Stimulus-dependent expression ofBdnfis mediated by ATF2, MYT1L, and EGR1 transcription factors

Neurotrophins like BDNF have a key role in the proper functioning of the central nervous system, influencing numerous processes like memory formation and behavior. An imbalance in BDNF levels can lead to a wide range of diseases, including depression and neurodevelopmental disorders. While the potential therapeutic effects of BDNF are well-recognized, there is a knowledge gap in understanding the mechanisms governing BDNF expression levels. Here, we focused on the regulation of Bdnf gene expression in response to different stimuli, specifically studying the effects of neuronal activity and BDNF-TrkB signaling on Bdnf transcription in cultured neurons from rats of either sex. We used in vitro DNA pulldown combined with mass spectrometry to determine transcription factors that interact with the Bdnf promoters upon different stimuli and validated numerous known regulators, such as USF and AP1 family, and novel candidate regulators using reporter assays. We show that the USF family of transcription factors is specifically recruited after membrane depolarization, whereas the AP1 family participates in Bdnf regulation only after BDNF-TrkB signaling. We further describe ATF2, MYT1L and EGR family as novel regulators of Bdnf expression by demonstrating their direct binding to Bdnf promoters using chromatin immunoprecipitation assays both in vitro and in vivo, showing their functional role in Bdnf gene expression, and ultimately identifying their regulatory cis -elements in Bdnf promoters. Furthermore, our results show competition between ATF2, CREB, and AP1 family in regulating Bdnf levels. Collectively, our results provide insight into the regulation of Bdnf expression upon different stimuli. Significance statement Membrane depolarization and neurotrophin BDNF (brain-derived neurotrophic factor) signaling via its receptor TrkB (tropomyosin receptor kinase B) are critical processes for proper neuronal functions. Here, we studied how these two stimuli regulate the expression of two main Bdnf transcripts - Bdnf exon I- and IV-containing transcripts. Our results reveal a remarkable overlap of regulators that are recruited to both Bdnf promoters I and IV and after both stimuli. Overall, our results shed light to the complex regulation of Bdnf expression highlighting a dynamic interplay of cooperative and competitive mechanisms among transcription factors. Understanding the regulatory mechanisms beyond single transcription factors and considering the combinatorial effects could pave the way for specifically modulating Bdnf levels as therapeutic interventions.