Publications de l'équipe - Programme de réplication et instabilité du génome - Centre de Recherche Institut Curie
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Publications de l’équipe
Programme de réplication et instabilité du génome
Année de publication : 2020
Arijita Chakraborty, Piroon Jenjaroenpun, Jing Li, Sami El Hilali, Andrew McCulley, Brian Haarer,
Elizabeth A Hoffman, Aimee Belak, Audrey Thorland, Heidi Hehnly, Carl Schildkraut, Chun-Long
Chen, Vladimir A Kuznetsov, Wenyi Feng (2020 Sep 23)
Replication Stress Induces Global Chromosome Breakage in the Fragile X
Genome.
Cell reports : 108179 : DOI : S2211-1247(20)31168-2
Résumé
Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by mutations in the FMR1
gene and deficiency of a functional FMRP protein. FMRP is known as a translation repressor
whose nuclear function is not understood. We investigated the global impact on genome
stability due to FMRP loss. Using Break-seq, we map spontaneous and replication stress-
induced DNA double-strand breaks (DSBs) in an FXS patient-derived cell line. We report that
the genomes of FXS cells are inherently unstable and accumulate twice as many DSBs as
those from an unaffected control. We demonstrate that replication stress-induced DSBs in
FXS cells colocalize with R-loop forming sequences. Exogenously expressed FMRP in FXS
fibroblasts ameliorates DSB formation. FMRP, not the I304N mutant, abates R-loop-induced
DSBs during programmed replication-transcription conflict. These results suggest that FMRP
is a genome maintenance protein that prevents R-loop accumulation. Our study provides
insights into the etiological basis for FXS.
Zhiming Li, Xu Hua, Albert Serra-Cardona, Xiaowei Xu, Songlin Gan, Hui Zhou, Wen-Si Yang,
Chun-Long Chen, Rui-Ming Xu, Zhiguo Zhang (2020 Sep 14)
DNA polymerase α interacts with H3-H4 and facilitates the transfer of parental
histones to lagging strands.
Science advances : eabb5820 : DOI : 10.1126/sciadv.abb5820
Résumé
How parental histones, the carriers of epigenetic modifications, are deposited onto
replicating DNA remains poorly understood. Here, we describe the eSPAN method
(enrichment and sequencing of protein-associated nascent DNA) in mouse embryonic stem
(ES) cells and use it to detect histone deposition onto replicating DNA strands with a
relatively small number of cells. We show that DNA polymerase α (Pol α), which synthesizes
short primers for DNA synthesis, binds histone H3-H4 preferentially. A Pol α mutant defective
in histone binding in vitro impairs the transfer of parental H3-H4 to lagging strands in both
yeast and mouse ES cells. Last, dysregulation of both coding genes and noncoding
endogenous retroviruses is detected in mutant ES cells defective in parental histone transfer.
Together, we report an efficient eSPAN method for analysis of DNA replication-linked
processes in mouse ES cells and reveal the mechanism of Pol α in parental histone transfer.
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 1Publications de l’équipe
Programme de réplication et instabilité du génome
Gnan Stefano, Liu Yaqun, Spagnuolo Manuela, Chen Chun-Long (2020 Aug 31)
The impact of transcription-mediated replication stress on genome instability
and human disease
Genome Instability & Disease : 1 : 207-234 : DOI : 10.1007/s42764-020-00021-y
Résumé
DNA replication is a vital process in all living organisms. At each cell division, > 30,000
replication origins are activated in a coordinated manner to ensure the duplication of > 6
billion base pairs of the human genome. During differentiation and development, this
program must adapt to changes in chromatin organization and gene transcription: its
deregulation can challenge genome stability, which is a leading cause of many diseases
including cancers and neurological disorders. Over the past decade, great progress has been
made to better understand the mechanisms of DNA replication regulation and how its
deregulation challenges genome integrity and leads to human disease. Growing evidence
shows that gene transcription has an essential role in shaping the landscape of genome
replication, while it is also a major source of endogenous replication stress inducing genome
instability. In this review, we discuss the current knowledge on the various mechanisms by
which gene transcription can impact on DNA replication, leading to genome instability and
human disease.
