Transgenic reporter analysis of ChIP-Seq-defined enhancers identifies novel target genes for the terminal selector UNC-3/Collier/Ebf

Terminal selector-type transcription factors are key regulators of neuronal identity and function (Hobert and Kratsios, 2019; Kratsios and Hobert, 2018). Mechanistically, terminal selectors are thought to act directly through binding at the cis-regulatory region of genes (termed “terminal identity genes”) that encode, among others, neurotransmitter [NT] synthesis proteins, ion channels, neuropeptides, and cell adhesion molecules (Hobert and Kratsios, 2019; Kratsios and Hobert, 2018). Although dozens of terminal selectors have been described thus far for individual neuron types of the nematode C. elegans (Hobert, 2016), the identification of their target genes has primarily relied on candidate approaches and availability of markers for neuronal terminal identity. Hence, unbiased methods are needed to identify the full spectrum of terminal selector target genes in individual neuron types. This study focuses on the phylogenetically conserved terminal selector UNC-3/Ebf (member of the Collier/Olf/Ebf family), which controls cholinergic motor neuron (MN) identity in the ventral nerve cord of the nematode C. elegans. To identify novel UNC-3 target genes, we took advantage of the genome-wide binding map of UNC-3 from our previous Chromatin Immunoprecipitation followed by Sequencing (ChIP-Seq) analysis (Li et al., 2020). We generated transgenic reporter lines for ten putative terminal identity genes (pxd-1, cal-2, lgc-4, ldb-1, nep-21, D2007.2, dmsr-2, ncs-2, npr-29, drn-1), whose expression patterns were largely unknown in C. elegans. Six of these reporter lines showed expression in ventral nerve cord MNs (nep-21, D2007.2, dmsr-2, ncs-2, npr-29, drn-1), whereas the remaining four (pxd-1, cal-2, lgc-4, ldb-1) showed expression in head and tail neurons, as well as some non-neuronal cells. Importantly, the number of ventral nerve cord MNs showing expression of the nep-21, D2007.2, and dmsr-2 reporters was significantly reduced in unc-3 null mutant animals, thereby expanding the repertoire of known UNC-3 target genes in these cells. Altogether, this study demonstrates that transgenic reporter analysis guided by ChIP-Seq results is a relatively efficient approach for the identification and validation of transcription factor target genes.