Serotonergic transcriptional regulatory logic in caenorhabditis elegans

Resumen

Neuronal diversity in the nervous system is generated through the activation of multiple unique batteries of terminal differentiation genes, which determine the functional properties of the distinct mature neurons. It is generally accepted that transcription factors (TFs) bind in a combinatorial and cooperative manner to DNA sequences of the genome called enhancers, placing TFs as the main regulators of gene expression. However, how these combinations of TFs identify and activate their target sequences is poorly understood. In this work we use as a paradigm the serotonergic neurons to unravel the regulatory rules that select a cell type-specific transcriptome during terminal differentiation. Serotonergic neurons are present in all eumetazoan groups and are universally defined by their ability to synthesise and release serotonin (5-HT), which is achieved by the expression of the ‘5-HT pathway genes’. Taking advantage of this phylogenetic conservation, we use the simple model organism Caenorhabditis elegans to dissect the transcriptional regulatory logic of serotonergic neurons. C. elegans hermaphrodites have three functionally different serotonergic subclasses: the HSN motorneuron, the ADF sensory neuron and the NSM neurosecretory motorneuron. All three neuron subtypes express the 5-HT pathway genes. Through an in vivo cis-regulatory analysis of these genes we have identified independent cis-regulatory modules (CRM) responsible for their expression in each serotonergic neuron subtype. This modular organisation suggests that different regulatory logics are employed in each neuron subclass to activate its terminal transcriptome. To deepen in our understanding of how cell type-specific transcriptional programs are implemented we decided to focus the rest of our work on the best characterized serotonergic neuron subtype, the HSN neuron, and carried out an extensive dissection of HSN terminal differentiation transcriptional rules. Loss of function mutant and cis-regulatory analyses reveal that direct activation of the HSN transcriptome is orchestrated by a code of six TFs, that we have termed HSN TF collective. This TF code is composed by AST-1 (ETS TF family), UNC-86 (POU TF family), SEM-4 (SPALT TF family), HLH-3 (bHLH TF family), EGL-46 (INSM TF family) and EGL-18 (GATA TF family). The expression of the HSN TF collective is sufficient to induce serotonergic fate in some specific contexts and is required throughout the life of the animal in order to maintain the identity of the HSN neuron. Bioinformatically identified binding site clusters for the six TFs of the HSN TF collective are enriched in known HSN expressed genes compared to a random set of genes. Through in vivo reporter analysis, we demonstrate that this clustering constitutes a regulatory signature that is sufficient for de novo identification of HSN neuron functional enhancers. This regulatory signature contains certain syntactic constrains that further improve the prediction of enhancer expression in the cell. Mouse orthologues of most members of the HSN TF collective are known regulators of the mammalian serotonergic differentiation program. This homology in both serotonergic regulatory programs allows for the identification of an additional candidate TF in the worm (PHA-4), orthologue to the mouse FOXA2, and a mouse TF (SALL2), orthologue of the worm SEM-4. Moreover, we prove that mouse orthologues can functionally substitute for their worm counterparts. Finally, Principal Coordinates Analysis suggests that, among C. elegans neurons, the HSN transcriptome most closely resembles that of mouse serotonergic neurons, which reveals deep homology. Our results show that a regulatory signature based on a defined set of TFs is sufficient for enhancer identification using primary DNA sequence. Moreover, our results identify rules governing the transcriptional regulatory code of a critically important neuronal type in two species separated by over 700 million years.