Figure 5.

Phosphorylation at H3S28 increases following GnRH, as does K27ac, and this plays a role in GnRH-induced gene expression. A and B, levels of H3S28p were analyzed in GnRH-treated αT3-1 cells as in Fig. 3. In A, aT3–1 cells (same blot as in Fig 3A, hence same H3 panel; H328p was analyzed following stripping), or in B, LbT2 cells which were treated, analyzed, and presented similarly. Averages from repeated experiments in LβT2 cells are shown graphically; t test compared mean values with untreated controls, as described in the legend to Fig. 3. C, Cga mRNA levels were measured by qPCR as described in the legend to Fig. 3, in cells treated with GnRH (6 h) with or without H89 added 60 min prior to the GnRH. The mRNA levels are expressed as fold of the mean levels in untreated control cells and presented as described in the legend to Fig. 4D. D, high resolution ChIP was carried out for H3 in cells treated with GnRH (2 h); alternatively, for E, H3S28p or F, H3K27ac in untreated or GnRH-treated (5 h) cells to examine these modifications at the −1 and +1 nucleosomes on the Cga. Data were analyzed and are presented as described in the legend to Fig. 3. *, p < 0.05; **, p < 0.01.