Publications by authors named "Xuewen Pan"

37 Publications

Optimization of AsCas12a for combinatorial genetic screens in human cells.

Nat Biotechnol 2021 01 13;39(1):94-104. Epub 2020 Jul 13.

Genetic Perturbation Platform, Broad Institute of MIT and Harvard, Cambridge, MA, USA.

Cas12a RNA-guided endonucleases are promising tools for multiplexed genetic perturbations because they can process multiple guide RNAs expressed as a single transcript, and subsequently cleave target DNA. However, their widespread adoption has lagged behind Cas9-based strategies due to low activity and the lack of a well-validated pooled screening toolkit. In the present study, we describe the optimization of enhanced Cas12a from Acidaminococcus (enAsCas12a) for pooled, combinatorial genetic screens in human cells. By assaying the activity of thousands of guides, we refine on-target design rules and develop a comprehensive set of off-target rules to predict and exclude promiscuous guides. We also identify 38 direct repeat variants that can substitute for the wild-type sequence. We validate our optimized AsCas12a toolkit by screening for synthetic lethalities in OVCAR8 and A375 cancer cells, discovering an interaction between MARCH5 and WSB2. Finally, we show that enAsCas12a delivers similar performance to Cas9 in genome-wide dropout screens but at greatly reduced library size, which will facilitate screens in challenging models.
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http://dx.doi.org/10.1038/s41587-020-0600-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854777PMC
January 2021

Optimized precooling combined with SO-released paper treatment improves the storability of longan ( Lour.) fruits stored at room temperature.

Food Sci Nutr 2020 Jun 16;8(6):2827-2838. Epub 2020 Apr 16.

Institute of Fruit Tree Research Guangdong Academy of Agricultural Sciences/Key Laboratory of South Subtropical Fruit Biology and Genetic Resource Utilization Ministry of Agriculture/Guangdong Province Key Laboratory of Tropical and Subtropical Fruit Tree Research Guangzhou China.

Precooling and sulfur dioxide fumigation were proved as effective methods for the preservation of longan ( Lour.) fruits. However, inadequate precooling and sulfur dioxide fumigation resulted in unexpected losses and short shelf life. A L(3) orthogonal test was conducted to screen out ideal dosage of sodium metabisulfite (factor A), precooling method (factor B), and precooling duration (factor C) to improve the storability of longan fruit stored for 48 hr at room temperature (RT) (25℃). The overall qualities of all of the treated longan fruits after a 48-hr storage (OQST) and during the 5-day shelf at 25℃ (OQSF) were better than those of the control fruits. The treated fruits showed brighter fresh color (higher L*, b*, C*, and h° values but lower a* value), higher flavonoid, and chlorophyll contents. Moreover, the SO residue was concentrated in pericarp but little in aril for any of the 12 treatments. The multivariate variance analysis showed that factor A was dominant to determine both of the OQST and OQSF, while factor B affected the OQST, and factor C affected the OQSF. In total, "0.22% sodium metabisulfite + 4 hr precooling + uncovered precooling" was considered to be an ideal treatment. These results would contribute to improving longan postharvest preservation technology.
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http://dx.doi.org/10.1002/fsn3.1577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7300044PMC
June 2020

Puupehenone, a Marine-Sponge-Derived Sesquiterpene Quinone, Potentiates the Antifungal Drug Caspofungin by Disrupting Hsp90 Activity and the Cell Wall Integrity Pathway.

mSphere 2020 01 8;5(1). Epub 2020 Jan 8.

National Center for Natural Products Research, School of Pharmacy, University of Mississippi, Oxford, Mississippi, USA

The cell wall-targeting echinocandin antifungals, although potent and well tolerated, are inadequate in treating fungal infections due to their narrow spectrum of activity and their propensity to induce pathogen resistance. A promising strategy to overcome these drawbacks is to combine echinocandins with a molecule that improves their activity and also disrupts drug adaptation pathways. In this study, we show that puupehenone (PUUP), a marine-sponge-derived sesquiterpene quinone, potentiates the echinocandin drug caspofungin (CAS) in CAS-resistant fungal pathogens. We have conducted RNA sequencing (RNA-seq) analysis, followed by genetic and molecular studies, to elucidate PUUP's CAS-potentiating mechanism. We found that the combination of CAS and PUUP blocked the induction of CAS-responding genes required for the adaptation to cell wall stress through the cell wall integrity (CWI) pathway. Further analysis showed that PUUP inhibited the activation of Slt2 (Mpk1), the terminal mitogen-activated protein (MAP) kinase in this pathway. We also found that PUUP induced heat shock response genes and inhibited the activity of heat shock protein 90 (Hsp90). Molecular docking studies predicted that PUUP occupies a binding site on Hsp90 required for the interaction between Hsp90 and its cochaperone Cdc37. Thus, we show that PUUP potentiates CAS activity by a previously undescribed mechanism which involves a disruption of Hsp90 activity and the CWI pathway. Given the requirement of the Hsp90-Cdc37 complex in Slt2 activation, we suggest that inhibitors of this complex would disrupt the CWI pathway and synergize with echinocandins. Therefore, the identification of PUUP's CAS-potentiating mechanism has important implications in the development of new antifungal combination therapies. Fungal infections cause more fatalities worldwide each year than malaria or tuberculosis. Currently available antifungal drugs have various limitations, including host toxicity, narrow spectrum of activity, and pathogen resistance. Combining these drugs with small molecules that can overcome these limitations is a useful strategy for extending their clinical use. We have investigated the molecular mechanism by which a marine-derived compound potentiates the activity of the antifungal echinocandin caspofungin. Our findings revealed a mechanism, different from previously reported caspofungin potentiators, in which potentiation is achieved by the disruption of Hsp90 activity and signaling through the cell wall integrity pathway, processes that play important roles in the adaptation to caspofungin in fungal pathogens. Given the importance of stress adaptation in the development of echinocandin resistance, this work will serve as a starting point in the development of new combination therapies that will likely be more effective and less prone to pathogen resistance.
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http://dx.doi.org/10.1128/mSphere.00818-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952202PMC
January 2020

