Although epigenetic control of the cell fate choice in embryonic stem cells is well established, little is known about the importance of epigenetic regulation during the terminal differentiation of neurons. We found that the subtype diversification of VC neurons requires H3K9 trimethylation, which restrained the expression of functionally important transcription factor UNC-4 in the vulval subtype of VC neurons. Using a rapidly degraded form of GFP, We found that only the vulval VC neurons (VC4 and VC5) but not the nonvulval subtype (VC1-3 and VC6) expressed
unc-4. Mutations in H3K9 methyltransferases (MET-2/SETDB1 and MET-1) and H3K9me3-binding proteins (CEC-3/Chromodomain protein, HPL-2/HP1, LIN-13/HP1-interacting protein, and LIN-65/MBT domain-containing protein), as well as a Q/N-rich domain-containing protein PQE-1, caused ectopic expression of
unc-4 in all six VC neurons, suggesting that H3K9 trimethylation was essential for silencing
unc-4 expression. We then found that
unc-4 expression in VC neurons depended on signal from the vulval tissue, since Muv mutants showed ectopic expression of
unc-4 in extra VC neurons flanking the pseudo vulvae. EGF signal secreted from the vulF cells triggered
unc-4 expression in the vulval VC neurons through the EGFR signaling, because either ablation of vulF cells or mutations in the EGFR/RAS/RAF/MAPK pathway eliminated
unc-4 expression in VC4 and VC5 neurons. Epigenetic silencing of
unc-4 was established in all six VC neurons prior to the differentiation cue. EGF signal from the developing vulva induced derepression of
unc-4 gene in the proximal VC4 and VC5 neurons, but the signal could not reach the distant nonvulval VC cells, which kept
unc-4 silenced. Downstream of the EGFR signaling in vulval VC neurons, the transcription factor LIN-11 and H3K9-specific histone demethylases removed the suppressive histone marks and derepressed
unc-4 transcription. Behaviorally, expression of UNC-4 in all the VC neurons caused an imbalance in the egg-laying circuit and resulted in egg-laying defects. Thus, epigenetic mechanisms by establishing subtype-specific gene expression regulate the terminal neuronal differentiation needed for optimal activity of a neural circuit.