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.     

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 ~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 ( ). We report that 11 different serpin genes reside within the 14q32.1 cluster, including two novel 1-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. These authors (Stephanie J. Namciu and Richard D. Friedman) contributed equally to this work.     

Physical and genetic mapping of the serpin gene cluster at 14q32.1: allelic association and a unique haplotype associated with alpha 1-antitrypsin deficiency.

The alpha 1-antitrypsin (PI) gene is part of a cluster of structurally related serine protease inhibitor genes localized at chromosome 14q32.1, a cluster that includes the alpha 1-antichymotrypsin (AACT), protein C inhibitor (PCI), and corticosteroid-binding globulin (CBG) genes and the alpha 1-antitrypsin-like pseudogene (PIL). The order of the genes is refined here by genetic mapping using simple tandem repeat polymorphisms (STRPs) and by physical mapping in YACs. The order of the genes is (centromere)-CBG-PIL-PI-PCI-AACT-(telomere). Analysis of DNA haplotypes comprising STRP and RFLP markers in the serpin genes reveals considerable allelic association throughout the cluster. Furthermore, the common alpha 1-antitrypsin deficiency allele, PI*Z, has a unique DNA haplotype at the CBG, PIL, and PI loci, which extends over 60 kb in 97% of cases and in 44% of cases includes the PCI and AACT loci. This unique haplotype will be of use in examining a number of other diseases, particularly those with an inflammatory component, thought to be associated with alpha 1-antitrypsin deficiency or partial deficiency.     

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.     

Chromosomal localization of the gene for human glucosamine-6-sulphatase to 12q14

Summary Glucosamine-6-sulphatase (G6S), a lysosomal enzyme found in all cells, is involved in the catabolism of heparin, heparan sulphate, and keratan sulphate. Deficiency of G6S results in the accumulation of undegraded substrate and the lysosomal storage disorder mucopolysaccharidosis type IIID (Sanfilippo D syndrome). Regional mapping by in situ hybridization of a ³H-labelled human G6S cDNA probe to human metaphase chromosomes indicated that the G6S gene is localized to chromosome 12 at q14. The localization of the G6S gene to chromosome 12 was confirmed using the G6S cDNA clone in Southern blot hybridization analysis of DNA from human x mouse hybrid cell lines.     

Chromosomal localization of the human placental lactogen-growth hormone gene cluster to 17q22-24.

Recombinant plasmid HCS-pBR322 containing a 550-base-pair (bp) insert of cDNA to human placental lactogen (hPL) mRNA was 3H-labeled by nick translation and hybridized in situ to human chromosome preparations in the presence of 10% dextran sulfate. A high percentage of cells (80%) were found to exhibit label on the distal end of the long arm of chromosome 17. Silver grains on this region constituted 25.5% of all labeled sites, allowing assignment of the hPL and growth hormone (hGH) genes, which have over 90% nucleotide homology in their coding sequences, to 17q22-24. A gene copy number experiment showed that both genes are present in approximately three copies per haploid genome.     

Comparative genomic analysis of the clade B serpin cluster at human chromosome 18q21: amplification within the mouse squamous cell carcinoma antigen gene locus

The human clade B serpins neutralize serine or cysteine proteinases and reside predominantly within the intracellular compartment. Genomic analysis shows that the 13 human clade B serpins map to either 6p25 (n = 3) or 18q21 (n = 10). Similarly, the mouse clade B serpins map to syntenic loci at 13A3.2 and 1D, respectively. The mouse clade B cluster at 13A3.2 shows a marked expansion in the number of serpin genes (n = 15). The purpose of this study was to determine whether a similar expansion occurred at 1D. Using STS-content mapping, comparative genomic DNA sequence analysis, and cDNA cloning, we found that the mouse clade B cluster at 1D showed nearly complete conservation of gene number, order, and orientation relative to those of 18q21. The only exception was the squamous cell carcinoma antigen (SCCA) locus. The human SCCA locus contains two genes, SERPINB3 (SCCA1) and SERPINB4 (SCCA2), whereas the mouse locus contains four serpins and three pseudogenes. Based on phylogenetic analysis and predicted amino acid sequences, amplification of the mouse SCCA locus occurred after rodents and primates diverged and was associated with some diversification of proteinase inhibitory activity relative to that of humans.     

DNA methylation and chromatin structure regulate T cell perforin gene expression

Perforin is a cytotoxic effector molecule expressed in NK cells and a subset of T cells. The mechanisms regulating its expression are incompletely understood. We observed that DNA methylation inhibition could increase perforin expression in T cells, so we examined the methylation pattern and chromatin structure of the human perforin promoter and upstream enhancer in primary CD4(+) and CD8(+) T cells as well as in an NK cell line that expresses perforin, compared with fibroblasts, which do not express perforin. The entire region was nearly completely unmethylated in the NK cell line and largely methylated in fibroblasts. In contrast, only the core promoter was constitutively unmethylated in primary CD4(+) and CD8(+) cells, and expression was associated with hypomethylation of an area residing between the upstream enhancer at -1 kb and the distal promoter at -0.3 kb. Treating T cells with the DNA methyltransferase inhibitor 5-azacytidine selectively demethylated this area and increased perforin expression. Selective methylation of this region suppressed promoter function in transfection assays. Finally, perforin expression and hypomethylation were associated with localized sensitivity of the 5' flank to DNase I digestion, indicating an accessible configuration. These results indicate that DNA methylation and chromatin structure participate in the regulation of perforin expression in T cells.     

