Multiple elements in human beta-globin locus control region 5' HS 2 are involved in enhancer activity and position-independent, transgene expression.

The human beta-globin Locus Control Region (LCR) has two important activities. First, the LCR opens a 200 kb chromosomal domain containing the human epsilon-, gamma- and beta-globin genes and, secondly, these sequences function as a powerful enhancer of epsilon-, gamma- and beta-globin gene expression. Erythroid-specific, DNase I hypersensitive sites (HS) mark sequences that are critical for LCR activity. Previous experiments demonstrated that a 1.9 kb fragment containing the 5' HS 2 site confers position-independent expression in transgenic mice and enhances human beta-globin gene expression 100-fold. Further analysis of this region demonstrates that multiple sequences are required for maximal enhancer activity; deletion of SP1, NF-E2, GATA-1 or USF binding sites significantly decrease beta-globin gene expression. In contrast, no single site is required for position-independent transgene expression; all mice with site-specific mutations in 5' HS 2 express human beta-globin mRNA regardless of the site of transgene integration. Apparently, multiple combinations of protein binding sites in 5' HS 2 are sufficient to prevent chromosomal position effects that inhibit transgene expression.     

The 5'HS4 core element of the human beta-globin locus control region is required for high-level globin gene expression in definitive but not in primitive erythropoiesis.

To assess the contribution of DNase I-hypersensitive site 4 (HS4) of the beta-globin locus control region (LCR) to overall LCR function we deleted a 280 bp fragment encompassing the core element of 5'HS4 from a 248 kb beta-globin locus yeast artificial chromosome (beta-YAC) and analyzed globin gene expression during development in beta-YAC transgenic mice. Four transgenic lines were established; each contained at least one intact copy of the beta-globin locus. The deletion of the 5'HS4 core element had no effect on globin gene expression during embryonic erythropoiesis. In contrast, deletion of the 5'HS4 core resulted in a significant decrease of gamma and beta-globin gene expression during definitive erythropoiesis in the fetal liver and a decrease of beta-globin gene expression in adult blood. We conclude that the core element of 5'HS4 is required for globin gene expression only in definitive erythropoiesis. Absence of the core element of HS4 may limit the ability of the LCR to provide an open chromatin domain and/or enhance gamma and beta-globin gene expression in the adult erythroid cells.     

A dominant chromatin-opening activity in 5' hypersensitive site 3 of the human beta-globin locus control region.

Single-copy human beta-globin transgenes are very susceptible to suppression by position effects of surrounding closed chromatin. However, these position effects are overcome by a 20 kbp DNA fragment containing the locus control region (LCR). Here we show that the 6.5 kbp microlocus LCR cassette reproducibly directs full expression from independent single-copy beta-globin transgenes. By testing individual DNase I-hypersensitive sites (HS) present in the microlocus cassette, we demonstrate that the 1.5 kbp 5'HS2 enhancer fragment does not direct beta-globin expression from single-copy transgenes. In contrast, the 1.9 kbp 5'HS3 fragment directs beta-globin expression in five independent single-copy transgenic mouse lines. Moreover, the 5'HS3 core element and beta-globin proximal promoter sequences are DNase I hypersensitive in fetal liver nuclei of these expressing transgenic lines. Taken together, these results demonstrate that LCR activity is the culmination of at least two separable functions including: (i) a novel activity located in 5'HS3 that dominantly opens and remodels chromatin structure; and (ii) a recessive enhancer activity residing in 5'HS2. We postulate that the different elements of the LCR form a 'holocomplex' that interacts with the individual globin genes.     

Analysis of mice containing a targeted deletion of beta-globin locus control region 5' hypersensitive site 3.