Previously Uncharacterized Vacuolar-type ATPase Binding Site Discovered from Structurally Similar Compounds with Distinct Mechanisms of Action.

ACS Chem Biol 2019 01 18;14(1):20-26. Epub 2018 Dec 18.

Novartis Institutes for BioMedical Research , 250 Massachusetts Avenue , Cambridge , Massachusetts 02139 , United States.

Using a comprehensive chemical genetics approach, we identified a member of the lignan natural product family, HTP-013, which exhibited significant cytotoxicity across various cancer cell lines. Correlation of compound activity across a panel of reporter gene assays suggested the vacuolar-type ATPase (v-ATPase) as a potential target for this compound. Additional cellular studies and a yeast haploinsufficiency screen strongly supported this finding. Competitive photoaffinity labeling experiments demonstrated that the ATP6V0A2 subunit of the v-ATPase complex binds directly to HTP-013, and further mutagenesis library screening identified resistance-conferring mutations in ATP6V0A2. The positions of these mutations suggest the molecule binds a novel pocket within the domain of the v-ATPase complex responsible for proton translocation. While other mechanisms of v-ATPase regulation have been described, such as dissociation of the complex or inhibition by natural products including bafilomycin A1 and concanamycin, this work provides detailed insight into a distinct binding pocket within the v-ATPase complex.
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http://dx.doi.org/10.1021/acschembio.8b00656DOI Listing
January 2019

Two plant-derived aporphinoid alkaloids exert their antifungal activity by disrupting mitochondrial iron-sulfur cluster biosynthesis.

J Biol Chem 2017 10 18;292(40):16578-16593. Epub 2017 Aug 18.

From the National Center for Natural Products Research,

Eupolauridine and liriodenine are plant-derived aporphinoid alkaloids that exhibit potent inhibitory activity against the opportunistic fungal pathogens and However, the molecular mechanism of this antifungal activity is unknown. In this study, we show that eupolauridine 9591 (E9591), a synthetic analog of eupolauridine, and liriodenine methiodide (LMT), a methiodide salt of liriodenine, mediate their antifungal activities by disrupting mitochondrial iron-sulfur (Fe-S) cluster synthesis. Several lines of evidence supported this conclusion. First, both E9591 and LMT elicited a transcriptional response indicative of iron imbalance, causing the induction of genes that are required for iron uptake and for the maintenance of cellular iron homeostasis. Second, a genome-wide fitness profile analysis showed that yeast mutants with deletions in iron homeostasis-related genes were hypersensitive to E9591 and LMT. Third, treatment of wild-type yeast cells with E9591 or LMT generated cellular defects that mimicked deficiencies in mitochondrial Fe-S cluster synthesis including an increase in mitochondrial iron levels, a decrease in the activities of Fe-S cluster enzymes, a decrease in respiratory function, and an increase in oxidative stress. Collectively, our results demonstrate that E9591 and LMT perturb mitochondrial Fe-S cluster biosynthesis; thus, these two compounds target a cellular pathway that is distinct from the pathways commonly targeted by clinically used antifungal drugs. Therefore, the identification of this pathway as a target for antifungal compounds has potential applications in the development of new antifungal therapies.
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http://dx.doi.org/10.1074/jbc.M117.781773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5633121PMC
October 2017

Identification of a novel NAMPT inhibitor by CRISPR/Cas9 chemogenomic profiling in mammalian cells.

Sci Rep 2017 02 16;7:42728. Epub 2017 Feb 16.

Novartis Institutes for BioMedical Research, Novartis Pharma AG, Forum 1 Novartis Campus, CH-4056 Basel, Switzerland.

Chemogenomic profiling is a powerful and unbiased approach to elucidate pharmacological targets and the mechanism of bioactive compounds. Until recently, genome-wide, high-resolution experiments of this nature have been limited to fungal systems due to lack of mammalian genome-wide deletion collections. With the example of a novel nicotinamide phosphoribosyltransferase (NAMPT) inhibitor, we demonstrate that the CRISPR/Cas9 system enables the generation of transient homo- and heterozygous deletion libraries and allows for the identification of efficacy targets and pathways mediating hypersensitivity and resistance relevant to the compound mechanism of action.
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http://dx.doi.org/10.1038/srep42728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5311948PMC
February 2017

Comprehensive functional analysis of the tousled-like kinase 2 frequently amplified in aggressive luminal breast cancers.

