hnRNP U and DDX47 Are Novel FANCD2 Interactors That May Help to Resolve R-Loops during Mild Replication Stress

Abstract Background: Fanconi anemia proteins, encoded by at least 22genes (FANCA-W), constitute the Interstrand Cross Link (ICL) repair pathway. While FANCD2 is a master regulator of ICL repair, it accumulates at common fragile sites (CFS) during mild replication stress stimulated by low-dose Aphidicolin (APH) treatment. A recent study indicated that FANCD2 is required for efficient genome replication across the CFS regions. FANCD2 is also implicated in the regulation of R-loops levels. R-loops, which consist of DNA: RNA hybrids and displaced single-stranded DNA, are physiologically relevant in the genome and associate with immunoglobulin class switching, replication of mitochondrial DNA as well as transcriptional promoters or terminators. However, in any case, untimely formation of R-loops is a major threat to genome instability. Furthermore, it has been reported that R-loops which are induced by common slicing factor mutations in cases with myelodysplastic syndrome are linked to compromised proliferation of hematopoietic progenitors. It is also interesting to note that a recent study shows an interaction of FANCD2 with splicing factor 3B1 (SF3B1) and proposes their role in organizing chromatin domains to ensure coordination of replication and co-transcriptional processes. Methods: To examine the genome-wide distribution of FANCD2 protein, we set out to create a derivative of human osteosarcoma cell line, U2OS, which incorporated a 3×FLAG tag into the FANCD2 termination codon by genome editing. We performed chromatin-immunoprecipitation and sequencing (ChIP-Seq) analysis, and provide a genome-wide landscape of replication stress response involving FANCD2 in this cell line. Moreover, we purified the FANCD2 complex and analyzed by liquid chromatography-tandem mass spectrometry, and confirmed this interaction by co-immunoprecipitation (Co-IP) and proximal ligation assay (PLA) with FANCD2-3xFLAG. R-loops levels were assayed as the number of S9.6 (anti DNA:RNA hybrid antibody) stained foci per nucleus. Results: FANCD2 accumulation mostly occurs in the central portion of large transcribed genes, including CFS, and its accumulation appeared to be dependent on R-loop formation induced by transcription-replication collisions during mild replication stress. Moreover, our mass spectrometry analysis identified that FANCD2 interacts with several RNA processing factors including heterogeneous nucleoprotein U (hnRNP U), or DEAD box protein 47 (DDX47). We confirmed the interaction of these factors with FANCD2 by Co-IP as well as PLA. It was previously reported that defects in RNA-processing factors result in R-loop accumulation associated genome instability. Indeed, we found that treatment with siRNA against hnRNP U or DDX47 resulted in the increased number of the S9.6 foci. Furthermore, FANCD2 and hnRNP U or DDX47 appeared to function in an epistatic manner in suppressing APH-induced transcription-replication collisions as detected by PLA between PCNA and RNA polymerase II. Conclusion: We suggest that FANCD2 protects genome stability by recruiting RNA processing enzymes, including hnRNP U or DDX47, to resolve or prevent accumulation of R-loops induced by transcription-replication collisions during mild replication stress. Thus, our study may provide a novel insight to understand the mechanism of bone marrow failure and leukemogenesis in Fanconi anemia patients. Disclosures Takaori-Kondo: Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria; Janssen Pharmaceuticals: Honoraria.

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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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Mass spectrometry and CLIP-seq analysis reveal BCL11b interactions with RNA processing pathways

10.1101/2020.11.05.369959 ◽

2020 ◽

Author(s):

Haitham Sobhy ◽

Marco De Rovere ◽

Amina Ait-Ammar ◽

Clementine Wallet ◽

Fadoua Daouad ◽

...

