Targeted deletion of 5′HS1 and 5′HS4 of the β-globin locus control region reveals additive activity of the DNaseI hypersensitive sites

Abstract

The mammalian ␤-globin locus is a multigenic, developmentally regulated, tissuespecific locus from which gene expression is regulated by a distal regulatory region, the locus control region (LCR). The functional mechanism by which the ␤-globin LCR stimulates transcription of the linked ␤-like globin genes remains unknown. The LCR is composed of a series of 5 DNaseI hypersensitive sites (5HSs) that form in the nucleus of erythroid precursors. These HSs are conserved among mammals, bind transcription factors that also bind to other parts of the locus, and compose the functional components of the LCR. To test the hypothesis that individual HSs have unique properties, homologous recombination was used to construct 5 lines of mice with individual deletions of each of the 5HSs of the endogenous murine ␤-globin LCR. Here it is reported that deletion of 5HS1 reduces expression of the linked genes by up to 24%, while deletion of 5HS4 leads to reductions of up to 27%. These deletions do not perturb the normal stagespecific expression of genes from this multigenic locus. In conjunction with previous studies of deletions of the other HSs and studies of deletion of the entire LCR, it is concluded that (1) none of the 5HSs is essential for nearly normal expression; (2) none of the HSs is required for proper developmental expression; and (3) the HSs do not appear to synergize either structurally or functionally, but rather form independently and appear to contribute additively to the overall expression from the locus. IntroductionThe multigene ␤-globin locus has been intensively studied as a model for the tissue-specific and developmental regulation of gene expression. 1 Transcription of the locus is restricted to erythroid cells, and each gene is expressed during specific developmental stages. Analysis of naturally occurring deletions in the human locus revealed that regions far upstream of the ␤-like globin genes are important for regulation of the locus. 2-4 These regions encompass major DNase I hypersensitive sites (5ЈHS1 to 5 in human, 5ЈHS1 to 6 in mouse) located upstream of the embryonic globin genes. [5][6][7][8][9] The smallest and most well studied of these deletions, the Hispanic thalassemia deletion, 4 removes 5ЈHS2 to 5 as well as 27 kilobases (kb) upstream of this region and leads to an alteration of the generalized DNase I sensitivity of the locus, alterations in the timing of replication and origin used, and the transcriptional silencing of the locus. 10 Linkage of large restriction fragments containing these 5ЈHSs to human ␤-globin genes leads to high-level expression in transgenic mice (reviewed in Grosveld et al 11 and Townes and Behringer 12 ) and contrasts with earlier studies in which isolated globin transgenes were expressed at low levels or not at all. 13 This region, the locus control region (LCR), is defined by its ability to mediate high-level, erythroidspecific expression of linked transgenes and to significantly reduce the influence of integration site on the expression of linked genes. Extensive ...

Deletion of the core region of 5′ HS2 of the mouse β-globin locus control region reveals a distinct effect in comparison with human β-globin transgenes

“…The corresponding studies of the endogenous murine LCR have to this point been limited to large deletions encompassing both HS cores and flanking regions. The resulting phenotypes have resembled the milder phenotypes of full HS deletions within transgenic human loci, including moderate suppression of gene expression [14][15][16]26 and little or no effect on formation of the remaining HSs. 17 We have performed a deletion of the core region of 5ЈHS2 of the mouse ␤-globin LCR to more broadly test the hypothesis that deletion of a core is more suppressive than deletion of an entire HS.…”

“…This model is supported by deletion studies of individual HSs at the mouse locus. [14][15][16] Deletion of individual HSs from the endogenous mouse locus lead to a 0% to 40% reduction in gene expression. If the suppressive activities of the individual site deletions are added together it equals the 95% reduction of gene expression seen with deletion of the entire LCR.…”

Dynamic GATA Factor Interplay at a Multicomponent Regulatory Region of the GATA-2 Locus

“…Deletion of individual HSs of the endogenous ␤-globin locus control region yielded incremental decreases in ␤-globin expression, consistent with a mechanism involving additivity (46,47). In contrast, deletion of HSs from the intact ␤-globin locus within a yeast artificial chromosome (48,49) and transfection experiments (50,51) reveal synergism between the HSs.…”

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

“…25 The implication of the finding that loss of mHS Ϫ26 results in only about a 50% decrease in mouse ␣ gene expression is that additional elements are required for full regulation of the cluster in mice. This in turn implies that there are important differences between mice and humans in ␣-globin gene regulation.…”