Nat Commun 2016 10 3;7:12991. Epub 2016 Oct 3.

Lester &Sue Smith Breast Center, Baylor College of Medicine, Houston, Texas 77030, USA.

More aggressive and therapy-resistant oestrogen receptor (ER)-positive breast cancers remain a great clinical challenge. Here our integrative genomic analysis identifies tousled-like kinase 2 (TLK2) as a candidate kinase target frequently amplified in ∼10.5% of ER-positive breast tumours. The resulting overexpression of TLK2 is more significant in aggressive and advanced tumours, and correlates with worse clinical outcome regardless of endocrine therapy. Ectopic expression of TLK2 leads to enhanced aggressiveness in breast cancer cells, which may involve the EGFR/SRC/FAK signalling. Conversely, TLK2 inhibition selectively inhibits the growth of TLK2-high breast cancer cells, downregulates ERα, BCL2 and SKP2, impairs G1/S cell cycle progression, induces apoptosis and significantly improves progression-free survival in vivo. We identify two potential TLK2 inhibitors that could serve as backbones for future drug development. Together, amplification of the cell cycle kinase TLK2 presents an attractive genomic target for aggressive ER-positive breast cancers.
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http://dx.doi.org/10.1038/ncomms12991DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5064015PMC
October 2016

Enrichment of Cdk1-cyclins at DNA double-strand breaks stimulates Fun30 phosphorylation and DNA end resection.

Nucleic Acids Res 2016 Apr 21;44(6):2742-53. Epub 2016 Jan 21.

Department of Molecular ad Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA

DNA double-strand breaks (DSBs) are one of the most cytotoxic types of DNA lesion challenging genome integrity. The activity of cyclin-dependent kinase Cdk1 is essential for DSB repair by homologous recombination and for DNA damage signaling. Here we identify the Fun30 chromatin remodeler as a new target of Cdk1. Fun30 is phosphorylated by Cdk1 on Serine 28 to stimulate its functions in DNA damage response including resection of DSB ends. Importantly, Cdk1-dependent phosphorylation of Fun30-S28 increases upon DNA damage and requires the recruitment of Fun30 to DSBs, suggesting that phosphorylation increases in situ at the DNA damage. Consistently, we find that Cdk1 and multiple cyclins become highly enriched at DSBs and that the recruitment of Cdk1 and cyclins Clb2 and Clb5 ensures optimal Fun30 phosphorylation and checkpoint activation. We propose that the enrichment of Cdk1-cyclin complexes at DSBs serves as a mechanism for enhanced targeting and modulating of the activity of DNA damage response proteins.
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http://dx.doi.org/10.1093/nar/gkv1544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4824098PMC
April 2016

Nannocystin A: an Elongation Factor 1 Inhibitor from Myxobacteria with Differential Anti-Cancer Properties.

Angew Chem Int Ed Engl 2015 Aug 15;54(35):10149-54. Epub 2015 Jul 15.

Novartis Institutes for BioMedical Research, Novartis Campus, 4056 Basel (Switzerland).

Cultivation of myxobacteria of the Nannocystis genus led to the isolation and structure elucidation of a class of novel cyclic lactone inhibitors of elongation factor 1. Whole genome sequence analysis and annotation enabled identification of the putative biosynthetic cluster and synthesis process. In biological assays the compounds displayed anti-fungal and cytotoxic activity. Combined genetic and proteomic approaches identified the eukaryotic translation elongation factor 1α (EF-1α) as the primary target for this compound class. Nannocystin A (1) displayed differential activity across various cancer cell lines and EEF1A1 expression levels appear to be the main differentiating factor. Biochemical and genetic evidence support an overlapping binding site of 1 with the anti-cancer compound didemnin B on EF-1α. This myxobacterial chemotype thus offers an interesting starting point for further investigations of the potential of therapeutics targeting elongation factor 1.
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http://dx.doi.org/10.1002/anie.201505069DOI Listing
August 2015

Identification of a New Class of Antifungals Targeting the Synthesis of Fungal Sphingolipids.

mBio 2015 Jun 23;6(3):e00647. Epub 2015 Jun 23.

Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York, USA

Unlabelled: Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [N'-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo-N'-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5, COS111, MKK1, and STE2, which are involved in vesicular transport and cell cycle progression, are targeted by BHBM.

Importance: Fungal infections are a significant cause of morbidity and mortality worldwide. Current antifungal drugs suffer from various drawbacks, including toxicity, drug resistance, and narrow spectrum of activity. In this study, we have demonstrated that pharmaceutical inhibition of fungal glucosylceramide presents a new opportunity to treat cryptococcosis and various other fungal infections. In addition to being effective against pathogenic fungi, the compounds discovered in this study were well tolerated by animals and additive to current antifungals. These findings suggest that these drugs might pave the way for the development of a new class of antifungals.
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http://dx.doi.org/10.1128/mBio.00647-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4479701PMC
June 2015

Synthesis and antifungal activities of miltefosine analogs.

Bioorg Med Chem Lett 2013 Sep 6;23(17):4828-31. Epub 2013 Jul 6.

