Bertrand Ducos

Impaired PRC2 activity promotes transcriptional instability and favors breast tumorigenesis

Alterations of chromatin modifiers are frequent in cancer, but their functional consequences often remain unclear. Focusing on the Polycomb protein EZH2 that deposits the H3K27me3 (trimethylation of Lys27 of histone H3) mark, we showed that its high expression in solid tumors is a consequence, not a cause, of tumorigenesis. In mouse and human models, EZH2 is dispensable for prostate cancer development and restrains breast tumorigenesis. High EZH2 expression in tumors results from a tight coupling to proliferation to ensure H3K27me3 homeostasis. However, this process malfunctions in breast cancer. Low EZH2 expression relative to proliferation and mutations in Polycomb genes actually indicate poor prognosis and occur in metastases. We show that while altered EZH2 activity consistently modulates a subset of its target genes, it promotes a wider transcriptional instability. Importantly, transcriptional changes that are consequences of EZH2 loss are predominantly irreversible. Our study provides an unexpected understanding of EZH2s contribution to solid tumors with important therapeutic implications.

Translation in astrocyte distal processes sets molecular heterogeneity at the gliovascular interface

Astrocytes send out long processes that are terminated by endfeet at the vascular surface and regulate vascular functions as well as homeostasis at the vascular interface. To date, the astroglial mechanisms underlying these functions have been poorly addressed. Here we demonstrate that a subset of messenger RNAs is distributed in astrocyte endfeet. We identified, among this transcriptome, a pool of messenger RNAs bound to ribosomes, the endfeetome, that primarily encodes for secreted and membrane proteins. We detected nascent protein synthesis in astrocyte endfeet. Finally, we determined the presence of smooth and rough endoplasmic reticulum and the Golgi apparatus in astrocyte perivascular processes and endfeet, suggesting for local maturation of membrane and secreted proteins. These results demonstrate for the first time that protein synthesis occurs in astrocyte perivascular distal processes that may sustain their structural and functional polarization at the vascular interface.

Spatiotemporal manipulation of retinoic acid activity in zebrafish hindbrain development via photo-isomerization

All-trans retinoic acid (RA) is a key player in many developmental pathways. Most methods used to study its effects in development involve continuous all-trans RA activation by incubation in a solution of all-trans RA or by implanting all-trans RA-soaked beads at desired locations in the embryo. Here we show that the UV-driven photo-isomerization of 13-cis RA to the trans-isomer (and vice versa) can be used to non-invasively and quantitatively control the concentration of all-trans RA in a developing embryo in time and space. This facilitates the global or local perturbation of developmental pathways with a pulse of all-trans RA of known concentration or its inactivation by UV illumination. In zebrafish embryos in which endogenous synthesis of all-trans RA is impaired, incubation for as little as 5 minutes in 1 nM all-trans RA (a pulse) or 5 nM 13-cis RA followed by 1-minute UV illumination is sufficient to rescue the development of the hindbrain if performed no later than bud stage. However, if subsequent to this all-trans RA pulse the embryo is illuminated (no later than bud stage) for 1 minute with UV light (to isomerize, i.e. deactivate, all-trans RA), the rescue of hindbrain development is impaired. This suggests that all-trans RA is sequestered in embryos that have been transiently exposed to it. Using 13-cis RA isomerization with UV light, we further show that local illumination at bud stage of the head region (but not the tail) is sufficient to rescue hindbrain formation in embryos whose all-trans RA synthetic pathway has been impaired.

Single molecule studies of helicases with magnetic tweezers

Helicases are a broad family of enzymes that perform crucial functions in DNA replication and in the maintenance of DNA and RNA integrity. A detailed mechanical study of helicases on DNA and RNA is possible using single molecule manipulation methods. Among those, magnetic tweezers (or traps) present a convenient, moderate throughput assay (tens of enzymes can be monitored simultaneously) that allow for high resolution (single base-pair) studies of these enzymes in various conditions and on various substrates (double and single stranded DNA and RNA). Here we discuss various implementation of the basic assay relevant for these studies.

Optical Control of Tumor Induction in the Zebrafish

The zebrafish has become an increasingly popular and valuable cancer model over the past few decades. While most zebrafish cancer models are generated by expressing mammalian oncogenes under tissue-specific promoters, here we describe a method that allows for the precise optical control of oncogene expression in live zebrafish. We utilize this technique to transiently or constitutively activate a typical human oncogene, kRASG12V, in zebrafish embryos and investigate the developmental and tumorigenic phenotypes. We demonstrate the spatiotemporal control of oncogene expression in live zebrafish, and characterize the different tumorigenic probabilities when kRASG12V is expressed transiently or constitutively at different developmental stages. Moreover, we show that light can be used to activate oncogene expression in selected tissues and single cells without tissue-specific promoters. Our work presents a novel approach to initiate and study cancer in zebrafish, and the high spatiotemporal resolution of this method makes it a valuable tool for studying cancer initiation from single cells.

