Exploiting a PAX3-FOXO1-induced synthetic lethal ATR dependency for rhabdomyosarcoma therapy

Pathognomonic PAX3-FOXO1 fusion oncogene expression is associated with poor outcome in rhabdomyosarcoma. Combining genome-wide CRISPR screening with cell-based functional genetic approaches, we here provide evidence that PAX3-FOXO1 induces replication stress, resulting in a synthetic lethal dependency to ATR-mediated DNA damage-response signaling in rhabdomyosarcoma. Expression of PAX3-FOXO1 in muscle progenitor cells was not only sufficient to induce hypersensitivity to ATR inhibition, but PAX3-FOXO1-expressing rhabdomyosarcoma cells also exhibited increased sensitivity to structurally diverse inhibitors of ATR, a dependency that could be validated genetically. Mechanistically, ATR inhibition led to replication stress exacerbation, decreased BRCA1 phosphorylation and reduced homologous recombination-mediated DNA repair pathway activity. Consequently, ATR inhibitor treatment increased sensitivity of rhabdomyosarcoma cells to PARP inhibition in vitro, and combined ATR and PARP inhibition induced regression of primary patient-derived alveolar rhabdomyosarcoma xenografts in vivo. Moreover, a genome-wide CRISPR activation screen (CRISPRa) identified FOS gene family members as inducers of resistance against ATR inhibitors. Mechanistically, FOS gene family members reduced replication stress in rhabdomyosarcoma cells. Lastly, compassionate use of ATR inhibitors in two pediatric patients suffering from relapsed PAX3-FOXO1-expressing alveolar rhabdomyosarcoma showed signs of tolerability, paving the way to clinically exploit this novel synthetic lethal dependency in rhabdomyosarcoma.

Download Full-text

A Comprehensive Survey on the Terpene Synthase Gene Family Provides New Insight into Its Evolutionary Patterns

Genome Biology and Evolution ◽

10.1093/gbe/evz142 ◽

2019 ◽

Vol 11 (8) ◽

pp. 2078-2098 ◽

Cited By ~ 8

Author(s):

Shu-Ye Jiang ◽

Jingjing Jin ◽

Rajani Sarojam ◽

Srinivasan Ramachandran

Keyword(s):

Gene Family ◽

Large Scale ◽

Family Members ◽

Terpene Synthase ◽

Limited Information ◽

Terpene Synthases ◽

Genome Wide ◽

A Genome ◽

Family Expansion ◽

Insight Into

Abstract Terpenes are organic compounds and play important roles in plant growth and development as well as in mediating interactions of plants with the environment. Terpene synthases (TPSs) are the key enzymes responsible for the biosynthesis of terpenes. Although some species were employed for the genome-wide identification and characterization of the TPS family, limited information is available regarding the evolution, expansion, and retention mechanisms occurring in this gene family. We performed a genome-wide identification of the TPS family members in 50 sequenced genomes. Additionally, we also characterized the TPS family from aromatic spearmint and basil plants using RNA-Seq data. No TPSs were identified in algae genomes but the remaining plant species encoded various numbers of the family members ranging from 2 to 79 full-length TPSs. Some species showed lineage-specific expansion of certain subfamilies, which might have contributed toward species or ecotype divergence or environmental adaptation. A large-scale family expansion was observed mainly in dicot and monocot plants, which was accompanied by frequent domain loss. Both tandem and segmental duplication significantly contributed toward family expansion and expression divergence and played important roles in the survival of these expanded genes. Our data provide new insight into the TPS family expansion and evolution and suggest that TPSs might have originated from isoprenyl diphosphate synthase genes.

Download Full-text

Inhibition of the ubiquitin-proteasome system by an NQO1-activatable compound

Cell Death and Disease ◽

10.1038/s41419-021-04191-9 ◽

2021 ◽

Vol 12 (10) ◽

Author(s):

Tatiana A. Giovannucci ◽

Florian A. Salomons ◽

Martin Haraldsson ◽

Lotta H. M. Elfman ◽

Malin Wickström ◽

...