To examine the function of murine beta-globin locus region (LCR) 5' hypersensitive site 3 (HS3) in its native chromosomal context, we deleted this site from the mouse germ line by using homologous recombination techniques. Previous experiments with human 5' HS3 in transgenic models suggested that this site independently contains at least 50% of total LCR activity and that it interacts preferentially with the human gamma-globin genes in embryonic erythroid cells. However, in this study, we demonstrate that deletion of murine 5' HS3 reduces expression of the linked embryonic epsilon y- and beta H 1-globin genes only minimally in yolk sac-derived erythroid cells and reduces output of the linked adult beta (beta major plus beta minor) globin genes by approximately 30% in adult erythrocytes. When the selectable marker PGK-neo cassette was left within the HS3 region of the LCR, a much more severe phenotype was observed at all developmental stages, suggesting that PGK-neo interferes with LCR activity when it is retained within the LCR. Collectively, these results suggest that murine 5' HS3 is not required for globin gene switching; importantly, however, it is required for approximately 30% of the total LCR activity associated with adult beta-globin gene expression in adult erythrocytes.     

人β—簇珠蛋白基因LCR调控元件中HS3与反式调控因子相互作用的研究

Upstream of the human epsilon-globin gene is the Locus Control Region (LCR) of the human beta-globin cluster, which consists of four DNase-I hypersensitive sites(HS1-HS4). It has been reported in transgenic experiments that HS3 preferentially regulates epsilon-globin gene expression. In order to elucidate the regulatory function of HS3 in the expression of globin gene, nuclear extracts from mouse hematopoietic tissues at several developmental stages were prepared and the binding of the nuclear factors to HS3 was analysed by using electrophoresis mobility shift assay(EMSA). Our results showed that the binding patterns of HS3 with nuclear extracts of mouse hematopoietic tissues at day 13 and day 18 of gestation were completely different; furthermore, by Southwestern Blot, the distinction between both stages was also demonstrated. It has been known that GATA and CACCC binding motifs are contained within HS3 core region. Using competitive gel-retardation assay, we found that no shift bands could be competed by using CACCC motif as a competitor. However one shift band at day 13 and day 18 of gestation could be competed respectively by using GATA motif as a competitor. We suggested that the shift bands, which could not be competed by both motifs, might be novel and stage-specific factors. In addition, by using Western Blot, we demonstrated that the two shift bands at day 13 and day 18 of gestation, competed by GATA motif, were GATA-2 and GATA-1 respectively: GATA-1 was expressed in mouse hematopoietic tissues at day 18 of gestation and not expressed at day 13 of gestation; however, GATA-2 was only expressed in mouse hematopoietic tissues at day 13 of gestation. According to these results, we speculated that HS3 might play an important role in regulation of stage-specific expression of globin genes through interaction between stage-specific nuclear factors and HS3.     

Nuclear proteins preferentially bound to the beta-globin gene: cellular specificity and variation during terminal differentiation of mouse erythroblasts

Restriction fragments of the mouse beta major globin gene and of the long terminal repeat (LTR) DNA fragment of the mouse mammary tumor provirus as a control, were used to analyze the specificity of DNA-protein interactions in nuclear extracts of mouse erythroleukemia (MEL) cells and of other differentiated mouse cultured cell lines. After gel electrophoresis and transfer to nitrocellulose, DNA-binding proteins with a preferential affinity for the cloned beta-globin genomic sequence were characterized and related to the level of globin gene expression during induction of differentiating mouse erythroblasts. Two proteins (110 K and 75 K) appear in differentiated MEL cells while another one (100 K), for which we have localized the binding site on the beta-globin gene, is present only in immature MEL cells.     

Detection of two tissue-specific DNA-binding proteins with affinity for sites in the mouse beta-globin intervening sequence 2.

To identify proteins from uninduced murine erythroleukemia nuclear extracts which specifically bind to sequences from the DNase I-hypersensitive region within the mouse beta-globin intervening sequence 2 (IVS2), a gel electrophoretic mobility shift assay was used. Two distinct sequence-specific binding proteins were detected. The specific binding sites for these factors were delineated by both DNase I protection footprinting and methylation interference. Factor B1 bound specifically to two homologous sites, B1-A and B1-B, approximately 100 base pairs apart within the IVS2 and on opposite strands. These two regions could interact with factor B1 independently. Factor B1 was limited to cells of hematopoietic lineages. Factor B2 bound to a site approximately 5 base pairs away from the B1-A site and was limited to cells of the erythroid lineage. The limited tissue distribution of these factors and the locations of their binding sites suggest that one or both of these factors may be involved in the formation of the tissue-specific DNase I-hypersensitive site in the IVS2 of the mouse beta-globin gene.