TDP1 mutation causing SCAN1 neurodegenerative syndrome hampers the repair of transcriptional DNA double-strand breaks.

TDP1 removes transcription-blocking topoisomerase I cleavage complexes (TOP1ccs), and its inactivating H493R mutation causes the neurodegenerative syndrome SCAN1. However, the molecular mechanism underlying the SCAN1 phenotype is unclear. Here, we generate human SCAN1 cell models using CRISPR-Cas9 and show that they accumulate TOP1ccs along with changes in gene expression and genomic distribution of R-loops. SCAN1 cells also accumulate transcriptional DNA double-strand breaks (DSBs) specifically in the G1 cell population due to increased DSB formation and lack of repair, both resulting from abortive removal of transcription-blocking TOP1ccs. Deficient TDP1 activity causes increased DSB production, and the presence of mutated TDP1 protein hampers DSB repair by a TDP2-dependent backup pathway. This study provides powerful models to study TDP1 functions under physiological and pathological conditions and unravels that a gain of function of the mutated TDP1 protein, which prevents DSB repair, rather than a loss of TDP1 activity itself, could contribute to SCAN1 pathogenesis.

Small Interfering RNAs 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, including plants and yeast. In mammals, its extent remains somewhat debated. Previous studies showed that siRNAs targeting gene promoters could induce the silencing of the targeted promoter, although the involvement of off-target mechanisms was also suggested. Here, by using nascent RNA capture and RNA polymerase II chromatin immunoprecipitation, we show that siRNAs targeting a chromatin-associated noncoding RNA induced its transcriptional silencing. Deletion of the sequence targeted by one of these siRNAs on the two alleles by genome editing further showed that this silencing was due to base-pairing of the siRNA to the target. Moreover, by using cells with heterozygous deletion of the target sequence, we showed that only the wild-type allele, but not the deleted allele, was silenced by the siRNA, indicating that transcriptional silencing occurred only in cis. Finally, we demonstrated that both Ago1 and Ago2 are involved in this transcriptional silencing. Altogether, our data demonstrate that siRNAs targeting a chromatin-associated RNA at a distance from its promoter induce its transcriptional silencing. Our results thus extend the possible repertoire of endogenous or exogenous interfering RNAs.

RNA polymerase II speed: a key player in controlling and adapting transcriptome composition.

RNA polymerase II (RNA Pol II) speed or elongation rate, i.e., the number of nucleotides synthesized per unit of time, is a major determinant of transcriptome composition. It controls co-transcriptional processes such as splicing, polyadenylation, and transcription termination, thus regulating the production of alternative splice variants, circular RNAs, alternatively polyadenylated transcripts, or read-through transcripts. RNA Pol II speed itself is regulated in response to intra- and extra-cellular stimuli and can in turn affect the transcriptome composition in response to these stimuli. Evidence points to a potentially important role of transcriptome composition modification through RNA Pol II speed regulation for adaptation of cells to a changing environment, thus pointing to a function of RNA Pol II speed regulation in cellular physiology. Analyzing RNA Pol II speed dynamics may therefore be central to fully understand the regulation of physiological processes, such as the development of multicellular organisms. Recent findings also raise the possibility that RNA Pol II speed deregulation can be detrimental and participate in disease progression. Here, we review initial and current approaches to measure RNA Pol II speed, as well as providing an overview of the factors controlling speed and the co-transcriptional processes which are affected. Finally, we discuss the role of RNA Pol II speed regulation in cell physiology.

KDM5A and KDM5B histone-demethylases contribute to HU-induced 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 levels of activated Chk1, a major player of the intra-S phase checkpoint that protects cells from RS. We also found that 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. 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.

Cytolethal Distending Toxin Promotes Replicative Stress Leading to Genetic Instability Transmitted to Daughter Cells.

The cytolethal distending toxin (CDT) is produced by several Gram-negative pathogenic bacteria. In addition to inflammation, experimental evidences are in favor of a protumoral role of CDT-harboring bacteria such as Escherichia coli, Campylobacter jejuni, or Helicobacter hepaticus. CDT may contribute to cell transformation in vitro and carcinogenesis in mice models, through the genotoxic action of CdtB catalytic subunit. Here, we investigate the mechanism of action by which CDT leads to genetic instability in human cell lines and colorectal organoids from healthy patients' biopsies. We demonstrate that CDT holotoxin induces a replicative stress dependent on CdtB. The slowing down of DNA replication occurs mainly in late S phase, resulting in the expression of fragile sites and important chromosomic aberrations. These DNA abnormalities induced after CDT treatment are responsible for anaphase bridge formation in mitosis and interphase DNA bridge between daughter cells in G1 phase. Moreover, CDT-genotoxic potential preferentially affects human cycling cells compared to quiescent cells. Finally, the toxin induces nuclear distension associated to DNA damage in proliferating cells of human colorectal organoids, resulting in decreased growth. Our findings thus identify CDT as a bacterial virulence factor targeting proliferating cells, such as human colorectal progenitors or stem cells, inducing replicative stress and genetic instability transmitted to daughter cells that may therefore contribute to carcinogenesis. As some CDT-carrying bacterial strains were detected in patients with colorectal cancer, targeting these bacteria could be a promising therapeutic strategy.

