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Xiaoyang Yang Department of Physiology, University of Manitoba, 745 Bannatyne Avenue, Winnipeg, Manitoba, Canada R3E 0J9

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Yan Jin Department of Physiology, University of Manitoba, 745 Bannatyne Avenue, Winnipeg, Manitoba, Canada R3E 0J9

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Peter A Cattini Department of Physiology, University of Manitoba, 745 Bannatyne Avenue, Winnipeg, Manitoba, Canada R3E 0J9

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Expression of pituitary and placental members of the human GH and chorionic somatomammotropin (CS) gene family is directed by an upstream remote locus control region (LCR). Pituitary-specific expression of GH requires direct binding of Pit-1 (listed as POU1F1 in the HUGO database) to sequences marked by a hypersensitive site (HS) region (HS I/II) 14.6 kb upstream of the GH-N gene (listed as GH1 in the HUGO database). We used human embryonic kidney 293 (HEK293) cells overexpressing wild-type and mutant Pit-1 proteins as a model system to gain insight into the mechanism by which Pit-1 gains access to the GH LCR. Addition of Pit-1 to these cells increased DNA accessibility at HS III, located 28 kb upstream of the human GH-N gene, in a POU homeodomain-dependent manner, as reflected by effects on histone hyperacetylation and RNA polymerase II activity. Direct binding of Pit-1 to HS III sequences is not supported. However, the potential for binding of ETS family members to this region has been demonstrated, and Pit-1 association with this ETS element in HS III sequences requires the POU homeodomain. Also, both ETS1 and ELK1 co-precipitate from human pituitary extracts using two independent sources of Pit-1 antibodies. Finally, overexpression of ELK1 or Pit-1 expression in HEK293 cells increased GH-N RNA levels. However, while ELK1 overexpression also stimulated placental CS RNA levels, the effect of Pit-1 appeared to correlate with ETS factor levels and target GH-N preferentially. These data are consistent with recruitment and an early role for Pit-1 in remodeling of the GH LCR at the constitutively open HS III through protein–protein interaction.

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Aristides Lytras Department of Physiology, University of Manitoba, Room 444, 745 Bannatyne Avenue, Winnipeg, Manitoba, Canada R3E 0J9

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Karen Detillieux Department of Physiology, University of Manitoba, Room 444, 745 Bannatyne Avenue, Winnipeg, Manitoba, Canada R3E 0J9

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Peter A Cattini Department of Physiology, University of Manitoba, Room 444, 745 Bannatyne Avenue, Winnipeg, Manitoba, Canada R3E 0J9

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The human chorionic somatomammotropin (CS) A and B genes (listed as CSH1 and CSH2 in the HUGO database) are highly expressed in placenta. A 241 bp potent enhancer, nucleotides (nts) 1–241, located at the 3′ end of the CS-B gene (CS-Benh) stimulates promoter activity specifically in placental trophoblast cells in vitro. Strong activity is exerted by a 23 bp element within the CS-Benh (nts 117–139), shown to interact with transcription enhancer factor (TEF) members of the transcription enhancer activator (TEA) DNA-binding domain-containing family. An identical TEF element is present in the homologous (97.5%) CS-Aenh; however, a few nucleotide differences suppress its activity. Previously, we identified regulatory sequences distinct from the TEF element within an 80 bp modulatory domain (nts 1–80) in the CS-Benh. Using structural and functional assays we now show that CCAAT/enhancer-binding protein (C/EBP) binding sites exist in the 80 bp modulatory domains of both enhancers, and an Elk-1 binding site exists in the modulatory domain of the CS-Aenh. C/EBPα or C/EBPβ strongly repressed CSp.CAT activity but stimulated CSp.CAT.CS-Benh activity. In contrast, the equivalent CS-A enhancer sequences were unable to relieve promoter repression. Elk-1 overexpression also resulted in differential effects on the CS-Aenh versus CS-Benh. Finally, we provide evidence for the association of C/EBPβ with the CS-A and CS-B genes in human placental chromatin, including differential involvement of C/EBPβ with the CS-Aenh versus the CS-Benh, and therefore consistent with the notion that these are regions of regulatory significance in vivo. We conclude that members of the C/EBP and Ets families can differentially modulate CS-Benh and CS-Aenh activity.

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Yan Jin Department of Physiology and Pathophysiology, University of Manitoba, Winnipeg, Manitoba, Canada

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Jessica S Jarmasz Department of Biochemistry and Medical Genetics, University of Manitoba, Winnipeg, Manitoba, Canada

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Shakila Sultana Department of Physiology and Pathophysiology, University of Manitoba, Winnipeg, Manitoba, Canada

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Luis Cordero-Monroy Canadian Centre for Agri-Food Research in Health and Medicine, St. Boniface Albrechtsen Research Centre, Winnipeg, Manitoba, Canada

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Carla G Taylor Department of Physiology and Pathophysiology, University of Manitoba, Winnipeg, Manitoba, Canada
Canadian Centre for Agri-Food Research in Health and Medicine, St. Boniface Albrechtsen Research Centre, Winnipeg, Manitoba, Canada
Department of Food and Human Nutritional Sciences, University of Manitoba, Winnipeg, Manitoba, Canada

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Peter Zahradka Department of Physiology and Pathophysiology, University of Manitoba, Winnipeg, Manitoba, Canada
Canadian Centre for Agri-Food Research in Health and Medicine, St. Boniface Albrechtsen Research Centre, Winnipeg, Manitoba, Canada

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Elissavet Kardami Department of Human Anatomy and Cell Science, University of Manitoba, Winnipeg, Manitoba, Canada

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Peter A Cattini Department of Physiology and Pathophysiology, University of Manitoba, Winnipeg, Manitoba, Canada

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The objective was to assess the potential differential effects of human versus mouse growth hormone in vivo, given that human unlike mouse growth hormone can bind prolactin as well as the growth hormone receptor. To this end, a transgenic CD-1 mouse expressing human but not mouse growth hormone was generated, and the phenotypes of male mice fed with a regular chow or high-fat diet were assessed. Pancreas and epididymal white adipose tissue gene expression and/or related function were targeted as the pancreas responds to both prolactin and growth hormone receptor signaling, and catabolic effects like lipolytic activity are more directly attributable to growth hormone and growth hormone receptor signaling. The resulting human growth hormone-expressing mice are smaller than wild-type CD-1 mice, despite higher body fat and larger adipocytes, but both mouse types grow at the same rate with similar bone densities. Unlike wild-type mice, there was no significant delay in glucose clearance in human growth hormone-expressing mice when assessed at 8 versus 24 weeks on a high-fat diet. However, both mouse types showed signs of hepatic steatosis that correlated with elevated prolactin but not growth hormone RNA levels. The larger adipocytes in human growth hormone-expressing mice were associated with modified leptin (higher) and adiponectin (lower) RNA levels. Thus, while limited to observations in the male, the human growth hormone-expressing mice exhibit signs of growth hormone insufficiency and adipocyte dysfunction as well as an initial resistance to the negative effects of high-fat diet on glucose clearance.

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