Specification of unique Pit-1 activity in the hGH locus control region

Abstract

The human GH (hGH) gene cluster is regulated by a remote 5 locus control region (LCR). HSI, an LCR component located 14.5 kb 5 to the hGH-N promoter, constitutes the primary determinant of highlevel hGH-N activation in pituitary somatotropes. HSI encompasses an array of three binding sites for the pituitary-specific POU homeodomain factor Pit-1. In the present report we demonstrate that all three Pit-1 sites in the HSI array contribute to LCR activity in vivo. Furthermore, these three sites as a unit are fully sufficient for position-independent and somatotrope-restricted hGH-N transgene activation. In contrast, the hGH-N transgene is not activated by Pit-1 sites native to either the hGH-N or rat (r)GH gene promoters. These findings suggest that the structures of the Pit-1 binding sites at HSI specify distinct chromatin-dependent activities essential for LCR-mediated activation of hGH in the developing pituitary.T he expression of the GH gene has been the focus of intense study over the past 20 years. A consistent feature of all GH promoters is a highly conserved pair of AT-rich binding sites for the pituitary-restricted trans factor Pit-1 (1, 2). Between these two Pit-1 elements is a conserved binding site for a ubiquitous DNA-binding protein, Zn15 (3). Additional, less conserved cis elements have been identified in the vicinity of the GH promoter in various species. Studies carried out in vitro and in celltransfection models indicate that the Pit-1 binding sites are essential to the expression of the GH gene (4-6). However, studies of human (h)GH gene expression in transgenic settings demonstrate that promoter-proximal elements including the Pit-1 sites are not sufficient to activate hGH gene expression in vivo. For example, in transgenic mice, the hGH gene with its full promoter and as much as 7.5 kb of 5Ј-flanking sequences is either not expressed or expressed at very low levels and does not maintain somatotrope cell specificity (7,8). These data suggest that regulatory sequences quite distant from the hGH gene are necessary to establish a transcriptionally active chromatin domain.The hGH gene cluster spans 48 kb on chromosome 17q22-24 (for details see Fig. 1; ref. 9). This cluster contains the pituitaryexpressed hGH-N and four genes expressed in the placenta:, and hCS-B (Fig. 1). A search for distal regulatory determinants of hGH-N gene expression revealed DNaseI hypersensitive sites (HSs) in pituitary chromatin and a partially overlapping set of HSs in placental chromatin (ref. 8; Fig. 1). HSI and HSII, located between Ϫ14.5 kb and Ϫ15 kb relative to the hGH-N promoter, are specific to pituitary chromatin. HSIV, at Ϫ30 kb, is specific to the placenta and HSIII and HSV, at Ϫ27.5 and Ϫ32 kb, respectively, are shared in both expressing tissues. The five HSs together comprise a putative hGH locus control region (LCR). Significantly, when hGH-N transgene DNA is extended to encompass the entire set of HSs, hGH gene expression is rendered pituitary-specific, copy number-dependent, and robust in every line generat...

Molecular Physiology of Pituitary Development: Signaling and Transcriptional Networks

“…All three Pit1 binding sites within the 404-bp fragment contribute to the LCR activity. Furthermore, these three Pit1 sites are sufficient to confer position-independent and somatotropes-restricted Ϫ0.5hGH-N transgene activation (261,262). In pituitary gland of transgenic mice carrying the intact hGH locus, ChIP assays reveal a 32-kb domain of pituitary-specific histone hyperacetylation extending from the LCR to the hGH-N promoter with peaks at HSI and HSII sites.…”

A 914-bp promoter is sufficient to reproduce the endogenous prolyl oligopeptidase gene localization in the mouse placenta if not subject to position effect

“…If the transgene contains an insulator activity, it shows site of integration independent and copy-number dependent expression (Kellum and Schedl, 1991;Li et al, 2002b). Alternatively, when the transgene contained a strong enhancer activity, it could be expressed in a site of integration independent manner (Shewchuk et al, 2002). In case of the POP promoter, we could not detect any EGFP expression in four lines out of six (Table 1), which indicated that the transgene expression was dependent on its integration site.…”

“…To analyze the regulatory mechanism of transcription, a reporter gene assay is often used as a powerful technique, and it can uncover the in vitro activity of a regulatory sequence. However, to assess the in vivo activity, transgenic mice have to be generated and analyzed, and the result could be different from that of the reporter gene assay (Shewchuk et al, 2002). This is because a transgene is integrated into the mouse genome and influenced by the adjacent chromosomal environment in transgenic mice.…”

Identifying growth hormone-regulated enhancers in the Igf1 locus

“…Although there are other examples in which several enhancers that bind the same transcription factor are associated with a single target gene, in few cases are these enhancers as prevalent or as dispersed as seen in the Igf1 locus. For example, the mouse Rankl gene encodes some distal elements that bind c-fos (6), the ␤-hemoglobin locus contains sites for GATA transcription factors (35), and the GH locus, sequences for Pou1f1 (47), but these potential enhancers are fewer in number than the Stat5b elements in Igf1. As there appear to be no other genes within ϳ150 kb of rat Igf1 on chromosome 7q22 or within ϳ200 kb of human IGF-I on chromosome 12q23.2 (Ensemble release 78, December 2014), it seems reasonable to assume that these elements are involved primarily with Igf1 [although ENCODE data for several human cell lines indicate that only some identified interactions between distal elements and promoters map to the nearest gene from the element (46)].…”