Alexy Promonet, Ismaël Padioleau, Yaqun Liu, Lionel Sanz, Anna Biernacka, Anne-Lyne Schmitz,
Magdalena Skrzypczak, Amélie Sarrazin, Clément Mettling, Maga Rowicka, Krzysztof Ginalski,
Frédéric Chedin, Chun-Long Chen, Yea-Lih Lin, Philippe Pasero (2020 Aug 10)
Topoisomerase 1 prevents replication stress at R-loop-enriched transcription
termination sites.
Nature communications : 3940 : DOI : 10.1038/s41467-020-17858-2
Résumé
R-loops have both positive and negative impacts on chromosome functions. To identify toxic
R-loops in the human genome, here, we map RNA:DNA hybrids, replication stress markers
and DNA double-strand breaks (DSBs) in cells depleted for Topoisomerase I (Top1), an
enzyme that relaxes DNA supercoiling and prevents R-loop formation. RNA:DNA hybrids are
found at both promoters (TSS) and terminators (TTS) of highly expressed genes. In contrast,
the phosphorylation of RPA by ATR is only detected at TTS, which are preferentially
replicated in a head-on orientation relative to the direction of transcription. In Top1-depleted
cells, DSBs also accumulate at TTS, leading to persistent checkpoint activation, spreading of
γ-H2AX on chromatin and global replication fork slowdown. These data indicate that fork
pausing at the TTS of highly expressed genes containing R-loops prevents head-on conflicts
between replication and transcription and maintains genome integrity in a Top1-dependent
manner.
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 2Publications de l’équipe
Programme de réplication et instabilité du génome
Année de publication : 2019
Olivier Brison, Sami El-Hilali, Dana Azar, Stéphane Koundrioukoff, Mélanie Schmidt, Viola Nähse,
Yan Jaszczyszyn, Anne-Marie Lachages, Bernard Dutrillaux, Claude Thermes, Michelle Debatisse,
Chun-Long Chen (2019 Dec 15)
Transcription-mediated organization of the replication initiation program across
large genes sets common fragile sites genome-wide.
Nature communications : 5693 : DOI : 10.1038/s41467-019-13674-5
Résumé
Common fragile sites (CFSs) are chromosome regions prone to breakage upon replication
stress known to drive chromosome rearrangements during oncogenesis. Most CFSs nest in
large expressed genes, suggesting that transcription could elicit their instability; however,
the underlying mechanisms remain elusive. Genome-wide replication timing analyses here
show that stress-induced delayed/under-replication is the hallmark of CFSs. Extensive
genome-wide analyses of nascent transcripts, replication origin positioning and fork
directionality reveal that 80% of CFSs nest in large transcribed domains poor in initiation
events, replicated by long-travelling forks. Forks that travel long in late S phase explains CFS
replication features, whereas formation of sequence-dependent fork barriers or head-on
transcription-replication conflicts do not. We further show that transcription inhibition during
S phase, which suppresses transcription-replication encounters and prevents origin resetting,
could not rescue CFS stability. Altogether, our results show that transcription-dependent
suppression of initiation events delays replication of large gene bodies, committing them to
instability.