Chromosomal localization of the human alpha 1-antitrypsin gene (PI) to 14q31-32.

In situ hybridization of a recombinant cDNA probe containing the human alpha 1-antitrypsin gene to metaphase chromosomes demonstrated significant hybridization to chromosomal segment 14q31-32. A high percentage of cells analyzed (31%) displayed labeling on chromosome 14. Of all labeled sites on chromosome 14, 60% were found on segment 14q31-32. These results refine the previous assignment of the human alpha 1-antitrypsin gene to segment 14q24.1-32.1.     

Exploring CTCF and cohesin related chromatin architecture at HOXA gene cluster in primary human fibroblasts

Spatial expression patterns of homeobox(HOX) genes delineate positional identity of primary fibroblasts from different topographic sites. The molecular mechanism underlying the establishing or maintaining of HOX gene expression pattern remains an attractive developmental issue to be addressed. Our previous work suggested a critical role of CTCF/cohesin-mediated higher-order chromatin structure in RA-induced HOXA activation in human teratocarcinoma NT2/D1 cells. This study investigated the recruitment of CTCF and cohesin, and the higher-order chromatin structure of the HOXA locus in fetal lung and adult foreskin fibroblasts, which display complementary HOXA gene expression patterns. Chromatin contacts between the CTCF-binding sites were observed with lower frequency in human foreskin fibroblasts. This observation is consistent with the lower level of cohesin recruitment and 5′ HOXA gene expression in the same cells. We also showed that CTCF-binding site A56(CBSA56) related chromatin structures exhibit the most notable changes in between the two types of cell, and hence may stand for one of the key CTCF-binding sites for cell-type specific chromatin structure organization. Together, these results imply that CTCF/cohesin coordinates HOXA cluster higher-order chromatin structure and expression during development, and provide insight into the relationship between cell-type specific chromatin organization and the spatial collinearity.     

Characterisation of the NUCKS gene on human chromosome 1q32.1 and the presence of a homologous gene in different species

The NUCKS gene is located on human chromosome 1q32.1 and consists of seven exons and six introns. The gene lacks a TATA box but contains two Inr elements, two GC boxes, and one consensus-binding site for E2F-1. NUCKS is expressed in all human adult and foetal tissues investigated, and has all the features of being a housekeeping gene. Both data searches and Western immunoblotting experiments show that a homologous protein is present in fish, amphibians, and birds but not in insects and yeast, suggesting that NUCKS is a vertebrate specific gene. In all the species investigated, the protein contains several consensus phosphorylation sites for cyclin-dependent kinases and CK-2, and we have shown that the fish protein (like mammalian NUCKS) indeed is a substrate for CDK1 and CK-2 in vitro. The NUCKS protein is also conserved with respect to a DNA-binding domain previously characterised in mammals, and two putative bipartite nuclear localisation signals.     

Chromosomal localization and structure of the human P1 protamine gene

The human P1 protamine gene and mRNA were amplified with the use of the polymerase chain reaction and cloned into PTZ19R. The sequences were determined which revealed the presence of an intron. Southern and Northern hybridization analyses showed that the gene was single copy and that the mRNA was approximately 450 bases long. The gene was mapped to chromosome 16 with the use of a somatic cell hybrid panel and localized to the 21 region of the q arm by in situ hybridization of the human P1 protamine probe to human metaphase chromosomes.     

DNase I-defined chromatin configuration of the human CD3 gene cluster.

The three CD3 genes on human chromosome 11q23 encode proteins (gamma, delta and epsilon) which form part of the antigen receptor on T lymphocytes. All three genes are clustered within 50 kb and are activated approximately contemporaneously during the early stages of T cell ontogeny. In order to pinpoint potential regulatory sequences important for locus activation and tissue-specific gene expression, the chromatin structure of almost 90 kb of this region has been probed in five cell lines using the endonuclease pancreatic DNase I. A set of DNase I hypersensitive (HS) sites has been defined in T cell chromatin, five of which were strong and not found in non-T cells, with the exception of the erythroleukaemia cell line K562, in which three sites were weakly expressed, correlating with a low level of delta mRNA. The subset of five HS sites map close to the CD3 genes and lie in regions which contain elements of defined function: the gamma promoter; the delta promoter and its 3' enhancer; and the epsilon promoter and its 3' enhancer. Since no further major T cell-restricted HS sites lie within the 90kb of the CD3 locus analysed, these five regions may contain all the sequences important for CD3 gene expression.     

The human cytochrome b5 gene and two of its pseudogenes are located on chromosomes 18q23, 14q31-32.1 and 20p11.2, respectively

Using very high stringency hybridization conditions for the Southern blot hybridization analysis of hamster-human cell hybrid DNA, we were able to map the human cytochrome b₅ gene and two of its pseudogenes (psgb₅1 and psgb₅2) unambiguously to chromosomes 18, 14, and 20. These localizations were confirmed and extended to 18q23, 14q31-32.1, and 20p11.2 by using a combination of nonisotopic in situ hybridization of chromosomal spreads and the polymerase chain reaction analysis of DNA samples isolated from somatic cell hybrids retaining deletions or translocations of chromosome 18.