Replication timing analysis in polyploid cells reveals Rif1 uses multiple mechanisms to promote underreplication in Drosophila

Abstract Regulation of DNA replication and copy number is necessary to promote genome stability and maintain cell and tissue function. DNA replication is regulated temporally in a process known as replication timing (RT). Rap1-interacting factor 1 (Rif1) is a key regulator of RT and has a critical function in copy number control in polyploid cells. Previously, we demonstrated that Rif1 functions with SUUR to inhibit replication fork progression and promote underreplication (UR) of specific genomic regions. How Rif1-dependent control of RT factors into its ability to promote UR is unknown. By applying a computational approach to measure RT in Drosophila polyploid cells, we show that SUUR and Rif1 have differential roles in controlling UR and RT. Our findings reveal that Rif1 acts to promote late replication, which is necessary for SUUR-dependent underreplication. Our work provides new insight into the process of UR and its links to RT.

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Replication timing analysis in polyploid cells reveals Rif1 uses multiple mechanisms to promote underreplication in Drosophila.

10.1101/2021.04.19.440528 ◽

2021 ◽

Author(s):

Souradip Das ◽

Madison Caballero ◽

Tatyana Kolesnikova ◽

Igor Zhimulev ◽

Amnon Koren ◽

...

Keyword(s):

Dna Replication ◽

Copy Number ◽

Genome Stability ◽

Timing Analysis ◽

Replication Timing ◽

Critical Function ◽

Replication Fork Progression ◽

Polyploid Cells ◽

Genomic Regions ◽

Insight Into

Regulation of DNA replication and copy number are necessary to promote genome stability and maintain cell and tissue function. DNA replication is regulated temporally in a process known as replication timing (RT). Rif1 is key regulator of RT and has a critical function in copy number control in polyploid cells. In a previous study (Munden et al., 2018), we demonstrated that Rif1 functions with SUUR to inhibit replication fork progression and promote underreplication (UR) of specific genomic regions. How Rif1-dependent control of RT factors into its ability to promote UR is unknown. By applying a computational approach to measure RT in Drosophila polyploid cells, we show that SUUR and Rif1 have differential roles in controlling UR and RT. Our findings reveal that Rif1 functions both upstream and downstream of SUUR to promote UR. Our work provides new mechanistic insight into the process of UR and its links to RT.

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SAF-A promotes origin licensing and replication fork progression to ensure robust DNA replication

10.1101/2021.03.22.436394 ◽

2021 ◽

Author(s):

Caitlin Connolly ◽

Saori Takahashi ◽

Hisashi Miura ◽

Ichiro Hiratani ◽

Nick Gilbert ◽

...

Keyword(s):

Dna Replication ◽

Biological Activities ◽

Timing Analysis ◽

Replication Timing ◽

Synthesis Rate ◽

Open Chromatin ◽

Replication Fork Progression ◽

Chromosome Domains ◽

Origin Licensing ◽

In Cells

The organisation of chromatin is closely intertwined with biological activities of chromosome domains, including transcription and DNA replication status. Scaffold attachment factor A (SAF-A), also known as Heteronuclear Ribonucleoprotein Protein U (HNRNPU), contributes to the formation of open chromatin structure. Here we demonstrate that SAF-A promotes the normal progression of DNA replication, and enables resumption of replication after inhibition. We report that cells depleted for SAF-A show reduced origin licensing in G1 phase, and consequently reduced origin activation frequency in S phase. Replication forks progress slowly in cells depleted for SAF-A, also contributing to reduced DNA synthesis rate. Single-cell replication timing analysis revealed that the boundaries between early- and late- replicating domains are blurred in cells depleted for SAF-A. Associated with these defects, SAF-A-depleted cells show elevated gH2A phosphorylation and tend to enter quiescence. Overall we find that SAF-A protein promotes robust DNA replication to ensure continuing cell proliferation.

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SAF-A promotes origin licensing and replication fork progression to ensure robust DNA replication

Journal of Cell Science ◽

10.1242/jcs.258991 ◽

2021 ◽

Author(s):

Caitlin Connolly ◽

Saori Takahashi ◽

Hisashi Miura ◽

Ichiro Hiratani ◽

Nick Gilbert ◽

...

