Decoding natural direct reprogramming : cellular permissivity and core nuclear mechanisms – CELLSwitch

- Dynamic transcriptomic analysis of transdifferentiation at the single cell level
- Gene regulatory network governing the initiation of transdifferentiation
- Single cell imaging

With its 100% efficiency and reproducibility our model has allowed us to address the mechanisms underlying the invariance and efficiency observed in natural cell type conversions, at the single cell level. Through unbiased approaches, we have found that the balance between different histone H3 methylation marks is determinant for invariant progression of Td. The SET1 and JMJD3 histone-modifying activities are partitioned through dynamic regulation of nuclear JMJD-3 protein levels and association with phase-specific conserved TFs. Finally, we found that H3K4me3 methylation and H3K27me3/me2 demethylation are also critical to provide protection against environmental variations or stresses, thus ensuring the robustness of Td under sub-optimal conditions. This study is the first to show that step-wise histone modifications are key to provide robustness to a multiple steps biological process such as natural direct reprogramming. Together with our previous findings that TFs are critical for the process, our results suggest a model where key TFs drive the conversion of cell identity through the de-differentiation and re-differentiation steps while the interplay of different histone modifications ensures its invariance. Finally, our work further suggested conserved roles for these histone modifiers in mammals (Zuryn et al, Science 2014).

The expected results have the potential to provide ground breaking molecular and functional insights into the conserved machinery that allows a differentiated cell to change its identity.

• We first expect to engineer new empowering tools to further carry on large scale, state-of-the-art approaches by purifying single cells from whole animals or automatising screens. We also expect to establish the 2A multiproteic expression technology in worms.

• Through transcriptomic analysis, we expect to identify factors that make the Y cell permissive to reprogramming, and to highlight the contribution of Notch signalling to this property.

• We expect from these experiments to define the core nuclear network that is required for the initiation of different direct reprogramming events in vivo.

• Through transcriptomic and functional analyses, we expect to unravel how epigenetic activities impact on plasticity at the nuclear level.

1. Zuryn S., Ahier A., Portoso M., Redhouse White E., Margueron R., Morin M.C. & Jarriault S. (2014) Sequential histone-modifying activities determine the robustness of transdifferentiation. Science 345(6198):826-829.

2. Ahier A. & Jarriault S.* (2014) Simultaneous Expression of Multiple Proteins Under a Single Promoter in Caenorhabditis elegans via a Versatile 2A-Based Toolkit. Genetics 196(3):605-13.

+ commentary on our work :
« Worming toward transdifferentiation, one (Epigenetic) step at a time« Beyret E. & Izpisua Belmonte J.C. (2014) Developmental Cell 30(6):641-2.

How cells can change their identity is a fascinating question that has attracted much attention in the last decade. Differentiated cells can be forced to directly adopt another differentiated identity, or to revert to a pluripotent state. Remarkably, direct reprogramming events can also occur naturally. Our goal is to determine the cellular requirements and the molecular circuitry controlling cell plasticity in vivo in an integrated context. Indeed, although cell identity switches have been described in various settings, the mechanisms underlying this phenomenon are largely unknown. Understanding cellular plasticity will have a tremendous impact on our perception of developmental and cancerous processes and could open new avenues for regenerative medicine strategies.

To systematically identify the molecules, genetic cascades and cellular requirements underlying cell plasticity, we turned to a simple model organism, the nematode C. elegans. The determination of the cellular lineage of C. elegans suggested that a few cells could change their identity during development, an observation not investigated further. Focusing on one particular identity switch that occurs in absence of cell division, we have established the worm as a unique and much expected model to study the mechanisms underlying cell plasticity, where a given cell can be unambiguously identified and its change of identity predicted and followed in vivo in a physiological context. In addition, the powerful C. elegans genetics allowed us to genetically challenge such phenomenon at the single cell level to systematically identify the molecules, genetic cascades and cellular requirements.

Using this model we have uncovered unexpected cellular transitions including a dedifferentiation step during a natural direct reprogramming in absence of cellular division. We have further unravelled a conserved NODE-like complex, composed of factors essentials for ES cells pluripotency, that potentiates cell plasticity in C. elegans, suggesting that conserved mechanisms underlie the control of cellular potency, both in natural or ex vivo settings. Interestingly, we have identified epigenetic activities acting on histone methylation and will test their role in this control. Altogether, these important findings show that i) the study of a direct reprogramming event in vivo in the worm gives access to the intricate details of the early steps of direct reprogramming; and that ii) it has and will lead to important concepts and mechanisms conserved with other cell plasticity events across phyla.

Building on these new research avenues that our early work has opened, we will pursue 3 original aims to take this research to the next level and determine what makes defined cells permissive to reprogramming in vivo, and what common molecular basis may underlie different reprogramming events: 1) The determination of the mechanisms that make one cell, and not its neighbours, able to be reprogrammed (permissivity); 2) To uncover the core nuclear networks that promote natural direct reprogramming; 3) To unravel the contribution of the epigenome in the implementation of the transition states during direct reprogramming. For this, we will continue to take advantage of the power of genetic and cellular tools in C. elegans. We will also develop new tools to go deeper at the molecular level and uncover the molecular machineries controlling each step.

We project that our work will likely provide novel insights into the cellular and molecular mechanisms involved in cellular plasticity. We expect our results to be of interest to a broad audience of scientists working in the cell plasticity, stem cell biology, developmental biology, cancer or regenerative medicine fields.