Progress 07/01/01 to 12/31/04
Outputs
This project contained two objectives. The first objective was to determine the role of a putative transcription factor HNF-4a in the regulation of the liver-specific growth hormone receptor promoter 1 (GHR P1), which transcribes GHR 1A mRNA in cattle; the second objective was to identify additional transcriptional factors interacting with the bovine GHR P1. Both objectives have been successfully completed. In objective 1, using electrophoretic mobility shift assays, transient transfection analyses, and ribonuclease protection assays, we have obtained evidence strongly supporting a role of HNF-4a in activating GHR P1 and hence GHR 1A mRNA expression in the liver of adult cattle (Jiang and Lucy, 2001). In objective 2, using the yeast one-hybrid system, we have identified chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) and hepatocyte nuclear factor 4 gamma (HNF-4g) as two additional binding proteins to the GHR P1 -196/-178 region, which was
initially identified as a HNF-4a binding site. Electrophoretic mobility shift assays confirm the binding of HNF-4g and COUP-TFII to this GHR P1 DNA region in vitro. Chromatin immunoprecipitation assays (ChIP) further demonstrate that HNF-4g and COUP-TFII as well as HNF-4a also bind to this GHR promoter region in vivo. Cotransfection analyses suggest that binding of each of these three transcription factors to the GHR P1 -196/-178 region activates GHR P1 (Xu et al., 2004). We have also found that food deprivation-caused decreases in GHR 1A mRNA expression in the liver of steers are not accompanied by decreased expression of HNF-4a, HNF-4g or COUP-TFII mRNA in the liver (Wang et al., 2003; Jiang et al., unpublished data). Interestingly, parturition-associated decreases in GHR 1A mRNA expression in the liver of dairy cows are associated with increased expression of HNF-4g mRNA (Jiang et al., unpublished data). These results indicate that HNF-4a, HNF-4g and COUP-TFII are transcription
factors interacting with bovine GHR P1 at a common DNA element and that these transcription factors may play a role in regulating GHR P1 activity and GHR 1A mRNA expression under different physiological conditions through yet unclear mechanisms.
Impacts
Growth hormone plays a central role in controlling growth and milk production in cattle. The results of this project suggest that HNF-4a, HNF-4g and COUP-TFII are also important genes controlling cattle growth and milk production because they regulate growth hormone receptor gene expression. The role of these genes in cattle growth and lactation should be further studied and the study could lead to the identification of cattle with better genetic potential for growth and (or) lactation or to the development of novel strategies to enhance growth and lactation in cattle.
Publications
- Xu, Q., Walther, N. and Jiang, H. 2004. Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) and hepatocyte nuclear factor 4g (HNF-4g) and HNF-4a regulate the bovine growth hormone receptor 1A promoter through a common DNA element. Journal of Molecular Endocrinology. 32:947-961.
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Progress 10/01/02 to 09/30/03
Outputs
The original objectives of this project were to determine whether transcription factor hepatocyte-4 (HNF-4) is involved in the regulation of the liver-specific growth hormone receptor promoter 1 (GHR P1) in cattle and to identify additional transcription factors for GHR P1. Our work during 10/2002-09/2003 was focused on the second objective. Using the -218/-151 region of the GHR P1 as bait to screen a bovine liver cDNA library in a yeast one-hybrid analysis, we identified chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) and HNF-4g as well as HNF-4a as potential binding proteins to the GHR -218/-151 region. These three factors were also identified as binding proteins in a second yeast one-hybrid analysis, in which the bait was the GHR -196/-178 region that had previously been mapped as the binding site for HNF-4a. Electrophoretic mobility shift assays confirmed the interactions of these three factors in bovine liver nuclear extracts with the GHR
-196/-178 region. Ribonuclease protection assays revealed that HNF-4g, HNF-4a and COUP-TFII mRNAs were expressed at high levels in the liver. In transient transfection analyses, each transcription factor enhanced GHR 1A promoter activity through binding to the -196/-178 region. These results together suggest that COUP-TFII, HNF-4g and HNF-4a may regulate GHR P1 activity through binding to a common DNA element.
Impacts
Growth hormone and growth hormone receptor are major regulators of growth, metabolism and milk production in cattle. This project is aimed to increase our understanding of how growth hormone receptor gene expression in cattle is regulated. A better understanding of the regulation of growth hormone receptor gene expression will provide a basis for developing novel biotechnical or genetic approaches to increase meat or milk production or productive efficiency.
Publications
- Wang Y, Eleswarapu S, Beal WE, Swecker WS, Akers RM, and Jiang H 2003 Reduced serum insulin-like growth factor (IGF) I is associated with reduced liver IGF-I mRNA and liver growth hormone receptor mRNA in food-deprived cattle. Journal of Nutrition. 133:2555-2560.
- Jiang H, Lucy MC, and Xu Q 2003 The bovine growth hormone receptor promoter 1 is positively regulated by hepatocyte nuclear factor 4gamma via the same element for hepatocyte nuclear factor 4alpha. Journal of Animal Science. 81(Supplement 1): 312.
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Progress 10/01/01 to 09/30/02
Outputs
This project had two original objectives. The first objective was to determine whether transcription factor hepatocyte nuclear factor-4 (HNF-4) is involved in the regulation of the liver-specific growth hormone receptor promoter 1 (GHR P1) in cattle; the second objective was to identify additional transcriptional factors for GHR P1 and also the second growth hormone receptor promoter named GHR P2. Since the beginning of this project, we have cloned the bovine HNF-4 cDNA and compared its expression levels between different tissues and between different developmental stages in cattle. Our results suggest that HNF-4 plays a role in liver- and postnatal stage-specific activation of GHR P1 in cattle but that HNF-4 alone is not sufficient to activate this promoter in the liver of postnatal cattle. We have also done some work for the second objective. Using yeast-one hybrid system, we have identified several candidate transcription factors for GHR P1 or GHR P2. We are
currently testing the functional importance of these candidate transcription factors in regulating the activity of GHR P1 or GHR P2.
Impacts
Growth hormone plays an important role in animal growth and metabolism and its action is intimately linked to the expression level of its specific receptor, growth hormone receptor. The results of this project will increase our understanding of how the expression of the growth hormone receptor gene is regulated in animals and this understanding will help to develop new biotechnologies to increase meat production to meet the nutritional needs of a rapidly increasing world population and to improve meat quality (e.g., reducing fat content) to meet the needs of people who wish to eat healthier.
Publications
- Jiang H and Lucy MC. 2001. Involvement of hepatocyte nuclear factor-4 in the expression of the growth hormone receptor 1A messenger ribonucleic acid in bovine liver. Mol Endocrinol. 15:1023-1034.
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