POU1F1-mediated activation of hGH-N by deoxyribonuclease I hypersensitive site II of the human growth hormone locus control region

The human growth hormone gene (hGH-N) is regulated by a distal locus control region (LCR) composed of five deoxyribonuclease I hypersensitive sites (HSs). The region encompassing HSI and HSII contains the predominant pituitary somatotrope-specific hGH-N activation function of the LCR. This activity was attributed primarily to POU1F1 (Pit-1) elements at HSI, as linkage to HSI was sufficient for properly regulated hGH-N expression in transgenic mice, while HSII alone had no activity. However, the presence of HSII in conjunction with HSI further enhanced hGH-N transgene expression, indicating additional determinants of pituitary hGH-N activation in the HSII region, but limitations of transgenic models and previous ex vivo systems have prevented the characterization of HSII. In the present study, we employ a novel minichromosome model of the hGH-N regulatory domain and show that HSII confers robust POU1F1-dependent activation of hGH-N in this system. This effect was accompanied by POU1F1-dependent histone acetylation and methylation throughout the minichromosome LCR/hGH-N domain. A series of in vitro DNA binding experiments revealed that POU1F1 binds to multiple sites at HSII, consistent with a direct role in HSII function. Remarkably, POU1F1 binding was localized in part to the 3' untranslated region of a primate-specific LINE-1 (long interspersed nuclear element 1) retrotransposon, suggesting that its insertion during primate evolution may have conferred function to the HSII region in the context of pituitary GH gene regulation. These observations clarify the function of HSII, expanding the role of POU1F1 in hGH LCR activity, and provide insight on the molecular evolution of the LCR.     

The human growth hormone gene cluster locus control region supports position-independent pituitary- and placenta-specific expression in the transgenic mouse

The human growth hormone (hGH) cluster contains five genes. The hGH-N gene is predominantly expressed in pituitary somatotropes, whereas the remaining four genes, the chorionic somatomammotropin genes (hCS-L, hCS-A, and hCS-B) and hGH-V, are expressed selectively in the placenta. In contrast, the mouse genome contains a single pituitary-specific GH gene and lacks any GH-related CS genes. Activation of the hGH transgene in the mouse is dependent on its linkage to a previously described locus control region (LCR) located -15 to -32 kilobases upstream of the hGH cluster. The sporadic, nonreproducible expression of hCS transgenes lacking the LCR suggests that they may be dependent on hGH LCR activity as well. To determine whether the hCS genes could be expressed with appropriate placental specificity, a series of five transgenic mouse lines carrying an 87-kilobase human genomic insert encompassing the majority of the hGH gene cluster and the entire contiguous LCR was established. All of the hGH cluster genes were appropriately expressed in each of these lines. High level expression of hGH was restricted to the pituitary and hCS to the labyrinthine layer of the placenta. The expression of the GH cluster genes in their respective tissues paralleled transgene copy numbers irrespective of the transgene insertion site in the host mouse genome. These studies have extended the utility of the transgenic mouse model for the analysis of the full spectrum of hGH gene cluster activation. Further, they support a role for the hGH LCR in placental hCS, as well as pituitary hGH gene activation, and expression.     

N-glycosylated variants of growth hormone in human pituitary extracts

This study was designed to investigate the existence, in human pituitary extracts, of growth hormone (GH) variants not encoded by the hGH-N gene. Using anion exchange-fast protein liquid chromatography followed by SDS-PAGE, we isolated several basic forms of pituitary GH. Incubation of these basic forms with endoglycosidase F/N-glycosidase F revealed that two of them (about 34 and 12 kD) were N-glycosylated. In contrast, no changes were found when samples were incubated with the O-linked glycosylation-specific O-glycosidase. Since the GH-N molecule lacks consensus sequences for N-linked glycosylation, our findings suggest that GH genes other than hGH-N are expressed in the human pituitary gland.