Analysis of DNA sequences required for pituitary-specific expression of the glycoprotein hormone alpha-subunit gene.

Abstract

Transient transfection studies have been used to determine the DNA sequences of the glycoprotein hormone alpha-subunit gene that are required for tissue-specific expression. In the initial phase of these studies, a variant mouse alpha gene was identified which contains a fully palindromic cAMP response element (CRE). The corresponding region of a previously cloned and sequenced mouse alpha gene contains a single point mutation that disrupts the symmetrical nature of this element. DNase footprint studies demonstrate that the fully palindromic CRE binds the CRE-binding protein with much higher affinity than the imperfect palindrome. Transfection experiments using both mouse alpha gene variants demonstrate differences in basal and cAMP-induced expression. Studies of the cAMP response of the human alpha gene indicated that this gene contains sequences other than the known CRE that are sufficient to permit a transcriptional response to cAMP in both placental and pituitary cells. Expression of human and mouse alpha-subunit genes has been examined in cells of the gonadotrope, thyrotrope, and trophoblast lineages to identify DNA sequences that mediate selective transcription of the alpha gene in these cells. The results demonstrate that sequences between about -500 and -200 are important for expression in the pituitary, but not the placenta. Clustered point mutations were used to further characterize sequences required for expression in the pituitary. Two regions, one at positions -445 to -438 and one at positions -337 to -330, were required for expression in cells of the gonadotrope lineage. One of these regions, at -337 to -330, is also important for expression in thyrotropes. When linked to a minimal promoter, multiple copies of the -344 to -300 region had transcriptional enhancer activity in gonadotropes and thyrotropes, but not in several other cell types. These results are consistent with a model involving different combinations of regulatory elements that determine cell-specific alpha expression in gonadotropes and thyrotropes.

The cAMP Response Elements of the α Subunit Gene Bind Similar Proteins in Trophoblasts and Gonadotropes but Have Distinct Functional Sequence Requirements

“…By exchanging the palindromic CREs of the human ␣ subunit promoter with a species variant of the element, we demonstrated that the sequence requirements for this element are different between the two cell types, an observation supported by a number of earlier findings (7,15,26). Together, these reports support the conclusion that the variant CRE is compatible with expression of the ␣ subunit gene in gonadotropes but not in trophoblasts.…”

“…Earlier studies using DNase footprinting or EMSA disclosed that the ␣ subunit CREs are occupied by proteins in both trophoblasts and gonadotropes, but the proteins were not identified (8,5,12,15,16,21). Steger et al (29) showed that purified CREB binds to the human ␣ subunit promoter and, with much lower affinity, to the mouse and equine ␣ subunit promoters.…”

“…These include the CREs, a specific sub-region in the URE, as well as the gonadotrope-specific element (GSE), the pituitary glycoprotein hormone basal element (PGBE), and the ␣ basal element (␣BE, Fig. 1B) (15)(16)(17). Mutational analysis of multiple response elements suggested that interactions occur between proteins binding PGBE, ␣BE, and the CREs (17).…”

Activation of the glycoprotein hormone alpha-subunit promoter by a LIM-homeodomain transcription factor.

“…Recently, we identified DNA sequences which are important for pituitary-specific expression of the mouse a promoter (43,44). These studies found that sequences between -344 to -300 were sufficient and necessary to direct maximal expression of the mouse a-subunit promoter to cells of the gonadotrope and thyrotrope lineages.…”

Lentiviral vectors efficiently transduce human gonadotroph and somatotroph adenomas in vitro. Targeted expression of transgene by pituitary hormone promoters

“…However, a shorter fragment ( 500 bp) seemed to be sufficient for full expression of the human -subunit gene in pituitary cell lines: the basal expression was not affected by deletion from 1800 to 442 bp upstream of the transcription start site of the gene (Horn et al 1992), but expression was reduced with loss of promoter sequences between -442 and -391 bp (Schoderbek et al 1992). This part of the promoter contains a pituitary glycoprotein hormone basal element (PGBE) which is able to function as a thyrotroph/ gonadotroph-specific enhancer (Aylwin & Burin 1995).…”