Figure 7.

GnRH induction of Cga expression requires MSK induced S28p at the +1 nucleosome. A and C–E, αT3-1 cells or B, LβT2 cells were treated with GnRH and/or RMM-64 added 2 h prior to the GnRH, whereas some of the cells in C were transfected with a RMM-64 resistant MSK1 mutant, C440V, before (A–C) qPCR analysis of the Cga mRNA levels (GnRH 8 h); or (D and E) ChIP analysis (GnRH 5 h) to determine the levels of H3S28p at the nucleosomes upstream and downstream of the Cga TSS. The qPCR and ChIP were carried out and data presented as described in the legend to Fig. 1, with levels shown relative to mean values in untreated control cells; the control values with/without GnRH are from the experiment shown in Fig. 5E. F, schematic model showing the GnRH-induced pathways leading to phosphorylation of H3S28 at the +1 nucleosome of the Cga gene, which is required for the transcriptional response, possibly mediated also through H3K27ac both of which modifications would facilitate RNAPII transition of this well positioned nucleosome for transcriptional elongation. Although GnRH also activates JNK, which is essential for H3S10p, this modification does not affect H3K9ac and plays little effect in basal or GnRH-induced transcription of this gene. *, p < 0.05; **, p < 0.01; ***, p < 0.001.