Biochemical characterization of diverse Stat5b-binding enhancers that mediate growth hormone-activated insulin-like growth factor-I gene transcription.

Many of the biological effects of growth hormone (GH) are mediated by insulin-like growth factor I (IGF-I), a 70-amino acid secreted peptide whose gene expression is rapidly induced by GH via the Stat5b transcription factor. We previously identified multiple evolutionarily conserved GH-activated chromosomal binding domains for Stat5b within the rat Igf1 locus, and proposed that they could regulate IGF-I gene activity. Here we investigate the biochemical and functional characteristics of these putative long-range transcriptional enhancers. Each element contained 2 or 3 individual Stat5b recognition sequences that could bind Stat5b in vitro, but with affinities that varied over a >100-fold range. Full transcriptional responsiveness to GH required that all Stat5b sites be intact within an individual enhancer. Replacement of a single lower-affinity Stat5b sequence with a higher-affinity one increased in vitro binding of Stat5b, and boosted transcriptional potency of the entire element to GH. As enhanced transcriptional activity involved changes in only one or two nucleotides within an enhancer DNA segment, there appears to be remarkable specificity and sensitivity in the ability of Stat5b to transform DNA binding activity into transcriptional function. Stat5b was able to stimulate the transcriptional activity of two enhancers in the absence of GH, indicating that individual Stat5b-regulated elements possess distinct functional features. We conclude that combinatorial interplay among multiple Stat5b-binding response elements with distinguishable biochemical properties is responsible for highly regulated control of IGF-I gene activity by GH

Stat5b binding sites are required to confer GH-responsiveness to <i>Igf1</i> promoter 2 in promoter-reporter assays.

<p>Results of luciferase assays in Cos-7 cells transiently transfected with reporter plasmids containing <i>Igf1</i> P2 and exon 2, plus wild type (WT) or mutated versions of individual Stat5b binding elements, and expression plasmids encoding the mouse GH receptor and rat Stat5b, and incubated with vehicle (dark bars) or rat GH [40 nM] (light bars) for 18 h. KO signifies knockout of a Stat5b binding site, with DKO representing double knockout and TKO, triple knockout. See ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050278#s2" target="_blank">Materials and Methods</a>’ for details. Bars represent the mean ± S.E. of 4–10 independent experiments (*, p<0.007; **, p<0.0007; #, p<0.017; ##, p<0.0017; ^∧, p<0.013 <i>vs.</i> WT with GH [unpaired t-test]). In each graph, relative luciferase values obtained using the WT Stat5b element in the absence of GH were set to 1. <b>A</b>. R2–4. <b>B</b>. R13. <b>C</b>. R34–35. <b>D</b>. R53–54. <b>E</b>. R57–59. <b>F</b>. R60–61.</p

Stat5b binding elements in the rat <i>Igf1</i> locus confer GH-responsiveness to <i>Igf1</i> promoter 2 in promoter-reporter assays. A

<p>. Diagram of the rat <i>Igf1</i> locus showing seven conserved Stat5b binding elements, R2–4, R8–9, R13, R34–35, R53–54, R57–59, and R60–61. Each element is depicted as a gray oval and the six <i>Igf1</i> exons are shown as black boxes. <b>B</b>. <u>Top</u> - schematic of luciferase reporter plasmids containing rat <i>Igf1</i> promoter 2 (P2) and exon 2, and individual Stat5b binding elements (Enhancer). <u>Bottom</u> - Results of luciferase activity assays in Cos-7 cells transiently transfected with expression plasmids encoding the mouse GH receptor and rat Stat5b and reporter plasmids containing each putative enhancer region diagramed to the <u>left</u> (with each Stat5b site indicated as a white oval, and R13.5 as a gray curved shape), after incubation of cells with vehicle (dark bars) or rat GH [40 nM] (light bars) for 18 h. The graph presents results of 4 independent experiments for each promoter plasmid (mean ± S.E.; *, p<0.01; **, p<0.001 <i>vs.</i> R34–35 without GH [unpaired t-test]). Other p values are indicated, and compare ± GH treatment [paired t-test]. Luciferase counts for R34–35 without GH ranged from 3.5 to 5.5×10³ relative light units/sec.</p

Stat5b differentially regulates the transcriptional activity of individual Stat5b elements in promoter-reporter assays.