National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, The University of Mississippi, MS 38677, USA.

Miltefosine is an alkylphosphocholine that shows broad-spectrum in vitro antifungal activities and limited in vivo efficacy in mouse models of cryptococcosis. To further explore the potential of this class of compounds for the treatment of systemic mycoses, nine analogs (3a-3i) were synthesized by modifying the choline structural moiety and the alkyl chain length of miltefosine. In vitro testing of these compounds against the opportunistic fungal pathogens Candida albicans, Candida glabrata, Candida krusei, Aspergillus fumigatus, and Cryptococcus neoformans revealed that N-benzyl-N,N-dimethyl-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3a), N,N-dimethyl-N-(4-nitrobenzyl)-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3d), and N-(4-methoxybenzyl)-N,N-dimethyl-2-{[(hexadecyloxy)hydroxyphosphinyl]oxy}ethanaminium inner salt (3e) exhibited minimum inhibitory concentrations (MIC) of 2.5-5.0 μg/mL against all tested pathogens, when compared to miltefosine with MICs of 2.5-3.3 μg/mL. Compound 3a showed low in vitro cytotoxicity against three mammalian cell lines similar to miltefosine. In vivo testing of 3a and miltefosine against C. albicans in a mouse model of systemic infection did not demonstrate efficacy. The results of this study indicate that further investigation will be required to determine the potential usefulness of the alkylphosphocholines in the treatment of invasive fungal infections.
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http://dx.doi.org/10.1016/j.bmcl.2013.06.096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3760157PMC
September 2013

A functional variomics tool for discovering drug-resistance genes and drug targets.

Cell Rep 2013 Feb 14;3(2):577-85. Epub 2013 Feb 14.

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.

Comprehensive discovery of genetic mechanisms of drug resistance and identification of in vivo drug targets represent significant challenges. Here we present a functional variomics technology in the model organism Saccharomyces cerevisiae. This tool analyzes numerous genetic variants and effectively tackles both problems simultaneously. Using this tool, we discovered almost all genes that, due to mutations or modest overexpression, confer resistance to rapamycin, cycloheximide, and amphotericin B. Most significant among the resistance genes were drug targets, including multiple targets of a given drug. With amphotericin B, we discovered the highly conserved membrane protein Pmp3 as a potent resistance factor and a possible target. Widespread application of this tool should allow rapid identification of conserved resistance mechanisms and targets of many more compounds. New genes and alleles that confer resistance to other stresses can also be discovered. Similar tools in other systems, such as human cell lines, will also be useful.
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http://dx.doi.org/10.1016/j.celrep.2013.01.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3594652PMC
February 2013

Discovering thiamine transporters as targets of chloroquine using a novel functional genomics strategy.

PLoS Genet 2012 29;8(11):e1003083. Epub 2012 Nov 29.

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, United States of America.

Chloroquine (CQ) and other quinoline-containing antimalarials are important drugs with many therapeutic benefits as well as adverse effects. However, the molecular targets underlying most such effects are largely unknown. By taking a novel functional genomics strategy, which employs a unique combination of genome-wide drug-gene synthetic lethality (DGSL), gene-gene synthetic lethality (GGSL), and dosage suppression (DS) screens in the model organism Saccharomyces cerevisiae and is thus termed SL/DS for simplicity, we found that CQ inhibits the thiamine transporters Thi7, Nrt1, and Thi72 in yeast. We first discovered a thi3Δ mutant as hypersensitive to CQ using a genome-wide DGSL analysis. Using genome-wide GGSL and DS screens, we then found that a thi7Δ mutation confers severe growth defect in the thi3Δ mutant and that THI7 overexpression suppresses CQ-hypersensitivity of this mutant. We subsequently showed that CQ inhibits the functions of Thi7 and its homologues Nrt1 and Thi72. In particular, the transporter activity of wild-type Thi7 but not a CQ-resistant mutant (Thi7(T287N)) was completely inhibited by the drug. Similar effects were also observed with other quinoline-containing antimalarials. In addition, CQ completely inhibited a human thiamine transporter (SLC19A3) expressed in yeast and significantly inhibited thiamine uptake in cultured human cell lines. Therefore, inhibition of thiamine uptake is a conserved mechanism of action of CQ. This study also demonstrated SL/DS as a uniquely effective methodology for discovering drug targets.
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http://dx.doi.org/10.1371/journal.pgen.1003083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3510038PMC
May 2013

DANPOS: dynamic analysis of nucleosome position and occupancy by sequencing.

Genome Res 2013 Feb 28;23(2):341-51. Epub 2012 Nov 28.

Division of Biostatistics, Dan L Duncan Cancer Center and Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.

