Dynamic Chromatin Modifications Control GnRH Gene Expression during Neuronal Differentiation and Protein Kinase C Signal Transduction

Abstract

Abstract GnRH, a neuropeptide produced by rare, specialized hypothalamic secretory neurons, is critical for reproduction. During development, GnRH gene expression increases as neurons migrate from the olfactory placode to the hypothalamus, with highest levels in the mature, postmitotic state. While neuronal differentiation is known to be controlled by chromatin modulations, the role of chromatin dynamics in GnRH gene regulation has not been studied. Here, we use mature and immature GnRH neuronal cell models to show that both neuron-specific and protein kinase C regulation of GnRH expression are mediated by chromatin structure and histone modifications. Only in GT1-7 mature GnRH neuronal cells did GnRH regulatory elements display high sensitivity to DNase and enrichment of active histone markers histone-H3 acetylation and H3 lysine 4 trimethylation (H3K4-Me3), as well as RNA Polymerase II (RNAPII) binding and enhancer RNA transcription. In contrast, H3K9-Me2, a marker of inactive chromatin, was highest in nonneuronal cells, low in GT1-7 cells, and intermediate in immature GnRH neuronal cells. The chromatin of the GnRH gene was therefore active in mature GnRH neuronal cells, inactive in nonneuronal cells, but not fully inactive in immature GnRH neuronal cells. Activation of protein kinase C (PKC) potently represses GnRH expression. PKC activation caused closing of the chromatin and decreased RNAPII occupancy at the GnRH minimal promoter (−278/−97). At GnRH-Enhancer-1 (−2404/−2100), PKC activation decreased phosphorylated-RNAPII binding, enhancer RNA transcription, and H3 acetylation, and reciprocally increased H3K9-Me2. Chromatin modifications therefore participate in the dynamic regulation and specification of GnRH expression to differentiated hypothalamic neurons.