Brison O., EL-Hilali S., Azar D., Koundrioukoff S., Schmidt M., Naehse-Kumpf V., Jaszczyszyn Y.,
Lachages A.M., Dutrillaux B., Thermes C., Debatisse M., Chen C.L. (2019 Jul 1)
TRANSCRIPTION-MEDIATED ORGANIZATION OF THE REPLICATION INITIATION
PROGRAM ACROSS LARGE GENES SETS UP COMMON FRAGILE SITES GENOME-
WIDE
bioRxiv : 714717 : DOI : 10.1101/714717
Résumé
Common Fragile Sites (CFSs) are chromosome regions prone to breakage under replication
stress, known to drive chromosome rearrangements during oncogenesis. Most CFSs nest in
large expressed genes, suggesting that transcription elicits their instability but the
underlying mechanisms remained elusive. Analyses of genome-wide replication timing of
human lymphoblasts here show that stress-induced delayed/under-replication is the hallmark
of CFSs. Extensive genome-wide analyses of nascent transcripts, replication origin
positioning and fork directionality reveal that 80% of CFSs nest in large transcribed domains
poor in initiation events, thus replicated by long-traveling forks. In contrast to formation of
sequence-dependent fork barriers or head-on transcription-replication conflicts, traveling-
long in late S phase explains CFS replication features. We further show that transcription
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 3Publications de l’équipe
Programme de réplication et instabilité du génome
inhibition during the S phase, which excludes the setting of new replication origins, fails to
rescue CFS stability. Altogether, results show that transcription-dependent suppression of
initiation events delays replication of large gene body, committing them to instability.
Chakraborty A., Jenjaroenpun P., McCulley A., Li J., Hilali S.E., Haarer B., Hoffman E.A., Belak A.,
Thorland A., Hehnly H., Chen C.l., Kuznetsov V., Feng W. (2019 Jun 1)
Fragile X Mental Retardation Protein regulates R-loop formation and prevents
global chromosome fragility
bioRxiv : 601906 : DOI : 10.1101/601906
Résumé
Fragile X syndrome (FXS) is the most prevalent inherited intellectual disability caused by
mutations in the Fragile X Mental Retardation gene (FMR1) and deficiency of its product,
FMRP. FMRP is a predominantly cytoplasmic protein thought to bind specific mRNA targets
and regulate protein translation. Its potential role in the nucleus is not well understood. We
are interested in the global impact on chromosome stability due to FMRP loss. Here we
report that compared to an FMRP-proficient normal cell line, cells derived from FXS patients
exhibit increased chromosome breaks upon DNA replication stress induced by a DNA
polymerase inhibitor, aphidicolin. Moreover, cells from FXS individuals fail to protect genomic
regions containing R-loops (co-transcriptional DNA:RNA hybrids) from aphidicolin-induced
chromosome breaks. We demonstrate that FMRP is important for abating R-loop
accumulation during transcription, particularly in the context of head-on collision with a
replication fork, and thereby preventing chromosome breakage. By identifying those FMRP-
bound chromosomal loci with overlapping R-loops and fragile sites, we report a list of novel
FMRP target loci, many of which have been implicated in neurological disorders. We show
that cells from FXS patients have reduced expression of xenobiotics metabolic enzymes,
suggesting defective xenobiotics metabolism/excretion might contribute to disease
development. Our study provides new insights into the etiological basis of, and enables the
discovery of new therapeutic targets for, the FXS.
Ming-Jun Shi, Xiang-Yu Meng, Philippe Lamy, A Rouf Banday, Jie Yang, Aura Moreno-Vega, Chun-
Long Chen, Lars Dyrskjøt, Isabelle Bernard-Pierrot, Ludmila Prokunina-Olsson, François Radvanyi
(2019 Apr 13)
APOBEC-mediated Mutagenesis as a Likely Cause of FGFR3 S249C Mutation
Over-representation in Bladder Cancer.
European urology : 9-13 : DOI : S0302-2838(19)30261-1
Résumé
FGFR3 is one of the most frequently mutated genes in bladder cancer and a driver of an
oncogenic dependency. Here we report that only the most common recurrent FGFR3
mutation, S249C (TCC→TGC), represents an APOBEC-type motif and is probably caused by
the APOBEC-mediated mutagenic process, accounting for its over-representation. We
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 4Publications de l’équipe
Programme de réplication et instabilité du génome
observed significant enrichment of the APOBEC mutational signature and overexpression of
AID/APOBEC gene family members in bladder tumors with S249C compared to tumors with
other recurrent FGFR3 mutations. Analysis of replication fork directionality suggests that the
coding strand of FGFR3 is predominantly replicated as a lagging strand template that could
favor the formation of hairpin structures, facilitating mutagenic activity of APOBEC enzymes.