Recent developments in next-generation sequencing have enabled whole-genome profiling of nucleosome organizations. Although several algorithms for inferring nucleosome position from a single experimental condition have been available, it remains a challenge to accurately define dynamic nucleosomes associated with environmental changes. Here, we report a comprehensive bioinformatics pipeline, DANPOS, explicitly designed for dynamic nucleosome analysis at single-nucleotide resolution. Using both simulated and real nucleosome data, we demonstrated that bias correction in preliminary data processing and optimal statistical testing significantly enhances the functional interpretation of dynamic nucleosomes. The single-nucleotide resolution analysis of DANPOS allows us to detect all three categories of nucleosome dynamics, such as position shift, fuzziness change, and occupancy change, using a uniform statistical framework. Pathway analysis indicates that each category is involved in distinct biological functions. We also analyzed the influence of sequencing depth and suggest that even 200-fold coverage is probably not enough to identify all the dynamic nucleosomes. Finally, based on nucleosome data from the human hematopoietic stem cells (HSCs) and mouse embryonic stem cells (ESCs), we demonstrated that DANPOS is also robust in defining functional dynamic nucleosomes, not only in promoters, but also in distal regulatory regions in the mammalian genome.
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http://dx.doi.org/10.1101/gr.142067.112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3561875PMC
February 2013

The Fun30 nucleosome remodeller promotes resection of DNA double-strand break ends.

Nature 2012 Sep 9;489(7417):576-80. Epub 2012 Sep 9.

Department of Molecular & Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA.

Chromosomal double-strand breaks (DSBs) are resected by 5' nucleases to form 3' single-stranded DNA substrates for binding by homologous recombination and DNA damage checkpoint proteins. Two redundant pathways of extensive resection have been described both in cells and in vitro, one relying on Exo1 exonuclease and the other on Sgs1 helicase and Dna2 nuclease. However, it remains unknown how resection proceeds within the context of chromatin, where histones and histone-bound proteins represent barriers for resection enzymes. Here we identify the yeast nucleosome-remodelling enzyme Fun30 as a factor promoting DSB end resection. Fun30 is the major nucleosome remodeller promoting extensive Exo1- and Sgs1-dependent resection of DSBs. The RSC and INO80 chromatin-remodelling complexes and Fun30 have redundant roles in resection adjacent to DSB ends. ATPase and helicase domains of Fun30, which are needed for nucleosome remodelling, are also required for resection. Fun30 is robustly recruited to DNA breaks and spreads along the DSB coincident with resection. Fun30 becomes less important for resection in the absence of the histone-bound Rad9 checkpoint adaptor protein known to block 5' strand processing and in the absence of either histone H3 K79 methylation or γ-H2A, which mediate recruitment of Rad9 (refs 9, 10). Together these data suggest that Fun30 helps to overcome the inhibitory effect of Rad9 on DNA resection.
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http://dx.doi.org/10.1038/nature11355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3640768PMC
September 2012

The Saccharomyces cerevisiae Nrd1-Nab3 transcription termination pathway acts in opposition to Ras signaling and mediates response to nutrient depletion.

Mol Cell Biol 2012 May 19;32(10):1762-75. Epub 2012 Mar 19.

Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

The Saccharomyces cerevisiae Nrd1-Nab3 pathway directs the termination and processing of short RNA polymerase II transcripts. Despite the potential for Nrd1-Nab3 to affect the transcription of both coding and noncoding RNAs, little is known about how the Nrd1-Nab3 pathway interacts with other pathways in the cell. Here we present the results of a high-throughput synthetic lethality screen for genes that interact with NRD1 and show roles for Nrd1 in the regulation of mitochondrial abundance and cell size. We also provide genetic evidence of interactions between the Nrd1-Nab3 and Ras/protein kinase A (PKA) pathways. Whereas the Ras pathway promotes the transcription of genes involved in growth and glycolysis, the Nrd1-Nab3 pathway appears to have a novel role in the rapid suppression of some genes when cells are shifted to poor growth conditions. We report the identification of new mRNA targets of the Nrd1-Nab3 pathway that are rapidly repressed in response to glucose depletion. Glucose depletion also leads to the dephosphorylation of Nrd1 and the formation of novel nuclear speckles that contain Nrd1 and Nab3. Taken together, these results indicate a role for Nrd1-Nab3 in regulating the cellular response to nutrient availability.
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http://dx.doi.org/10.1128/MCB.00050-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3347421PMC
May 2012

Sampangine inhibits heme biosynthesis in both yeast and human.

Eukaryot Cell 2011 Nov 9;10(11):1536-44. Epub 2011 Sep 9.

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.

The azaoxoaporphine alkaloid sampangine exhibits strong antiproliferation activity in various organisms. Previous studies suggested that it somehow affects heme metabolism and stimulates production of reactive oxygen species (ROS). In this study, we show that inhibition of heme biosynthesis is the primary mechanism of action by sampangine and that increases in the levels of reactive oxygen species are secondary to heme deficiency. We directly demonstrate that sampangine inhibits heme synthesis in the yeast Saccharomyces cerevisiae. It also causes accumulation of uroporphyrinogen and its decarboxylated derivatives, intermediate products of the heme biosynthesis pathway. Our results also suggest that sampangine likely works through an unusual mechanism-by hyperactivating uroporhyrinogen III synthase-to inhibit heme biosynthesis. We also show that the inhibitory effect of sampangine on heme synthesis is conserved in human cells. This study also reveals a surprising essential role for the interaction between the mitochondrial ATP synthase and the electron transport chain.
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http://dx.doi.org/10.1128/EC.05170-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3209050PMC
November 2011

Making temperature-sensitive mutants.

Methods Enzymol 2010 1;470:181-204. Epub 2010 Mar 1.

