Deletion of the mouse alpha -globin regulatory element (HS -26) has an unexpectedly mild phenotype (original) (raw)
Related papers
Nucleic Acids Research, 1994
We have ligated two cosmids through an oligonucleotide linker to produce a single fragment spanning 70 kb of the human a-globin cluster, in which the alike globin genes (p2, a2 and a1), their regulatory element (HS-40) and erythrold-specific DNase I hypersensitive sites accurately retain their normal genomic organization. The r (embryonic) and ax (embryonic, fetal and adult) globin genes were expressed In all 17 transgenic embryos. Similarly, all fetal and adult mice from seven transgenic lines that contained one or more copies of the fragment, produced up to 66% of the level of endogenous mouse a-globin mRNA. However, as for smaller constructs containing these elements, human a-globin expression was not copy number dependent and decreased by 1.5-9.0 fold during development. These findings suggest that either it is not possible to obtain full regulation of human a-globin expression in transgenic mice or, more likely, that additional a-globin regulatory elements lie beyond the 70 kb segment of DNA analysed.
Blood, 1995
We have examined the role of the major positive upstream regulatory element of the human alpha-globin gene locus (HS-40) in its natural chromosomal context. Using homologous recombination, HS-40 was replaced by a neo marker gene in a mouse erythroleukemia hybrid cell line containing a single copy of human chromosome 16. In clones from which HS-40 had been deleted, human alpha-globin gene expression was severely reduced, although basal levels of alpha 1 and alpha 2-globin mRNA expression representing less than 3% of the level in control cell lines were detected. Deletion of the neo marker gene, by using FLP recombinase/FLP recombinase target system, proved that the phenotype observed was not caused by the regulatory elements of this marker gene. In the targeted clones, deletion of HS-40 apparently does not affect long-range or local chromatin structure at the alpha promoters. Therefore, these results indicate that, in the experimental system used, HS-40 behaves as a strong inducible ...
Analysis of enhancer function of the HS-40 core sequence of the human α-globin cluster
1997
Abstract HS-40 is the major regulatory element of the human α-globin locus, located 40 kb upstream of the ζ-globin gene. To test for potential interactions between HS-40 and the β-or the γ-globin gene promoters in stable transfection assays, the HS-40 core sequence was cloned upstream of either the β promoter or the γ promoter driving the neomycin phosphotransferase gene and enhancer activity was measured using a colony assay.
Annals of the New York Academy of Sciences, 1998
To gain insights into the functions of individual DNA'se hypersensitive sites within the β globin locus control region (LCR), we deleted the endogenous 5′ HS-2 and HS-3 regions from the mouse germline using homologous recombination techniques. We demonstrated that the deletion of either murine 5′ HS-2 or 5′ HS-3 reduced the expression of the embryonic εy and βh1 globin genes minimally in yolk sac-derived erythrocytes, but that both knockouts reduced the output of the adult β (β-Major + β-Minor) globin genes by approximately 30% in adult erythrocytes. When the selectable marker PGK-Neo cassette was retained within either the HS-2 or HS-3 region, a much more severe reduction in globin gene expression was observed at all developmental stages. PGK-Neo was shown to be expressed in an erythroid-specific fashion when it was retained in the HS-3 position. These results show that neither 5′ HS-2 nor HS-3 is required for the activity of embryonic globin genes, nor are these sites required for correct developmental switching. However, each site is required for approximately 30% of the total LCR activity associated with adult βglobin gene expression in adult red blood cells. Each site therefore contains some nonredundant information that contributes to adult globin gene function.
1995
Harris et al., 1990), and direct genomic analysis (Ger-We have determined the cDNA and genomic strucmino et al., 1992; Vyas et al., 1992; European Polycystic ture of a gene (014 gene) that lies adjacent to the hu-Kidney Disease Consortium, 1994) this Ç2-Mb teman a-globin cluster. Although it is expressed in a lomeric region has previously been shown to be highly wide range of cell lines and tissues, a previously de-GC rich and to contain a high density of CpG islands scribed erythroid-specific regulatory element that and genes. To obtain a more precise description of this controls expression of the a-globin genes lies within type of area, we have studied in detail the organization intron 5 of this gene. Analysis of the 014 gene promoter and chromatin structure of the most distal 200 kb of shows that it is GC rich and associated with a constitu-16p (Fig. 1). tively expressed DNase 1 hypersensitive site; unlike Proximal to the simple sequence telomeric repeats the a-globin promoter, it does not contain a TATA or (TTAGGG) n lies a complex, polymorphic, subtelomeric CCAAT box. These and other differences in promoter region that contains sequences shared with the ends structure may explain why the erythroid regulatory of other, nonhomologous chromosomes (Wilkie et al., element interacts specifically with the a-globin pro-1991). Proximal to this, within a segment of 150 kb, moters and not the 014 gene promoter, which lies between the a promoters and their regulatory element. we have identified nine CpG islands, each associated Interspecies comparisons demonstrate that the sewith a DNase 1 hypersensitive site (Vyas et al., 1992). quence and location of the 014 gene adjacent to the a The most distal islands (at coordinates 014, 074, 089/ cluster have been maintained since the bird/mammal 090, and 099) are associated with widely expressed divergence, 270 million years ago. ᭧ 1995 Academic Press, Inc. genes, one of which (074 gene) encodes a DNA repair enzyme (3-methyl purine DNA glycosylase, MPG)
Analysis of the human alpha-globin gene cluster in transgenic mice
Proceedings of the National Academy of Sciences, 1993
A 350-bp segment of DNA associated with an erythroid-specific DNase I-hypersensitive site (HS-40), upstream of the alpha-globin gene cluster, has been identified as the major tissue-specific regulator of the alpha-globin genes. However, this element does not direct copy number-dependent or developmentally stable expression of the human genes in transgenic mice. To determine whether additional upstream hypersensitive sites could provide more complete regulation of alpha gene expression we have studied 17 lines of transgenic mice bearing various DNA fragments containing HSs -33, -10, -8, and -4, in addition to HS -40. Position-independent, high-level expression of the human zeta- and alpha-globin genes was consistently observed in embryonic erythroid cells. However, the additional HSs did not confer copy-number dependence, alter the level of expression, or prevent the variable down-regulation of expression in adults. These results suggest that the region upstream of the human alpha-gl...