Epigenetic activation of Sox2 gene in the developing vertebrate neural plate

Abstract

One of the earliest manifestations of neural induction is onset of expression of the neural marker Sox2, mediated by the activation of the enhancers N1 and N2. By using loss and gain of function, we find that Sox2 expression requires the activity of JmjD2A and the Msk1 kinase, which can respectively demethylate the repressive H3K9me3 mark and phosphorylate the activating H3S10 (H3S10ph) mark. Bimolecular fluorescence complementation reveals that the adaptor protein 14-3-3, known to bind to H3S10ph, interacts with JMJD2A and may be involved in its recruitment to regulatory regions of the Sox2 gene. Chromatin immunoprecipitation reveals dynamic binding of JMJD2A to the Sox2 promoter and N-1 enhancer at the time of neural plate induction. Finally, we show a clear temporal antagonism on the occupancy of H3K9me3 and H3S10ph modifications at the promoter of the Sox2 locus before and after the neural plate induction. Taken together, our results propose a series of epigenetic events necessary for the early activation of the Sox2 gene in neural progenitor cells.

FIGURE 1:

JmjD2A expression precedes that of Sox2 in the neural territory. Expression pattern in the early chick embryo by whole-mount ISH at stages 3+, 4, and 5 revealed that JmjD2A precedes that of Sox2 in the nascent neural plate.

FIGURE 2:

Loss of JmjD2A causes reduced expression of Sox2. Sox2 ISH of chick embryos electroporated with control-MO (A), JmjD2A-tbMO (B), or JmjD2A-sbMO (C). Loss of JmjD2A causes a clear reduction in the expression of Sox2 all along the neural plate. (D) Electroporation of JmjD2A-sbMO together with a vector containing the coding region of JmjD2A (pCI-JmjD2A) rescues the depletion of Sox2 expression as assayed by ISH. Insets, the distribution of fluorescently labeled MO (green). (E) Quantification of embryos showing wild-type (WT; white), mild (black), or strong (red) Sox2 reduction on electroporated embryos. Asterisk indicates significant difference (p < 0.01) by contingency table followed by chi-square test. Numbers represent individual embryos. See Supplemental Figure S2 for phenotype description.

FIGURE 3:

JmD2A and Msk1 gain of function induces ectopic Sox2 expression. ISH of Sox2 gene expression after electroporation with control empty vector (A), JmjD2A (B), MSK1 (C), or a combination of both JmjD2A + Msk1 (D) overexpression vectors. Ectopic Sox2 expression (white arrowhead) was clearly evident when both JmjD2A and Msk1 overexpression vectors were coelectroporated. Top, left fluorescent electroporated area. (E) Ratio of embryos with normal or ectopic Sox2 expression phenotype. *p < 0.05 vs. control MO by contingency table followed by chi-square.

FIGURE 4:

JmjD2A directly interacts with the adaptor protein 14-3-3 as revealed by BiFC. (A) Schematic representation of JmjD2A and 14-3-3 constructs used for the BiFC assay. (B–D) Chick embryos were coelectroporated with JmjD2A-VN/14-3-3-VC or (E-G) JmjD2A-VN/mock-VC vectors at stage 5 and collected 16 h later. Embryos were then cryostat sectioned, and the electroporated cells (red) were analyzed for Venus fluorescence, reflecting positive protein interaction (green). White arrowheads indicate nuclear interaction. VC, C-terminus of Venus; VN, N-terminus of Venus; IRES, internal ribosome entry site.

FIGURE 5:

Endogenous JMJD2A protein is recruited to Sox2 promoter concomitantly with dynamic and opposite H3K9me3 and H3S10ph occupancy. ChIP-qPCR on dissected neural plate tissue from stage 3 and stage 5 embryos. (A) Schematic diagram representing the position where the primers were designed on the Sox2 locus: N1 and N2 enhancers, the promoter region at −0.5 kb, and the gene body at +1.0 kb from the TSS. (B) Endogenous JMJD2A binds to Sox2 promoter and N1 enhancer at stage 5 (St5) but was undetected at stage 3 (St3). (C) In vivo ChIP-qPCR for H3K9me3 and H3S10ph at Sox2 promoter (−0.5) and N1 and N2 enhancers before (St3) and after (St5) neural plate induction. IgG antibody was used as a mock antibody. Graphs represent the fold enrichment with respect to IgG immunoprecipitation, and error bars show SDs.

FIGURE 6:

Model for Sox2 enhancer activation on the neural plate. (A) The interaction between HP1 and H3K9me3 maintains the repressive state of Sox2. (B) The phosphorylation of H3S10 by the kinase MSK1 destabilizes the interaction between HP1 and H3K9me3. (C) The adaptor protein 14-3-3 interacts with JmjD2A and recruits it to the H3S10ph mark. (D) JMJD2A demethylates the H3K9me3 mark, activating Sox2 expression in the nascent neural plate.