Control of Protein Activity and Gene Expression by Cyclofen‐OH Uncaging

The use of light to control the expression of genes and the activity of proteins is a rapidly expanding field. Whereas many of these approaches use fusion between a light‐activable protein and the protein of interest to control the activity of the latter, it is also possible to control the activity of a protein by uncaging a specific ligand. In that context, controlling the activation of a protein fused to the modified estrogen receptor (ERT) by uncaging its ligand cyclofen‐OH has emerged as a generic and versatile method to control the activation of proteins quantitatively, quickly, and locally in a live organism. We present that approach and its uses in a variety of physiological contexts.

A mechanistic study of helicases with magnetic traps

Helicases are a broad family of enzymes that separate nucleic acid double strand structures (DNA/DNA, DNA/RNA, or RNA/RNA) and thus are essential to DNA replication and the maintenance of nucleic acid integrity. We review the picture that has emerged from single molecule studies of the mechanisms of DNA and RNA helicases and their interactions with other proteins. Many features have been uncovered by these studies that were obscured by bulk studies, such as DNA strands switching, mechanical (rather than biochemical) coupling between helicases and polymerases, helicase‐induced re‐hybridization and stalled fork rescue.

Quantitative study of the somitogenetic wavefront in zebrafish

A quantitative description of the molecular networks that sustain morphogenesis is one of the challenges of developmental biology. Specifically, a molecular understanding of the segmentation of the antero-posterior axis in vertebrates has yet to be achieved. This process known as somitogenesis is believed to result from the interactions between a genetic oscillator and a posterior-moving determination wavefront. Here we quantitatively study and perturb the network in zebrafish that sustains this wavefront and compare our observations to a model whereby the wavefront is due to a switch between stable states resulting from reciprocal negative feedbacks of Retinoic Acid (RA) on the activation of ERK and of ERK on RA synthesis. This model quantitatively accounts for the near linear shortening of the post-somitic mesoderm (PSM) in response to the observed exponential decrease during somitogenesis of the mRNA concentration of a morphogen (Fgf8). It also accounts for the observed dynamics of the PSM when the molecular components of the network are perturbed. The generality of our model and its robustness allows for its test in other model organisms.

Longitudinal Analyses of Blood Transcriptome During Conversion to Psychosis

The biological processes associated with the onset of schizophrenia remain largely unknown. Current hypotheses favor gene × environment interactions as supported by our recent report about DNA methylation changes during the onset of psychosis. Here, we conducted the first longitudinal transcriptomic analysis of blood samples from 31 at-risk individuals who later converted to psychosis and 63 at-risk individuals who did not. Individuals were followed for a maximum of 1 year. Blood samples were collected at baseline and at the end of follow-up and individuals served as their own controls. Differentially expressed genes between the 2 groups were identified using the RNA sequencing of an initial discovery subgroup (n = 15 individuals). The most promising results were replicated using high-throughput real-time qPCR in the whole cohort (n = 94 individuals). We identified longitudinal changes in 4 brain-expressed genes based on RNAseq analysis. One of these genes (CPT1A) was replicated in the whole cohort. The previously observed hypermethylation in NRP1 and GSTM5 during the onset of psychosis correlated with a decrease in corresponding gene expression. RNA sequencing also identified 2 co-expression networks that were impaired after conversion compared with baseline-the Wnt pathway including AKT1, CPT1A and semaphorins, and the Toll-like receptor pathway, related to innate immunity. This longitudinal study of transcriptomic changes in individuals with at-risk mental state revealed alterations during conversion to psychosis in pathways and genes relevant to schizophrenia. These results may be a first step toward better understanding psychosis onset. They may also help to identify new biomarkers and targets for disease-modifying therapeutic strategies.

Single molecule kinetics uncover roles for E. coli RecQ DNA helicase domains and interaction with SSB

Abstract Most RecQ DNA helicases share a conserved domain arrangement that mediates their activities in genomic stability. This arrangement comprises a helicase motor domain, a RecQ C-terminal (RecQ-C) region including a winged-helix (WH) domain, and a ‘Helicase and RNase D C-terminal’ (HRDC) domain. Single-molecule real-time translocation and DNA unwinding by full-length Escherichia coli RecQ and variants lacking either the HRDC or both the WH and HRDC domains was analyzed. RecQ operated under two interconvertible kinetic modes, ‘slow’ and ‘normal’, as it unwound duplex DNA and translocated on single-stranded (ss) DNA. Consistent with a crystal structure of bacterial RecQ bound to ssDNA by base stacking, abasic sites blocked RecQ unwinding. Removal of the HRDC domain eliminates the slow mode while preserving the normal mode of activity. Unexpectedly, a RecQ variant lacking both the WH and HRDC domains retains weak helicase activity. The inclusion of E. coli ssDNA-binding protein (SSB) induces a third ‘fast’ unwinding mode four times faster than the normal RecQ mode and enhances the overall helicase activity (affinity, rate, and processivity). SSB stimulation was, furthermore, observed in the RecQ deletion variants, including the variant missing the WH domain. Our results support a model in which RecQ and SSB have multiple interacting modes.