In vitro APOBEC deamination assays confirmed S249 as an APOBEC target. We also found
that the FGFR3 S249C mutation was common in three other cancer types with an APOBEC
mutational signature, but rare in urothelial tumors without APOBEC mutagenesis and in two
diseases probably related to aging. PATIENT SUMMARY: We propose that APOBEC-mediated
mutagenesis can generate clinically relevant driver mutations even within suboptimal motifs,
such as in the case of FGFR3 S249C, one of the most common mutations in bladder cancer.
Knowledge about the etiology of this mutation will improve our understanding of the
molecular mechanisms of bladder cancer.
Année de publication : 2017
Klein K., Wang W., Borrman T., Chan S., Zhang D., Weng Z., Hastie A., Chen C., Gilbert D.M.,
Rhind N. (2017 Jan 1)
Genome-Wide Identification of Early-Firing Human Replication Origins by Optical
Replication Mapping
bioRxiv : 214841 : DOI : 10.1101/214841
Résumé
The timing of DNA replication is largely regulated by the location and timing of replication
origin firing. Therefore, much effort has been invested in identifying and analyzing human
replication origins. However, the heterogeneous nature of eukaryotic replication kinetics and
the low efficiency of individual origins in metazoans has made mapping the location and
timing of replication initiation in human cells difficult. We have mapped early-firing origins in
HeLa cells using Optical Replication Mapping, a high-throughput single-molecule approach
based on Bionano Genomics genomic mapping technology. The single-molecule nature and
290-fold coverage of our dataset allowed us to identify origins that fire with as little as 1%
efficiency. We find sites of human replication initiation in early S phase are not confined to
well-defined efficient replication origins, but are instead distributed across broad initiation
zones consisting of many inefficient origins. These early-firing initiation zones co-localize with
initiation zones inferred from Okazaki-fragment-mapping analysis and are enriched in ORC1
binding sites. Although most early-firing origins fire in early-replication regions of the
genome, a significant number fire in late-replicating regions, suggesting that the major
difference between origins in early and late replicating regions is their probability of firing in
early S-phase, as opposed to qualitative differences in their firing-time distributions. This
observation is consistent with stochastic models of origin timing regulation, which explain
the regulation of replication timing in yeast.
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 5Publications de l’équipe
Programme de réplication et instabilité du génome
Année de publication : 2016
Nataliya Petryk, Malik Kahli, Yves d'Aubenton-Carafa, Yan Jaszczyszyn, Yimin Shen, Maud Silvain,
Claude Thermes, Chun-Long Chen, Olivier Hyrien (2016 Jan 12)
Replication landscape of the human genome.
Nature communications : 10208 : DOI : 10.1038/ncomms10208
Résumé
Despite intense investigation, human replication origins and termini remain elusive. Existing
data have shown strong discrepancies. Here we sequenced highly purified Okazaki
fragments from two cell types and, for the first time, quantitated replication fork
directionality and delineated initiation and termination zones genome-wide. Replication
initiates stochastically, primarily within non-transcribed, broad (up to 150 kb) zones that
often abut transcribed genes, and terminates dispersively between them. Replication fork
progression is significantly co-oriented with the transcription. Initiation and termination
zones are frequently contiguous, sometimes separated by regions of unidirectional
replication. Initiation zones are enriched in open chromatin and enhancer marks, even when
not flanked by genes, and often border ‘topologically associating domains’ (TADs). Initiation
zones are enriched in origin recognition complex (ORC)-binding sites and better align to
origins previously mapped using bubble-trap than λ-exonuclease. This novel panorama of
replication reveals how chromatin and transcription modulate the initiation process to create
cell-type-specific replication programs.
Année de publication : 2013
Olivier Hyrien, Aurélien Rappailles, Guillaume Guilbaud, Antoine Baker, Chun-Long Chen, Arach
Goldar, Nataliya Petryk, Malik Kahli, Emilie Ma, Yves d'Aubenton-Carafa, Benjamin Audit, Claude
Thermes, Alain Arneodo (2013 Oct 8)
From simple bacterial and archaeal replicons to replication N/U-domains.
