Epigenetic activation of Sox2 enhancer on embryonic neural plate

MARINI, M.; BOUZAS, S.; BUZZI, A. L.; GULLERMO, E.; BRONNER, M.

Seattle

Simposio; Society for Developmental Biology 73rd Annual meeting; 2014

      During vertebrate development, the first and definitive neural marker is the Sox2 gene. The spatiotemporal control of Sox2 expression is regulated by multiple enhancers. The N-1 enhancer initially activates Sox2 expression in response to inducing signals from Hensen?s node; then the N-2 maintains Sox2 expression in regions rostral to the node. Here, we characterize the in vivo function of the H3K9me3 histone demethylase, JmjD2A, during Sox2 activation in the early neural plate. In situ hybridization analysis reveals that JmjD2A expression precedes that of Sox2 in the neural territory. To study the effects of JmjD2A knock down, we performed gain- and loss-of-function experiments, by electroporating either the full-length enzyme or a translation-blocking morpholino unilaterally into stages 3-5 embryos that were then collected at stage 7+. Gain of JmjD2A function induces ectopic Sox2 expression outside of the neural plate territory. Reciprocally, loss of JmjD2A at stage 3 causes a clear diminution in the expression of Sox2 all along the neural tube (N-1 and N-2 domains). In contrast, electroporations performed at stage 5 only affect Sox2 expression around the node (N-1 domain). The heterochromatin protein HP1, which typically interacts with the H3K9me3 mark, has previously been shown to act as a repressor until Sox2 induction. Interestingly, the observed effects were potentiated by co-electroporating JmjD2A morpholino with the kinase Msk1, which can phosphorylate the H3S10 (H3S10ph) necessary for the HP1-H3K9me3 dissociation. Consistent with this, we show by Bimolecular Fluorescense Complementation that the adaptor protein 14-3-3, reported to bind to H3S10ph, can interact with JmjD2A. Finally, chromatin immunoprecipitation reveals binding of JmjD2A to the N-1, but not N-2, enhancer in dissected neural plate tissues. Taken together, these results suggest that sequential epigenetic events are necessary for early activation of Sox2 N-1 enhancer in neural progenitor cells.