Molecular Regulation of a Transcriptionally Poised State in Neurons and its Role in Learning

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PROJECT SUMMARY/ABSTRACT Rapid and transient induction of gene expression in neurons is necessary for memory formation. Genes that are activated in the brain in response to stimuli are regulated by the release of a poised transcriptional state, where RNA polymerase II (RNAP2) pauses just downstream of the gene promoter after initiating transcription. When calcium-dependent signaling cascades are triggered, the RNAP2 pause is released. This release allows RNAP2 to elongate across the length of the gene, and messenger RNA (mRNA) transcripts are generated. There is a transcriptional refractory period that lasts for hours after a stimulus when neurons are transcriptionally unresponsive to subsequent stimuli that may be linked to the time it takes to reset poised RNAP2. This period of dampened transcriptional response may explain the phenomenon where animals that have little time between training sessions do not learn as well as animals with more spaced out training session schedules, despite total training time being equal between groups. Preliminary experiments point to Hexamethylene bisacetamide inducible protein 1 (HEXIM1) as a critical factor for setting up and resetting the poised state in neurons due to its ability to sequester the positive transcription elongation factor b (P-TEFb) protein complex, which is responsible for releasing the RNAP2 transcriptional pause. While other regulators of P-TEFb have been linked to human cognitive diseases including intellectual disability, Alzheimer’s, mood disorders, and others, very little is known about HEXIM1 in the brain. The central hypothesis of this project is that suppression of P-TEFb by HEXIM1 in neurons is required for RNAP2 to set up a poised state so a burst of gene expression can be induced in response to a stimulus, and that while the poised state is getting set up following a transcriptional burst, learning is impaired. I will test this hypothesis with three specific aims. Aim 1 will explore the regulation of the P-TEFb/HEXIM1 complex by calcium channels to identify which memory-associated calcium signaling pathways impact P-TEFb activity. Molecular associations between P-TEFb and HEXIM1, and their dependence on calcium-associated phosphorylation events will be tested using biochemical assays in primary neuron cultures. Aim 2 will test how RNAP2 cycles through a set of steps (poise, elongate, disengage, then poise again) that dictate levels of gene inducibility following neuronal depolarization. Confocal microscopy, immunocytochemistry, and florescence in situ hybridization will be combined to ascertain the association of inducible genes with nuclear subcompartments in containing poised, elongating, and inactive RNAP2 during and after neuronal stimulation. Aim 3 will probe the role of HEXIM1 in memory and determining the rate of learning. Behavioral tests will be conducted in mice after knockdown or overexpression of the Hexim1 gene in the hippocampus. This career development award will further develop the translation of my skillsets in studying transcriptional regulation mechanisms to the study of learning and memory.