Journal of molecular biology : 4673-89 : DOI : 10.1016/j.jmb.2013.09.021
Résumé
The Replicon Theory proposed 50 years ago has proven to apply for replicons of the three
domains of life. Here, we review our knowledge of genome organization into single and
multiple replicons in bacteria, archaea and eukarya. Bacterial and archaeal
replicator/initiator systems are quite specific and efficient, whereas eukaryotic replicons
show degenerate specificity and efficiency, allowing for complex regulation of origin firing
time. We expand on recent evidence that ~50% of the human genome is organized as
~1,500 megabase-sized replication domains with a characteristic parabolic (U-shaped)
replication timing profile and linear (N-shaped) gradient of replication fork polarity. These
N/U-domains correspond to self-interacting segments of the chromatin fiber bordered by
open chromatin zones and replicate by cascades of origin firing initiating at their borders and
propagating to their center, possibly by fork-stimulated initiation. The conserved occurrence
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 6Publications de l’équipe
Programme de réplication et instabilité du génome
of this replication pattern in the germline of mammals has resulted over evolutionary times
in the formation of megabase-sized domains with an N-shaped nucleotide compositional
skew profile due to replication-associated mutational asymmetries. Overall, these results
reveal an evolutionarily conserved but developmentally plastic organization of replication
that is driving mammalian genome evolution.
Année de publication : 2012
Benjamin Audit, Antoine Baker, Chun-Long Chen, Aurélien Rappailles, Guillaume Guilbaud,
Hanna Julienne, Arach Goldar, Yves d'Aubenton-Carafa, Olivier Hyrien, Claude Thermes, Alain
Arneodo (2012 Dec 15)
Multiscale analysis of genome-wide replication timing profiles using a wavelet-
based signal-processing algorithm.
Nature protocols : 98-110 : DOI : 10.1038/nprot.2012.145
Résumé
In this protocol, we describe the use of the LastWave open-source signal-processing
command language (http://perso.ens-lyon.fr/benjamin.audit/LastWave/) for analyzing cellular
DNA replication timing profiles. LastWave makes use of a multiscale, wavelet-based signal-
processing algorithm that is based on a rigorous theoretical analysis linking timing profiles to
fundamental features of the cell’s DNA replication program, such as the average replication
fork polarity and the difference between replication origin density and termination site
density. We describe the flow of signal-processing operations to obtain interactive visual
analyses of DNA replication timing profiles. We focus on procedures for exploring the space-
scale map of apparent replication speeds to detect peaks in the replication timing profiles
that represent preferential replication initiation zones, and for delimiting U-shaped domains
in the replication timing profile. In comparison with the generally adopted approach that
involves genome segmentation into regions of constant timing separated by timing transition
regions, the present protocol enables the recognition of more complex patterns of the spatio-
temporal replication program and has a broader range of applications. Completing the full
procedure should not take more than 1 h, although learning the basics of the program can
take a few hours and achieving full proficiency in the use of the software may take days.
A Baker, C L Chen, H Julienne, B Audit, Y d'Aubenton-Carafa, C Thermes, A Arneodo (2012 Nov
27)
Linking the DNA strand asymmetry to the spatio-temporal replication program:
