Dynamic in Vivo Binding of STAT5 to Growth Hormone-Regulated Genes in Intact Rat Liver. Sex-Specific Binding at Low- But Not High-Affinity STAT5 Sites

Abstract

Phylogenetic footprinting was used to predict functional transcription factor binding sites (TFBS) for signal transducer and activator of transcription (STAT) 5, a GH-activated transcription factor, in the GH-responsive genes IGF-I, SOCS2, and HNF6. Each gene, including upstream (100 kb) and downstream regions (25 kb), was aligned across four species and searched for conserved STAT5-binding sites using TFBS matrices. Predicted sites were classified as paired or single and whether or not they matched the STAT5 consensus sequence TTCN(3)GAA. Fifty-seven of the predicted genomic regions were assayed by chromatin immunoprecipitation from male rat liver with high STAT5 activity. STAT5 binding was enriched (up to 24-fold) at eight genomic regions of IGF-I, including three novel regions in the second intron, and at four regions of SOCS2, including three novel upstream sites. STAT5 binding to HNF6 was modestly enriched (up to 3-fold) at one consensus site and two novel, nonconsensus sites. Overall, 14 of 17 identified sites were paired STAT5 sites. STAT5 binding to these sites was dynamic in male rat liver, cycling on and off in response to each plasma GH pulse. Moreover, sex-specific STAT5 binding was apparent; in female rat liver, where nuclear STAT5 activity is generally low, STAT5 binding to IGF-I and SOCS2 was limited to high-affinity sites. Analysis of the verified STAT5 binding sites indicated that STAT5 TFBS matrix 459 in combination with a STAT5 consensus sequence was the best predictor of STAT5 binding to these three genes. Using these criteria, multiple novel STAT5 binding sites were identified and then verified in several other GH-inducible genes, including MUP genes, where male-specific gene expression was associated with male-specific STAT5 binding to multiple low-affinity STAT5 sites.

Dispersed Chromosomal Stat5b-binding Elements Mediate Growth Hormone-activated Insulin-like Growth Factor-I Gene Transcription

“…In further support of this hypothesis, two distinct GH-inducible Stat5b binding domains have been mapped to chromatin in human IGF1 and rat Igf1 loci (28 -30), and both appear able to act as GH-and Stat5b-dependent regulators of IGF-I promoter function in cultured cells (28,30). As results of more recent experiments have suggested the potential existence of multiple Stat5b-binding elements in the IGF-I gene (31,32), we sought to identify and characterize putative GH-regulated and Stat5b-dependent chromosomal enhancers responsible for GH-activated IGF-I gene transcription. Our results show that GH acutely stimulates recruitment of Stat5b to a least seven distinct chromosomal domains found throughout the Igf1 locus coincident with induction of IGF-I gene transcription and that each can function in vitro to augment IGF-I promoter activity.…”

“…Locus-Since our discovery of HS7 as a GH-inducible Stat5b-binding element in IGF-I intron 2, and our demonstration that it could activate GH-mediated IGF-I gene transcription in transgenic mice and in tissue culture (28,37), several other putative Stat5b-binding sites have been mapped to the Igf1 locus (29,31,32), although only one other region, termed 5Ј distal, has been found to regulate IGF-I promoter activity in reconsti- tution experiments in cultured cells (29,30). We thus devised a multistep approach combining genomics, in vivo protein-DNA binding assays in hepatic chromatin, and functional studies to identify and characterize putative Stat5b-binding sequences, including assessing their ability to regulate IGF-I gene expression.…”

Gender-specific postnatal demethylation and establishment of epigenetic memory

“…3C) that are unique to nonpromoter regulatory sites (Supplemental Fig. S3A), with some of these known to be associated with sexual dimorphism in the liver (Laz et al 2009). Furthermore, by using previously published data (Zhang et al 2012;Kosters et al 2013), we were able to demonstrate that some of these factors actually bind physically to their target sequences but only in the male liver, (Fig.…”

Identifying growth hormone-regulated enhancers in the Igf1 locus

“…Analogous regions have been identified near the mouse Igf1 gene (17,26) and to a far more limited extent near human IGF-I (55). The most remarkable feature of these chromosomal regions, perhaps besides their dynamic plasticity in chromatin, is that there are so many of them.…”

“…Unlike other GH-activated and Stat5b-dependent genes, in which critical transcriptional regulatory elements are located within the proximal promoters (12,61), there are no Stat5b binding sites near either of the two IGF-I gene promoters (12). Although several dispersed GHinducible STAT5b elements have been mapped in human IGF-I and rodent Igf1 gene chromatin (10,12,26,55,60), it is unknown whether these binding domains or others function as potential transcriptional enhancers (11,43).…”