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.     

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 ~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-1α (HNF-1α) or HNF-4. Taken together, these results suggest that histone acetylation depends on interactions between the HNF-1α/HNF-4 signaling cascade and the serpin LCR.     

Physical mapping of four serpin genes: alpha 1-antitrypsin, alpha 1-antichymotrypsin, corticosteroid-binding globulin, and protein C inhibitor, within a 280-kb region on chromosome I4q32.1.

Alpha 1-antitrypsin (alpha 1AT; protease inhibitor [PI] locus), alpha 1-antichymotrypsin (alpha 1ACT; AACT locus), corticosteroid-binding globulin (CBG; CBG locus), and protein C inhibitor (PCI; PCI locus) are members of the serine protease inhibitor (serpin) superfamily. A noncoding PI-like (PIL) gene has been located 12 kb 3' of the PI gene. The PI, PIL, and AACT loci have been localized to 14q32.1, the CBG locus has been localized to 14q31-14q32.1, and PCI has been mapped to chromosome 14. Genetic linkage analysis suggests tight linkage between PI and AACT. We have used pulsed-field gel electrophoresis to generate a physical map linking these five serpin genes. The order of the genetic loci is AACT/PCI-PI-PIL-CBG, with a maximum distance of about 220 kb between the AACT/PCI and PI genes. These genes form a PI cluster at 14q32.1, similar to that of the homologous genes on murine chromosome l2. The close proximity of these genes has implications for disease-association studies.     

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.     

X-chromosome localization of the functional gene for the E1 alpha subunit of the human pyruvate dehydrogenase complex

The functional gene locus for the E1 alpha subunit of the human pyruvate dehydrogenase complex has been localized to the p22.1-22.2 region of the X chromosome by in situ hybridization and analysis of somatic cell hybrids with various human X-chromosome rearrangements. Another locus showing significant cross-hybridization with an E1 alpha cDNA probe was detected on chromosome 4, in the region q22. The X-chromosome localization of the pyruvate dehydrogenase E1 alpha subunit gene provides a number of possible explanations for the clinical and biochemical variability which is a major feature of human pyruvate dehydrogenase deficiency.