scholarly journals A genome-wide portrait of pervasive drug contaminants

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Joseph Uche Ogbede ◽  
Guri Giaever ◽  
Corey Nislow
Keyword(s):  
Ammonium Sulfate ◽  
Homozygous Deletion ◽  
Growth Defect ◽  
Ammonium Ions ◽  
Cellular Effects ◽  
Genome Wide ◽  
A Genome ◽  
Homozygous Diploid ◽  
Vacuolar Protein ◽  
Related Compounds

AbstractUsing a validated yeast chemogenomic platform, we characterized the genome-wide effects of several pharmaceutical contaminants, including three N-nitrosamines (NDMA, NDEA and NMBA), two related compounds (DMF and 4NQO) and several of their metabolites. A collection of 4800 non-essential homozygous diploid yeast deletion strains were screened in parallel and the strain abundance was quantified by barcode sequencing. These data were used to rank deletion strains representing genes required for resistance to the compounds to delineate affected cellular pathways and to visualize the global cellular effects of these toxins in an easy-to-use searchable database. Our analysis of the N-nitrosamine screens uncovered genes (via their corresponding homozygous deletion mutants) involved in several evolutionarily conserved pathways, including: arginine biosynthesis, mitochondrial genome integrity, vacuolar protein sorting and DNA damage repair. To investigate why NDMA, NDEA and DMF caused fitness defects in strains lacking genes of the arginine pathway, we tested several N-nitrosamine metabolites (methylamine, ethylamine and formamide), and found they also affected arginine pathway mutants. Notably, each of these metabolites has the potential to produce ammonium ions during their biotransformation. We directly tested the role of ammonium ions in N-nitrosamine toxicity by treatment with ammonium sulfate and we found that ammonium sulfate also caused a growth defect in arginine pathway deletion strains. Formaldehyde, a metabolite produced from NDMA, methylamine and formamide, and which is known to cross-link free amines, perturbed deletion strains involved in chromatin remodeling and DNA repair pathways. Finally, co-administration of N-nitrosamines with ascorbic or ferulic acid did not relieve N-nitrosamine toxicity. In conclusion, we used parallel deletion mutant analysis to characterize the genes and pathways affected by exposure to N-nitrosamines and related compounds, and provide the data in an accessible, queryable database.

scholarly journals Genome-Wide Analysis Identifies an Essential Human TBX3 Pacemaker Enhancer

2020 ◽  
Vol 127 (12) ◽  
pp. 1522-1535 ◽  
Author(s):  
Vincent W.W. van Eif ◽  
Stephanie I. Protze ◽  
Fernanda M. Bosada ◽  
Xuefei Yuan ◽  
Tanvi Sinha ◽  
...  
Keyword(s):  
Heart Rate ◽  
Heart Rate Recovery ◽  
Homozygous Deletion ◽  
Pacemaker Cell ◽  
Pacemaker Cells ◽  
Selective Loss ◽  
Adult Mice ◽  
Genome Wide ◽  
A Genome ◽  
And Function

Rationale: The development and function of the pacemaker cardiomyocytes of the sinoatrial node (SAN), the leading pacemaker of the heart, are tightly controlled by a conserved network of transcription factors, including TBX3 (T-box transcription factor 3), ISL1 (ISL LIM homeobox 1), and SHOX2 (short stature homeobox 2). Yet, the regulatory DNA elements (REs) controlling target gene expression in the SAN pacemaker cells have remained undefined. Objective: Identification of the regulatory landscape of human SAN-like pacemaker cells and functional assessment of SAN-specific REs potentially involved in pacemaker cell gene regulation. Methods and Results: We performed Assay for Transposase-Accessible Chromatin using sequencing on human pluripotent stem cell–derived SAN-like pacemaker cells and ventricle-like cells and identified thousands of putative REs specific for either human cell type. We validated pacemaker cell–specific elements in the SHOX2 and TBX3 loci. CRISPR-mediated homozygous deletion of the mouse ortholog of a noncoding region with candidate pacemaker-specific REs in the SHOX2 locus resulted in selective loss of Shox2 expression from the developing SAN and embryonic lethality. Putative pacemaker-specific REs were identified up to 1 Mbp upstream of TBX3 in a region close to MED13L harboring variants associated with heart rate recovery after exercise. The orthologous region was deleted in mice, which resulted in selective loss of expression of Tbx3 from the SAN and (cardiac) ganglia and in neonatal lethality. Expression of Tbx3 was maintained in other tissues including the atrioventricular conduction system, lungs, and liver. Heterozygous adult mice showed increased SAN recovery times after pacing. The human REs harboring the associated variants robustly drove expression in the SAN of transgenic mouse embryos. Conclusions: We provided a genome-wide collection of candidate human pacemaker-specific REs, including the loci of SHOX2 , TBX3 , and ISL1 , and identified a link between human genetic variants influencing heart rate recovery after exercise and a variant RE with highly conserved function, driving SAN expression of TBX3 .

scholarly journals NRF2 Activation Confers Resistance to eIF4A Inhibitors in Cancer Therapy

