Identification of novel factors involved in or regulating initiation of DNA replication by a genome-wide phenotypic screen inSaccharomyces cerevisiae

Abstract Background Functional genomics uses unbiased systematic genome-wide gene disruption or analyzes natural variations such as gene expression profiles of different tissues from multicellular organisms to link gene functions to particular phenotypes. Functional genomics approaches are of particular importance to identify large sets of genes that are specifically important for a particular biological process beyond known candidate genes, or when the process has not been studied with genetic methods before. Results Here, we present a large set of genes whose disruption interferes with the function of the odoriferous defensive stink glands of the red flour beetle Tribolium castaneum. This gene set is the result of a large-scale systematic phenotypic screen using a reverse genetics strategy based on RNA interference applied in a genome-wide forward genetics manner. In this first-pass screen, 130 genes were identified, of which 69 genes could be confirmed to cause knock-down gland phenotypes, which vary from necrotic tissue and irregular reservoir size to irregular color or separation of the secreted gland compounds. The knock-down of 13 genes caused specifically a strong reduction of para-benzoquinones, suggesting a specific function in the synthesis of these toxic compounds. Only 14 of the 69 confirmed gland genes are differentially overexpressed in stink gland tissue and thus could have been detected in a transcriptome-based analysis. Moreover, of the 29 previously transcriptomics-identified genes causing a gland phenotype, only one gene was recognized by this phenotypic screen despite the fact that 13 of them were covered by the screen. Conclusion Our results indicate the importance of combining diverse and independent methodologies to identify genes necessary for the function of a certain biological tissue, as the different approaches do not deliver redundant results but rather complement each other. The presented phenotypic screen together with a transcriptomics approach are now providing a set of close to hundred genes important for odoriferous defensive stink gland physiology in beetles.

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Dbf4-Dependent Kinase (DDK)-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A in Saccharomyces cerevisiae

G3 Genes|Genome|Genetics ◽

10.1534/g3.120.401131 ◽

2020 ◽

Vol 10 (6) ◽

pp. 2057-2068 ◽

Cited By ~ 3

Author(s):

Jessica R. Eisenstatt ◽

Lars Boeckmann ◽

Wei-Chun Au ◽

Valerie Garcia ◽

Levi Bursch ◽

...

Keyword(s):

Dna Replication ◽

Replication Initiation ◽

Centromeric Chromatin ◽

Dna Replication Initiation ◽

Link Type ◽

Genome Wide ◽

A Genome ◽

Evolutionarily Conserved ◽

Histone H3 Variant

The evolutionarily conserved centromeric histone H3 variant (Cse4 in budding yeast, CENP-A in humans) is essential for faithful chromosome segregation. Mislocalization of CENP-A to non-centromeric chromatin contributes to chromosomal instability (CIN) in yeast, fly, and human cells and CENP-A is highly expressed and mislocalized in cancers. Defining mechanisms that prevent mislocalization of CENP-A is an area of active investigation. Ubiquitin-mediated proteolysis of overexpressed Cse4 (GALCSE4) by E3 ubiquitin ligases such as Psh1 prevents mislocalization of Cse4, and psh1Δ strains display synthetic dosage lethality (SDL) with GALCSE4. We previously performed a genome-wide screen and identified five alleles of CDC7 and DBF4 that encode the Dbf4-dependent kinase (DDK) complex, which regulates DNA replication initiation, among the top twelve hits that displayed SDL with GALCSE4. We determined that cdc7-7 strains exhibit defects in ubiquitin-mediated proteolysis of Cse4 and show mislocalization of Cse4. Mutation of MCM5 (mcm5-bob1) bypasses the requirement of Cdc7 for replication initiation and rescues replication defects in a cdc7-7 strain. We determined that mcm5-bob1 does not rescue the SDL and defects in proteolysis of GALCSE4 in a cdc7-7 strain, suggesting a DNA replication-independent role for Cdc7 in Cse4 proteolysis. The SDL phenotype, defects in ubiquitin-mediated proteolysis, and the mislocalization pattern of Cse4 in a cdc7-7 psh1Δ strain were similar to that of cdc7-7 and psh1Δ strains, suggesting that Cdc7 regulates Cse4 in a pathway that overlaps with Psh1. Our results define a DNA replication initiation-independent role of DDK as a regulator of Psh1-mediated proteolysis of Cse4 to prevent mislocalization of Cse4.

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Alterations in cellular metabolism triggered by URA7 or GLN3 inactivation cause imbalanced dNTP pools and increased mutagenesis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1618714114 ◽

2017 ◽

Vol 114 (22) ◽

pp. E4442-E4451 ◽

Cited By ~ 14

Author(s):

Tobias T. Schmidt ◽

Gloria Reyes ◽

Kerstin Gries ◽

Cemile Ümran Ceylan ◽

Sushma Sharma ◽

...