Michael Smith Laboratories, Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada.

The study of temperature-sensitive (Ts) mutant phenotypes is fundamental to gene identification and for dissecting essential gene function. In this chapter, we describe two "shuffling" methods for producing Ts mutants using a combination of PCR, in vivo recombination, and transformation of diploid strains heterozygous for a knockout of the desired mutation. The main difference between the two methods is the type of strain produced. In the "plasmid" version, the product is a knockout mutant carrying a centromeric plasmid carrying the Ts mutant. In the "chromosomal" version, The Ts alleles are integrated directly into the endogenous locus, albeit not in an entirely native configuration. Both variations have their strengths and weaknesses, which are discussed here.
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http://dx.doi.org/10.1016/S0076-6879(10)70008-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2957654PMC
January 2011

Trivalent arsenic inhibits the functions of chaperonin complex.

Genetics 2010 Oct 26;186(2):725-34. Epub 2010 Jul 26.

Department of Biology and Integrated Imaging Center, The Johns Hopkins University, Baltimore, MD 21218, USA.

The exact molecular mechanisms by which the environmental pollutant arsenic works in biological systems are not completely understood. Using an unbiased chemogenomics approach in Saccharomyces cerevisiae, we found that mutants of the chaperonin complex TRiC and the functionally related prefoldin complex are all hypersensitive to arsenic compared to a wild-type strain. In contrast, mutants with impaired ribosome functions were highly arsenic resistant. These observations led us to hypothesize that arsenic might inhibit TRiC function, required for folding of actin, tubulin, and other proteins postsynthesis. Consistent with this hypothesis, we found that arsenic treatment distorted morphology of both actin and microtubule filaments. Moreover, arsenic impaired substrate folding by both bovine and archaeal TRiC complexes in vitro. These results together indicate that TRiC is a conserved target of arsenic inhibition in various biological systems.
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http://dx.doi.org/10.1534/genetics.110.117655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2954479PMC
October 2010

Genome-wide reinforcement of cohesin binding at pre-existing cohesin sites in response to ionizing radiation in human cells.

J Biol Chem 2010 Jul 25;285(30):22784-92. Epub 2010 May 25.

Center for Molecular Discovery, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030, USA.

The cohesin complex plays a central role in genome maintenance by regulation of chromosome segregation in mitosis and DNA damage response (DDR) in other phases of the cell cycle. The ATM/ATR phosphorylates SMC1 and SMC3, two core components of the cohesin complex to regulate checkpoint signaling and DNA repair. In this report, we show that the genome-wide binding of SMC1 and SMC3 after ionizing radiation (IR) is enhanced by reinforcing pre-existing cohesin binding sites in human cancer cells. We demonstrate that ATM and SMC3 phosphorylation at Ser(1083) regulate this process. We also demonstrate that acetylation of SMC3 at Lys(105) and Lys(106) is induced by IR and this induction depends on the acetyltransferase ESCO1 as well as the ATM/ATR kinases. Consistently, both ESCO1 and SMC3 acetylation are required for intra-S phase checkpoint and cellular survival after IR. Although both IR-induced acetylation and phosphorylation of SMC3 are under the control of ATM/ATR, the two forms of modification are independent of each other and both are required to promote reinforcement of SMC3 binding to cohesin sites. Thus, SMC3 modifications is a mechanism for genome-wide reinforcement of cohesin binding in response to DNA damage response in human cells and enhanced cohesion is a downstream event of DDR.
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http://dx.doi.org/10.1074/jbc.M110.134577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2906269PMC
July 2010

A microarray-based genetic screen for yeast chronological aging factors.

PLoS Genet 2010 Apr 22;6(4):e1000921. Epub 2010 Apr 22.

Department of Biochemistry and Molecular Genetics, University of Virginia Health System, School of Medicine, Charlottesville, Virginia, United States of America.

Model organisms have played an important role in the elucidation of multiple genes and cellular processes that regulate aging. In this study we utilized the budding yeast, Saccharomyces cerevisiae, in a large-scale screen for genes that function in the regulation of chronological lifespan, which is defined by the number of days that non-dividing cells remain viable. A pooled collection of viable haploid gene deletion mutants, each tagged with unique identifying DNA "bar-code" sequences was chronologically aged in liquid culture. Viable mutants in the aging population were selected at several time points and then detected using a microarray DNA hybridization technique that quantifies abundance of the barcode tags. Multiple short- and long-lived mutants were identified using this approach. Among the confirmed short-lived mutants were those defective for autophagy, indicating a key requirement for the recycling of cellular organelles in longevity. Defects in autophagy also prevented lifespan extension induced by limitation of amino acids in the growth media. Among the confirmed long-lived mutants were those defective in the highly conserved de novo purine biosynthesis pathway (the ADE genes), which ultimately produces IMP and AMP. Blocking this pathway extended lifespan to the same degree as calorie (glucose) restriction. A recently discovered cell-extrinsic mechanism of chronological aging involving acetic acid secretion and toxicity was suppressed in a long-lived ade4Delta mutant and exacerbated by a short-lived atg16Delta autophagy mutant. The identification of multiple novel effectors of yeast chronological lifespan will greatly aid in the elucidation of mechanisms that cells and organisms utilize in slowing down the aging process.
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http://dx.doi.org/10.1371/journal.pgen.1000921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2858703PMC
April 2010

The unfolded protein response is necessary but not sufficient to compensate for defects in disulfide isomerization.

