Serotonergic Transcription of HumanFEVReveals Direct GATA Factor Interactions and Fate ofPet-1-Deficient Serotonin Neuron Precursors

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Abstract
Altered expression of the humanFEV(fifth Ewing variant) ETS transcription factor gene impacts the level of CNS serotonin (5-HT) neuron gene expression and maternal nurturing. However, the regulatory mechanisms that determineFEVexpression are poorly understood. Here, we investigated thecis-regulatory control ofFEVto begin to identify the upstream transcription factors that restrictFEVexpression to 5-HT neurons. We find that sequences extending only 275 bp upstream of theFEV5′ untranslated region are sufficient to directFEVtransgene expression to embryonic 5-HT neurons, although sequences farther upstream are required for maintenance in adult 5-HT neurons. Two highly conserved consensus GATA factor binding sites within the 275 bp region interact with GATA factorsin vitro. Chromatin immunoprecipitations with embryonic hindbrain demonstrated Gata-2 interactions with the orthologous mousePet-1ETScis-regulatory region. Mutagenesis of GATA sites revealed that one or the other site is required for serotonergicFEVtransgene expression. Unexpectedly,FEV–LacZ transgenes enabled determination of 5-HT neuron precursor fate in the adultPet-1−/−dorsal and median raphe nuclei and thus provided additional insight intoFEV/Pet-1function. Comparable numbers ofFEV–LacZ-positive cells were detected inPet-1+/−andPet-1−/−adult dorsal raphe nuclei, indicating that the majority of mutant serotonergic precursors are not fated to apoptosis. However, B7 dorsal raphe cells were aberrantly distributed, suggesting a role forFEV/Pet-1in their midline organization. Our findings identify a direct transcriptional interaction between Gata-2 andFEVand a unique marker for new insight intoFEV/Pet-1function in 5-HT neuron development.