Biology subjects

Trouche, D.

Publications and source records attributed to Trouche, D..

6 recordsLinked to original sources

bioRxiv2026

Direct effects on gene transcription revealed by global and CRISPR-targeted oxidative DNA damage

Oxidative stress induces rapid expression changes of specific genes. However, whether these changes are due to direct and local DNA oxidation and whether they depend on the repair machinery is still debated. Here, we show by total RNA-Seq that upon oxidative stress, many genes are either rapidly repressed or activated. The early transcriptional response also includes the activation of many read-through RNAs. Importantly, degradation of the endogenous OGG1 DNA glycosylase showed that the early repression of these genes partially depends on OGG1, therefore demonstrating that these genome expression changes are due to the processing of oxidized DNA. We further demonstrate by ChIPseq against total or S5-phosphorylated RNA polymerase II that transcriptional regulation by oxidative stress involves changes in the recruitment of RNA polymerase II to transcription start sites. Finally, through the use of a chemoptogenetic system allowing the targeting of ROS to chosen genomic loci, we were able to reproduce repression of some genes through the local induction of oxidative DNA damage at their promoters, demonstrating that local DNA oxidation directly affects transcription of genes. Altogether, our results show that local oxidative DNA damage leads to alterations in the activity of specific promoters by a mechanism dependent on repair processes.

genomics↗

bioRxiv2023

Control of intestinal stemness and cell lineage by histone variant H2A.Z isoforms

The histone variant H2A.Z plays important functions in the regulation of gene expression. In mammals, it is encoded by two genes, giving raise to two highly related isoforms named H2A.Z.1 and H2A.Z.2, which can have similar or antagonistic functions depending on the promoter. Knowledge of the physiopathological consequences of such functions emerges, but how the balance between these isoforms regulates tissue homeostasis is not fully understood. Here, we investigated the relative role of H2A.Z isoforms in intestinal epithelial homeostasis. Through genome-wide analysis of H2A.Z genomic localization in differentiating Caco-2 cells, we uncovered an enrichment of H2A.Z isoforms on the bodies of genes which are induced during enterocyte differentiation, stressing the potential importance of H2A.Z isoforms dynamics in this process. Through a combination of in vitro and in vivo experiments, we further demonstrated the two isoforms cooperate for stem and progenitor cells proliferation, as well as for secretory lineage differentiation. However, we found that they antagonistically regulate enterocyte differentiation, with H2A.Z.1 preventing terminal differentiation and H2A.Z.2 favoring it. Altogether, these data indicate that H2A.Z isoforms are critical regulators of intestine homeostasis and may provide a paradigm of how the balance between two isoforms of the same chromatin structural protein can control physiopathological processes.

cell biology↗

bioRxiv2022

siRNAs targeting a chromatin-associated RNA induce its transcriptional silencing in human cells

Transcriptional gene silencing by small interfering RNAs (siRNAs) has been widely described in various species, such as plants or fission yeasts. In mammals, its extent remained somewhat debated. Previous studies showed that siRNAs targeting gene promoters can induce the silencing of the targeted promoter, although the involvement of off-target mechanisms was also suggested. Here, by nascent RNA capture and RNA Pol II ChIP, we show that siRNAs targeting a chromatin-associated non-coding RNA induce its transcriptional silencing. Deletion of the sequence targeted by one of these siRNAs on the two alleles by genome editing further show that this silencing is due to base pairing of the siRNA to the target. Moreover, by using cells with heterozygous deletion of the target sequence, we show that only the wild type allele, but not the deleted allele, is silenced by the siRNA, indicating that transcriptional silencing occurs only in cis. Finally, we demonstrate that both Ago1 and Ago2 are involved in this transcriptional silencing. Altogether, our data demonstrate that siRNAs targeting a chromatin-associated RNA at distance from its promoter induce its transcriptional silencing. Our results thus extend the possible repertoire of endogenous or exogenous interfering RNAs.

molecular biology↗

bioRxiv2020

Circular ANRIL isoforms switch from repressors to activators of p15/CDKN2B expression during RAF1 oncogene-induced senescence

