Dissertation Defense - Patricia Garay
Promoter Usage and Smith-Magenis Syndrome Protein Retinoic Acid Induced-1 in Neuronal Activity-Dependent Transcription
Dr. Shigeki Iwase, Chair
The brain adapts to the environment by converting signals of neural activity into altered synaptic connections. Long-term changes in synapses depend upon neural activity-dependent transcription. Activity-dependent transcription, in turn, requires proper packaging of the DNA through chromatin regulation. Genes encoding chromatin regulators, transcription factors, and synaptic proteins are associated disproportionately with autism spectrum conditions and intellectual disabilities. Such neurodevelopmental conditions may arise from the disruption of synaptic plasticity. While forms of synaptic plasticity such as long-term potentiation/depression and synaptic scaling are known to require synaptic proteins and transcription factors, the roles of chromatin regulating proteins in these processes remain poorly understood.
In part, this is because the commonly used method of seeking differentially expressed genes from steady-state mRNA profiles can obscure the direct effects of chromatin regulators on dynamic transcription. Therefore, I used the nascent RNA sequencing method BrU-seq and adapted analysis techniques to examine chromatin regulator function in the context of activity-dependent transcription during synaptic scaling. I then revealed roles for the Smith-Magenis Syndrome protein Retinoic Acid Induced-1 (RAI1) as a regulator of activity-dependent transcription and synaptic scaling in the baseline and low-activity states of neurons.
In a distinct project, I found that neural activity shifts alter not only gene expression, but also gene promoter selection. I determined that multi-promoter genes make up nearly 10% of expressed genes in neuronal cultures, and that neuronal activity guides differential promoter usage in ~10% of them. I also observed differential promoter/transcription start site usage in vivo in physiological models of neuronal activity induction and found evidence of excitatory-neuron-specific promoter switching. Differential promoter usage predominately predicts altered N-terminal protein sequences of synaptic and phosphodiesterase family genes. Promoter-specific isoforms of the phosphodiesterase PDE2A revealed differential organelle-targeting and altered electrophysiological properties, suggesting promoter usage can regulate subcellular protein localization and synaptic function.