Keyword(s):

Mass Spectrometry ◽

Rna Processing ◽

Rna Splicing ◽

Protein Complexes ◽

B Cell Lymphoma ◽

Mass Spectrometry Analysis ◽

Nonsense Mediated Decay ◽

Spectrometry Analysis ◽

Genome Wide ◽

Processing Pathways

AbstractAlthough BCL11b (B-cell lymphoma/leukemia 11B, CTIP2) is a well-known transcription repressor and tumor suppressor, its functions and cellular pathways are largely unknown. Here, we show that BCL11b interacts with RNA splicing/processing and nonsense-mediated decay (NMD) proteins, including FUS, SMN1, UPF1 and Drosha. Mass spectrometry analysis (LC-MS/MS) shows that BCL11b interacts with histones, polymerases, and chromatin remodeling (CHD, SWI/SNF, and topoisomerase) proteins. BCL11b-bound RNAs were UV cross-linked and sequenced (CLIP-seq) showing that BCL11b binds to coding and noncoding RNAs (ncRNAs). Surprisingly, RNA transcripts and proteins produced by the same genes like FUS, ESWR1, CHD and Tubulin, were found bound to BCL11b. Deeper analysis of the CLIP-seq data further suggested that BCL11b binds to nonsense mediated RNA decay and retained introns transcripts. Our study is the first genome-wide study of BCL11b-protein and BCL11b-RNA interactants. Our results suggest that the functions of BCL11b are not restricted to the regulation of gene transcription. BCL11b may also control physiologic and physiopathologic pathways by direct bindings to protein complexes, coding RNA and non-coding RNA.ImportanceFirst genome-wide BCL11b-proteins interactomicsFirst genome-wide BCL11b-RNA interactomicsBCL11b interacts with RNA processing and RNA splicing proteinsBCL11b interacts with neurodegenerative genes and sarcoma genes

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Intact-Cell MALDI-ToF Mass Spectrometry for the Authentication of Drug-Adapted Cancer Cell Lines

Cells ◽

10.3390/cells8101194 ◽

2019 ◽

Vol 8 (10) ◽

pp. 1194 ◽

Cited By ~ 1

Author(s):

Jane F. Povey ◽

Emily Saintas ◽

Adewale V. Aderemi ◽

Florian Rothweiler ◽

Richard Zehner ◽

...

Keyword(s):

Mass Spectrometry ◽

Cell Line ◽

Cell Lines ◽

Mammalian Cells ◽

Intact Cell ◽

Mass Spectrometry Analysis ◽

Genetic Origin ◽

Whole Cell ◽

Maldi Tof Mass Spectrometry ◽

Maldi Tof

The use of cell lines in research can be affected by cell line misidentification. Short tandem repeat (STR) analysis is an effective method, and the gold standard, for the identification of the genetic origin of a cell line, but methods that allow the discrimination between cell lines of the same genetic origin are lacking. Here, we use intact cell MALDI-ToF mass spectrometry analysis, routinely used for the identification of bacteria in clinical diagnostic procedures, for the authentication of a set of cell lines consisting of three parental neuroblastoma cell lines (IMR-5, IMR-32 and UKF-NB-3) and eleven drug-adapted sublines. Principal component analysis (PCA) of intact-cell MALDI-ToF mass spectrometry data revealed clear differences between most, but not all, of the investigated cell lines. Mass spectrometry whole-cell fingerprints enabled the separation of IMR-32 and its clonal subline IMR-5. Sublines that had been adapted to closely related drugs, for example, the cisplatin- and oxaliplatin-resistant UKF-NB-3 sublines and the vincristine- and vinblastine-adapted IMR-5 sublines, also displayed clearly distinctive patterns. In conclusion, intact whole-cell MALDI-ToF mass spectrometry has the potential to be further developed into an authentication method for mammalian cells of a common genetic origin.

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Genome-wide mapping and profiling of γH2AX binding hotspots in response to different replication stress inducers

10.1101/644500 ◽

2019 ◽

Author(s):

Xinxing Lyu ◽

Megan Chastain ◽

Weihang Chai

Keyword(s):

