Identification of a novel DNase I hypersensitive site within the far upstream region of the human HLA-DRA gene

The class II products of the major histocompatibility complex have a distribution restricted to certain tissues and cells. For instance, they are constitutively expressed by B lymphocytes, but not by resting T lymphocytes. In this study, we report the identification of a novel DNase I hypersensitive site within a putative regulatory region of the human HLA-DRA gene, the so-called far upstream region. This hypersensitive site was present in the genome of the DRalpha-positive human B-lymphoid Raji cell line, and absent in the DRalpha-negative T-lymphoid Jurkat cell line. In addition, this hypersensitive site was also present in transgenic B lymphocytes isolated from the murine transgenic line TG 53, carrying a single integrated copy of the human HLA-DRA gene per haploid genome. The correlation between DRA expression and the presence of this far upstream hypersensitive site suggests novel long distance chromatin remodeling mechanisms possibly shared by human and murine class II genes.     

Th2-specific chromatin remodeling and enhancer activity in the Th2 cytokine locus control region

We recently identified a 3' region of the rad50 gene possessing strong enhancer activity as well as activity consistent with function as a locus control region (LCR) for the flanking Th2 cytokine genes. In this study, we identify several functional elements within this region by examining chromatin changes as well as activity in transgenic mice. We find within this region four DNase I hypersensitive clusters, three of which are highly conserved and predominantly expressed in Th2 cells. Histone acetylation of this region is elevated in Th2 cells. Further, one of the hypersensitive sites (RHS7) is rapidly demethylated in Th2, but not Th1, cells. In transgenic mice, these hypersensitive sites impart strong, Th2-specific enhancer activity as well as copy number-dependent expression of the reporter gene, recapitulating LCR function. We postulate that these sites function alone or in combination with other regulatory elements to coordinate gene expression in the Th2 cytokine locus.     

A single erythroid-specific DNase I super-hypersensitive site activates high levels of human beta-globin gene expression in transgenic mice

Erythroid-specific DNase I super-hypersensitive (HS) sites that are normally located far upstream of the human beta-globin locus were inserted immediately upstream of a 4.1-kb fragment containing the human beta-globin gene. These constructs (HS beta) and a construct containing the beta-globin gene alone (beta) were microinjected into fertilized mouse eggs, and expression was analyzed in erythroid fetal liver and brain of day-16 embryos that developed. Only 7 of 23 animals that contained the beta gene alone expressed human beta-globin mRNA in erythroid tissue, and the average level of expression per gene copy was 0.3% of endogenous mouse beta-globin mRNA. In contrast, 50 of 51 transgenic mice that contained various HS beta constructs expressed the transgene specifically in erythroid tissue. The average level of expression per gene copy for constructs containing all five upstream HS sites was 109% of endogenous mouse beta-globin mRNA. Constructs that contained a single super-hypersensitive site (HS II beta) expressed 40% as much human beta-globin as mouse beta-globin mRNA per gene copy. These results demonstrate that the HS VI site, normally located downstream of the human beta-globin locus, is not required for high-level expression. Furthermore, the results demonstrate that high levels of human beta-globin gene expression can be obtained in transgenic mice even when a relatively small fragment of DNA (1.9 kb) containing erythroid-specific super-hypersensitive site II (HS II) is inserted upstream of the human beta-globin gene.     

Evidence for a complex regulatory array in the first intron of the human adenosine deaminase gene

Adenosine deaminase (ADA) is expressed ubiquitously by diverse mammalian cells and tissues but at levels that vary according to tissue and species. In humans, the thymus exhibits levels of the enzyme up to 100-fold higher than most other tissues. Using transgenic mice, we identified human ADA gene regulatory domains. Up to 3.7 kb of 5'-flanking and first exon DNA from the human ADA gene failed to promote the expression of a chloramphenicol acetyl transferase (CAT) reporter gene in an efficient, reproducible, or tissue-appropriate manner in transgenic mice. However, when 12.8 kb of DNA from the first intron of the human ADA gene was placed 3' of CAT-coding and -processing sequences, transgenic mice reproducibly expressed CAT activity in most tissues, with profoundly high levels in the thymus. DNase I hypersensitivity studies demonstrated that among transgenic mouse tissues, human thymus, and a variety of human cell lines, a region of the intron 4-10 kb downstream of the first exon exhibited an array of hypersensitive sites that varied according to tissue and cell type. Deletion of this region from the gene construction eliminated high-level expression in transgenic mice. In transfection-transient expression assays, the 12.8-kb intron fragment exhibited enhancer activity in several cell types. A 1.3-kb fragment encompassing two of the hypersensitive sites exhibited some of these activities. The results of these studies suggest that the diverse pattern of human ADA gene expression is determined, in part, by a cluster of cis-regulatory elements contained within its large first intron.     

