Identifying growth hormone-regulated enhancers in the Igf1 locus

Abstract

Growth hormone (GH) plays a central role in regulating somatic growth and in controlling multiple physiological processes in humans and other vertebrates. A key agent in many GH actions is the secreted peptide, IGF-I. As established previously, GH stimulates IGF-I gene expression via the Stat5b transcription factor, leading to production of IGF-I mRNAs and proteins. However, the precise mechanisms by which GH-activated Stat5b promotes IGF-I gene transcription have not been defined. Unlike other GH-regulated genes, there are no Stat5b sites near either of the two IGF-I gene promoters. Although dispersed GH-activated Stat5b binding elements have been mapped in rodent Igf1 gene chromatin, it is unknown how these distal sites might function as potential transcriptional enhancers. Here we have addressed mechanisms of regulation of IGF-I gene transcription by GH by generating cell lines in which the rat Igf1 chromosomal locus has been incorporated into the mouse genome. Using these cells we find that physiological levels of GH rapidly and potently activate Igf1 gene transcription while stimulating physical interactions in chromatin between inducible Stat5b-binding elements and the Igf1 promoters. We have thus developed a robust experimental platform for elucidating how dispersed transcriptional enhancers control Igf1 gene expression under different biological conditions.

Keywords: IGF-I; STAT; chromatin immunoprecipitation; epigenetics; gene transcription; growth hormone.

Fig. 1.

Mapping recombinant cell lines incorporating the rat Igf1 locus. A: schematic of rat Igf1 bacterial artificial chromosome (BAC) Ch230-113M6, which consists of 241 kb of rat chromosome 7, including the ∼80 kb 6-exon Igf1 gene, ∼110 kb of 5′-flanking, and ∼51 kb of 3′-flanking DNA. The location of the pGK-Neo selection cassette, which was introduced into the BAC by recombineering (28, 62), is indicated, as are the 7 previously identified growth hormone (GH)-regulated Stat5b-binding elements (red circles) and a scale bar. The enlargement below the main map depicts the two Igf1 promoters, P1 and P2, and adjacent exons 1–3. The horizontal arrow indicates the direction of gene transcription. The locations of the 11 PCR primer pairs used to characterize each clonal cell line are indicated (see Table 1 for DNA sequences). B: results of PCR mapping experiments using genomic DNA isolated from BAC cell lines 11 and 20 and primer pairs 1 and 11. These pairs amplify DNA from the 5′- and 3′-ends of the BAC, respectively. C: results of PCR mapping experiments with genomic DNA from each BAC cell line and primer pairs 2 through 10. Collectively, results in B and C demonstrate that the entire BAC has been incorporated into each recombinant cell line. D: results of copy number determinations for cell lines 11 and 20, using as a standard curve serial dilutions of BAC DNA (see materials and methods for details).

Fig. 2.

Analysis of recombinant cell lines incorporating the rat Igf1 locus. A: experimental plan to test responsiveness to Stat5b of the rat Igf1 gene in individual BAC-containing cell lines (Dox, doxycycline). B: results of RT-PCR experiments for rat Igf1 (primers span exons 3 and 4) or mouse Gapdh mRNA (see Table 2) using RNA isolated from the listed recombinant cell lines transduced with constitutively active rat Stat5b and tTA and treated ± Dox for 18 h. C: experimental plan to test GH responsiveness of the rat Igf1 gene in individual BAC-containing cell lines. D: results of RT-PCR experiments using RNA isolated from the listed recombinant cell lines transduced with the mouse GH receptor, rat Stat5b, and tTA, and treated with vehicle or recombinant rat GH [40 nM] for 18 h. Liver RNA from GH-deficient rats treated with vehicle or recombinant rat GH [1.5 mg/kg] for 6 h represents a negative and positive control respectively for GH responsiveness.

Fig. 3.

