Analysis of the human alpha-globin gene cluster in transgenic mice.

Abstract

A 350-bp segment of DNA associated with an erythroid-specific DNase I-hypersensitive site (HS -40), upstream of the a-globin gene cluster, has been identified as the major tissue-specific regulator of the a-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 a 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 C-and a-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 a-globin genes is not equivalent to that upstream of the f3 locus and that although the two clusters are coordinately expressed, there may be differences in their regulation.

Deletion of the mouse α-globin regulatory element (HS −26) has an unexpectedly mild phenotype

“…Analysis of -globin gene expression during development of the homozygous KO embryos showed a reduction in the / ϩ ␣ ratio from 50% at 9.5 days of gestation to 10% at 13.5 days to zero at 15.5 days (data not shown); this is similar to the pattern observed in normal animals, 7 suggesting that the loss of mHS Ϫ26 does not have a differential effect on the 2 genes.…”

“…To address this question directly, we compared the expression in MEL cells of an mHS Ϫ26␣ construct (distinguished from the endogenous genes by using the coding sequence of the human ␣ gene) with that of an HS Ϫ40␣ construct studied previously. 7,24 The mHS Ϫ26␣ construct was cotransfected into adenine phosphoribosyltransferase (APRT)-negative MEL cells with an APRT plasmid and selected in histone acetyltransferase medium. Clones positive for mHS Ϫ26␣ were expanded and induced to terminal differentiation.…”

“…3,[5][6][7] Even in combination, the other HS sites appear not to add substantially to this effect. Although a large (150 kb) fragment spanning all the HS sites and the ␣-globin cluster is expressed at relatively high levels (22%-66%) in a developmentally stable manner, 4 no fragment has so far been found consistently to provide fully regulated expression of the human ␣ cluster in the erythroid cells of transgenic mice.…”

The role of EKLF in human beta-globin gene competition.

“…Robertson et al (1995) described the silencing of expression of a construct containing an Escherichia coli lacZ gene under control of the human ~-globin HS-40 element in transgenic mice. It had been shown previously that HS-40 could not maintain highlevel copy number-dependent expression of the c~-globin gene throughout development as expression levels declined as development proceeded (Sharpe et al 1992(Sharpe et al , 1993. Histochemical staining for [3-gal showed that the lower expression levels seen late in development correlated with decreasing numbers of lacZ-expressing cells, and suggested that a decrease in the rate of transcription from individual promoters did not play a role.…”

Novel large deletions in the human α-globin gene cluster: Clarifying the HS-40 long-range regulatory role in the native chromosome environment

“…Lying 30-70 kb upstream of the α-globin genes, four highly conserved elements (multispecies conserved sequences, MCS-R1 to 4) corresponding to erythroid-specific DNase I hypersensitive sites (HS-48, HS-40, HS-33, HS-10, respectively) act as long-range regulatory elements of the α-like globin genes [4, reviewed in 5]. Several approaches like the analysis of interspecific hybrids [6] and stable transfectants [6,7], studies in transgenic mice [6,8] or in a humanized mouse model [9], provided evidence that the major regulatory element of this gene cluster in the human locus is MCS-R2 (which corresponds to HS-40). On their own, the other elements (MCS-R1, 3, 4) seem unable to induce substantial levels of α-globin gene expression.…”