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.

Chromatin Dynamics in Intestinal Epithelial Homeostasis: A Paradigm of Cell Fate Determination versus Cell Plasticity.

The rapid renewal of intestinal epithelium is mediated by a pool of stem cells, located at the bottom of crypts, giving rise to highly proliferative progenitor cells, which in turn differentiate during their migration along the villus. The equilibrium between renewal and differentiation is critical for establishment and maintenance of tissue homeostasis, and is regulated by signaling pathways (Wnt, Notch, Bmp…) and specific transcription factors (TCF4, CDX2…). Such regulation controls intestinal cell identities by modulating the cellular transcriptome. Recently, chromatin modification and dynamics have been identified as major actors linking signaling pathways and transcriptional regulation in the control of intestinal homeostasis. In this review, we synthesize the many facets of chromatin dynamics involved in controlling intestinal cell fate, such as stemness maintenance, progenitor identity, lineage choice and commitment, and terminal differentiation. In addition, we present recent data underlying the fundamental role of chromatin dynamics in intestinal cell plasticity. Indeed, this plasticity, which includes dedifferentiation processes or the response to environmental cues (like microbiota's presence or food ingestion), is central for the organ's physiology. Finally, we discuss the role of chromatin dynamics in the appearance and treatment of diseases caused by deficiencies in the aforementioned mechanisms, such as gastrointestinal cancer, inflammatory bowel disease or irritable bowel syndrome. Graphical abstract.

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 and rDNA repeats instability has been reported in cancer, aging and neurological diseases. Here, we describe that the histone demethylase JMJD6 is rapidly recruited to nucleolar DNA damage and is crucial for the relocalisation of rDNA in nucleolar caps. Yet, JMJD6 is dispensable for rDNA transcription inhibition. Mass spectrometry analysis 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 reveal that rDNA transcription inhibition is uncoupled from rDNA relocalisation into nucleolar caps and that JMJD6 is required for rDNA stability through its role in nucleolar caps formation.

Integrated analysis of H2A.Z isoforms function reveals a complex interplay in gene regulation.

The H2A.Z histone variant plays major roles in the control of gene expression. In human, H2A.Z is encoded by two genes expressing two isoforms, H2A.Z.1 and H2A.Z.2 differing by three amino acids. Here, we undertook an integrated analysis of their functions in gene expression using endogenously-tagged proteins. RNA-Seq analysis in untransformed cells showed that they can regulate both distinct and overlapping sets of genes positively or negatively in a context-dependent manner. Furthermore, they have similar or antagonistic function depending on genes. H2A.Z.1 and H2A.Z.2 can replace each other at Transcription Start Sites, providing a molecular explanation for this interplay. Mass spectrometry analysis showed that H2A.Z.1 and H2A.Z.2 have specific interactors, which can mediate their functional antagonism. Our data indicate that the balance between H2A.Z.1 and H2A.Z.2 at promoters is critically important to regulate specific gene expression, providing an additional layer of complexity to the control of gene expression by histone variants.

The H2A.Z histone variant integrates Wnt signaling in intestinal epithelial homeostasis.

The Tip60/p400 chromatin-modifying complex, which is involved in the incorporation and post-translational modification of the H2A.Z histone variant, regulates cell proliferation and important signaling pathways, such as Wnt. Here, we study the involvement of H2A.Z in intestinal epithelial homeostasis, which is dependent on the finely-tuned equilibrium between stem cells renewal and differentiation, under the control of such pathway. We use cell models and inducible knock-out mice to study the impact of H2A.Z depletion on intestinal homeostasis. We show that H2A.Z is essential for the proliferation of human cancer and normal intestinal crypt cells and negatively controls the expression of a subset of differentiation markers, in cultured cells and mice. H2A.Z impairs the recruitment of the intestine-specific transcription factor CDX2 to chromatin, is itself a target of the Wnt pathway and thus, acts as an integrator for Wnt signaling in the control of intestinal epithelial cell fate and homeostasis.

Control of Gene Expression in Senescence through Transcriptional Read-Through of Convergent Protein-Coding Genes.

