Neuronal Activity-Dependent Transcription Start Sites

In the brain, tightly controlled transcription is critical for proper neurodevelopment, learning, and memory. Since over half of mammalian genes are predicted to have multiple transcription start sites (TSS), the question arises: how do neurons selectively utilize alternative TSS? Studies have described steady-state, cell type-specific TSS usage in brain tissue, but recent evidence from plant biology hints that alternative TSS may also be regulated in a dynamic, stimulus-dependent manner. We therefore wondered if neuronal activity, a key signal in the brain, directs dynamic use of alternative TSS. To test this, we utilized a form of nascent RNA-sequencing, BrU-seq, which allows sensitive detection of short-term changes in transcription. We purified nascent RNA from mouse primary forebrain cultures treated with drugs to reduce or increase neuronal firing rate, and prepared next generation sequencing libraries using direct ligation of adapters to RNA, which enriches for read density at TSS. Our custom analysis pipeline revealed over 30 genes with activity-dependent usage of alternative TSS. Usage of these alternative TSS is predicted to alter the encoded protein primary structures, and in some cases to alter the protein’s sub-cellular localization. Using a CRISPR/Cas9-mediated protein-tagging technique (SLENDR), we tested if altered neuronal activity indeed results in a switch in sub-cellular localization of alternative TSS gene products. Lastly, we examined if these changes in TSS usage occurred in neurons in vivo, using next generation sequencing of ribosome-bound RNA after a paradigm of neuronal activation. Taken together, this work renders TSS selection as a key layer of gene regulation during the brain’s response to its environment. Future work aspires to illuminate mechanisms of TSS-specific transcriptional regulation and determine the influence of activity-dependent TSS on neuronal plasticity.