Cancers ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 639
Author(s):  
Viraj R. Sanghvi ◽  
Prathibha Mohan ◽  
Kamini Singh ◽  
Linlin Cao ◽  
Marjan Berishaj ◽  
...  
Keyword(s):  
Protein Production ◽  
Mechanisms Of Resistance ◽  
New Class ◽  
Translation Factors ◽  
Genome Wide ◽  
A Genome ◽  
Nrf2 Activation ◽  
Anti Cancer ◽  
New Strategy ◽  
Related Compounds

Inhibition of the eIF4A RNA helicase with silvestrol and related compounds is emerging as a powerful anti-cancer strategy. We find that a synthetic silvestrol analogue (CR-1-31 B) has nanomolar activity across many cancer cell lines. It is especially active against aggressive MYC+/BCL2+ B cell lymphomas and this likely reflects the eIF4A-dependent translation of both MYC and BCL2. We performed a genome-wide CRISPR/Cas9 screen and identified mechanisms of resistance to this new class of therapeutics. We identify three negative NRF2 regulators (KEAP1, CUL3, CAND1) whose inactivation is sufficient to cause CR1-31-B resistance. NRF2 is known to alter the oxidation state of translation factors and cause a broad increase in protein production. We find that NRF2 activation particularly increases the translation of some eIF4A-dependent mRNAs and restores MYC and BCL2 production. We know that NRF2 functions depend on removal of sugar adducts by the frutosamine-3-kinase (FN3K). Accordingly, loss of FN3K results in NRF2 hyper-glycation and inactivation and resensitizes cancer cells to eIF4A inhibition. Together, our findings implicate NRF2 in the translation of eIF4A-dependent mRNAs and point to FN3K inhibition as a new strategy to block NRF2 functions in cancer.

scholarly journals An RNA symbiont enhances heat tolerance and secondary homothallism in the oomycete Phytophthora infestans

Microbiology ◽  
2010 ◽  
Vol 156 (7) ◽  
pp. 2026-2034 ◽  
Author(s):  
Howard S. Judelson ◽  
Audrey M. V. Ah-Fong ◽  
Anna-Liisa Fabritius
Keyword(s):  
Phytophthora Infestans ◽  
Cellular Effects ◽  
Hsp70 Family ◽  
Genome Wide ◽  
A Genome ◽  
Hsp70 Chaperone ◽  
Genes Encoding ◽  
Genome Wide Search ◽  
Oospore Formation ◽  
Late Blight Pathogen

Some strains of Phytophthora infestans, the potato late blight pathogen, harbour a small extrachromosomal RNA called PiERE1. A previous study reported that this RNA symbiont does not noticeably affect its host. Here it is revealed that PiERE1 exerts subtle effects on P. infestans, which result in greater thermotolerance during growth and an increase in secondary homothallism, i.e. oospore formation in the absence of the opposite mating type. The interaction can be considered mutualistic since these traits may increase the fitness of P. infestans in nature. Assays of biomarkers for cellular stress revealed that an Hsp70 chaperone was upregulated by PiERE1. A genome-wide search for more members of the Hsp70 family identified ten belonging to the DnaK subfamily, one in the Hsp110/SSE subfamily, and pseudogenes. Four DnaK subfamily genes encoding predicted cytoplasmic or endoplasmic reticulum proteins were upregulated in strains harbouring PiERE1. This may explain the greater thermotolerance conferred by the RNA element, and suggests that Hsp70 may be a useful biomarker for testing organisms for the cellular effects of symbiotic elements.

scholarly journals Genome-wide Analysis of AP-3–dependent Protein Transport in Yeast

2009 ◽  
Vol 20 (5) ◽  
pp. 1592-1604 ◽  
Author(s):  
Vikram C. Anand ◽  
Lydia Daboussi ◽  
Todd C. Lorenz ◽  
Gregory S. Payne
Keyword(s):  
Protein Transport ◽  
Adaptor Protein ◽  
Subcellular Fractionation ◽  
Type I ◽  
Transport Pathways ◽  
Genome Wide ◽  
A Genome ◽  
Rapid Generation ◽  
Vacuolar Protein ◽  
Er Export

The evolutionarily conserved adaptor protein-3 (AP-3) complex mediates cargo-selective transport to lysosomes and lysosome-related organelles. To identify proteins that function in AP-3–mediated transport, we performed a genome-wide screen in Saccharomyces cerevisiae for defects in the vacuolar maturation of alkaline phosphatase (ALP), a cargo of the AP-3 pathway. Forty-nine gene deletion strains were identified that accumulated precursor ALP, many with established defects in vacuolar protein transport. Maturation of a vacuolar membrane protein delivered via a separate, clathrin-dependent pathway, was affected in all strains except those with deletions of YCK3, encoding a vacuolar type I casein kinase; SVP26, encoding an endoplasmic reticulum (ER) export receptor for ALP; and AP-3 subunit genes. Subcellular fractionation and fluorescence microscopy revealed ALP transport defects in yck3Δ cells. Characterization of svp26Δ cells revealed a role for Svp26p in ER export of only a subset of type II membrane proteins. Finally, ALP maturation kinetics in vac8Δ and vac17Δ cells suggests that vacuole inheritance is important for rapid generation of proteolytically active vacuolar compartments in daughter cells. We propose that the cargo-selective nature of the AP-3 pathway in yeast is achieved by AP-3 and Yck3p functioning in concert with machinery shared by other vacuolar transport pathways.