Antisense RNAs are non-coding RNAs that can regulate their corresponding sense RNAs and are generally produced from specific promoters. We uncover here a family of antisense RNAs, named START RNAs, produced during cellular senescence by transcriptional read-through at convergent protein-coding genes. Importantly, START RNAs repress the expression of their corresponding sense RNAs. In proliferative cells, we found that the Pol II elongation rate is limited downstream of TTS at START loci, allowing transcription termination to occur before Pol II reaches the convergent genes, thus preventing antisense RNA production and interference with the expression of the convergent genes. START RNAs are repressed by H2A.Z histone variant, whose local occupancy decreases in senescence. Our results thus uncover a mechanism of gene expression regulation relying on read-through antisense transcript production at convergent genes, underlining the functional importance of chromatin regulation in the control of RNA pol II elongation rate at intergenic regions.

The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability.

The interplay between methylation and demethylation of histone lysine residues is an essential component of gene expression regulation and there is considerable interest in elucidating the roles of proteins involved. Here we report that histone demethylase KDM4A/JMJD2A, which is involved in the regulation of cell proliferation and is overexpressed in some cancers, interacts with RNA Polymerase I, associates with active ribosomal RNA genes and is required for serum-induced activation of rDNA transcription. We propose that KDM4A controls the initial stages of transition from 'poised', non-transcribed rDNA chromatin into its active form. We show that PI3K, a major signalling transducer central for cell proliferation and survival, controls cellular localization of KDM4A and consequently its association with ribosomal DNA through the SGK1 downstream kinase. We propose that the interplay between PI3K/SGK1 signalling cascade and KDM4A constitutes a mechanism by which cells adapt ribosome biogenesis level to the availability of growth factors and nutrients.

Control of alternative end joining by the chromatin remodeler p400 ATPase.

Repair of DNA double-strand breaks occurs in a chromatin context that needs to be modified and remodeled to allow suitable access to the different DNA repair machineries. Of particular importance for the maintenance of genetic stability is the tight control of error-prone pathways, such as the alternative End Joining pathway. Here, we show that the chromatin remodeler p400 ATPase is a brake to the use of alternative End Joining. Using specific intracellular reporter susbstrates we observed that p400 depletion increases the frequency of alternative End Joining events, and generates large deletions following repair of double-strand breaks. This increase of alternative End Joining events is largely dependent on CtIP-mediated resection, indicating that it is probably related to the role of p400 in late steps of homologous recombination. Moreover, p400 depletion leads to the recruitment of poly(ADP) ribose polymerase (PARP) and DNA ligase 3 at DNA double-strand breaks, driving to selective killing by PARP inhibitors. All together these results show that p400 acts as a brake to prevent alternative End Joining-dependent genetic instability and underline its potential value as a clinical marker.

Control of genetic stability by a new heterochromatin compaction pathway involving the Tip60 histone acetyltransferase.

Pericentric heterochromatin is a highly compacted structure required for accurate chromosome segregation in mitosis. In mammals, it relies on methylation of histone H3K9 by Suv39H enzymes, which provides a docking site for HP1 proteins, therefore mediating heterochromatin compaction. Here we show that, when this normal compaction pathway is defective, the histone acetyltransferase Tip60 is recruited to pericentric heterochromatin, where it mediates acetylation of histone H4K12. Furthermore, in such a context, depletion of Tip60 leads to derepression of satellite transcription, decompaction of pericentric heterochromatin, and defects in chromosome segregation in mitosis. Finally, we show that depletion of BRD2, a double bromodomain-containing protein that binds H4K12ac, phenocopies the Tip60 depletion with respect to heterochromatin decompaction and defects in chromosome segregation. Taking the results together, we identify a new compaction pathway of mammalian pericentric heterochromatin relying on Tip60 that might be dependent on BRD2 recruitment by H4K12 acetylation. We propose that the underexpression of Tip60 observed in many human tumors can promote genetic instability via defective pericentric heterochromatin.

A vlincRNA participates in senescence maintenance by relieving H2AZ-mediated repression at the INK4 locus.

Non-coding RNAs (ncRNAs) play major roles in proper chromatin organization and function. Senescence, a strong anti-proliferative process and a major anticancer barrier, is associated with dramatic chromatin reorganization in heterochromatin foci. Here we analyze strand-specific transcriptome changes during oncogene-induced human senescence. Strikingly, while differentially expressed RNAs are mostly repressed during senescence, ncRNAs belonging to the recently described vlincRNA (very long intergenic ncRNA) class are mainly activated. We show that VAD, a novel antisense vlincRNA strongly induced during senescence, is required for the maintenance of senescence features. VAD modulates chromatin structure in cis and activates gene expression in trans at the INK4 locus, which encodes cell cycle inhibitors important for senescence-associated cell proliferation arrest. Importantly, VAD inhibits the incorporation of the repressive histone variant H2A.Z at INK4 gene promoters in senescent cells. Our data underline the importance of vlincRNAs as sensors of cellular environment changes and as mediators of the correct transcriptional response.

