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.
Highlights
Glucocorticoids (GCs) are involved in the regulation of almost all the biological processes ranging from growth and development to immunity and reproduction [1,2]
Binding of p-cAMP response element binding protein (CREB) and glucocorticoid receptor (GR) to GR exon 1–4 and 1–5 promoters was breed-specific, which was associated with breed difference in histone H3 acetylation (H3Ac) We identified one CREB and one specificity protein 1 (Sp1) binding motif in promoter 1–4, and one CREB, two Sp1 binding sites in promoter 1–5. 59-GGTACAnnnTGTTCT-39 has been recognized as a positively modulated glucocorticoid responsive element (GRE) [23], whereas the consensus sequence 59-tggacg-39 was suggested as negatively modulated glucocorticoid responsive element [12,24]
Our results agree with the previous notion that GR promoter is highly conserved among mammalian species studied, including human [4], rat [21], mice [26], etc
Summary
Glucocorticoids (GCs) are involved in the regulation of almost all the biological processes ranging from growth and development to immunity and reproduction [1,2]. The biological functions of GCs require the functionality of their intracellular binding protein, glucocorticoid receptor (GR) which is known as nuclear receptor subfamily 3, group C, member 1 (NR3C1). Processes that regulate the expression of GR gene are critical to the maintenance of appropriate functions of glucocorticoids. The 59-heterogeneity of GR mRNA transcripts and promoter usage represent complex mechanisms for the regulation of GR transcription in different tissues under different conditions. The proximal GR promoter comprises of a CpG island which shows high sequence homology between rats and humans [5]
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