Keyword(s):

Ubiquitin Proteasome System ◽

Malignant Cells ◽

Genome Wide ◽

A Genome ◽

Ubiquitin Proteasome ◽

Dependent Cell ◽

Increased Sensitivity ◽

The Impact

AbstractMalignant cells display an increased sensitivity towards drugs that reduce the function of the ubiquitin-proteasome system (UPS), which is the primary proteolytic system for destruction of aberrant proteins. Here, we report on the discovery of the bioactivatable compound CBK77, which causes an irreversible collapse of the UPS, accompanied by a general accumulation of ubiquitylated proteins and caspase-dependent cell death. CBK77 caused accumulation of ubiquitin-dependent, but not ubiquitin-independent, reporter substrates of the UPS, suggesting a selective effect on ubiquitin-dependent proteolysis. In a genome-wide CRISPR interference screen, we identified the redox enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) as a critical mediator of CBK77 activity, and further demonstrated its role as the compound bioactivator. Through affinity-based proteomics, we found that CBK77 covalently interacts with ubiquitin. In vitro experiments showed that CBK77-treated ubiquitin conjugates were less susceptible to disassembly by deubiquitylating enzymes. In vivo efficacy of CBK77 was validated by reduced growth of NQO1-proficient human adenocarcinoma cells in nude mice treated with CBK77. This first-in-class NQO1-activatable UPS inhibitor suggests that it may be possible to exploit the intracellular environment in malignant cells for leveraging the impact of compounds that impair the UPS.

Download Full-text

Genome-Wide Identification and Analysis of the Phosphoenolpyruvate Carboxylase Gene Family in Suaeda aralocaspica, an Annual Halophyte With Single-Cellular C4 Anatomy

Frontiers in Plant Science ◽

10.3389/fpls.2021.665279 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jing Cao ◽

Gang Cheng ◽

Lu Wang ◽

Tayier Maimaitijiang ◽

Haiyan Lan

Keyword(s):

Gene Family ◽

Phosphoenolpyruvate Carboxylase ◽

Abiotic Stresses ◽

Expression Patterns ◽

Substrate Affinity ◽

Photosynthetic Pathway ◽

Genome Wide ◽

A Genome ◽

Chlorenchyma Cell

Phosphoenolpyruvate carboxylase (PEPC) plays pivotal roles in the carbon fixation of photosynthesis and a variety of metabolic and stress pathways. Suaeda aralocaspica belongs to a single-cellular C4 species and carries out a photosynthetic pathway in an unusually elongated chlorenchyma cell, which is expected to have PEPCs with different characteristics. To identify the different isoforms of PEPC genes in S. aralocaspica and comparatively analyze their expression and regulation patterns as well as the biochemical and enzymatic properties in this study, we characterized a bacterial-type PEPC (BTPC; SaPEPC-4) in addition to the two plant-type PEPCs (PTPCs; SaPEPC-1 and SaPEPC-2) using a genome-wide identification. SaPEPC-4 presented a lower expression level in all test combinations with an unknown function; two SaPTPCs showed distinct subcellular localizations and different spatiotemporal expression patterns but positively responded to abiotic stresses. Compared to SaPEPC-2, the expression of SaPEPC-1 specifically in chlorenchyma cell tissues was much more active with the progression of development and under various stresses, particularly sensitive to light, implying the involvement of SaPEPC-1 in a C4 photosynthetic pathway. In contrast, SaPEPC-2 was more like a non-photosynthetic PEPC. The expression trends of two SaPTPCs in response to light, development, and abiotic stresses were also matched with the changes in PEPC activity in vivo (native) or in vitro (recombinant), and the biochemical properties of the two recombinant SaPTPCs were similar in response to various effectors while the catalytic efficiency, substrate affinity, and enzyme activity of SaPEPC-2 were higher than that of SaPEPC-1 in vitro. All the different properties between these two SaPTPCs might be involved in transcriptional (e.g., specific cis-elements), posttranscriptional [e.g., 5′-untranslated region (5′-UTR) secondary structure], or translational (e.g., PEPC phosphorylation/dephosphorylation) regulatory events. The comparative studies on the different isoforms of the PEPC gene family in S. aralocaspica may help to decipher their exact role in C4 photosynthesis, plant growth/development, and stress resistance.

