scholarly journals Computational prediction of CRISPR-impaired non-coding regulatory regions

2020 ◽  
Author(s):  
Nina Baumgarten ◽  
Florian Schmidt ◽  
Martin Wegner ◽  
Marie Hebel ◽  
Manuel Kaulich ◽  
...  
Keyword(s):  
Computational Prediction ◽  
Regulatory Elements ◽  
Cell Type ◽  
Coding Regions ◽  
Gene Coding ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen ◽  
Genomic Regions ◽  
Made In

AbstractGenome-wide CRISPR screens are becoming more widespread and allow the simultaneous interrogation of thousands of genomic regions. Although recent progress has been made in the analysis of CRISPR screens, it is still an open problem how to interpret CRISPR mutations in non-coding regions of the genome. Most of the tools concentrate on the interpretation of mutations introduced in gene coding regions. We introduce a computational pipeline that uses epigenomic information about regulatory elements for the interpretation of CRISPR mutations in non-coding regions. We illustrate our approach on the analysis of a genome-wide CRISPR screen in hTERT-RPE-1 cells and reveal novel regulatory elements that mediate chemoresistance against doxorubicin in these cells. We infer links to established and to novel chemoresistance genes. Our approach is general and can be applied on any cell type and with different CRISPR enzymes.

scholarly journals Computational prediction of CRISPR-impaired non-coding regulatory regions

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Nina Baumgarten ◽  
Florian Schmidt ◽  
Martin Wegner ◽  
Marie Hebel ◽  
Manuel Kaulich ◽  
...  
Keyword(s):  
Computational Prediction ◽  
Regulatory Elements ◽  
Coding Regions ◽  
Gene Coding ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen ◽  
Genomic Regions ◽  
Analysis Protocol ◽  
Made In

Abstract Genome-wide CRISPR screens are becoming more widespread and allow the simultaneous interrogation of thousands of genomic regions. Although recent progress has been made in the analysis of CRISPR screens, it is still an open problem how to interpret CRISPR mutations in non-coding regions of the genome. Most of the tools concentrate on the interpretation of mutations introduced in gene coding regions. We introduce a computational pipeline that uses epigenomic information about regulatory elements for the interpretation of CRISPR mutations in non-coding regions. We illustrate our analysis protocol on the analysis of a genome-wide CRISPR screen in hTERT-RPE1 cells and reveal novel regulatory elements that mediate chemoresistance against doxorubicin in these cells. We infer links to established and to novel chemoresistance genes. Our analysis protocol is general and can be applied on any cell type and with different CRISPR enzymes.

scholarly journals Genome-Wide CRISPR Screen Reveals Autophagy Disruption as the Convergence Mechanism That Regulates the NRF2 Transcription Factor

2019 ◽  
Vol 39 (13) ◽  
Author(s):  
Michael J. Kerins ◽  
Pengfei Liu ◽  
Wang Tian ◽  
William Mannheim ◽  
Donna D. Zhang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Negative Regulator ◽  
Cell Type ◽  
Genome Wide ◽  
A Genome ◽  
Nrf2 Activation ◽  
Crispr Screen ◽  
Short Palindromic Repeat ◽  
Intimate Link ◽  
Convergence Mechanism

ABSTRACT The nuclear factor (erythroid 2)-like 2 (NRF2 or NFE2L2) transcription factor regulates the expression of many genes that are critical in maintaining cellular homeostasis. Its deregulation has been implicated in many diseases, including cancer and metabolic and neurodegenerative diseases. While several mechanisms by which NRF2 can be activated have gradually been identified over time, a more complete regulatory network of NRF2 is still lacking. Here we show through a genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screen that a total of 273 genes, when knocked out, will lead to sustained NRF2 activation. Pathway analysis revealed a significant overrepresentation of genes (18 of the 273 genes) involved in autophagy. Molecular validation of a subset of the enriched genes identified 8 high-confidence genes that negatively regulate NRF2 activity irrespective of cell type: ATG12, ATG7, GOSR1, IFT172, NRXN2, RAB6A, VPS37A, and the well-known negative regulator of NRF2, KEAP1. Of these, ATG12, ATG7, KEAP1, and VPS37A are known to be involved in autophagic processes. Our results present a comprehensive list of NRF2 negative regulators and reveal an intimate link between autophagy and NRF2 regulation.

scholarly journals Identification of genomic regions affecting grain peroxidase activity in bread wheat using genome-wide association study

