A novel, highly regulated, rapidly inducible system for the expression of chicken progesterone receptor, cPRA, in Saccharomyces cerevisiae

A rapidly inducible and tightly regulated system for the expression of protein in yeast is based on a chimeric promoter constructed of two copies of a vitellogenin-estrogen-response element (ERE) which are inserted upstream from the promoter of the yeast gene encoding iso-1-cytochrome c. The chimeric promoter was inserted in a yeast expression plasmid upstream from the coding sequence of ubiquitin fused in frame to a cDNA encoding the full-length chicken progesterone receptor A (cPRA). The resultant plasmid (YEpA2) was co-transformed in Saccharomyces cerevisiae with a plasmid which encodes the human estrogen receptor. Estradiol (E2)-induced transactivation of the chimeric promoter results in transcription of the cPRA gene from YEpA2, and synthesis of cPRA. The fusion protein, ubiquitin-cPRA, is rapidly cleaved in vivo to produce cPRA. Analysis of samples by Western immunoblot shows that cPRA is almost undetectable in the absence of E2, and that treatment with 50 nM E2 results in a 500-1000-fold induction of cPRA (0.06-0.3% of the total protein) after 1 h. The plasmid-expressed soluble receptor is stable and demonstrates the correct affinity for its ligand. We have prepared yeast extracts using enzymatic digestion of the cell wall with oxalyticase followed by hypotonic shock. This has resulted in a dramatic increase in the % of receptor which binds hormone compared to previous studies which used mechanical disruption techniques. The cPRA is biologically active since it activates transcription of a co-transformed reporter gene containing its response element.(ABSTRACT TRUNCATED AT 250 WORDS

Specificity of thyroid hormone receptor subtype and steroid receptor coactivator-1 on thyroid hormone action

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Haploinsufficiency of the corepressor of estrogen receptor activity (REA) enhances estrogen receptor function in the mammary gland

Estrogen receptor (ER)-mediated gene expression plays an essential role in mammary gland morphogenesis, function, and carcinogenesis. The repressor of ER activity (REA) is an ER-interactive protein that counterbalances estrogen-induced ER transcriptional activity. Our previous study showed that genetic deletion of both REA alleles resulted in embryonic lethality. This study demonstrates that REA and ERalpha are coexpressed in mammary epithelial cells. REA heterozygous (REA(+/-)) mutant mice exhibit faster mammary ductal elongation in virgin animals, increased lobuloalveolar development during pregnancy, and delayed mammary gland involution after weaning. These morphological phenotypes of REA(+/-) mice are associated with significantly increased cell proliferation and ER transcriptional activities, as indicated by the estrogen response element (ERE)-luciferase reporter in the WT/ERE-Luc and REA(+/-)/ERE-Luc bigenic mice and by the higher expression levels of estrogen-responsive genes such as progesterone receptor and cyclin D1 in the mammary gland. Our analysis also revealed that REA is an important repressor of ER transcriptional activity in the mammary gland under natural, as well as ovariectomized and estrogen-replaced, hormonal conditions. Our results indicate that REA is a physiological modulator of ER function in the mammary gland and that its correct gene dosage is required for maintenance of normal ER activity and normal mammary gland development. Consequently, a reduction or loss of REA function may cause overactivation of ER and increase breast cancer risk in human

Gonadotropin-releasing hormone-regulated prohibitin mediates apoptosis of the gonadotrope cells

10.1210/me.2013-1210Molecular Endocrinology27111856-1870MOEN

Regulation of expression of thyroid hormone receptor isoforms and coactivators in liver and heart by thyroid hormone

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