Early-life m6A RNA demethylation by fat mass and obesity-associated protein (FTO) influences resilience or vulnerability to heat stress later in life.

Early life heat stress leads to either resilience or vulnerability to a similar stress later in life. We have previously shown that this tuning of the stress response depends on neural network organization in the hypothalamic PO/AH thermal response center, and is regulated by epigenetic mechanisms. Here we expand our understanding of stress response establishment describing a role for epitranscriptomic regulation of the epigenetic machinery. Specifically, we explore the role of N6-methyladenosine (m6A) RNA methylation in long-term response to heat stress. Heat conditioning of 3-day-old chicks diminished m6A RNA methylation in the hypothalamus, simultaneously with an increase in the mRNA levels of the m6A demethylase, fat mass and obesity-associated protein (FTO). Moreover, a week later, methylation of two heat-stress-related transcripts, Histone 3 Lysine 27 (H3K27) Methyltransferase, Enhancer of zeste homolog 2 (EZH2) and Brain-derived Neurotrophic Factor (BDNF), were down-regulated in harsh-heat-conditioned chicks. During heat challenge a week after conditioning, there was a reduction of m6A levels in mild-heat-conditioned chicks and an elevation in harsh-heat-conditioned ones. This increase in m6A modification was negatively correlated with the expression levels of both BDNF and EZH2. Antisense “knockdown” of FTO caused an elevation of global m6A RNA methylation, reduction of EZH2 and BDNF mRNA levels and decrease in global H3K27 dimethylation as well as dimethyl H3K27 level along BDNF coding region and, finally, led to heat vulnerability. These findings emphasize the multilevel regulation of gene expression, including both epigenetic and epitranscriptomic regulatory mechanisms, fine-tuning the neural network organization in a response to stress. SIGNIFICANCE STATEMENTExposure to different levels of stress during the critical period of thermal-control establishment confers future vulnerability or resilience and depends on epigenetic regulation. Tuning the stress-response set-point is crucial because of its implications for psychopathologies. Here, we demonstrate a cross talk between the epitranscriptomic and epigenetic systems in stress response establishment. Specifically, early-life heat conditioning diminished m6A RNA methylation in the hypothalamus, simultaneously with an increased expression of the m6A demethylase, FTO. Antisense “knockdown” of FTO, resulted in heat vulnerability. Moreover, this dual-level regulation is also demonstrated on BDNF expression in heat stress, including m6A marks on BDNF transcript and H3K27me2 modifications on the BDNF gene. Crosstalk between epigenetic and epitranscriptomic regulation can balance the response to future heat challenges.