Locus control region elements HS2 and HS3 in combination with chromatin boundaries confer high-level expression of a human beta-globin transgene in a centromeric region

Expression constructs are subject to position-effects in transgenic assays unless they harbour elements that protect them from negative or positive influences exerted by chromatin at the site of integration. Locus control regions (LCRs) and boundary elements are able to protect from position effects by preventing heterochromatization of linked genes. The LCR in the human beta-globin gene locus is located far upstream of the genes and composed of several erythroid specific DNase I hypersensitive (HS) sites. Previous studies demonstrated that the LCR HS sites act synergistically to confer position-independent and high-level globin gene expression at different integration sites in transgenic mice. Here we show that LCR HS sites 2 and 3, in combination with boundary elements derived from the chicken beta-globin gene locus, confer high-level human beta-globin gene expression in different chromosomal integration sites in transgenic mice. Moreover, we found that the construct is accessible to nucleases and highly expressed when integrated in a centromeric region. These results demonstrate that the combination of enhancer, chromatin opening and boundary activities can establish independent expression units when integrated into chromatin.     

Aberrant tissue specific expression of the transgene in transgenic mice that carry the hepatitis B virus genome defective in the X gene

Summary The control mechanisms for the transgene expression in mice that carry the hepatitis B virus genome defective in the polymerase and X genes were analyzed. Ten lines of transgenic mouse were established, and in seven lines the surface and e antigens were detected in the serum. In transgenic mice from five lines examined, the transgene was markedly expressed in a broad spectrum of tissues including the kidney, heart, brain, muscle and intestine, but only poorly in the liver. In the kidney and heart the 3.5 kb and 2.1 kb mRNAs were expressed, whereas only the 0.8 kb and 4.0 kb mRNAs were detected in the testis and brain, respectively, suggesting that each of the mRNAs was transcribed through a different control mechanism. The surface, e and core antigens accumulated in the kidney and heart. DNA was hypomethylated at a region closely downstream of the enhancer in the liver, kidney and heart, and a DNase I hypersensitive site was detected upstream of the enhancer in these tissues. In the testis, however, the whole transgene was hypomethylated and the DNase I hypersensitive site was closer to the enhancer. These differences may be relevant to the preferential expression of the 0.8 kb mRNA in the testis, but cannot explain the inefficiency of transgene expression in the liver. Our observations suggest that the X protein is required for efficient expression of the viral gene in the liver but not in other tissues.     

Th2-specific DNase I-hypersensitive sites in the murine IL-13 and IL-4 intergenic region

IL-4 and IL-13 are cytokines preferentially produced by Th2 cells, and their genes are located in close proximity on human chromosome 5 and mouse chromosome 11. To identify potential regulatory elements that confer Th2-specific expression of IL-4 and IL-13 genes, we constructed a physical map of the IL-13/IL-4 locus and conducted DNase I- hypersensitive (DH) site analysis using Th clones and in vitro- differentiated effector Th cells obtained from TCR transgenic mice. Three DH sites, HSS1, HSS2 and HSS3, were identified within the intergenic region between IL-13 and IL-4 genes. HSS3 was observed both in Th1 and Th2 cells as well as CD4+ naive T cells, while HSS1 and HSS2 were detected exclusively in Th2 cells. The correlation between differentiation into Th2 subtype and the appearance of HSS1 and HSS2 suggests that these regions may play a role in subtype-specific expression of the IL-13/IL-4 locus.