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Zhengfu Zhou ◽  
Huiyue Guan ◽  
Congcong Liu ◽  
Ziwei Zhang ◽  
Shenghui Geng ◽  
...  
Keyword(s):  
Association Study ◽  
Candidate Gene ◽  
Genome Wide Association Study ◽  
Significant Snps ◽  
Regulatory Elements ◽  
Genome Wide Association ◽  
Genetic Loci ◽  
Genome Wide ◽  
A Genome ◽  
Genomic Regions

Abstract Background Peroxidase (POD) activity plays an important role in flour-based product quality, which is mainly associated with browning and bleaching effects of flour. Here, we performed a genome-wide association study (GWAS) on POD activity using an association population consisted with 207 wheat world-wide collected varieties. Our study also provide basis for the genetic improvement of flour color-based quality in wheat. Results Twenty quantitative trait loci (QTLs) were detected associated with POD activity, explaining 5.59–12.67% of phenotypic variation. Superior alleles were positively correlated with POD activity. In addition, two SNPs were successfully developed to KASP (Kompetitive Allele-Specific PCR) markers. Two POD genes, TraesCS2B02G615700 and TraesCS2D02G583000, were aligned near the QTLs flanking genomic regions, but only TraesCS2D02G583000 displayed significant divergent expression levels (P < 0.001) between high and low POD activity varieties in the investigated association population. Therefore, it was deduced to be a candidate gene. The expression level of TraesCS2D02G583000 was assigned as a phenotype for expression GWAS (eGWAS) to screen regulatory elements. In total, 505 significant SNPs on 20 chromosomes (excluding 4D) were detected, and 9 of them located within 1 Mb interval of TraesCS2D02G583000. Conclusions To identify genetic loci affecting POD activity in wheat grain, we conducted GWAS on POD activity and the candidate gene TraesCS2D02G583000 expression. Finally, 20 QTLs were detected for POD activity, whereas two QTLs associated SNPs were converted to KASP markers that could be used for marker-assisted breeding. Both cis- and trans-acting elements were revealed by eGWAS of TraesCS2D02G583000 expression. The present study provides genetic loci for improving POD activity across wide genetic backgrounds and largely improved the selection efficiency for breeding in wheat.

scholarly journals Fruitless decommissions regulatory elements to implement cell-type-specific neuronal masculinization

2020 ◽  
Author(s):  
Margarita V. Brovkina ◽  
Rachel Duffié ◽  
Abbigayl E. C. Burtis ◽  
E. Josephine Clowney
Keyword(s):  
Molecular Mechanisms ◽  
Fruit Fly ◽  
Cell Types ◽  
Regulatory Elements ◽  
Cell Type ◽  
A Genome ◽  
Bona Fide ◽  
Candidate Regions ◽  
Male Specific ◽  
Genomic Regions

AbstractIn the fruit fly Drosophila melanogaster, male-specific splicing and translation of the Fruitless transcription factor (FruM) alters the presence, anatomy, and/or connectivity of >60 types of central brain neurons that interconnect to generate male-typical behaviors. While the indispensable function of FruM in sex-specific behavior has been understood for decades, the molecular mechanisms underlying its activity remain unknown. Here, we take a genome-wide, brain-wide approach to identifying regulatory elements whose activity depends on the presence of FruM. We identify 436 high-confidence genomic regions differentially accessible in male fruitless neurons, validate candidate regions as bona-fide, differentially regulated enhancers, and describe the particular cell types in which these enhancers are active. We find that individual enhancers are not activated universally but are dedicated to specific fru+ cell types. Aside from fru itself, genes are not dedicated to or common across the fru circuit; rather, FruM appears to masculinize each cell type differently, by tweaking expression of the same effector genes used in other circuits. Finally, we find FruM motifs enriched among regulatory elements that are open in the female but closed in the male. Together, these results suggest that FruM acts cell-type-specifically to decommission regulatory elements in male fruitless neurons.

scholarly journals Fruitless decommissions regulatory elements to implement cell-type-specific neuronal masculinization

PLoS Genetics ◽  
2021 ◽  
Vol 17 (2) ◽  
pp. e1009338
Author(s):  
Margarita V. Brovkina ◽  
Rachel Duffié ◽  
Abbigayl E. C. Burtis ◽  
E. Josephine Clowney
Keyword(s):  
Molecular Mechanisms ◽  
Fruit Fly ◽  
Cell Types ◽  
Regulatory Elements ◽  
Cell Type ◽  
A Genome ◽  
Bona Fide ◽  
Candidate Regions ◽  
Male Specific ◽  
Genomic Regions

