferase (gpt) gene was inserted by homologous recombination into the chromosomal alpha 1-antitrypsin (alpha 1AT) gene of HPRT-deficient human hepatoma cells. These insertions encoded chimeric alpha 1AT-gpt mRNAs that were expressed in the modified cells. Six targeted integrations were obtained, but only two of these harbored simple insertion events. The remaining four homologous insertions contained additional DNA sequences 3' of the gpt coding cassette. Variant cell lines deficient for gpt expression were isolated from transfectants containing either homologous or non-homologous gpt insertions by selection in media containing 6-thioguanine. These variant cell lines expressed alpha 1AT but not alpha 1AT-gpt mRNAs, indicating that they contained expression defects in cis. Genotypic analyses suggested that the predominant mechanism by which the variants were generated was by nondisjunctive loss of chromosomes containing the modified alpha 1AT-gpt alleles. Somatic cell hybrids formed by fusing hepatoma cells containing targeted alpha 1AT-gpt insertions with fibroblasts exhibited extinction of both modified and unmodified alpha 1AT alleles.

Isolation and characterization of HPRT-deficient human hepatoma cells.

Human hepatoma cells deficient in HPRT activity were isolated by challenging HepG2 cells with 6-thioguanine (6TG). Three 6TG-resistant isolates were plated in selective media, and each clonal line displayed an 8-azaguanine-resistant, HAT-sensitive phenotype. The HPRT-deficient phenotype of one of these clones, H30-1, was confirmed in genetic tests: the HAT-sensitivity of H30-1 cells was complemented by fusion by HPRT+ (Ltk-) but not HPRT- (A9) cells. Furthermore, transfection of the bacterial xanthine-guanine phosphoribosyl transferase (gpt) gene into H30-1 cells rendered them HAT-resistant. H30-1 cells maintained the differentiated morphology, growth characteristics, fusion properties, and transfection efficiencies typical of parental HepG2 cells, and they expressed several liver-specific genes. Finally, the H30-1 cell line contained a modal number of 50 chromosomes. Therefore, H30-1 cells represent an HPRT-deficient HepG2 derivative that retains its differentiated phenotype in vitro.