Breed-Dependent Transcriptional Regulation of 5′-Untranslated GR (NR3C1) Exon 1 mRNA Variants in the Liver of Newborn Piglets

Abstract

Glucocorticoids are vital for life and regulate an array of physiological functions by binding to the ubiquitously expressed glucocorticoid receptor (GR, also known as NR3C1). Previous studies demonstrate striking breed differences in plasma cortisol levels in pigs. However, investigation into the breed-dependent GR transcriptional regulation is hampered by lacking porcine GR promoter information. In this study, we sequenced 5.3 kb upstream of the translation start codon of the porcine GR gene, and identified seven alternative 5′-untranslated exons 1–4, 1–5, 1–6, 1–7, 1–8, 1–9,10 and 1–11. Among all these mRNA variants, exons 1–4 and 1–5, as well as the total GR were expressed significantly (P<0.05) higher in the liver of newborn piglets of Large White (LW) compared with Erhualian, a Chinese indigenous breed. Overall level of CpG methylation in the region flanking exons 1–4 and 1–5 did not show breed difference. However, nuclear content of Sp1, p-CREB and GR in the liver was significantly (P<0.05) higher in LW piglets, associated with enhanced binding of p-CREB, and higher level of histone H3 acetylation in 1–4 and 1–5 promoters. In contrast, GR binding to promoters of exons 1–4 and 1–5 was significantly diminished in LW piglets, implicating the presence of negative GREs. These results indicate that the difference in the hepatic expression of GR transcript variants between two breeds of pigs is determined, at least partly, by the disparity in the binding of transcription factors and the enrichment of histone H3 acetylation to the promoters.

Breed-specific expression of GR exon 1 mRNA variants and profile of GR promoter CpG methylation in the hippocampus of newborn piglets

“…However, we observed a positive correlation between serum cortisol level and the relative hippocampal weight in neonatal piglets. EHL piglets, which had two-folds higher serum cortisol level (Zou et al, 2012), demonstrated significantly higher hippocampus weight relative to BW as compared with LW piglets. It is noted that the higher serum cortisol level in EHL pigs is a breed characteristic that is evolved over generations of domestication and selection for this breed.…”

“…GR is expressed in a tissue-specific manner with multiple GR exon 1 mRNA variants in human (Turner et al, 2006), rodents (Yau et al, 2004) and pigs (Zou et al, 2012).…”

“…Western blotting analysis for total GR protein Total protein was extracted from hippocampus samples as previously described (Zou et al, 2012). The concentrations were determined with a Pierce BCA Protein Assay kit (23225; Thermo, Rockford, USA).…”

“…Previous studies described the breed-dependent pattern of total GR expression in hippocampus (Wei et al, 2010), liver and muscle (Li et al, 2009) in pigs showing different stress coping style, hepatic gluconeogenesis, and intramuscular fat deposition, respectively. Recently, we cloned a 5.3 kb proximal promoter sequence of porcine GR gene, and identified seven un-translated alternative first exons each preceded by a distinct promoter (Zou et al, 2012). Transcript variants of the porcine GR gene were reported (Reyer et al, 2013), and GR exon 1 to 4 and 1 to 5 transcripts were found to be differentially expressed in the liver between Large White (LW) and Erhualian (EHL) pigs (Zou et al, 2012).…”

“…Recently, we cloned a 5.3 kb proximal promoter sequence of porcine GR gene, and identified seven un-translated alternative first exons each preceded by a distinct promoter (Zou et al, 2012). Transcript variants of the porcine GR gene were reported (Reyer et al, 2013), and GR exon 1 to 4 and 1 to 5 transcripts were found to be differentially expressed in the liver between Large White (LW) and Erhualian (EHL) pigs (Zou et al, 2012). However, the breed-dependent expression of GR and its 5′ exon 1 mRNA variants in porcine hippocampus has not been described.…”