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Cancer Susceptibility ◽

Dntp Pool ◽

Dna Mismatch ◽

Genome Wide ◽

A Genome ◽

Damage Response ◽

Eukaryotic Dna ◽

Active Site Mutants

Eukaryotic DNA replication fidelity relies on the concerted action of DNA polymerase nucleotide selectivity, proofreading activity, and DNA mismatch repair (MMR). Nucleotide selectivity and proofreading are affected by the balance and concentration of deoxyribonucleotide (dNTP) pools, which are strictly regulated by ribonucleotide reductase (RNR). Mutations preventing DNA polymerase proofreading activity or MMR function cause mutator phenotypes and consequently increased cancer susceptibility. To identify genes not previously linked to high-fidelity DNA replication, we conducted a genome-wide screen in Saccharomyces cerevisiae using DNA polymerase active-site mutants as a “sensitized mutator background.” Among the genes identified in our screen, three metabolism-related genes (GLN3, URA7, and SHM2) have not been previously associated to the suppression of mutations. Loss of either the transcription factor Gln3 or inactivation of the CTP synthetase Ura7 both resulted in the activation of the DNA damage response and imbalanced dNTP pools. Importantly, these dNTP imbalances are strongly mutagenic in genetic backgrounds where DNA polymerase function or MMR activity is partially compromised. Previous reports have shown that dNTP pool imbalances can be caused by mutations altering the allosteric regulation of enzymes involved in dNTP biosynthesis (e.g., RNR or dCMP deaminase). Here, we provide evidence that mutations affecting genes involved in RNR substrate production can cause dNTP imbalances, which cannot be compensated by RNR or other enzymatic activities. Moreover, Gln3 inactivation links nutrient deprivation to increased mutagenesis. Our results suggest that similar genetic interactions could drive mutator phenotypes in cancer cells.

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MRE11-RAD50-NBS1 activates Fanconi Anemia R-loop suppression at transcription-replication conflicts

10.1101/472654 ◽

2018 ◽

Author(s):

Emily Yun-chia Chang ◽

James P. Wells ◽

Shu-Huei Tsai ◽

Yan Coulombe ◽

Yujia A. Chan ◽

...

Keyword(s):

Dna Replication ◽

Fanconi Anemia ◽

Replication Fork ◽

Genome Instability ◽

Human Cancer ◽

Replication Stress ◽

Maintenance Factors ◽

Genome Wide ◽

A Genome ◽

Dna Replication Stress

SUMMARYEctopic R-loop accumulation causes DNA replication stress and genome instability. To avoid these outcomes, cells possess a range of anti-R-loop mechanisms, including RNaseH that degrades the RNA moiety in R-loops. To comprehensively identify anti-R-loop mechanisms, we performed a genome-wide trigenic interaction screen in yeast lacking RNH1 and RNH201. We identified >100 genes critical for fitness in the absence of RNaseH, which were enriched for DNA replication fork maintenance factors such as RAD50. We show in yeast and human cells that R-loops accumulate during RAD50 depletion. In human cancer cell models, we find that RAD50 and its partners in the MRE11-RAD50-NBS1 complex regulate R-loop-associated DNA damage and replication stress. We show that a non-nucleolytic function of MRE11 is important for R-loop suppression via activation of PCNA-ubiquitination by RAD18 and recruiting anti-R-loop helicases in the Fanconi Anemia pathway. This work establishes a novel role for MRE11-RAD50-NBS1 in directing tolerance mechanisms of transcription-replication conflicts.

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A genome-wide inducible phenotypic screen identifies antisense RNA constructs silencing Escherichia coli essential genes

FEMS Microbiology Letters ◽

10.1111/j.1574-6968.2012.02503.x ◽

2012 ◽

Vol 329 (1) ◽

pp. 45-53 ◽

Cited By ~ 29

Author(s):

Jia Meng ◽

Gregory Kanzaki ◽

Diane Meas ◽

Christopher K. Lam ◽

Heather Crummer ◽

...

Keyword(s):

Escherichia Coli ◽

Antisense Rna ◽

Essential Genes ◽

Genome Wide ◽

Phenotypic Screen ◽

A Genome

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A genome-wide CRISPR/Cas phenotypic screen for modulators of DUX4 cytotoxicity reveals screen complications

10.1101/2020.07.27.223420 ◽

2020 ◽

Author(s):

Ator Ashoti ◽

Francesco Limone ◽

Melissa van Kranenburg ◽

Anna Alemany ◽

Mirna Baak ◽

...

