MiR-138 inhibits EZH2 methyltransferase expression and methylation of histone H3 at lysine 27, and affects thermotolerance acquisition.

Thermotolerance acquisition involves neuronal network remodeling and, hence, alteration in the repertoire of expressed proteins. We have previously demonstrated the role of histone H3 methylation at lysine 27 (H3K27) by EZH2 methyltransferase in the regulation of gene expression during the critical period for the establishment of thermal control in chicks. Here we describe another level of biological regulation, demonstrating the inhibitory role of microRNAs (miRNAs) in the regulation of EZH2 expression in thermoregulatory system development and functioning. During heat conditioning in the critical period for the establishment of thermal control, a decrease in expression of the EZH2-targeting miR-138 occurred simultaneously with an increase in EZH2 levels in the preoptic anterior hypothalamus. Intracranial injection of miR-138 during the critical period led to a transient reduction in EZH2 levels, which was accompanied by a decrease in H3K27 methylation. Injection of miR-138 followed by heat conditioning also abolished EZH2 induction during heat conditioning. Moreover, this miR-138-induced inhibition of EZH2 during the critical period resulted in a long-term effect on EZH2 expression. A week after the treatment, the EZH2 protein levels in conditioned and in nonconditioned chicks were different from those in their saline-injected counterparts and the directions of change were opposite to each other. Finally, miR-138 injection during the critical period disrupted the establishment of thermoregulation, manifested as a defective body temperature response to heat. These data demonstrate a role for miRNAs in regulating the expression of histone-modifying enzymes, and thus emphasize the multilevel regulation mechanism which includes both epigenetic and miRNA regulatory mechanisms in neuronal network organization during the critical period of sensory development.