J Biol Chem 2009 Apr 20;284(16):10400-8. Epub 2009 Feb 20.

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Houston, Texas, USA.

Pdi1p (protein-disulfide isomerase) is a folding assistant of the endoplasmic reticulum (ER) that catalyzes disulfide formation and the isomerization of incorrect disulfides. Its disulfide forming activity is its essential function in Saccharomyces cerevisiae. A truncation mutant (Pdi1a') that is competent in disulfide formation but deficient in catalyzing isomerization has only a small effect on growth, although the maturation of isomerase-requiring substrates (carboxypeptidase Y) is impaired (Xiao, R., Wilkinson, B., Solovyov, A., Winther, J. R., Holmgren, A., Lundstrom-Ljung, J., and Gilbert, H. F. (2004) J. Biol. Chem. 279, 49780-49786). We show here that there are multiple ways to compensate for defects in disulfide formation and isomerization in the ER. Genes of the unfolded protein response are induced, and deletions of the nonessential IRE1 or HAC1 genes are synthetically lethal. Diploid synthetic lethality analysis by microarray (dSLAM) using PDIa' and a temperature-sensitive mutant of PDIa' as query mutations reveals a group of 130 synthetically lethal genes. Only 10 of these correspond to genes clearly associated with the unfolded protein response. More than half are involved in vesicle traffic, not only out of and into the ER but anterograde and retrograde traffic from most cellular compartments. This suggests that defects in protein maturation in one intracellular compartment may be compensated for by adjusting vesicular traffic patterns throughout the cell.
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http://dx.doi.org/10.1074/jbc.M900377200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2667727PMC
April 2009

A comprehensive synthetic genetic interaction network governing yeast histone acetylation and deacetylation.

Genes Dev 2008 Aug;22(15):2062-74

High-Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

Histone acetylation and deacetylation are among the principal mechanisms by which chromatin is regulated during transcription, DNA silencing, and DNA repair. We analyzed patterns of genetic interactions uncovered during comprehensive genome-wide analyses in yeast to probe how histone acetyltransferase (HAT) and histone deacetylase (HDAC) protein complexes interact. The genetic interaction data unveil an underappreciated role of HDACs in maintaining cellular viability, and led us to show that deacetylation of the histone variant Htz1p at Lys 14 is mediated by Hda1p. Studies of the essential nucleosome acetyltransferase of H4 (NuA4) revealed acetylation-dependent protein stabilization of Yng2p, a potential nonhistone substrate of NuA4 and Rpd3C, and led to a new functional organization model for this critical complex. We also found that DNA double-stranded breaks (DSBs) result in local recruitment of the NuA4 complex, followed by an elaborate NuA4 remodeling process concomitant with Rpd3p recruitment and histone deacetylation. These new characterizations of the HDA and NuA4 complexes demonstrate how systematic analyses of genetic interactions may help illuminate the mechanisms of intricate cellular processes.
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http://dx.doi.org/10.1101/gad.1679508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2492751PMC
August 2008

Acetylation of Smc3 by Eco1 is required for S phase sister chromatid cohesion in both human and yeast.

Mol Cell 2008 Jul;31(1):143-51

Center for Molecular Discovery, Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030, USA.

Sister chromatid cohesion is normally established in S phase in a process that depends on the cohesion establishment factor Eco1, a conserved acetyltransferase. However, due to the lack of known in vivo substrates, how Eco1 regulates cohesion is not understood. Here we report that yeast Eco1 and its human ortholog, ESCO1, both acetylate Smc3, a component of the cohesin complex that physically holds the sister chromatid together, at two conserved lysine residues. Mutating these lysine residues to a nonacetylatable form leads to increased loss of sister chromatid cohesion and genome instability in both yeast and human. In addition, we clarified that the acetyltransferase activity of Eco1 is essential for its function. Our study thus identified a molecular target for the acetyltransferase Eco1 and revealed that Smc3 acetylation is a conserved mechanism in regulating sister chromatid cohesion.
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http://dx.doi.org/10.1016/j.molcel.2008.06.006DOI Listing
July 2008

Microarray-based genetic screen defines SAW1, a gene required for Rad1/Rad10-dependent processing of recombination intermediates.

Mol Cell 2008 May;30(3):325-35

Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245, USA.

Elimination of a double-strand break (DSB) flanked by direct repeat sequences is mediated by single-strand annealing (SSA), which relies on a distinct set of gene products involving recombination, mismatch repair, and nucleotide excision repair. Here, we screened for yeast mutants defective in SSA with a plasmid-based SSA assay coupled to a barcode microarray readout. The screen identified Yal027Wp/Saw1 (single-strand annealing weakened 1) and Slx4 besides other known SSA proteins. Saw1 interacts physically with Rad1/Rad10, Msh2/Msh3, and Rad52 proteins, and cells lacking SLX4 or SAW1 accumulate recombination intermediates blocked at the Rad1/Rad10-dependent 3' flap cleavage step. Slx4 and Saw1 also contribute to the integrity of ribosomal DNA arrays. Saw1 mutants that fail to interact with Rad1, but retain interaction with Rad52 and Msh2, are defective in 3' flap removal and SSA repair. Deletion of SAW1 abolished association of Rad1 at SSA intermediates in vivo. We propose that Saw1 targets Rad1/Rad10 to Rad52-coated recombination intermediates.
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http://dx.doi.org/10.1016/j.molcel.2008.02.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2398651PMC
May 2008

Analysis of genetic interactions on a genome-wide scale in budding yeast: diploid-based synthetic lethality analysis by microarray.

