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

Signaling, chromatin and TFs in the control of pluripotency in ES cells:

Nanog is a major pluripotency TF that is especially important in the control of self-renewal. Yet, the exact mechanisms by which Nanog fosters self-renewal were relatively ill-defined. Using a Crispr-based techniques we showed that Nanog function depends on signaling: in the presence of the pro-self-renewal LIF citokyne, Nanog rewires the pluripotency network by controlling Brg1 recruitment, chromatin accessibility and binding of additional TFs; in the absence of LIF, however, Nanog promotes H3K27me3 at developmental genes. In addition, self-renewing ES cells display dramatic cell-to-cell variability in the expression level of 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. Our work places Nanog at the center of the regulatory pathway linking signaling and chromatin states, such that self-renewal and differentiation can be appropriately orchestrated.

Inheritance of the pluripotency network's activity:

The preservation of pluripotency throughout cell division - self-renewal -, is strictly dependent on the highly efficient action of the pluripotency gene regulatory network. However, how TFs cope with replication and mitosis, two major challenges to the chromatin were completely unknown. We hypothesized that some pluripotency TFs may act as bookmarking factors by remaining bound to critical target genes during mitosis; subsequently, we also proposed that some TFs may be capable of binding rapidly upon passage of the replication fork. This would enable the rapid and efficient re-establishment of the network in nascent chromatids and in daughter cells, Thereby preserving pluripotency. Using a combination of genome-wide and imaging approaches, we first showed that one pluripotency TF, Esrrb, but not others like Oct4 or Sox2, bind to a subset of its interphase targets during mitosis. In doing so, Esrrb enables the preservation of nucleosome positioning during mitosis. We could then show that immediately after replication, within minutes of the passage of the fork, Esrrb binding regions are fully organised regarding nucleosome positioning. A major caveat of this work, however, was the lack of direct functional evidence of the nucleosome organisation capacity of a TF, especially during mitosis. We recently provided such evidence by identifying CTCF as a major TF conferring chromatin resiliency immediately after replication and during mitosis. Altogether, our work highlights that selected TFs, such as Esrrb and CTCF, execute specific nucleosome positioning functions during replication and mitosis, thereby contributing to the inheritance of gene regulatory information. We call these TFs, resilient TFs.

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