In the fruit fly Drosophila melanogaster, male-specific splicing and translation of the Fruitless transcription factor (FruM) alters the presence, anatomy, and/or connectivity of >60 types of central brain neurons that interconnect to generate male-typical behaviors. While the indispensable function of FruM in sex-specific behavior has been understood for decades, the molecular mechanisms underlying its activity remain unknown. Here, we take a genome-wide, brain-wide approach to identifying regulatory elements whose activity depends on the presence of FruM. We identify 436 high-confidence genomic regions differentially accessible in male fruitless neurons, validate candidate regions as bona-fide, differentially regulated enhancers, and describe the particular cell types in which these enhancers are active. We find that individual enhancers are not activated universally but are dedicated to specific fru+ cell types. Aside from fru itself, genes are not dedicated to or common across the fru circuit; rather, FruM appears to masculinize each cell type differently, by tweaking expression of the same effector genes used in other circuits. Finally, we find FruM motifs enriched among regulatory elements that are open in the female but closed in the male. Together, these results suggest that FruM acts cell-type-specifically to decommission regulatory elements in male fruitless neurons.

scholarly journals Genome-wide screens identify Toxoplasma gondii determinants of parasite fitness in IFNγ-activated murine macrophages

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Yifan Wang ◽  
Lamba Omar Sangaré ◽  
Tatiana C. Paredes-Santos ◽  
Musa A. Hassan ◽  
Shruthi Krishnamurthy ◽  
...  
Keyword(s):  
Dense Granule ◽  
Cell Type ◽  
Murine Macrophages ◽  
Parasite Fitness ◽  
Early Immune Response ◽  
Host Cytoplasm ◽  
Genome Wide ◽  
A Genome ◽  
Crispr Screen

Abstract Macrophages play an essential role in the early immune response against Toxoplasma and are the cell type preferentially infected by the parasite in vivo. Interferon gamma (IFNγ) elicits a variety of anti-Toxoplasma activities in macrophages. Using a genome-wide CRISPR screen we identify 353 Toxoplasma genes that determine parasite fitness in naїve or IFNγ-activated murine macrophages, seven of which are further confirmed. We show that one of these genes encodes dense granule protein GRA45, which has a chaperone-like domain, is critical for correct localization of GRAs into the PVM and secretion of GRA effectors into the host cytoplasm. Parasites lacking GRA45 are more susceptible to IFNγ-mediated growth inhibition and have reduced virulence in mice. Together, we identify and characterize an important chaperone-like GRA in Toxoplasma and provide a resource for the community to further explore the function of Toxoplasma genes that determine fitness in IFNγ-activated macrophages.

scholarly journals Assessing genome-wide dynamic changes in enhancer activity during early mESC differentiation by FAIRE-STARR-seq

2021 ◽  
Author(s):  
Laura V Glaser ◽  
Mara Steiger ◽  
Alisa Fuchs ◽  
Alena van Boemmel ◽  
Edda Einfeldt ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Embryonic Stem ◽  
Computational Prediction ◽  
Regulatory Elements ◽  
Activity Levels ◽  
Sequence Motifs ◽  
Enhancer Activity ◽  
Early Stages ◽  
Genome Wide ◽  
A Genome

Embryonic stem cells (ESCs) can differentiate into any given cell type and therefore represent a versatile model to study the link between gene regulation and differentiation. To quantitatively assess the dynamics of enhancer activity during the early stages of murine ESC differentiation, we analyzed accessible genomic regions using STARR-seq, a massively parallel reporter assay. This resulted in a genome-wide quantitative map of active mESC enhancers, in pluripotency and during the early stages of differentiation. We find that only a minority of accessible regions is active and that such regions are enriched near promoters, characterized by specific chromatin marks, enriched for distinct sequence motifs, and modeling shows that active regions can be predicted from sequence alone. Regions that change their activity upon retinoic acid-induced differentiation are more prevalent at distal intergenic regions when compared to constitutively active enhancers. Further, analysis of differentially active enhancers verified the contribution of individual TF motifs toward activity and inducibility as well as their role in regulating endogenous genes. Notably, the activity of retinoic acid receptor alpha (RARα) occupied regions can either increase or decrease upon the addition of its ligand, retinoic acid, with the direction of the change correlating with spacing and orientation of the RARα consensus motif and the co-occurrence of additional sequence motifs. Together, our genome-wide enhancer activity map elucidates features associated with enhancer activity levels, identifies regulatory regions disregarded by computational prediction tools, and provides a resource for future studies into regulatory elements in mESCs.