Binding Sites ◽

Genome Stability ◽

Genome Instability ◽

Genetic Diseases ◽

Cpg Islands ◽

Replication Stress ◽

Common Features ◽

Genome Wide ◽

Different Types ◽

Fork Stalling

AbstractBackgroundReplication stress (RS) gives rise to DNA damage that threatens genome stability. RS can originate from different sources that stall replication by diverse mechanisms. However, the mechanism underlying how different types of RS contribute to genome instability is unclear, in part due to the poor understanding of the distribution and characteristics of damage sites induced by different RS mechanisms.ResultsWe use ChIP-seq to map γH2AX binding sites genome-wide caused by aphidicolin (APH), hydroxyurea (HU), and methyl methanesulfonate (MMS) treatments in human lymphocyte cells. Mapping of γH2AX ChIP-seq reveals that APH, HU, and MMS treatments induce non-random γH2AX chromatin binding at discrete regions, suggesting that there are γH2AX binding hotspots in the genome. Characterization of the distribution and sequence/epigenetic features of γH2AX binding sites reveals that the three treatments induce γH2AX binding at largely non-overlapping regions, suggesting that RS may cause damage at specific genomic loci in a manner dependent on the fork stalling mechanism. Nonetheless, γH2AX binding sites induced by the three treatments share common features including compact chromatin, coinciding with larger-than-average genes, and depletion of CpG islands and transcription start sites. Moreover, we observe significant enrichment of SINEs in γH2AX sites in all treatments, indicating that SINEs may be a common barrier for replication polymerases.ConclusionsOur results identify the location and common features of genome instability hotspots induced by different types of RS, and help in deciphering the mechanisms underlying RS-induced genetic diseases and carcinogenesis.

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Identification of the Fanconi Anemia Complementation Group I Gene, FANCI

Analytical Cellular Pathology ◽

10.1155/2007/151968 ◽

2007 ◽

Vol 29 (3) ◽

pp. 211-218

Author(s):

Josephine C. Dorsman ◽

Marieke Levitus ◽

Davy Rockx ◽

Martin A. Rooimans ◽

Anneke B. Oostra ◽

...

Keyword(s):

Cell Line ◽

Fanconi Anemia ◽

Complementation Group ◽

Chromosomal Breakage ◽

Western Blots ◽

Nuclear Localization Signals ◽

Group I ◽

Genome Wide ◽

A Genome ◽

Candidate Regions

To identify the gene underlying Fanconi anemia (FA) complementation group I we studied informative FA-I families by a genome-wide linkage analysis, which resulted in 4 candidate regions together encompassing 351 genes. Candidates were selected via bioinformatics and data mining on the basis of their resemblance to other FA genes/proteins acting in the FA pathway, such as: degree of evolutionary conservation, presence of nuclear localization signals and pattern of tissue-dependent expression. We found a candidate, KIAA1794 on chromosome 15q25-26, to be mutated in 8 affected individuals previously assigned to complementation group I. Western blots of endogenous FANCI indicated that functionally active KIAA1794 protein is lacking in FA-I individuals. Knock-down of KIAA1794 expression by siRNA in HeLa cells caused excessive chromosomal breakage induced by mitomycin C, a hallmark of FA cells. Furthermore, phenotypic reversion of a patient-derived cell line was associated with a secondary genetic alteration at the KIAA1794 locus. These data add up to two conclusions. First, KIAA1794 is a FA gene. Second, this gene is identical to FANCI, since the patient cell lines found mutated in this study included the reference cell line for group I, EUFA592.

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Genome-Wide Analysis of the Effects of Heat Shock on aSaccharomyces cerevisiaeMutant With a Constitutively Activated cAMP-Dependent Pathway

Comparative and Functional Genomics ◽

10.1002/cfg.415 ◽

2004 ◽

Vol 5 (5) ◽

pp. 419-431 ◽

Cited By ~ 20

Author(s):

Dawn L. Jones ◽

June Petty ◽

David C. Hoyle ◽

Andrew Hayes ◽

Stephen G. Oliver ◽

...

Keyword(s):