Keyword(s):

Dna Replication ◽

Biological Activities ◽

Timing Analysis ◽

Replication Timing ◽

Synthesis Rate ◽

Open Chromatin ◽

Replication Fork Progression ◽

Chromosome Domains ◽

Origin Licensing ◽

Attachment Factor

The organisation of chromatin is closely intertwined with biological activities of chromosome domains, including transcription and DNA replication status. Scaffold attachment factor A (SAF-A), also known as Heteronuclear Ribonucleoprotein Protein U (HNRNPU), contributes to the formation of open chromatin structure. Here we demonstrate that SAF-A promotes the normal progression of DNA replication, and enables resumption of replication after inhibition. We report that cells depleted for SAF-A show reduced origin licensing in G1 phase, and consequently reduced origin activation frequency in S phase. Replication forks also progress less consistently in cells depleted for SAF-A, contributing to reduced DNA synthesis rate. Single-cell replication timing analysis revealed two distinct effects of SAF-A depletion: first, the boundaries between early- and late-replicating domains become more blurred; and second, SAF-A depletion causes replication timing changes that tend to bring regions of discordant domain compartmentalisation and replication timing into concordance. Associated with these defects, SAF-A-depleted cells show elevated -H2AX formation and tend to enter quiescence. Overall we find that SAF-A protein promotes robust DNA replication to ensure continuing cell proliferation.

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Rif1 inhibits replication fork progression and controls DNA copy number in Drosophila

10.1101/346650 ◽

2018 ◽

Author(s):

Alexander Munden ◽

Zhan Rong ◽

Rama Gangula ◽

Simon Mallal ◽

Jared T. Nordman

Keyword(s):

Copy Number ◽

Replication Fork ◽

Genome Stability ◽

Replication Timing ◽

Physical Interaction ◽

Dependent Manner ◽

Replication Forks ◽

Dna Copy Number ◽

Replication Fork Progression ◽

Maintain Genome Stability

ABSTRACTControl of DNA copy number is essential to maintain genome stability and ensure proper cell and tissue function. In Drosophila polyploid cells, the SNF2-domain-containing SUUR protein inhibits replication fork progression within specific regions of the genome to promote DNA underreplication. While dissecting the function of SUUR’s SNF2 domain, we identified a physical interaction between SUUR and Rif1. Rif1 has many roles in DNA metabolism and regulates the replication timing program. We demonstrate that repression of DNA replication is dependent on Rif1. Rif1 localizes to active replication forks in an SUUR-dependent manner and directly regulates replication fork progression. Importantly, SUUR associates with replication forks in the absence of Rif1, indicating that Rif1 acts downstream of SUUR to inhibit fork progression. Our findings uncover an unrecognized function of the Rif1 protein as a regulator of replication fork progression.

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Rif1 inhibits replication fork progression and controls DNA copy number in Drosophila

eLife ◽

10.7554/elife.39140 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 14

Author(s):

Alexander Munden ◽

Zhan Rong ◽

Amanda Sun ◽

Rama Gangula ◽

Simon Mallal ◽

...

Keyword(s):

Copy Number ◽

Replication Fork ◽

Genome Stability ◽

Replication Timing ◽

Dependent Manner ◽

Replication Forks ◽

Dna Copy Number ◽

Replication Fork Progression ◽

Suur Protein ◽

Maintain Genome Stability

Control of DNA copy number is essential to maintain genome stability and ensure proper cell and tissue function. In Drosophila polyploid cells, the SNF2-domain-containing SUUR protein inhibits replication fork progression within specific regions of the genome to promote DNA underreplication. While dissecting the function of SUUR’s SNF2 domain, we identified an interaction between SUUR and Rif1. Rif1 has many roles in DNA metabolism and regulates the replication timing program. We demonstrate that repression of DNA replication is dependent on Rif1. Rif1 localizes to active replication forks in a partially SUUR-dependent manner and directly regulates replication fork progression. Importantly, SUUR associates with replication forks in the absence of Rif1, indicating that Rif1 acts downstream of SUUR to inhibit fork progression. Our findings uncover an unrecognized function of the Rif1 protein as a regulator of replication fork progression.

