Figure 3.

Exposure to GnRH elevates levels of H3S10p and H3ac globally and at the Cga gene, correlating with its increase in expression. A, cells treated with GnRH for 0–6 h were harvested at the same time, and Western blot analysis was carried out on the histone fraction using antibody to H3S10p, H3K9ac, H3K18ac, and H3K27ac, following stripping of the membrane after each reaction. Total histone H3 was used as the loading control. (The experiment was performed several times and blotting was performed in various sequences, but for the results shown here, the sequence was K27ac, H3, K18ac, S10p, and K9ac.) Relative levels of each modified protein signal are shown below the blot as fold over the level in untreated cells, after normalization to H3. B, serum-starved αT3-1 cells were treated with GnRH for 8 h after which total RNA was extracted for qPCR. Cga mRNA levels are shown after normalization with those of Rplp0, and relative to the mean in untreated control cells, as described in the legend to Fig. 1. Statistical analysis was Student's t test: **, p < 0.01. C–F, ChIP analysis at the Cga gene promoter/5′ end was carried out in untreated or GnRH-treated αT3–1 cells using antibodies for total H3 and: C, H3K9ac; D, H3K18ac; E, H3K27ac; or F, H3S10p. Levels of precipitated DNA were quantified by qPCR and analyzed as described in the legend to Fig. 1, presented relative to the mean levels in the untreated control; *, p < 0.05; **, p < 0.01; ***, p < 0.001.