Pablo NAVARRO Lab: Our projects

« Je ne puis pas donner la réalité des faits, je n’en puis présenter que l’ombre » 

Henri Beyle - Stendhal

The epigenetic foundation of gene expression heterogeneity:

Self-renewing ES cells display dramatic cell-to-cell variability in the expression level of key transcription factors such as Nanog. Whereas Nanog-positive cells self-renew efficiently, Nanog-negative cells are prone to differentiate. Although these two populations can dynamically inter-convert, very little is known about the underlying mechanisms. It is believed that Nanog fluctuations result from stochastic modifications of the activity of the pluripotency network, which is inhibited by Erk and Gsk3b signalling pathways. However, the Nanog-negative state can be inherited during several cell divisions. Thus, yet to describe mechanisms of mitotic inheritance may be involved in Nanog heterogeneity. We are currently exploring this possibility and have shown that a histone mark associated with gene repression is enriched at Nanog in undifferentiated ES cells. This enrichment is maintained during mitosis and lost upon inhibition of Erk and Gsk3b, correlating with the loss of Nanog-negative cells. This indicates that the mark that we have identified may be instrumental to maintain the negative state.

Epigenetic inheritance of the network's activity:

To maintain the undifferentiated state, ES cells need to keep silent differentiation genes. However, the genetic invalidation of key systems of epigenetic repression (Polycomb, H3K9 and CpG methylation) does not impair ES cells self-renewal. This suggests that silencing of developmental genes may be reestablished after each division owing to the highly efficient action of the pluripotency gene regulatory network. Under this context, the existence of a memory of gene activation would be particularly interesting to accelerate the re-establishment of the network upon re-entry into G1. We hypothesized that some pluripotency regulators may act as bookmarking factors by remaining bound to critical target genes during mitosis. This would enable the rapid and efficient re-establishment of the network after mitosis such that self-renewal is efficiently preserved. Using imaging and ChIP-Seq approaches we have identified one such pluripotency transcription factor, Esrrb. We are now pursuing our efforts to identify additional bookmarking factors in ES cells and to establish the regulatory principles underlying the mitotic epigenome. More recently, we have also started to address how replication impacts gene regulation in ES cells, and whether specific pluripotency regulators enable the reassembly of functional regulatory complexes after the passage of the fork.

Esrrb on metaphase-stage ES cells
Mouse ES cell karyotype stained for Esrrb
Esrrb ChIP-Seq in interphase (blue) and mitosis (red)
RNA-Seq of one of our LASR candidates

The contribution of long non-coding RNAs to the network’s activity:

The study of ES cells has not escaped the recent explosion of regulatory lncRNA biology: from the thousands of lncRNAs that have been identified across the mouse genome a large subset is expressed in ES cells. Our goal is to identify “pluripotency lncRNAs” that would impact on ES cell biology as strongly as well established pluripotency transcription factors. We will focus on Nanog-mediated regulation as a tool to discover relevant lncRNAs. Indeed, among the three main pluripotency factors (Oct4, Sox2 and Nanog), only Nanog can be manipulated without dramatically affecting the ES cell transcriptome. Yet, within the very limited number of genes strictly dependent upon Nanog, highly relevant transcription factors such as Esrrb, Klf4, Rex1 and Prdm14 are found. We therefore postulate that within the potentially small number of Nanog-dependent lncRNAs, molecules as important as the aforementioned transcription factors should be easily identified. We are using RNA-Seq approaches to identify lncRNAs (LASR for Long-non-coding Associated to Self-Renewal) that respond to Nanog levels and are associated to the ground state of pluripotency. We are testing their functions using a combination of CRISPR-activation and -deletion studies.
RNA-FISH of one of our candidates (green), nextC1
Created by Pablo Navarro