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The Temporal Order of DNA Replication Shaped by Mammalian DNA Methyltransferases

Cells ◽

10.3390/cells10020266 ◽

2021 ◽

Vol 10 (2) ◽

pp. 266

Author(s):

Shin-ichiro Takebayashi ◽

Tyrone Ryba ◽

Kelsey Wimbish ◽

Takuya Hayakawa ◽

Morito Sakaue ◽

...

Keyword(s):

Dna Replication ◽

Temporal Order ◽

Expression System ◽

Embryonic Stem ◽

Replication Timing ◽

Dna Methyltransferases ◽

Dna Hypomethylation ◽

Transcriptional Changes ◽

A Cell ◽

Genomic Regions

Multiple epigenetic pathways underlie the temporal order of DNA replication (replication timing) in the contexts of development and disease. DNA methylation by DNA methyltransferases (Dnmts) and downstream chromatin reorganization and transcriptional changes are thought to impact DNA replication, yet this remains to be comprehensively tested. Using cell-based and genome-wide approaches to measure replication timing, we identified a number of genomic regions undergoing subtle but reproducible replication timing changes in various Dnmt-mutant mouse embryonic stem (ES) cell lines that included a cell line with a drug-inducible Dnmt3a2 expression system. Replication timing within pericentromeric heterochromatin (PH) was shown to be correlated with redistribution of H3K27me3 induced by DNA hypomethylation: Later replicating PH coincided with H3K27me3-enriched regions. In contrast, this relationship with H3K27me3 was not evident within chromosomal arm regions undergoing either early-to-late (EtoL) or late-to-early (LtoE) switching of replication timing upon loss of the Dnmts. Interestingly, Dnmt-sensitive transcriptional up- and downregulation frequently coincided with earlier and later shifts in replication timing of the chromosomal arm regions, respectively. Our study revealed the previously unrecognized complex and diverse effects of the Dnmts loss on the mammalian DNA replication landscape.

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The Protective Role of Dormant Origins in Response to Replicative Stress

International Journal of Molecular Sciences ◽

10.3390/ijms19113569 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3569 ◽

Cited By ~ 11

Author(s):

Lilas Courtot ◽

Jean-Sébastien Hoffmann ◽

Valérie Bergoglio

Keyword(s):

Dna Replication ◽

Mammalian Cells ◽

Genome Stability ◽

Replication Timing ◽

Protective Role ◽

Entire Genome ◽

Replicative Stress ◽

A Cell ◽

The Many

Genome stability requires tight regulation of DNA replication to ensure that the entire genome of the cell is duplicated once and only once per cell cycle. In mammalian cells, origin activation is controlled in space and time by a cell-specific and robust program called replication timing. About 100,000 potential replication origins form on the chromatin in the gap 1 (G1) phase but only 20–30% of them are active during the DNA replication of a given cell in the synthesis (S) phase. When the progress of replication forks is slowed by exogenous or endogenous impediments, the cell must activate some of the inactive or “dormant” origins to complete replication on time. Thus, the many origins that may be activated are probably key to protect the genome against replication stress. This review aims to discuss the role of these dormant origins as safeguards of the human genome during replicative stress.

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Efficient, quick and easy-to-use DNA replication timing analysis with START-R suite

NAR Genomics and Bioinformatics ◽

10.1093/nargab/lqaa045 ◽

2020 ◽

Vol 2 (2) ◽

Cited By ~ 1

Author(s):

Djihad Hadjadj ◽

Thomas Denecker ◽

Eva Guérin ◽

Su-Jung Kim ◽

Fabien Fauchereau ◽

...

Keyword(s):

Dna Replication ◽

Cell Fate ◽

Web Application ◽

Timing Analysis ◽

Mutant Cell ◽

Replication Timing ◽

Experimental Conditions ◽

Replication Process ◽

Time Required ◽

Dna Replication Timing

Abstract DNA replication must be faithful and follow a well-defined spatiotemporal program closely linked to transcriptional activity, epigenomic marks, intranuclear structures, mutation rate and cell fate determination. Among the readouts of the spatiotemporal program of DNA replication, replication timing analyses require not only complex and time-consuming experimental procedures, but also skills in bioinformatics. We developed a dedicated Shiny interactive web application, the START-R (Simple Tool for the Analysis of the Replication Timing based on R) suite, which analyzes DNA replication timing in a given organism with high-throughput data. It reduces the time required for generating and analyzing simultaneously data from several samples. It automatically detects different types of timing regions and identifies significant differences between two experimental conditions in ∼15 min. In conclusion, START-R suite allows quick, efficient and easier analyses of DNA replication timing for all organisms. This novel approach can be used by every biologist. It is now simpler to use this method in order to understand, for example, whether ‘a favorite gene or protein’ has an impact on replication process or, indirectly, on genomic organization (as Hi-C experiments), by comparing the replication timing profiles between wild-type and mutant cell lines.