Long non-coding RNAs (ncRNAs) are major regulators of gene expression and cell fate. The INK4 locus encodes the tumour suppressor proteins p15INK4b, p16INK4a and p14ARF required for cell cycle arrest and whose expression increases during senescence. ANRIL is a ncRNA antisense to the p15 gene. In proliferative cells, ANRIL prevents senescence by repressing INK4 genes through the recruitment of Polycomb-group proteins. In models of replicative and RASval12 oncogene-induced senescence (OIS), the expression of ANRIL and Polycomb proteins decreases, thus allowing INK4 derepression. Here, we found in a model of RAF1 OIS that ANRIL expression rather increases, due in particular to an increased stability. This led us to search for circular ANRIL isoforms, as circular RNAs are rather stable species. We found that the expression of two circular ANRIL increases in several OIS models (RAF1, MEK1 and BRAF). In proliferative cells, they repress p15 expression, while in RAF1 OIS, they promote full induction of p15, p16 and p14ARF expression. Further analysis of one of these circular ANRIL shows that it interacts with Polycomb proteins and decreases EZH2 Polycomb protein localization and H3K27me3 at the p15 and p16 promoters, respectively. We propose that changes in the ratio between Polycomb proteins and circular ANRIL isoforms allow these isoforms to switch from repressors of p15 gene to activators of all INK4 genes in RAF1 OIS. Our data reveal that regulation of ANRIL expression depends on the senescence inducer and underline the importance of circular ANRIL in the regulation of INK4 gene expression and senescence.

molecular biology↗

bioRxiv2019

KDM5 histone-demethylases contribute to replication stress response and tolerance.

KDM5A and KDM5B histone-demethylases are overexpressed in many cancers and have been involved in drug tolerance. Here, we describe that KDM5A, together with KDM5B, contribute to replication stress (RS) response and tolerance. First, they positively regulate RRM2, the regulatory subunit of Ribonucleotide Reductase. Second, they are required for optimal activation of Chk1, a major player of the intra-S phase checkpoint that protects cells from RS. This role in Chk1 activation is probably direct since KDM5A is enriched at ongoing replication forks and associates with both PCNA and Chk1. Because RRM2 is a major determinant of replication stress tolerance, we developed cells resistant to HU, and show that KDM5A/B proteins are required for both RRM2 overexpression and tolerance to HU, in a manner that is independent of their demethylase activity. Altogether, our results indicate that KDM5A/B are major players of RS management. They also show that drugs targeting the enzymatic activity of KDM5 proteins may not affect all cancer-related consequences of KDM5A/B overexpression.

cancer biology↗

bioRxiv2019

JMJD6 participates in the maintenance of ribosomal DNA integrity in response to DNA damage

Ribosomal DNA (rDNA) is the most transcribed genomic region and contains hundreds of tandem repeats. Maintaining these rDNA repeats as well as the level of rDNA transcription is essential for cellular homeostasis. DNA damages generated in rDNA need to be efficiently and accurately repaired as rDNA repeats instability has been reported in cancer, aging and neurological diseases. Here, we describe that the histone demethylase JMJD6 is rapidly recruited at nucleolar DNA damage and is crucial for the relocalisation of rDNA in nucleolar caps. Yet, JMJD6 is dispensable for rDNA transcription inhibition. Mass spectrometry study revealed that JMJD6 interacts with the nucleolar protein Treacle and modulates its interaction with NBS1. Moreover, cells deficient for JMJD6 show increased sensitivity to nucleolar DNA damage as well as loss and rearrangements of rDNA repeats upon irradiation. Altogether our data reveals that rDNA transcription inhibition is uncoupled from rDNA relocalisation into nucleolar caps and that JMJD6 is required for rDNA stability upon the rDNA damage response through its role in nucleolar caps formation.\n\nAuthor summaryRibosomal DNA is the most transcribed genomic region composed of repeated sequences. Transcribed rDNA is essential for cellular homeostasis and cell proliferation. Numerous pathologies such as cancer and neurological disorders are described to present defective rDNA repeats maintenance. The mechanisms involved in the control of rDNA integrity involve major DNA repair pathways such as NonHomologous End-Joining and Homologous Recombination. However, how they are controlled and orchestrated is poorly understood. Here, we identified JMJD6 as a new member of the maintenance of rDNA integrity. We observed that JMJD6 controls the recruitment of NBS1 in the nucleolus in order to lead to the proper response to DNA damage at rDNA repeats.\n\nAuthor contributionsYC and DT wrote the manuscript with input from their coauthors. JF, CC, JH, S-KM, JL, J-PL and YC performed the experiments. JC supervised JF and JH during purification and mass spectrometry analysis. YC and DT conceived the project, designed research and coordinated the studies.

cell biology↗