Mass Spectrometry ◽

Heat Shock ◽

Mass Spectrometry Analysis ◽

Ergosterol Biosynthesis ◽

Wild Type ◽

Altered Expression ◽

Expression Levels ◽

Genome Wide ◽

A Genome ◽

Dependent Pathway

We have used DNA microarray technology and 2-D gel electrophoresis combined with mass spectrometry to investigate the effects of a drastic heat shock from 30℃ to 50℃ on a genome-wide scale. This experimental condition is used to differentiate between wild-type cells and those with a constitutively active cAMP-dependent pathway inSaccharomyces cerevisiae. Whilst more than 50% of the former survive this shock, almost all of the latter lose viability. We compared the transcriptomes of the wildtype and a mutant strain deleted for the genePDE2, encoding the high-affinity cAMP phosphodiesterase before and after heat shock treatment. We also compared the two heat-shocked samples with one another, allowing us to determine the changes that occur in thepde2Δ mutant which cause such a dramatic loss of viability after heat shock. Several genes involved in ergosterol biosynthesis and carbon source utilization had altered expression levels, suggesting that these processes might be potential factors in heat shock survival. These predictions and also the effect of the different phases of the cell cycle were confirmed by biochemical and phenotypic analyses. 146 genes of previously unknown function were identified amongst the genes with altered expression levels and deletion mutants in 13 of these genes were found to be highly sensitive to heat shock. Differences in response to heat shock were also observed at the level of the proteome, with a higher level of protein degradation in the mutant, as revealed by comparing 2-D gels of wild-type and mutant heat-shocked samples and mass spectrometry analysis of the differentially produced proteins.

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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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MetaNovo: a probabilistic approach to peptide and polymorphism discovery in complex metaproteomic datasets

10.1101/605550 ◽

2019 ◽

Cited By ~ 3

Author(s):

Matthys G Potgieter ◽

Andrew JM Nel ◽

Suereta Fortuin ◽

Shaun Garnett ◽

Jerome M. Wendoh ◽

...

Keyword(s):

Mass Spectrometry ◽

Protein Expression ◽

Cell Line ◽

Mass Spectrometry Analysis ◽

Metagenomic Data ◽

Human Neuroblastoma Cell ◽

Microbial Protein ◽

Microbial Composition ◽

Human Neuroblastoma ◽

Microbial Ecosystems

AbstractMetagenome-driven microbiome research is providing important new insights in fields as diverse as the pathogenesis of human disease, the metabolic interactions of complex microbial ecosystems involved in agriculture, and climate change. However, poor correlations typically observed between RNA and protein expression datasets even for single organisms make it hard to infer microbial protein expression with any accuracy from metagenomic data, thus restricting movement beyond microbial catalogues and into functional analysis of microbial effector molecules. By contrast, mass spectrometry analysis of microbiome data at the protein level in theory allows direct measurement of dynamic changes in microbial protein composition, localisation and modification that may mediate host/pathogen interactions in complex microbial ecosystems, but analysis of such metaproteomic datasets remains challenging.Here we describe a novel data analysis approach, MetaNovo, that searches complex datasets against the entire known protein universe, whilst still controlling false discovery rates, thus enabling metaproteomic data analyses without requiring prior expectation of likely sample composition or metagenomic data generation that classically inform construction of focussed, relatively small search libraries. MetaNovo directly identifies and quantifies the expressed metaproteomes, and estimates the microbial composition present in complex microbiome samples, using scalable de novo sequence tag matching and probabilistic optimization of very large, unbiased sequence databases prior to target-decoy search. We validated MetaNovo against the results obtained from the recently published MetaPro-IQ pipeline on 8 human mucosal-luminal interface samples, with comparable numbers of peptide and protein identifications being found when searching relatively small databases. We then showed that using an unbiased search of the entire release of UniProt (ca. 90 million protein sequences1) MetaNovo was able to identify a similar bacterial taxonomic distribution compared to that found using a small, focused matched metagenome database, but now also simultaneously identified proteins present in the samples that are derived from other organisms missed by 16S or shotgun sequencing and by previous metaproteomic methods. Using MetaNovo to analyze a set of single-organism human neuroblastoma cell-line samples (SH-SY5Y) against UniProt we achieved a comparable MS/MS identification rate during target-decoy search to using the UniProt human Reference proteome, with 22583 (85.99 %) of the total set of identified peptides shared in common. Taxonomic analysis of 612 peptides not found in the canonical set of human proteins yielded 158 peptides unique to the Chordata phylum as potential human variant identifications. Of these, 40 had previously been predicted and 9 identified using whole genome sequencing in a proteogenomic study of the same cell-line.By estimating taxonomic and peptide level information on microbiome samples directly from tandem mass spectrometry data, MetaNovo enables simultaneous identification of human, bacterial, helminth, fungal, viral and other eukaryotic proteins in a sample, thus allowing correlations between changes in microbial protein abundance and change in the host proteome to be drawn based on a single analysis.Data are available via ProteomeXchange with identifier PXD014214. The MetaNovo software is available from GitHub2 and can be run as a standalone Singularity or Docker container available from the Docker Hub3.

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