Transcriptional promiscuity of the human alpha-globin gene.

Abstract

The human aL-globin gene displays the unusual property of transcriptional promiscuity: that is, it functions in the absence of an enhancer when transfected into nonerythroid cell lines. It is also unusual in that its promoter region lies in a hypomethylated HpaII tiny fragment (HTF) island containing multiple copies of the consensus sequence for the SPl-binding site. We have investigated whether there is a relationship between these two observations. First, we investigated the mouse ac-globin gene since it does not lie in an HTF island. We have demonstrated that it was not transcriptionally promiscuous. Second, we studied the transcriptional activity of the human a-globin gene in the absence of the GC-rich region containing putative SPl-binding sites and found a small (two-to threefold) but consistent positive effect of this region on transcriptional activity in both nonerythroid and erythroid cell lines. However, this effect did not account for the promiscuous nature of the human a-globin gene. We found that in a nonreplicating system, the human a-globin gene, like that of the mouse, required a simian virus 40 enhancer in order to be transcriptionally active in nonerythroid and erythroid cell lines. Since we only observed enhancer independence of the human a-globin gene in a high-copy-number replicating system, we suggest that competition for trans-acting factors could explain these results. Finally, our experiments with the erythroid cell line Putko suggest that there are no tissue-specific enhancers within 1 kilobase 5' of the human a-globin cap site or within the gene itself.

Induction by HMBA and DMSO of genes introduced into mouse erythroleukemia and other cell lines by transient transfection.

“…The response of these constructs following transfection and induction was measured by both CAT and RNA analyses (not shown). Deletion of sequences from -1375 to -76 relative to the cap site gave a decrease in the level of expression similar to that described previously (Whitelaw et al 1989), but induction remained unaffected. Removal of an additional 8 bp 3' to position -7 6 destroyed the CAAT box and caused the loss of measurable expression.…”

“…Although the ~-and [3-globin genes were both induced in transiently transfected MELCs, expression of the c~-globin gene was much greater than that of the [3-globin gene, a situation similar to that in nonerythroid cells (Banerji et al 1981;Mellon et al 1981;Humphries et al 1982;Treisman et al 1983;Whitelaw et al 1989). This suggested that the transiently transfecting globin genes gained no advantage from being expressed in an erythroid environment.…”

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

“…pA and poly A, polyadenylation site; ex1 to ex3, exon 1 to exon 3. was found to confer a strong (ϳ30-fold) positive effect on expression, which shows that this rabbit ␣-globin 5Ј flanking DNA segment is capable of responding to an enhancer. This result is consistent with the results of earlier studies with human ␣-globin gene constructs (31,36,47). In the test shown in Fig.…”

“…However, when both more extensive 5Ј flanking and internal sequences are included in the construct (e.g., ␣-Luc), inclusion of this major regulatory region has at most a two-to fourfold effect. This is comparable to the effect of the SV40 enhancer on the transient expression of an intact rabbit ␣-globin gene in K562 cells (11,22) and a human ␣-globin gene in a replicating system in HeLa cells (47). One possible explanation for this a K562 cell clones were stably transfected with human ␣-globin-luciferase fusion constructs in CpG island and non-CpG island environments.…”

“…Whether the effect of CpG island sequences is seen with unintegrated, transiently transfected plasmids appears to correlate with the strength of the reporter gene in that cell type. Reporter genes with promoters that express at low levels and that are significantly augmented by known enhancers also show a positive effect of CpG island sequences in transiently transfected cells (7,22,36,47). In contrast, reporter genes with promoters that are readily expressed without an enhancer show a CpG island effect only after stable integration.…”