Brain serotonin neuron gene regulatory networks and chromatin architecture

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Precise spatiotemporal control of gene expression is crucial throughout life for maturation of postmitotic neuronal function and preservation of brain health. Although continuously expressed neuronal transcription factors termed terminal selectors, such as Pet1 and Lxm1b, are key regulators of gene expression across fetal and postnatal stages of life the underlying molecular mechanisms through which they control stage specific neuronal gene expression are poorly understood. Here, we aim to fill this gap by comprehensively investigating terminal selector function in postmitotic serotonin (5-HT) neurons. Pet1 and Lmx1b’s control of serotonergic gene expression is of broad interest as 5-HT has wide-ranging modulatory effects on central neural circuitry and altered serotonergic gene expression has been implicated in several developmental neuropsychiatric disorders including depression, stress-related anxiety, autism, OCD, and schizophrenia. The studies proposed here are motivated, in part, by our discovery of changing dependencies of continuously expressed 5-HT neuron terminal effector genes on Pet1 as 5-HT neurons mature. We unexpectedly found that Pet1’s terminal selector control of 5-HT synthesis genes is largely switched off in the early postnatal period and instead Pet1 switches to controlling the upregulation of neurotransmitter GPCR genes, Htr1a, Adra1b, that are needed for afferent synaptic modulation of 5-HT neuron excitability. These recent observations have led us to suggest a new principle that we have termed “terminal selector target switching”. We suggest that as postmitotic neurons progress through life, continuous terminal selector regulated transcription is not static, as is the prevailing assumption. Instead, we hypothesize that terminal selector regulated transcription is highly dynamic in which regulatory factor interactions with target genes are remodeled as postmitotic neurons mature. We hypothesize the remodeling of regulatory interactions is rooted in the temporal remodeling of postmitotic neuronal chromatin architecture. In Aim 1, we will investigate Lmx1b’s control of early postnatal 5-HT gene expression to determine whether postmitotic target switching is a general property of 5-HT terminal selectors. In Aim 2, we will use our newly developed 5HT-ATAC-seq protocol to uncover the temporal dynamics of 5-HT open chromatin as 5-HT neurons develop and mature from fetal to early postnatal stages of life. Specifically, we will address these questions: Do open chromatin states undergo maturational changes in parallel with the maturation of 5-HT neuron transcriptomes? Do switches in target dependence result from gene specific changes in open chromatin? Aim 3 will investigate a plausible potential mechanism through which developing 5-HT gene expression patterns are controlled: Terminal selectors, Pet1 and Lmx1b, directly act to control the maturation of 5-HT neuronal open chromatin states. Understanding how terminal selectors control postmitotic neuronal gene expression may illuminate potential neurodevelopmental disease mechanisms that cause aberrant neuronal gene expression and defects in neuronal maturation.