Bdnf encodes a neurotrophin with critical roles in brain development, neuronal regeneration and synaptic plasticity. I performed 4C-seq to understand which genomic regions interact with Bdnf in cortical neurons, as enhancers or co-regulated genes often loop to the genes they regulate. Excitingly, I identified a loop from Bdnf to an intergenic site 45 kb downstream, which we postulate is a novel enhancer. Experiments using a reciprocal viewpoint at the intergenic site confirm the interaction. Analysis of published data suggests that in neurons the region has peaks of enhancer-associated histone modifications, and displays DNase hypersensitivity. It is bound by coactivators and transcription factors, which are associated with enhancer activation, supporting our hypothesis that the region could act as an enhancer.
I wanted to investigate whether the putative enhancer plays a role during developmental Bdnf activation. I used a primary neuron differentiation system to isolate neural progenitor cells (NPCs) and postmitotic neurons (PMNs), and found significant upregulation of all Bdnf isoforms from NPCs to PMNs. Preliminary 4C-seq data suggests that the loop to the intergenic region is present in PMNs but not NPCs. Preliminary double DNA-FISH experiments labelling Bdnf and the putative enhancer suggest that the inter-probe distance decreases from NPCs to PMNs, in keeping with the population level looping data. I have also established that during neuronal development the Bdnf locus moves away from the repressive nuclear periphery, and towards RNA polymerase II foci.
Transcription from active enhancers is thought to be vital for their function. I mapped published data and found nascent transcription at the putative Bdnf enhancer in neurons. qRT-PCR confirms expression in the region, which increases from NPC to PMN concomitantly with Bdnf mRNA, and is sensitive to transcriptional inhibition. These data show that the intergenic region we have identified as a site of interaction with Bdnf bears many characteristics of a neuronal enhancer.
To functionally dissect how the putative enhancer influences Bdnf expression levels in vivo, I established lentiviral infection of CRISPR guide RNA (gRNA)-directed dCas9-KRAB. This transcriptional repression system attains >65% infected PMNs, which are notoriously hard to target. I am currently using this scheme to repress the enhancer and assess Bdnf transcription during development. Bdnf is an essential factor in neuronal growth, including axonal branching and dendrite arborization. I have analysed activity-induced dendritic growth and found that repression of the enhancer abrogates Bdnf-driven dendritic arborization. I am now performing rescue experiments to demonstrate that the effect of enhancer inhibition is mediated through failure to upregulate Bdnf. We further plan to assess the role of the enhancer on brain development in vivo using in utero electroporation.
We hope to finish this work and submit for open access publication by the end of 2020. During this Fellowship I published a review article on the regulation of neuronal gene expression through genome architecture (Current Opinion in Neurobiology, 2019). I presented the project in a selected short talk at the Academy of Medical Sciences ‘The Developing Brain in Heath and Disease’ conference (March 2019) as well as in internal seminars and lab meetings. No website has been developed for this project.