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Establishment of expression-state boundaries by Rif1 and Taz1 in fission yeast

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1614837114 ◽

2017 ◽

Vol 114 (5) ◽

pp. 1093-1098 ◽

Cited By ~ 12

Author(s):

Tea Toteva ◽

Bethany Mason ◽

Yutaka Kanoh ◽

Peter Brøgger ◽

Daniel Green ◽

...

Keyword(s):

Dna Replication ◽

Fission Yeast ◽

Replication Timing ◽

Genetic Screens ◽

Origin Firing ◽

Regulate Gene Expression ◽

Boundary Formation ◽

Amino Terminal ◽

Amino Terminal Domain ◽

Insight Into

The Shelterin component Rif1 has emerged as a global regulator of the replication-timing program in all eukaryotes examined to date, possibly by modulating the 3D-organization of the genome. In fission yeast a second Shelterin component, Taz1, might share similar functions. Here, we identified unexpected properties for Rif1 and Taz1 by conducting high-throughput genetic screens designed to identifycis-andtrans-acting factors capable of creating heterochromatin–euchromatin boundaries in fission yeast. The preponderance ofcis-acting elements identified in the screens originated from genomic loci bound by Taz1 and associated with origins of replication whose firing is repressed by Taz1 and Rif1. Boundary formation and gene silencing by these elements required Taz1 and Rif1 and coincided with altered replication timing in the region. Thus, small chromosomal elements sensitive to Taz1 and Rif1 (STAR) could simultaneously regulate gene expression and DNA replication over a large domain, at the edge of which they established a heterochromatin–euchromatin boundary. Taz1, Rif1, and Rif1-associated protein phosphatases Sds21 and Dis2 were each sufficient to establish a boundary when tethered to DNA. Moreover, efficient boundary formation required the amino-terminal domain of the Mcm4 replicative helicase onto which the antagonistic activities of the replication-promoting Dbf4-dependent kinase and Rif1-recruited phosphatases are believed to converge to control replication origin firing. Altogether these observations provide an insight into a coordinated control of DNA replication and organization of the genome into expression domains.

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Topologically associating domain boundaries are enriched in early firing origins and restrict replication fork progression

10.1101/2020.10.21.348946 ◽

2020 ◽

Author(s):

Emilia Puig Lombardi ◽

Madalena Tarsounas

Keyword(s):

Binding Sites ◽

Replication Fork ◽

Replication Timing ◽

Replication Initiation ◽

Ctcf Binding ◽

Origin Firing ◽

Replication Fork Progression ◽

Fork Stalling ◽

Genomic Regions ◽

The Impact

ABSTRACTTopologically associating domains (TADs) are units of the genome architecture defined by binding sites for the CTCF transcription factor and cohesin-mediated loop extrusion. Genomic regions containing DNA replication initiation sites have been mapped in the proximity of TAD boundaries. However, the factors that determine this positioning have not been identified. Moreover, the impact of TADs on the directionality of replication fork progression remains unknown. Here we use EdU-seq technology to map origin firing sites at 10 kb resolution and to monitor replication fork progression after restart from hydroxyurea arrest. We show that origins firing in early/mid S-phase within TAD boundaries map to two distinct peaks flanking the centre of the boundary, which is occupied by CTCF and cohesin. When transcription is inhibited chemically or deregulated by oncogene overexpression, replication origins become repositioned to the centre of the TAD. Furthermore, we demonstrate the strikingly asymmetric fork progression initiating from origins located within TAD boundaries. Divergent CTCF binding sites and neighbouring TADs with different replication timing (RT) cause fork stalling in regions external to the TAD. Thus, our work assigns for the first time a role to transcription within TAD boundaries in promoting replication origin firing and demonstrates how genomic regions adjacent to the TAD boundaries could restrict replication progression.

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