Reorganization of postmitotic neuronal chromatin accessibility for maturation of serotonergic identity

Assembly of transcriptomes encoding unique neuronal identities requires selective accessibility of transcription factors to cis-regulatory sequences in nucleosome-embedded postmitotic chromatin. Yet, the mechanisms controlling postmitotic neuronal chromatin accessibility are poorly understood. Here, we show that unique distal enhancers define the Pet1 neuron lineage that generates serotonin (5-HT) neurons. Heterogeneous single cell chromatin landscapes are established early in postmitotic Pet1 neurons and reveal the putative regulatory programs driving Pet1 neuron subtype identities. Distal enhancer accessibility is highly dynamic as Pet1 neurons mature, suggesting the existence of regulatory factors that reorganize postmitotic neuronal chromatin. We find that Pet1 and Lmx1b control chromatin accessibility to select Pet1-lineage specific enhancers for 5-HT neurotransmission. Additionally, these factors are required to maintain chromatin accessibility during early maturation suggesting that postmitotic neuronal open chromatin is unstable and requires continuous regulatory input. Together our findings reveal postmitotic transcription factors that reorganize accessible chromatin for neuron specialization. Overall design: 5-HT neuron ATAC-seq profiles of control (CON), Pet1 null, Lmx1bCKO, Pet1CKO, and Pet1/Lmx1b CKO (control and Pet1 null at E11.5, E14.5 and E17.5 (Pet1CKO, Lmx1bCKO, and DKO only at E14.5)) mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 550. The sequence reads that passed quality filters were processed using the ENCODE ATAC-seq pipeline and compared for differential accessibility using DESeq2. ChIPmentation for histone marks and CUT&RUN for the transcription factors Pet1 and Lmx1b were deep sequenced using an Illumina HiSeq 550. RNA-seq for control and DKO Pet1-neurons at E17.5 were deep sequenced using an Illumina HiSeq 550.