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

Abstract

In contrast to other globin genes, the human and rabbit ␣-globin genes are expressed in transfected erythroid and nonerythroid cells in the absence of an enhancer. This enhancer-independent expression of the ␣-globin gene requires extensive sequences not only from the 5 flanking sequence but also from the intragenic region. However, the features of these internal sequences that are responsible for their positive effect are unclear. We tested several possible determinants of this activity. One possibility is that a previously identified array of discrete binding sites for known and potential regulatory proteins within the ␣-globin gene comprise an intragenic enhancer specific for the ␣-globin promoter, but directed rearrangements of the sequences show that this is not the case. Alternatively, the promoter may extend into the gene, with the function of the discrete binding sites being dependent on maintenance of their proper positions and orientations relative to the 5 flanking sequence. However, the positive effects observed in gene fusions do not localize to a discrete region of the ␣-globin gene and the results of internal deletions and point mutations argue against a required role of the targeted discrete binding sites. A third possibility is that the CpG island, which includes both the 5 flanking and intragenic regions associated with the positive activity, may itself have a more general effect on expression in transfected cells. Indeed, we show that the size of the CpG island in constructs correlates with the level of gene expression. Furthermore, the ␣-globin promoter is more active in the context of a previously inactive CpG island than in an A؉T-rich context, showing that the CpG island provides an environment more permissive for expression. These effects are seen only after integration, suggesting a possible mechanism at the level of chromatin structure.

Long Human–Mouse Sequence Alignments Reveal Novel Regulatory Elements: A Reason to Sequence the Mouse Genome

“…This suggested that the effects of the CpG island outside the proximal promoter were to provide a more permissive environment for promoter activity than do bulk A+T-rich DNAs, but that this effect is not dependent on binding of specific transactivators at discrete locations. The postulated general effect of CpG islands is supported by three lines of evidence: (1) The level of gene expression increases with increasing size of the CpG island included in transfection constructs; (2) deletion of prominent binding sites for Sp1 and YY1 has no effect; and (3) addition of ␣-globin gene promoter fragments to a transcriptionally inactive CpG island gives a much higher level of expression after integration into the genome than does addition of these fragments to an A+T-rich DNA fragment (Shewchuk and Hardison 1997). This more permissive effect of CpG islands may be exerted at least in part at the level of chromatin structure, as CpG island DNA from the ␣-globin gene has a much lower affinity for nucleosome reconstitution in vitro than does the A+T-rich DNA fragments from the ␤-globin gene .…”

Biochemical and molecular investigations on qualitative and quantitative Hb polymorphism in the river buffalo (Bubalus bubalis L.) population reared in Southern Italy

“…This inhibition is modulated by transcriptional termination signals located between the two α genes. Recently, Shewchuk and Hardison (1997) have shown that the size of CpG islands in the α-globin gene cluster, which includes both 5' flanking and intragenic regions, positively correlates with the level of gene expression, also suggesting that a mechanism at the chromatin structure level may be involved.…”

Efficient and Reliable Transfection of Mouse Erythroleukemia Cells Using Cationic Lipids

“…Stably transfected cells carry the test plasmids integrated into a chromosome and hence the template DNA is in a normal chromatin structure. Different kinds of questions can be addressed with the two types of transfections; e.g., transient transfections can provide a rapid means for identifying promoters and enhancers (2) whereas stably transfected cells can reveal position effects (21,22) or integration-dependent enhancement (14,16).…”