<p><b>A</b>. Results of luciferase activity assays in Cos-7 cells transiently transfected with expression plasmids encoding the mouse GH receptor and either wild type (WT), dominant negative (DN), or constitutively active (CA) rat Stat5b, and reporter plasmids containing <i>Igf1</i> P2 and exon 2, and each individual intact or mutated (KO) Stat5b binding element after incubation in serum free medium for 18 h. The graph depicts results of 4 independent experiments for each promoter plasmid comparing Stat5b^CA or Stat5b^DN with Stat5b^WT (mean ± S.E.; *, p<0.025; **, p<0.0025) [unpaired t-test]. The inset shows a higher power view of R57–59. <b>B</b>. Detection of Stat5b, the Flag epitope (for transfected WT, DN, and CA Stat5b), phospho (p) Stat5b, and α-tubulin by immunoblotting in whole cell protein extracts (Con = non-transfected control cells). <b>C</b>. Detection of Flag, pStat5b, Creb and α-tubulin by immunoblotting in nuclear and cytoplasmic protein extracts. <b>D</b>. Immunocytochemistry of Cos7 cells transfected with WT, DN, or CA Stat5b using antibodies to pStat5 (top) or Flag (bottom), after incubation in serum-free medium without GH for 18 h. Nuclei were stained with Hoescht dye (blue).</p

Assessing binding affinity of Stat 5b for individual DNA sites.

<p>Quantitative DNA-protein binding was assessed by gel-mobility shift experiments as described in ‘<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050278#s2" target="_blank">Materials and Methods</a>’ using varying concentrations of Cy5.5-labeled double-stranded oligonucleotides for R58, R34, or R35, and 1 µg of nuclear protein from Cos-7 cells transfected with expression plasmids for the mouse GH receptor and wild-type rat Stat5b, and incubated with rat GH [40 nM] for 1 h. DNA binding was quantified with a LiCoR Odyssey infrared scanner and v3.0 analysis software, and results were plotted as shown. <u>Left panels</u>: representative results from individual experiments using nuclear proteins from cells expressing the mouse GH receptor and wild-type Stat5b after GH treatment. FP = unbound probe. The arrow indicates protein-DNA complexes. <u>Right panels</u>: binding curves with Kds listed (mean ± S.E., n = 3 experiments).</p

Defining a hierarchy of binding affinities of Stat5b for individual DNA sites within the rat <i>Igf1</i> locus. A

<p>. Gel-mobility shift experiments were performed with the Cy5.5-labeled double-stranded probe R34, 2 µg of nuclear protein from Cos-7 cells transfected with expression plasmids for the mouse GH receptor and rat Stat5b, and incubated with rat GH [40 nM] for 1 h, and various concentrations of competitor DNAs as indicated. Two representative individual competition experiments are shown. The arrow indicates the location of protein-DNA complexes (NS, no Stat5b in nuclear protein extract, FP = unbound probe). <b>B</b>. The graph illustrates results of competition experiments for 4 different unlabeled double-stranded competitor DNAs (mean ± S.E., n = 3 independent experiments, with 4 data points/experiment). <b>C</b>. Results for all probes have been tabulated (n = 3 independent experiments, with 4 data points/experiment) and are presented as IC50 values (DNA concentration at which binding of labeled probe is reduced to 50% of starting value). The 95% confidence interval (CI) also is indicated and each Stat5b core DNA binding sequence is listed.</p

DNA Sequences of Oligonucleotide Probes [core Stat5b binding site underlined].

<p>DNA Sequences of Oligonucleotide Probes [core Stat5b binding site underlined].</p