Rapid induction of Igf1 gene expression by GH in a BAC-containing cell line. A: experimental plan outlining steps to test Igf1 gene regulation in response to GH and Stat5b in 10T1/2 Igf1-BAC clone 11. B: time course of appearance of tyrosine phosphorylated Stat5b (pStat5), total Stat5b (using antibody to Flag), and α-tubulin, as assessed by immunoblotting after addition of vehicle (No GH) or recombinant rat GH [40 nM] to clone 11 cells. C: GH treatment causes the nuclear accumulation of Stat5b in the nucleus of 10T1/2 Igf1-BAC clone 11, as detected by immunocytochemistry. DAPI was used to stain nuclei. D: time course of accumulation of Igf1 and Gapdh mRNAs, as assessed by RT-PCR using RNA isolated at different times after GH addition to cells. Liver RNA from GH-deficient rats treated acutely with vehicle or a single injection of recombinant rat GH [1.5 mg/kg] serves as both negative and positive controls for GH-responsiveness. E: time course of appearance of mouse Igf1, Igfals, Socs2, and S17 mRNAs, as assessed by RT-PCR using RNA isolated at different times after GH addition to cells. Liver RNA from intact adult male mice serves as a positive control.

Fig. 4.

Rapid induction of Igf1 gene transcription by GH and Stat5b in a BAC-containing cell line. A: representative experiment showing time course of appearance of nascent transcripts for the rat Igf1 gene and for several mouse genes, as assessed by RT-PCR using nuclear RNA isolated at different times after GH addition to clone 11 cells. B: quantitative analysis of time course of accumulation of nascent transcripts from rat Igf1 promoters 1 and 2 and for mouse Igfals, Socs2, and Gapdh, measured at different times after addition of GH to clone 11 cells, as assessed by qRT-PCR. Primer sets are listed in Table 3 (means ± SD of n = 5 independent experiments; *P < 0.01; **P < 0.001 vs. time 0).

Fig. 5.

GH induces recruitment of Stat5b, RNA polymerase II, and p300 to potential binding elements in chromatin within the rat Igf1 locus. Results are shown of quantitative chromatin immunoprecipitation experiments using chromatin harvested from rat Igf1-BAC clone 11 after addition to cells of recombinant rat GH [40 nM] for 0, 1 or 4 h. Primer sets are listed in Table 4. All studies were performed by qPCR. A: analysis of binding of Stat5b to the 7 Stat5 binding elements depicted in Fig. 1 plus control primer pairs dispersed throughout the rat Igf1 locus (Neg). B: results of binding of RNA polymerase II (pol II) to the same elements. C: binding of p300 to the same elements. Results reflect the means ± SD of 3–5 independent experiments (*P < 0.05; **P < 0.01; ***P < 0.001 vs. time 0).

Fig. 6.

Identifying GH-induced interactions of Stat5b binding elements with Igf1 gene promoters by chromatin conformation and capture (3C) assay. A: schematic of rat Igf1 BAC CH230-113M6 depicting the ∼80 kb 6-exon Igf1 gene and flanking DNA, locations of 7 putative Stat5b-binding enhancers (circles), and locations of restriction sites for AflII (multiple small vertical lines). A scale bar and the insertion site of the pGK-Neo selection cassette also are illustrated. Below the main map the enlargement depicts the two Igf1 promoters, P1 and P2, and adjacent exons 1–3, and below that the line indicates the 3C “bait” DNA fragment and PCR primers. B: results of 3C experiments measured by semiquantitative PCR for each of the 7 putative Stat5b-binding enhancers studied before and 60 min after addition of recombinant rat GH [40 nM] to cells. The lane marked “BAC” represents a positive control, consisting of an aliquot of BAC DNA that was digested with AflII, and then religated. All positive results were confirmed by DNA sequencing. C: quantitative results of 3C experiments, measured by qPCR for each of the 7 putative Stat5b-binding enhancers studied in B (means ± SD of 3 independent experiments; *P < 0.05 vs. time 0). Primer sets are listed in Table 5 for parts B and C.