II. Accounting for neighbor-dependent substitution rates.
The European physical journal. E, Soft matter : 123 : DOI : 10.1140/epje/i2012-12123-9
Résumé
In paper I, we addressed the impact of the spatio-temporal program on the DNA composition
evolution in the case of time homogeneous and neighbor-independent substitution rates. But
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 7Publications de l’équipe
Programme de réplication et instabilité du génome
substitution rates do depend on the flanking nucleotides as exemplified in vertebrates where
CpG sites are hypermutable so that the substitution rate C –> T depends dramatically (ten
fold) on whether the cytosine belongs to a CG dinucleotide or not. With the specific goal to
account for neighbor-dependence, we revisit our minimal modeling of neutral substitution
rates in the human genome. When assuming that r = CpG –> TpG and its reverse
complement r(c) = CpG –> CpA are (by far) the main neighbor-dependent substitution rates,
we demonstrate, using perturbative analysis, that neighbor-dependence does not affect the
decomposition of the compositional asymmetry into a transcription- and a replication-
associated components, the former increases in magnitude with transcription rate and
changes sign with gene orientation, whereas the latter is proportional to the replication fork
polarity. Indeed the neighbor dependence case differs from the neighbor-independent model
by an additional source term related to the CG dinucleotide content in both the transcription
and replication-associated components. We finally discuss the case of time-dependent
substitution rates confirming as a very general result the fact that the skew can still be
decomposed into a transcription- and a replication-associated components.
Benjamin Audit, Lamia Zaghloul, Antoine Baker, Alain Arneodo, Chun-Long Chen, Yves
d'Aubenton-Carafa, Claude Thermes (2012 Nov 15)
Megabase replication domains along the human genome: relation to chromatin
structure and genome organisation.
Sub-cellular biochemistry : 57-80 : DOI : 10.1007/978-94-007-4525-4_3
Résumé
In higher eukaryotes, the absence of specific sequence motifs, marking the origins of
replication has been a serious hindrance to the understanding of (i) the mechanisms that
regulate the spatio-temporal replication program, and (ii) the links between origins
activation, chromatin structure and transcription. In this chapter, we review the partitioning
of the human genome into megabased-size replication domains delineated as N-shaped
motifs in the strand compositional asymmetry profiles. They collectively span 28.3% of the
genome and are bordered by more than 1,000 putative replication origins. We recapitulate
the comparison of this partition of the human genome with high-resolution experimental
data that confirms that replication domain borders are likely to be preferential replication
initiation zones in the germline. In addition, we highlight the specific distribution of
experimental and numerical chromatin marks along replication domains. Domain borders
correspond to particular open chromatin regions, possibly encoded in the DNA sequence, and
around which replication and transcription are highly coordinated. These regions also present
a high evolutionary breakpoint density, suggesting that susceptibility to breakage might be
linked to local open chromatin fiber state. Altogether, this chapter presents a
compartmentalization of the human genome into replication domains that are landmarks of
the human genome organization and are likely to play a key role in genome dynamics during
evolution and in pathological situations.
A Baker, H Julienne, C L Chen, B Audit, Y d'Aubenton-Carafa, C Thermes, A Arneodo (2012 Sep
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 8Publications de l’équipe
Programme de réplication et instabilité du génome
25)
Linking the DNA strand asymmetry to the spatio-temporal replication program.
I. About the role of the replication fork polarity in genome evolution.
The European physical journal. E, Soft matter : 92
Résumé
Two key cellular processes, namely transcription and replication, require the opening of the
DNA double helix and act differently on the two DNA strands, generating different mutational
patterns (mutational asymmetry) that may result, after long evolutionary time, in different
nucleotide compositions on the two DNA strands (compositional asymmetry). We elaborate
on the simplest model of neutral substitution rates that takes into account the strand
asymmetries generated by the transcription and replication processes. Using perturbation
theory, we then solve the time evolution of the DNA composition under strand-asymmetric
substitution rates. In our minimal model, the compositional and substitutional asymmetries
are predicted to decompose into a transcription- and a replication-associated components.
The transcription-associated asymmetry increases in magnitude with transcription rate and
changes sign with gene orientation while the replication-associated asymmetry is
proportional to the replication fork polarity. These results are confirmed experimentally in
the human genome, using substitution rates obtained by aligning the human and
chimpanzee genomes using macaca and orangutan as outgroups, and replication fork
polarity determined in the HeLa cell line as estimated from the derivative of the mean
replication timing. When further investigating the dynamics of compositional skew evolution,
we show that it is not at equilibrium yet and that its evolution is an extremely slow process
with characteristic time scales of several hundred Myrs.
INSTITUT CURIE, 20 rue d’Ulm, 75248 Paris Cedex 05, France | 9You can also read