scholarly journals A genome-wide map of regulatory elements in zebrafish

Lab Animal ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 17-17
Author(s):  
Alexandra Le Bras
Keyword(s):  
Regulatory Elements ◽  
Genome Wide ◽  
A Genome

scholarly journals Epigenome-Wide Study Identified Methylation Sites Associated with the Risk of Obesity

Nutrients ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1984
Author(s):  
Majid Nikpay ◽  
Sepehr Ravati ◽  
Robert Dent ◽  
Ruth McPherson
Keyword(s):  
Quantitative Trait Locus ◽  
Quantitative Trait ◽  
Rare Variants ◽  
Genome Wide ◽  
A Genome ◽  
Genome Wide Search ◽  
Risk Snps ◽  
Trait Locus ◽  
Genomic Regions ◽  
Eqtl Data

Here, we performed a genome-wide search for methylation sites that contribute to the risk of obesity. We integrated methylation quantitative trait locus (mQTL) data with BMI GWAS information through a SNP-based multiomics approach to identify genomic regions where mQTLs for a methylation site co-localize with obesity risk SNPs. We then tested whether the identified site contributed to BMI through Mendelian randomization. We identified multiple methylation sites causally contributing to the risk of obesity. We validated these findings through a replication stage. By integrating expression quantitative trait locus (eQTL) data, we noted that lower methylation at cg21178254 site upstream of CCNL1 contributes to obesity by increasing the expression of this gene. Higher methylation at cg02814054 increases the risk of obesity by lowering the expression of MAST3, whereas lower methylation at cg06028605 contributes to obesity by decreasing the expression of SLC5A11. Finally, we noted that rare variants within 2p23.3 impact obesity by making the cg01884057 site more susceptible to methylation, which consequently lowers the expression of POMC, ADCY3 and DNAJC27. In this study, we identify methylation sites associated with the risk of obesity and reveal the mechanism whereby a number of these sites exert their effects. This study provides a framework to perform an omics-wide association study for a phenotype and to understand the mechanism whereby a rare variant causes a disease.

scholarly journals A Genome-Wide Analysis of Pathogenesis-Related Protein-1 (PR-1) Genes from Piper nigrum Reveals Its Critical Role during Phytophthora capsici Infection

Genes ◽  
2021 ◽  
Vol 12 (7) ◽  
pp. 1007
Author(s):  
Divya Kattupalli ◽  
Asha Sreenivasan ◽  
Eppurathu Vasudevan Soniya
Keyword(s):  
Phytophthora Capsici ◽  
Regulatory Elements ◽  
Piper Nigrum ◽  
Binding Motif ◽  
Altered Expression ◽  
Genome Wide ◽  
A Genome ◽  
Pathogenesis Related Protein ◽  
Pathogenesis Related

Black pepper (Piper nigrum L.) is a prominent spice that is an indispensable ingredient in cuisine and traditional medicine. Phytophthora capsici, the causative agent of footrot disease, causes a drastic constraint in P. nigrum cultivation and productivity. To counterattack various biotic and abiotic stresses, plants employ a broad array of mechanisms that includes the accumulation of pathogenesis-related (PR) proteins. Through a genome-wide survey, eleven PR-1 genes that belong to a CAP superfamily protein with a caveolin-binding motif (CBM) and a CAP-derived peptide (CAPE) were identified from P. nigrum. Despite the critical functional domains, PnPR-1 homologs differ in their signal peptide motifs and core amino acid composition in the functional protein domains. The conserved motifs of PnPR-1 proteins were identified using MEME. Most of the PnPR-1 proteins were basic in nature. Secondary and 3D structure analyses of the PnPR-1 proteins were also predicted, which may be linked to a functional role in P. nigrum. The GO and KEGG functional annotations predicted their function in the defense responses of plant-pathogen interactions. Furthermore, a transcriptome-assisted FPKM analysis revealed PnPR-1 genes mapped to the P. nigrum-P. capsici interaction pathway. An altered expression pattern was detected for PnPR-1 transcripts among which a significant upregulation was noted for basic PnPR-1 genes such as CL10113.C1 and Unigene17664. The drastic variation in the transcript levels of CL10113.C1 was further validated through qRT-PCR and it showed a significant upregulation in infected leaf samples compared with the control. A subsequent analysis revealed the structural details, phylogenetic relationships, conserved sequence motifs and critical cis-regulatory elements of PnPR-1 genes. This is the first genome-wide study that identified the role of PR-1 genes during P. nigrum-P. capsici interactions. The detailed in silico experimental analysis revealed the vital role of PnPR-1 genes in regulating the first layer of defense towards a P. capsici infection in Panniyur-1 plants.

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