Dataset Information

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.

SUBMITTER: Laz EV

PROVIDER: S-EPMC2718744 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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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.

Laz Ekaterina V EV Sugathan Aarathi A Waxman David J DJ

Molecular endocrinology (Baltimore, Md.) 20090507 8

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 mat ...[more]

PMID: 19423653

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Project description:Sex-dependent pituitary growth hormone (GH) secretory patterns determine the sex-biased expression of >1,000 genes in mouse and rat liver, affecting lipid and drug metabolism, inflammation and disease. A fundamental biological question is how robust differential expression can be achieved for hundreds of sex-biased genes simply based on the GH input signal pattern: pulsatile GH stimulation in males vs. near-continuous GH exposure in females. STAT5 is an essential transcriptional mediator of the sex-dependent effects of GH in the liver, but the mechanisms that underlie its sex-dependent actions are obscure. Here we elucidate the dynamic, sex-dependent binding of STAT5 and the GH/STAT5-regulated repressor BCL6 to mouse liver chromatin, revealing the counteractive interplay between these two regulators of liver sex-specificity. Our findings establish a close correlation between sex-dependent STAT5 binding and sex-biased target gene expression. Moreover, sex-dependent STAT5 binding correlated positively with sex-biased DNase hypersensitivity and H3-K4me1 and H3-K4me3 (activating) marks, correlated negatively with sex-biased H3-K27me3 (repressive) marks, and was associated with sex-differentially enriched motifs for HNF6/CDP factors. Importantly, BCL6 binding was preferentially associated with repression of female-biased STAT5 targets in male liver. Furthermore, BCL6 and STAT5 common targets but not BCL6 unique targets showed strong enrichment for lipid and drug metabolism. These findings provide a comprehensive, genome-wide view of the mechanisms whereby these two GH-regulated transcription factors establish and maintain sex differences affecting liver physiology and disease. The approaches used here to characterize sex-dependent STAT5 and BCL6 binding can be applied to other condition-specific regulatory factors and binding sites and their interplay with co-operative chromatin-binding factors. Mouse livers were excised from individual male and female mice killed at either a peak of STAT5 binding activity, or during the growth hormone (GH) interpulse interval, when STAT5 activity is either low (females) or essentially undetectable (males). Sonicated, cross-linked liver nuclear chromatin was then used to identify STAT5 binding sites by ChIP-Seq.

Project description:Pituitary GH-secretory profiles are sex dependent and regulate the sexually dimorphic expression of a large number of genes in the liver. The slow response of many sex-specific liver genes to changes in plasma GH status suggests that GH acts in the liver via both direct and indirect mechanisms organized in a hierarchical regulatory network. Presently, genome-wide liver transcription profiling was conducted to elucidate the global impact of pituitary hormone ablation on the sex specificity of rat liver gene expression and to identify sex-specific genes that respond rapidly to GH as candidates for direct targets of GH action. Hypophysectomy abolished the sex specificity of approximately 90% of 1032 sex-dependent genes, consistent with the dominant role of pituitary GH in regulating liver sexual dimorphism. Two major classes of sex-specific genes were identified: genes that were down-regulated after hypophysectomy and may be subject to positive GH regulation (461 class I genes), and genes that were up-regulated after hypophysectomy and may be subject to negative GH regulation (224 class II genes). Fifty class I sex-specific genes were induced, and 38 class II sex-specific genes were suppressed within 90 min of a physiological GH pulse, suggesting they are primary GH response genes. A further 71 sex-specific genes responded after a second GH treatment and may correspond to secondary response genes. Twenty four DNA-binding proteins were identified as early GH response genes, of which 15 were induced and nine were suppressed by GH. Five of these 24 genes displayed sex-specific expression, consistent with a hierarchical transcriptional network controlling sex-specific liver gene expression. Class II male-specific genes, such as Cyp2a2 and Cyp2c13, were down-regulated within 30 min of GH pulse treatment, as determined by heterogeneous nuclear RNA analysis, suggesting that transcription of these genes is restricted to the GH-free interpulse period in adult male rat liver. We conclude that GH acts via both positive and negative regulatory mechanisms to establish and maintain the sex specificity of liver gene expression.

Dataset Information

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.

Publications

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.

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