scholarly journals Specialized Rap1p/Gcr1p Transcriptional Activation Through Gcr1p DNA Contacts Requires Gcr2p, as Does Hyperphosphorylation of Gcr1p

Genetics ◽  
1997 ◽  
Vol 147 (2) ◽  
pp. 493-505 ◽  
Author(s):  
Xiao Zeng ◽  
Stephen J Deminoff ◽  
George M Santangelo
Keyword(s):  
Transcriptional Activation ◽  
Growth Defect ◽  
Loop Formation ◽  
Dna Loop ◽  
Genome Wide ◽  
A Genome ◽  
Eukaryotic Gene ◽  
Dna Elements ◽  
Eukaryotic Gene Regulation ◽  
Hyperphosphorylated Form

The multifunctional regulatory factor Rap1p of Saccharomyces cerevisiae accomplishes one of its tasks, transcriptional activation, by complexing with Gcr1p. An unusual feature of this heteromeric complex is its apparent capacity to contact simultaneously two adjacent DNA elements (UASRPG and the CT box, bound specifically by Rap1p and Gcr1p, respectively). The complex can activate transcription through isolated UASRPG but not CT elements. In promoters that contain both DNA signals its activity is enhanced, provided the helical spacing between the two elements is appropriate; this suggests that at least transient DNA loop formation is involved. We show here that this CT box-dependent augmentation of Rap1p/Gcr1p activation requires the presence of a third protein Gcr2p; the Gcr2– growth defect appears to result from a genome-wide loss of the CT box effect. Interestingly, a hyperphosphorylated form of Gcr1p disappears in Δgcr2 cells but reappears if they harbor a doubly point-mutated GCR1 allele that bypasses the Gcr2– growth defect. Gcr2p therefore appears to induce a conformation change in Gcr1p and/or stimulate its hyperphosphorylation; one or both of these effects can be mimicked in the absence of GCR2 by mutation of GCR1. This improved view of Rap1p/Gcr1p/Gcr2p function reveals a new aspect of eukaryotic gene regulation: modification of an upstream activator, accompanied by at least transient DNA loop formation, mediates its improved capacity to activate transcription.

Establishment of a genome-wide RNAi screen; Identification of novel genes involved in clonal maintenance of ALL

2014 ◽  
Vol 226 (03) ◽  
Author(s):  
F Ponthan ◽  
D Pal ◽  
J Vormoor ◽  
O Heidenreich
Keyword(s):  
Rnai Screen ◽  
Novel Genes ◽  
Genome Wide ◽  
A Genome

A genome-wide linkage scan for familial partial lipodystrophy susceptibility genes in a German kindreds

2007 ◽  
Vol 30 (4) ◽  
pp. 86
Author(s):  
M. Lanktree ◽  
J. Robinson ◽  
J. Creider ◽  
H. Cao ◽  
D. Carter ◽  
...  
Keyword(s):  
Subcutaneous Fat ◽  
Visceral Obesity ◽  
Fat Distribution ◽  
Dominant Negative ◽  
Molecular Forms ◽  
Partial Lipodystrophy ◽  
Genome Wide ◽  
A Genome ◽  
Familial Partial Lipodystrophy ◽  
Promoter Mutations

Background: In Dunnigan-type familial partial lipodystrophy (FPLD) patients are born with normal fat distribution, but subcutaneous fat from extremities and gluteal regions are lost during puberty. The abnormal fat distribution leads to the development of metabolic syndrome (MetS), a cluster of phenotypes including hyperglycemia, dyslipidemia, hypertension, and visceral obesity. The study of FPLD as a monogenic model of MetS may uncover genetic risk factors of the common MetS which affects ~30% of adult North Americans. Two molecular forms of FPLD have been identified including FPLD2, resulting from heterozygous mutations in the LMNA gene, and FPLD3, resulting from both heterozygous dominant negative and haploinsufficiency mutations in the PPARG gene. However, many patients with clinically diagnosed FPLD have no mutation in either LMNA or PPARG, suggesting the involvement of additional genes in FPLD etiology. Methods: Here, we report the results of an Affymetrix 10K GeneChip microarray genome-wide linkage analysis study of a German kindred displaying the FPLD phenotype and no known lipodystrophy-causing mutations. Results: The investigation identified three chromosomal loci, namely 1q, 3p, and 9q, with non-parametric logarithm of odds (NPL) scores >2.7. While not meeting the criteria for genome-wide significance, it is interesting to note that the 1q and 3p peaks contain the LMNA and PPARG genes respectively. Conclusions: Three possible conclusions can be drawn from these results: 1) the peaks identified are spurious findings, 2) additional genes physically close to LMNA, PPARG, or within 9q, are involved in FPLD etiology, or 3) alternative disease causing mechanisms not identified by standard exon sequencing approaches, such as promoter mutations, alternative splicing, or epigenetics, are also responsible for FPLD.

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