Regulatory rules and evolution of neuronal gene expression

Project Information

NEUROCODE

Grant agreement ID: 101002203

DOI

10.3030/101002203

EC signature date 16 February 2021

Start date 1 June 2021

End date 31 May 2027

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 997 500,00

EU contribution

€ 1 997 500,00

Coordinated by

AGENCIA ESTATAL CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS
Spain

Periodic Reporting for period 3 - NEUROCODE (Regulatory rules and evolution of neuronal gene expression)

Reporting period: 2024-06-01 to 2025-11-30

Summary of the context and overall objectives of the project

To achieve complex functions, even simple nervous systems require a sophisticated
degree of specialization in labor giving rise to the most complex cellular diversity found in any
organ. Understanding the mechanisms guiding neuronal specification programs remains a major challenge. Importantly, failure to properly implement these genetic programs is linked to several neuropsychiatric and neurodegenerative diseases.
In this project we use the nematode C. elegans, an essential animal model for its simplicity and amenability to genetic modifications, for studying the general principles of neuron specification, to provide important insights to four fundamental questions:

1) First, how are neuron-type specific regulatory landscapes shaped in each type of neuron? We previously showed that complex combinations of transcription factors (TFs) implement neuron-type specific programs. TF behaviour at the genome-wide level is not well-understood. In this project we propose to develop a new tool to study in vivo TF binding profiles in a cell-type specific manner. This tool will allow us to better understand the function of TFs in different neuron types. We also expect it could be of great use to other groups in the C. elegans community.

2) Second, how are TF interactions mirrored by syntax rules of the regulatory genome? Our preliminary data
demonstrate extensive TF-TF cooperativity that is mirrored by specific syntax in the target enhancers. Once again, availability of tools limits our capability to move forward in the understanding of how information is stored in the non-coding genome. In this grant we are trying to develop massively parallel reporter assays and bioinformatics to begin decoding the rules of the neuronal
regulatory genome.

3) Third, we expand from transcriptional regulation to other layers of complexity and have started studying the involvement of the basic translational machinery in neuronal differentiation processes. The mechanisms regulating the basal translational machinery are emerging as a new dimension in the regulation of cell type specification. We recently found the conserved translation initiation factor eIF3A is required for serotonergic terminal differentiation, opening the door to the study of this novel regulatory role for eIF3 complex.

4) Finally, we are also studying how new types of neurons emerge during evolution. In this part of the project we use different species of Caenorhabditis nematodes to explore how the gene regulatory networks, that have been extensively characterized in C. elegans, are modified in other species.

We expect that results from this project will unravel novel principles on the regulation of neuronal specification and the evolution of cell types in the nervous system, fundamental for our understanding of brain development and function. This knowledge can also be important in the development of new therapeutic strategies to treat diseases of the nervous system.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

In the first half of the project we have allocated a great amount of effort in the development of a new tool that could allow us to interrogate the action of TFs in specific neuron types of C. elegans at a resolution that has not been achieved before. We have managed to endogenously tag TFs in a cell-type specific manner and are now working on obtaining genomic binding profiles for some of these factors using CUT and RUN technique. We expect to start obtaining relevant neuron-type specific TF binding profiles in the following months. In addition, we have teamed up with Christian Frokjaer-Jensen, at Kaust University (https://orcid.org/0000-0002-3178-0906) one of the world leading figures in developing new genomic tools for C. elegans research, to adapt a high throughput method, Massively Parallel Reporter Assays (MPRAs), to study the activity of cis regulatory modules in vivo in C. elegans.
Regarding the role of basal translational machinery, specifically eIF3A in neuron differentiation, we found that this factor might be particularly relevant to induce differentiation of neurons that remain in a quiescent undifferenciated state for prolonged periods of time, such as the HSN serotonergic hermaphroditic specific neuron and the CEM cholinergic, male specific neuron. We are currently working on the identification of the mechanisms used by eIF3A to control exit from quiescence.
Finally, studying additional Caenorhabiditis species we have identified the gene regulatory mechanisms that allowed the emergence of the serotonergic phenotype in a particupar neuron type in the group of Angaria species. These results are one of the few examples for which the molecular events underlying emergence of novelty has been identify and we expect to submit a manuscript for its publication in the next few months

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

As already mentioned, we are working on advancing some of the tools in the field that will allow us to interrogate the role of TFs in the specification of diverse neuron types with a resolution that has not been achieved before in C. elegans. We are also developing MPRAs to be used in vivo in C. elegans. These two technical improvements, together with the wealth of knowledge we already have from C. elegans nervous system specification mechanisms and the amenability to genetic modifications of C. elegans and other Caenorhabiditis species will allow us to better understand the gene regulatory networks controlling neuron type specification and differentiation. We will also use the same tools to identify how these gene regulatory networks are modified along evolution to provide neurons with new functions. For example, we have identified how terminal selectors (transcription factors that act in the postmitotic neurons to act as master regulators of specific fates) can acquire new direct targets to modify the functionalities of the neurons in different species.