CCAAT/enhancer binding protein beta2 is involved in growth hormone-regulated insulin-like growth factor-II gene expression in the liver of rainbow trout (Oncorhynchus mykiss).
Previously, we showed that levels of different CCAAT/enhancer binding protein (C/EBP) mRNAs in the liver of rainbow trout were modulated by GH and suggested that C/EBPs might be involved in GH-induced IGF-II gene expression. As a step toward further investigation, we have developed monospecific polyclonal antibodies to detect rainbow trout C/EBPalpha, -beta1, -beta2, and -delta2 isoform proteins. Injection of GH into adult rainbow trout resulted in a significant increase of C/EBPbeta1, C/EBPbeta2, and C/EBPdelta2 proteins in the liver. Chromatin immunoprecipitation analysis revealed that C/EBPbeta2 binds to multiple sites at the 5' promoter/regulatory region, introns, and the 3' untranslated region of the IGF-II gene. GH treatment reduced C/EBPbeta2 binding to several of these regions at 6 h after injection. The decreased occupancy of C/EBPbeta2 coincided well with an increase of histone H4 acetylation at the proximal promoter and elevation of the IGF-II mRNA level. Immunoblotting analysis showed that C/EBPbeta2 existed predominately as a truncated form in the liver, and cotransfection analysis further showed that the truncated C/EBPbeta2 acted as a negative regulator on IGF-II proximal promoter. GH treatment caused deacetylation of C/EBPbeta2 in the liver. In addition, we observed a GH-dependent interaction of C/EBPbeta2 with a complex involving histone H1. All together, these results suggest that C/EBPbeta2 was regulated at multiple levels by GH, and C/EBPbeta2 may play a suppressive role in mediating GH-induced IGF-II expression in the liver of rainbow trout.
Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269, USA.
Jay H
JH
Lo
Thomas T
TT
Chen
D000107:Acetylation / Q000187:drug effects; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D029941:Fish Proteins / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D013006:Growth Hormone / Q000494:pharmacology*; D006657:Histones / Q000378:metabolism; D015151:Immunoblotting; D007335:Insulin-Like Growth Factor II / Q000235:genetics*; D008099:Liver / Q000187:drug effects* / Q000378:metabolism; D017686:Oncorhynchus mykiss / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D022782:CCAAT-Enhancer-Binding Protein-beta; D029941:Fish Proteins; D006657:Histones; D007335:Insulin-Like Growth Factor II; D013006:Growth Hormone