Enhancer-Mediated Regulation of Bdnf

Brain Derived Neurotrophic Factor (BDNF) plays essential roles in neuronal survival, differentiation, and plasticity, and it contributes to learning and memory. The expression of BDNF is highly induced by neuronal activity and is mediated primarily at the transcriptional level, via the regulation of stimulus-modulated transcription factors. Although much is known about the mechanisms of Bdnf regulation at this gene’s promoters, we do not know about the presence and contribution of distal enhancers in regulating activity-dependent Bdnf expression. To address this, we first aligned a 5C-seq dataset to ChIP datasets for many biochemical features present at active enhancers. From this analysis, we identified six regions that show strong indications of being active enhancers upon neuronal stimulation. To functionally test the role of these specific putative gene regulatory elements in the regulation of Bdnf induction, we used dCas9-KRAB based CRISPRi tools in embryonic mouse cortical neuron cultures stimulated by KCl. When dCas9-KRAB was recruited to Bdnf promoters, more than a two-fold reduction in Bdnf expression was seen. However, when dCas9-KRAB was recruited to the putative enhancers, a weaker suppression in Bdnf expression was seen for two regions. RNA-seq data from these regions show low levels of eRNA expression upon neuronal activation, suggesting that these regions may not be enhancers. As it is possible that these regions may be stronger enhancers under different stimulation conditions, in the future we will interrogate these regions upon bicuculline stimulation. Also, since this suggests that eRNA may be a more robust marker for active enhancers, we will be using RNA-seq datasets and eRNA quantification methods to narrow in and identify Bdnf enhancers. However, with the data collected from the experiments thus far, it is still unclear if these regions are indeed distal Bdnf enhancers.