W. Clay Spencer and Evan S. Deneris
Department of Neurosciences, Case Western Reserve University, Cleveland, OH USA.
Serotonergic function requires precise spatiotemporal control of gene expression in 5-HT neurons and altered expression patterns in these neurons are thought to contribute to psychiatric disorders. Serotonergic gene expression depends on proper patterns of open and closed chromatin states to permit appropriate transcription factor interactions with the promoters and enhancers of 5-HT expressed genes. As is the case with virtually all neuron-types, nothing is known about how 5-HT neurons acquire patterns of open chromatin to enable expression of genes needed for 5-HT neuron function. 5-HT neurons mature upon their initial postmitotic differentiation at about embryonic day 11 through three weeks of postnatal life. During this prolonged stage, expression of hundreds of genes is upregulated to support acquisition of mature characteristics while other genes needed at earlier stages of development are turned off. We hypothesize that dynamic changes in patterns of 5-HT neuron open chromatin underlies the dynamic patterns of gene expression that support differentiation and maturation of 5-HT neurons. To investigate this idea, we developed a novel pipeline for assay of Tn5 transposase-mediated insertion of sequencing adaptors (ATAC-seq) into native chromatin using flow sorted fetal and early postnatal 5-HT neurons. Our pipeline, 5HT-ATAC-seq, has produced highly reproducible maps of the open chromatin landscape in maturing 5-HT neurons. Comparing 5-HT neurons and non-5-HT cells, over 20,000 5-HT neuron-specific open chromatin regions were identified across the genome. 5-HT-specific open regions mark putative transcriptional regulatory elements controlling 5-HT expressed genes, including Pet1, Tph2, Ddc, Gch1, Gchfr, Slc6a4, Slc22a3, Maoa, and Maob. Our working 5HT-ATAC-seq pipeline enables investigation of how maturation of 5-HT open chromatin relates to maturation of 5-HT gene expression patterns, what is the impact of environmental and genetic factors on maturation of 5-HT open chromatin patterns, and what are the regulatory mechanisms that establish 5-HT neuron-type open chromatin landscapes.