Quantifying DNA double-strand breaks induced by site-specific endonucleases in living cells by ligation-mediated purification.

Recent advances in our understanding of the management and repair of DNA double-strand breaks (DSBs) rely on the study of targeted DSBs that have been induced in living cells by the controlled activity of site-specific endonucleases, usually recombinant restriction enzymes. Here we describe a protocol for quantifying these endonuclease-induced DSBs; this quantification is essential to an interpretation of how DSBs are managed and repaired. A biotinylated double-stranded oligonucleotide is ligated to enzyme-cleaved genomic DNA, allowing the purification of the cleaved DNA on streptavidin beads. The extent of cleavage is then quantified either by quantitative PCR (qPCR) at a given site or at multiple sites by genome-wide techniques (e.g., microarrays or high-throughput sequencing). This technique, named ligation-mediated purification, can be performed in 2 d. It is more accurate and sensitive than existing alternative methods, and it is compatible with genome-wide analysis. It allows the amount of endonuclease-mediated breaks to be precisely compared between two conditions or across the genome, thereby giving insight into the influence of a given factor or of various chromatin contexts on local repair parameters.

Interplay between chromatin-modifying enzymes controls colon cancer progression through Wnt signaling.

Cancer progression is associated with epigenetic alterations, such as changes in DNA methylation, histone modifications or variants incorporation. The p400 ATPase, which can incorporate the H2A.Z variant, and the Tip60 histone acetyltransferase are interacting chromatin-modifying proteins crucial for the control of cell proliferation. We demonstrate here that Tip60 acts as a tumor suppressor in colon, since mice heterozygous for Tip60 are more susceptible to chemically induced preneoplastic lesions and adenomas. Strikingly, heterozygosity for p400 reverses the Tip60-dependent formation of preneoplastic lesions, uncovering for the first time pro-oncogenic functions for p400. By genome-wide analysis and using a specific inhibitor in vivo, we demonstrated that these effects are dependent on Wnt signaling which is antagonistically impacted by p400 and Tip60: p400 directly favors the expression of a subset of Wnt-target genes and regulators, whereas Tip60 prevents ß-catenin acetylation and activation. Taken together, our data underline the physiopathological importance of interplays between chromatin-modifying enzymes in the control of cancer-related signaling pathways.

H2A.Z depletion impairs proliferation and viability but not DNA double-strand breaks repair in human immortalized and tumoral cell lines.

In mammalian cells, DNA double-strand breaks (DSB) can be repaired by 2 main pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). To give access to DNA damage to the repair machinery the chromatin structure needs to be relaxed, and chromatin modifications play major roles in the control of these processes. Among the chromatin modifications, changes in nucleosome composition can influence DNA damage response as observed with the H2A.Z histone variant in yeast. In mammals, p400, an ATPase of the SWI/SNF family able to incorporate H2A.Z in chromatin, was found to be important for histone ubiquitination and BRCA1 recruitment around DSB or for HR in cooperation with Rad51. Recent data with 293T cells showed that mammalian H2A.Z is recruited to DSBs and is important to control DNA resection, therefore participating both in HR and NHEJ. Here we show that depletion of H2A.Z in the osteosarcoma U2OS cell line and in immortalized human fibroblasts does not change parameters of DNA DSB repair while affecting clonogenic ability and cell cycle distribution. In addition, no recruitment of H2A.Z around DSB can be detected in U2OS cells either after local laser irradiation or by chromatin immunoprecipitation. These data suggest that the role of H2A.Z in DSB repair is not ubiquitous in mammals. In addition, given that important cellular parameters, such as cell viability and cell cycle distribution, are more sensitive to H2A.Z depletion than DNA repair, our results underline the difficulty to investigate the role of versatile factors such as H2A.Z.

The chromatin remodeler p400 ATPase facilitates Rad51-mediated repair of DNA double-strand breaks.

DNA damage signaling and repair take place in a chromatin context. Consequently, chromatin-modifying enzymes, including adenosine triphosphate-dependent chromatin remodeling enzymes, play an important role in the management of DNA double-strand breaks (DSBs). Here, we show that the p400 ATPase is required for DNA repair by homologous recombination (HR). Indeed, although p400 is not required for DNA damage signaling, DNA DSB repair is defective in the absence of p400. We demonstrate that p400 is important for HR-dependent processes, such as recruitment of Rad51 to DSB (a key component of HR), homology-directed repair, and survival after DNA damage. Strikingly, p400 and Rad51 are present in the same complex and both favor chromatin remodeling around DSBs. Altogether, our data provide a direct molecular link between Rad51 and a chromatin remodeling enzyme involved in chromatin decompaction around DNA DSBs.