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 ␣1-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.Chromatin is the template for transcription in vivo. Alterations in the chromatin organization of specific loci generally accompany changes in gene activity, and these chromatin effects can be monitored in several ways. For example, the accessibility of specific chromosomal loci to exogenous nucleases generally reflects their transcriptional activity (48). Similarly, the formation of expression-associated DNase I-hypersensitive sites (DHSs) (39), covalent modifications of histones and other chromosomal proteins (25), and nuclear compartmentalization of specific loci (43) all change during eukaryotic gene activation. This suggests that regulatory mechanisms that govern gene activity are linked to those that control chromatin structure. It is a fundamental challenge for mammalian genetics to identify and characterize these regulatory interactions in their native genomic environments.The human serine protease inhibitor (serpin) gene cluster at 14q32.1 is a useful model system for studying the cell-specific regulation of gene expression and chromatin structure. The serpin locus is located at approximately 92.3 megabases (Mb) along chromosome 14q's 104-Mb length (http://www.ncbi.nlm .nih.gov/). There are 11 functionally diverse serpin genes within this ϳ400-kb region, and the genes are organized into three discrete subclusters of four, three, and four genes each. The proximal subcluster, about 107 kb in length, is the best characterized; it includes the well-studied ␣1-antitrypsin gene (␣1AT, SERPINA1), an antitrypsin-related pseudogene (ATR, SERPINA2) ϳ13 kb downstream, the corticosteroid-binding globulin gene (CBG, SERPINA6) ϳ68 kb downstream of ␣1AT (37), and the recently identified protein Z inhibitor gene (ZPI, SERPINA10) ϳ100 kb downstream of ␣1AT (20). These serpin genes are expressed in hepatic cells, but they are repressed in most other cell types.Regulation of human ␣1AT gene expression has been studied in detail (8,13,21,29,32,33,45). Human ␣1AT mRNA is one of the most abundant transcripts in hepatic cells, but the gene is not expressed in most other cell types. These large differences in the transcription...

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.

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