BDNF-AS regulates genes related to cell migration and neuronal function in mature human neurons

Abstract

Long thought to be transcriptional noise, long noncoding RNAs (lncRNAs) are emerging as important regulators of gene expression via their effects on both transcription and translation. Understanding the roles of these transcripts in regulation of gene expression has filled in long-standing gaps of knowledge of otherwise well-understood protein coding genes. Brain derived neurotrophic factor (BDNF) is arguably one of the most important neurotrophins in the development of the CNS. Highly spatiotemporally controlled, it has an intricate system of regulation that has been extensively studied but remains incompletely understood. Interestingly, the human genome, but not the mouse genome, encodes a lncRNA (BDNF AS) that is antisense to and overlapping the coding exon of the BDNF, gene raising the possibility of a species-specific mechanism of BDNF gene regulation given what we know about antisense lncRNAs. However, because BDNF expression is highly selective for stimulus-inducible neurons, experimentally addressing the functional impact of this antisense lncRNA requires an adequate human neuronal system, thus the role of the BDNF-AS remains largely unknown. To fill this gap in knowledge we have used human iPSC-derived stimulus-inducible neurons and dCas9-Krab/CRISPRi knockdown to disrupt the AS expression and probe the effects on BDNF and other genes via RNAseq, among other means. We use smFISH to characterize AS expression both alone and in relation to BDNF within single neurons. CUT&RUN is used to uncover the how changes in AS expression affect chromatin modifications at the BDNF locus and beyond. These data show that the BDNF AS not only regulates BDNF in human neurons, but also genes related to cell migration and neuronal differentiation. Some of these differentially expressed genes following AS KD overlap with differentially methylated regions following AS KD suggesting a mechanism of regulation for some, but not all, of the differentially expressed genes. We additionally use RNAseq to probe the activity-dependent lncRNA transcriptome of human glutamatergic neurons.