Promoters initiate RNA synthesis, and enhancers stimulate promoter activity. Whether promoter and enhancer activities are encoded distinctly in DNA sequences is unknown. We measured the enhancer and promoter activities of thousands of DNA fragments transduced into mouse neurons. We focused on genomic loci bound by the neuronal activity-regulated coactivator CREBBP, and we measured enhancer and promoter activities both before and after neuronal activation. We find that the same sequences typically encode both enhancer and promoter activities. However, gene promoters generate more promoter activity than distal enhancers, despite generating similar enhancer activity. Surprisingly, the greater promoter activity of gene promoters is not due to conventional core promoter elements or splicing signals. Instead, we find that particular transcription factor binding motifs are intrinsically biased toward the generation of promoter activity, whereas others are not. Although the specific biases we observe may be dependent on experimental or cellular context, our results suggest that gene promoters are distinguished from distal enhancers by specific complements of transcriptional activators.

Pharmacological perturbation is a powerful tool for understanding mRNA synthesis, but identification of the specific steps of this multi-step process that are targeted by small molecules remains challenging. Here we applied strand-specific total RNA sequencing (RNA-seq) to identify and distinguish specific pharmacological effects on transcription and pre-mRNA processing in human cells. We found unexpectedly that the natural product isoginkgetin, previously described as a splicing inhibitor, inhibits transcription elongation. Compared to well-characterized elongation inhibitors that target CDK9, isoginkgetin caused RNA polymerase accumulation within a broader promoter-proximal band, indicating that elongation inhibition by isoginkgetin occurs after release from promoter-proximal pause. RNA-seq distinguished isoginkgetin and CDK9 inhibitors from topoisomerase I inhibition, which alters elongation across gene bodies. We were able to detect these and other specific defects in mRNA synthesis at low sequencing depth using simple metagene-based metrics. These metrics now enable total-RNA-seq-based screening for high-throughput identification of pharmacological effects on individual stages of mRNA synthesis.

Topoisomerase I inhibition and peripheral nerve injury induce DNA breaks and ATF3-associated axon regeneration in sensory neurons Graphical abstract Highlights d Axonal injury rapidly induces ATF3-dependent expression of RAGs in DRG neurons d An ATF3 reporter is used to identify how ATF3 is induced in injured neurons d Topo I inhibition increases ATF3 expression and enhances axonal regeneration d DNA breaks are increased at the ATF3 locus in injured neurons by Topo I inhibition

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The proteosome inhibitor bortezomib has revolutionized the treatment of multiple hematologic malignancies, but in many cases its efficacy is limited by a dose-dependent peripheral neuropathy. We show that human induced pluripotent stem cell (hiPSC) derived motor neurons and sensory neurons provide a model system for the study of bortezomib-induced peripheral neuropathy with promising implications for furthering mechanistic understanding and developing treatments for preventing axonal damage. Human neurons in tissue culture display distal-to-proximal neurite degeneration when exposed to bortezomib. This process coincides with disruptions in mitochondrial function and energy homeostasis similar to those described in rodent models of bortezomib-induced neuropathy. Moreover, while the degenerative process is unaffected by inhibition of caspases, it is completely blocked by exogenous nicotinamide adenine dinucleotide (NAD+), a mediator of the SARM1-dependent axon degeneration pathway. We demonstrate that bortezomib-induced neurotoxicity in relevant human neurons proceeds through mitochondrial dysfunction and the NAD+ depletion mediated axon degeneration, raising the possibility that targeting these changes may provide effective therapeutics for the prevention of bortezomib-induced neuropathy and that modeling chemotherapy-induced neuropathy in human neurons has utility.