However, analysis of the top and bottom zones clearly showed that within the first 2 min following exposure to a novel environment, otpam866−/− animals spend significantly less time in the bottom tank zone and more time in the top zone when compared to their wild-type (WT) siblings, indicating that Otpa is necessary for normal behavioral response to novelty stress ( Figure 2C). Taken together, these results show that the adaptive response to stress is impaired in the absence of otpa gene activity. We next explored the mechanism

underlying the effect of Otp on stress adaptation. In order to identify the prime targets of Otp regulation in response to homeostatic challenge, we performed GDC-0199 chromatin immunoprecipitation (ChIP) assay using an anti-Otp antibody, followed by either promoter-specific quantitative PCR or high-throughput sequencing (ChIP-seq) (Figure 3A). We looked for genomic promoter regions

that showed enrichment of Otp binding following either physical or osmotic stress. A complete analysis of the ChIP-seq experiment will be published elsewhere (L.A.-Z. and G.L. in preparation). Our ChIP analyses showed that the Otp protein is recruited to the fish crh promoter following exposure to physical and osmotic stressors ( Figure 3B; Figure S3A). Otp was also found to form a complex with the crh promoter in the hypothalamic paraventricular nuclei dissected from mice that were subjected to a psychological MEK inhibition stressor ( Figure S4A). In agreement with tuclazepam the impaired stress response we observed in the otpam866 mutant ( Figure 1G), the association of Otp with the crh promoter was significantly diminished in these animals ( Figure S3B). Low-level enrichment of Otp binding to crh promoter in the otpam866 mutants is likely due to Otpa’s paralog Otpb, which is recognized by our

polyclonal antibody. These experiments demonstrate that recruitment of Otp to the crh promoter is triggered by stress challenges and that this process is conserved in fish and mammals. Another Otp target revealed by the ChIP-seq screen is the promoter of the a2bp1 gene (also known as rbfox1), which encodes a splicing factor known to regulate the alternative splicing of several neuronal transcripts linked to neuronal plasticity ( Lee et al., 2009). As with crh, Otp forms a complex with the a2bp1 promoter following physical and osmotic stress in fish and in response to psychological stress in mice ( Figures 3C and 3D; Figures S3C and S4B). In agreement with this finding, an acute foot shock stressor in mice, which induced a stereotypical crh transcription, led to a rapid increase in the levels of a2bp1 mRNA ( Figures 4A and 4B). Similar induction of a2bp1 mRNA expression was induced following exposure to stressors in the fish ( Figure 4C; Figure S2D). In contrast, the stressor-induced increase in a2bp1 mRNA was significantly reduced in zebrafish larvae homozygous for the otpam866 mutant allele ( Figure 4C; Figure S2D).