The human growth hormone locus control region mediates long-distance transcriptional activation independent of nuclear matrix attachment regions.

Expression of the human growth hormone (hGH-N) transgene in the mouse pituitary is dependent on a multicomponent locus control region (LCR). The primary determinant of hGH LCR function maps to the pituitary-specific DNase I hypersensitive sites (HS) HSI,II, located 15 kb 5' to the hGH-N gene. The mechanism by which HSI,II mediates long-distance activation of the hGH locus remains undefined. Matrix attachment regions (MARs) comprise a set of AT-rich DNA elements postulated to interact with the nuclear scaffold and to mediate long-distance interactions between LCR elements and their target promoters. Consistent with this model, sequence analysis strongly predicted a MAR determinant in close proximity to HSI,II. Surprisingly, cell-based analysis of nuclear scaffolds failed to confirm a MAR at this site, and extensive mapping demonstrated that the entire 87 kb region encompassing the hGH LCR and contiguous hGH gene cluster was devoid of MAR activity. Homology searches revealed that the predicted MAR reflected the recent insertion of a LINE 3'-UTR segment adjacent to HSI,II. These data point out discordance between sequence-based MAR predictions and in vivo MAR function and predict a novel MAR-independent mechanism for long-distance activation of hGH-N gene expression.

Pit-1 binding sites at the somatotrope-specific DNase I hypersensitive sites I, II of the human growth hormone locus control region are essential for in vivo hGH-N gene activation.

The human growth hormone gene cluster is composed of five closely related genes. The 5'-most gene in the cluster, hGH-N, is expressed exclusively in somatotropes and lactosomatotropes of the anterior pituitary. Although the hGH-N promoter contains functional binding sites for multiple transcription factors, including Sp1, Zn-15, and Pit-1, predictable and developmentally appropriate expression of hGH-N transgenes in the mouse pituitary requires the presence of a previously characterized locus control region (LCR) composed of multiple chromatin DNase I hypersensitive sites (HS). LCR determinant(s) necessary for hGH-N transgene activation are largely conferred by two closely spaced HS (HS I,II) located 14.5 kilobase pairs upstream of the hGH-N gene. The region sufficient to mediate this activity has recently been sublocalized to a 404-base pair segment of HS I,II (F14 segment). In the present study, we identified multiple binding sites for the pituitary POU domain transcription factor Pit-1 within this segment. Using a transgenic founder assay, these sites were shown to be required for high level, position-independent, and somatotrope-specific expression of a linked hGH-N transgene. Because the Pit-1 sites in the hGH-N gene promoter are insufficient for such gene activation in vivo, these data suggested a unique chromatin-mediated developmental role for Pit-1 in the hGH LCR.

CpG islands from the alpha-globin gene cluster increase gene expression in an integration-dependent manner.

In contrast to other globin genes, the human and rabbit alpha-globin genes are expressed in transfected erythroid and nonerythroid cells in the absence of an enhancer. This enhancer-independent expression of the alpha-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 alpha-globin gene comprise an intragenic enhancer specific for the alpha-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 alpha-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 alpha-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.