Sp1 functions in a chromatin-dependent manner to augment human alpha-globin promoter activity.

Abstract

The 5' flanking region ofthe human a-globin gene is highly G+C rich and contains multiple copies of the consensus sequence for the Spl binding site. We investigated the role of this G+C-rich region in augmenting a-globin promoter activity in the presence of the far-upstream a-globin enhancer, HS-40. We show that in transiently transfected erythroid cells, deletion of the a-globin G+C-rich 5' flanking region has no effect on a-globin promoter activity. However, upon stable integration into chromatin, deletion of this region causes a nearly 90% decrease in promoter activity compared with expression from an a-globin promoter retaining this region. These results suggest that the a-globin G+C-rich 5' flanking region augments a-globin promoter activity in a chromatin-dependent manner. We further show that this G+C-rich region is required for the activation of a-globin gene expression during erythroid differentiation. Finally, we show by both footprint analysis and functional assays that the ability of the G+C-rich region to increase ax-globin promoter activity from a stably integrated a-globin gene is mediated by its multiple binding sites for the transcription factor Spl.The human a-globin gene cluster lies within an early replicating G+C-rich isochore close to the telomere of the short arm of chromosome 16. It consists of three functional genes arranged 5'-;2-a2-a1-3'. As with the ,B-globin genes, the a-like globin genes are erythroid specific. However, they are structurally quite different to the f3-globin genes in that they are highly G+C rich, associated with CpG islands, and lack scaffold-associated regions (for review, see ref.

CpG Islands from the α-Globin Gene Cluster Increase Gene Expression in an Integration-Dependent Manner

“…First, the ␣-Luc plasmid, which has the entire CpG island, expresses at a high level after stable integration in the absence of HS Ϫ40, and the presence of this enhancer may be incapable of further substantial increases in expression. Consistent with this interpretation, constructs that do show a strong positive response to HS Ϫ40 in stably transfected K562 cells are not expressed at a detectable level in its absence (32). Second, the effect of HS Ϫ40 after stable integration in MEL cells is seen after induction (20,32), and in the absence of HS Ϫ40, expression of the human ␣-globin gene does not increase upon induction of stably transfected MEL cells (10,20).…”

“…This result contrasts with HS Ϫ40's strong positive effect on the expression of the human ␣-globin gene in stably transfected MEL cells upon induction (20,32). The very modest effect of HS Ϫ40 in stably transfected K562 cells may have multiple causes.…”

“…It is important to note that the constructs in these latter experiments did not contain internal sequences (the promoter fragments end at ϩ17 of exon 1), and only background levels of expression were observed in the absence of induction of MEL cells or in the absence of HS Ϫ40 in K562 cells. However, both the experiments reported here and those of Pondel et al (32), using significantly different constructs and assay systems, revealed integration-dependent and presumably chromatin-associated effects of the 5Ј flanking portion of the CpG island. Furthermore, we show that another CpG island from the inactive -globin gene also supports high-level expression of the ␣-globin gene promoter and this effect is seen only after integration.…”

“…Although only Sp1 was observed to bind to this segment in vitro and the effect of the sequences extending from positions Ϫ267 to Ϫ85 in stably transfected cells can be mimicked by a set of six Sp1 binding sites (32), integration-dependent activities of Sp1 binding sites in any context have yet to be demonstrated. In contrast, the ability of Sp1 to activate transcription of transiently transfected constructs is well established (e.g., 28,30,46,52).…”

“…This region is highly GϩC rich and comprises a third of the CpG island, but there is little sequence conservation beyond these features (19,51). Recently, Pondel et al (32) showed that part of this GϩC-rich region between Ϫ267 and Ϫ85 of the human ␣-globin gene is needed for inducible expression of constructs after stable integration into MEL cells when it is enhanced by HS Ϫ40. It is important to note that the constructs in these latter experiments did not contain internal sequences (the promoter fragments end at ϩ17 of exon 1), and only background levels of expression were observed in the absence of induction of MEL cells or in the absence of HS Ϫ40 in K562 cells.…”