Keyword(s):

Treatment Options ◽

Underlying Mechanism ◽

Facioscapulohumeral Muscular Dystrophy ◽

Knock Out ◽

Genome Wide ◽

Phenotypic Screen ◽

A Genome ◽

The Face ◽

Muscle Disorder ◽

Pioneer Transcription Factor

AbstractFacioscapulohumeral muscular dystrophy (FHSD), a fundamentally complex muscle disorder that thus far remains untreatable. As the name implies, FSHD starts in the muscles of the face and shoulder gridle. The main perturbator of the disease is the pioneer transcription factor DUX4, which is misexpressed in affected tissues due to a failure in epigenetic repressive mechanisms. In pursuit of unraveling the underlying mechanism of FSHD and finding potential therapeutic targets or treatment options, we performed an exhaustive genome-wide CRISPR/Cas9 phenotypic rescue screen to identify modulators of DUX4 cytotoxicity. We found no key effectors other than DUX4 itself, suggesting treatment efforts in FSHD should be directed towards its direct modulation.The screen did however reveal some rare and unexpected Cas9-induced genomic events, that may provide important considerations for planning future CRISPR/Cas9 knock-out screens.

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Chromatin structure-dependent histone incorporation revealed by a genome-wide deposition assay

eLife ◽

10.7554/elife.66290 ◽

2021 ◽

Vol 10 ◽

Author(s):

Hiroaki Tachiwana ◽

Mariko Dacher ◽

Kazumitsu Maehara ◽

Akihito Harada ◽

Yosuke Seto ◽

...

Keyword(s):

Dna Replication ◽

Chromatin Structure ◽

Histone Variant ◽

Condensed Chromatin ◽

Deposition Mechanism ◽

Permeabilized Cells ◽

Genome Wide ◽

A Genome ◽

Histone Deposition ◽

Epigenetic Feature

In eukaryotes, histone variant distribution within the genome is the key epigenetic feature. To understand how each histone variant is targeted to the genome, we developed a new method, the RhIP (Reconstituted histone complex Incorporation into chromatin of Permeabilized cell) assay, in which epitope-tagged histone complexes are introduced into permeabilized cells and incorporated into their chromatin. Using this method, we found that H3.1 and H3.3 were incorporated into chromatin in replication-dependent and -independent manners, respectively. We further found that the incorporation of histones H2A and H2A.Z mainly occurred at less condensed chromatin (open), suggesting that condensed chromatin (closed) is a barrier for histone incorporation. To overcome this barrier, H2A, but not H2A.Z, uses a replication-coupled deposition mechanism. Our study revealed that the combination of chromatin structure and DNA replication dictates the differential histone deposition to maintain the epigenetic chromatin states.

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MRE11-RAD50-NBS1 promotes Fanconi Anemia R-loop suppression at transcription–replication conflicts

Nature Communications ◽

10.1038/s41467-019-12271-w ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 16

Author(s):

Emily Yun-Chia Chang ◽

Shuhe Tsai ◽

Maria J. Aristizabal ◽

James P. Wells ◽

Yan Coulombe ◽

...

Keyword(s):

Dna Replication ◽

Fanconi Anemia ◽

Genome Instability ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Mrn Complex ◽

Genome Wide ◽

A Genome ◽

Dna Replication Stress

Abstract Ectopic R-loop accumulation causes DNA replication stress and genome instability. To avoid these outcomes, cells possess a range of anti-R-loop mechanisms, including RNaseH that degrades the RNA moiety in R-loops. To comprehensively identify anti-R-loop mechanisms, we performed a genome-wide trigenic interaction screen in yeast lacking RNH1 and RNH201. We identified >100 genes critical for fitness in the absence of RNaseH, which were enriched for DNA replication fork maintenance factors including the MRE11-RAD50-NBS1 (MRN) complex. While MRN has been shown to promote R-loops at DNA double-strand breaks, we show that it suppresses R-loops and associated DNA damage at transcription–replication conflicts. This occurs through a non-nucleolytic function of MRE11 that is important for R-loop suppression by the Fanconi Anemia pathway. This work establishes a novel role for MRE11-RAD50-NBS1 in directing tolerance mechanisms at transcription–replication conflicts.

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C17orf53 defines a novel pathway involved in inter-strand crosslink repair

10.1101/758722 ◽

2019 ◽

Author(s):

Chao Wang ◽

Zhen Chen ◽

Dan Su ◽

Mengfan Tang ◽

Litong Nie ◽

...

Keyword(s):

Cell Proliferation ◽

Dna Replication ◽

Repair Pathway ◽

Genome Maintenance ◽

Uncharacterized Gene ◽

Genome Wide ◽

A Genome ◽

Repair Pathways ◽

Repair Defect ◽

Atr Kinase

SummaryATR kinase is a master regulator of genome maintenance and participates in DNA replication and various DNA repair pathways. In a genome-wide screening for ATR-dependent fitness genes, we identified a previously uncharacterized gene, C17orf53, whose loss led to hypersensitivity to ATR inhibition. C17orf53 is conserved in vertebrate and is required for efficient cell proliferation. Loss of C17orf53 slowed down DNA replication and led to pronounced ICL repair defect. Further genetic analyses revealed that C17orf53 functions downstream in ICL repair pathway, probably by affecting the loading of repair factors such as RAD54. In addition, we showed that C17orf53 is a ssDNA- and RPA-binding protein, both of which are important for its functions in the cell. Taken together, C17orf53 is a novel component involved in ICL repair pathway.

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