Mechanisms of Neuronal Activity-Dependent Transcription

Abstract

Experience-dependent neural activity initiates cascades of activity-regulated gene expression that produce long-lasting changes in the adult brain. Different patterns of activity can produce opposing synaptic changes that each require transcription, but whether these different patterns of activity produce distinct programs of ARG transcription is not known. We addressed this question in Chapter 2, where we find that in both in vivo and in vitro studies, brief activity selectively induces a subset of the activity-regulated gene program that corresponds to the initial, rapid wave of transcription that is induced by sustained activity. This selective transcriptional response to brief activity requires MAPK/ERK signaling, which functions to mediate the rapid recruitment of RNAPII to promoters. Genes induced by brief activity are further differentiated from other inducible genes by a baseline open-chromatin state, higher levels of promoter- occupied RNAPII, binding of the MAPK-sensitive transcription factor SRF, and proximity to rapidly activated enhancers. These rapid enhancers are occupied at baseline by SRF and activate in response to brief depolarization by increasing histone acetylation and eRNA transcription, as detected by eRNA-sequencing and H3K27ac ChIP-seq. MAPK/ERK is required for eRNA synthesis but not increases in enhancer histone acetylation, suggesting that MAPK/ERK acts on a specific stage of enhancer activation and implicating MAPK/ERK-dependent eRNA synthesis in the transcriptional response to brief activity. Overall, these results identify changes in the epigenome that enable different neuronal activity patterns to induce distinct gene expression programs. In Chapter three, we further evaluate candidate transcriptional regulatory mechanisms differentiating differently patterned genes. In using small-molecule inhibitors to test the contribution of DNA-damage response proteins to activity-dependent transcription, we find that NMS-873, an allosteric inhibitor of the AAA ATP-ase, p97/VCP, acts acutely and selectively to inhibit stimulus-dependent transcription. This transcription inhibition proceeds through a mechanism that is upstream of RNAPII initiation. We next generate and validate inhibitor- resistant mutant cell lines to test the on-target activity of NMS-873. Experiments in these cell lines suggest that NMS-873 suppresses transcription through an off-target mechanism of action.