Methods Mol Biol 2008 ;416:221-47

Department of Molecular Biology and Genetics, The High Throughput Biology Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Comprehensive collections of open reading frame (ORF) deletion mutant strains exist for the budding yeast Saccharomyces cerevisiae. With great prescience, these strains were designed with short molecular bar codes or TAGs that uniquely mark each deletion allele, flanked by shared priming sequences. These features have enabled researchers to handle yeast mutant collections as complex pools of approximately 6000 strains. The presence of any individual mutant within a pool can be assessed indirectly by measuring the relative abundance of its corresponding TAG(s) in genomic DNA prepared from the pool. This is readily accomplished by wholesale polymerase chain reaction (PCR) amplification of the TAGs using fluorescent oligonucleotide primers that recognize the common flanking sequences, followed by hybridization of the labeled PCR products to a TAG oligonucleotide microarray. Here we describe a method-diploid-based synthetic lethality analysis by microarray (dSLAM)-whereby such pools can be manipulated to rapidly construct and assess the fitness of 6000 double-mutant strains in a single experiment. Analysis of double-mutant strains is of growing importance in defining the spectrum of essential cellular functionalities and in understanding how these functionalities interrelate.
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http://dx.doi.org/10.1007/978-1-59745-321-9_15DOI Listing
June 2008

Plasmid-chromosome shuffling for non-deletion alleles in yeast.

Nat Methods 2008 Feb 13;5(2):167-9. Epub 2008 Jan 13.

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA.

Here we describe a facile plasmid-chromosome shuffling technique for generating and analyzing non-deletion alleles in the yeast Saccharomyces cerevisiae. This technique takes advantage of an existing set of genome-wide haploid-convertible heterozygous diploid yeast knockout mutants. This simple method will facilitate characterization of essential gene functions and genome-wide investigation of protein structure-function relationships.
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http://dx.doi.org/10.1038/nmeth.1173DOI Listing
February 2008

Efficient Tor signaling requires a functional class C Vps protein complex in Saccharomyces cerevisiae.

Genetics 2007 Aug 11;176(4):2139-50. Epub 2007 Jun 11.

Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA.

The Tor kinases regulate responses to nutrients and control cell growth. Unlike most organisms that only contain one Tor protein, Saccharomyces cerevisiae expresses two, Tor1 and Tor2, which are thought to share all of the rapamycin-sensitive functions attributable to Tor signaling. Here we conducted a genetic screen that defined the global TOR1 synthetic fitness or lethal interaction gene network. This screen identified mutations in distinctive functional categories that impaired vacuolar function, including components of the EGO/Gse and PAS complexes that reduce fitness. In addition, tor1 is lethal in combination with mutations in class C Vps complex components. We find that Tor1 does not regulate the known function of the class C Vps complex in protein sorting. Instead class C vps mutants fail to recover from rapamycin-induced growth arrest or to survive nitrogen starvation and have low levels of amino acids. Remarkably, addition of glutamate or glutamine restores viability to a tor1 pep3 mutant strain. We conclude that Tor1 is more effective than Tor2 at providing rapamycin-sensitive Tor signaling under conditions of amino acid limitation, and that an intact class C Vps complex is required to mediate intracellular amino acid homeostasis for efficient Tor signaling.
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http://dx.doi.org/10.1534/genetics.107.072835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1950620PMC
August 2007

dSLAM analysis of genome-wide genetic interactions in Saccharomyces cerevisiae.

Methods 2007 Feb;41(2):206-21

Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, MD 21205, USA.

Analysis of genetic interactions has been extensively exploited to study gene functions and to dissect pathway structures. One such genetic interaction is synthetic lethality, in which the combination of two non-lethal mutations leads to loss of organism viability. We have developed a dSLAM (heterozygote diploid-based synthetic lethality analysis with microarrays) technology that effectively studies synthetic lethality interactions on a genome-wide scale in the budding yeast Saccharomyces cerevisiae. Typically, a query mutation is introduced en masse into a population of approximately 6000 haploid-convertible heterozygote diploid Yeast Knockout (YKO) mutants via integrative transformation. Haploid pools of single and double mutants are freshly generated from the resultant heterozygote diploid double mutant pool after meiosis and haploid selection and studied for potential growth defects of each double mutant combination by microarray analysis of the "molecular barcodes" representing each YKO. This technology has been effectively adapted to study other types of genome-wide genetic interactions including gene-compound synthetic lethality, secondary mutation suppression, dosage-dependent synthetic lethality and suppression.
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http://dx.doi.org/10.1016/j.ymeth.2006.07.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2491713PMC
February 2007