Download Full-text

Inhibition of the ubiquitin-proteasome system by a bioactivatable compound

10.1101/2021.03.16.435589 ◽

2021 ◽

Author(s):

Tatiana A. Giovannucci ◽

Florian A. Salomons ◽

Martin Haraldsson ◽

Lotta H. M. Elfman ◽

Malin Wickström ◽

...

Keyword(s):

Ubiquitin Proteasome System ◽

Malignant Cells ◽

Genome Wide ◽

A Genome ◽

Ubiquitin Proteasome ◽

Dependent Cell ◽

Increased Sensitivity ◽

The Impact

SummaryMalignant cells display an increased sensitivity towards drugs that reduce the function of the ubiquitin-proteasome system (UPS), which is the primary proteolytic system for destruction of aberrant proteins. Here, we report on the discovery of the bioactivatable compound CBK77, which causes an irreversible collapse of the UPS, accompanied by a general accumulation of ubiquitylated proteins and caspase-dependent cell death. CBK77 caused accumulation of ubiquitin-dependent, but not ubiquitin-independent, reporter substrates of the UPS, suggesting a selective effect on ubiquitin-dependent proteolysis. In a genome-wide CRISPR interference screen, we identified the redox enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) as a critical mediator of CBK77 activity, and further demonstrated its role as the compound bioactivator. Through affinity-based proteomics, we found that CBK77 covalently interacts with ubiquitin. In vitro experiments showed that CBK77-treated ubiquitin conjugates were less susceptible to disassembly by deubiquitylating enzymes. In vivo efficacy of CBK77 was validated by reduced growth of NQO1-proficient human adenocarcinoma cells in nude mice treated with CBK77. This first-in-class NQO1-activatable UPS inhibitor suggests that it may be possible to exploit the intracellular environment in malignant cells for leveraging the impact of compounds that impair the UPS.

Download Full-text

Genome-wide identification of cyclin-dependent kinase (CDK) genes affecting adipocyte differentiation in cattle

BMC Genomics ◽

10.1186/s12864-021-07653-8 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Cuili Pan ◽

Zhaoxiong Lei ◽

Shuzhe Wang ◽

Xingping Wang ◽

Dawei Wei ◽

...

Keyword(s):

Gene Family ◽

Expression Analysis ◽

Adipocyte Differentiation ◽

Bos Taurus ◽

Adipogenic Differentiation ◽

Critical Role ◽

Bos Indicus ◽

Specific Expression ◽

Genome Wide ◽

A Genome

Abstract Background Cyclin-dependent kinases (CDKs) are protein kinases regulating important cellular processes such as cell cycle and transcription. Many CDK genes also play a critical role during adipogenic differentiation, but the role of CDK gene family in regulating bovine adipocyte differentiation has not been studied. Therefore, the present study aims to characterize the CDK gene family in bovine and study their expression pattern during adipocyte differentiation. Results We performed a genome-wide analysis and identified a number of CDK genes in several bovine species. The CDK genes were classified into 8 subfamilies through phylogenetic analysis. We found that 25 bovine CDK genes were distributed in 16 different chromosomes. Collinearity analysis revealed that the CDK gene family in Bos taurus is homologous with Bos indicus, Hybrid-Bos taurus, Hybrid Bos indicus, Bos grunniens and Bubalus bubalis. Several CDK genes had higher expression levels in preadipocytes than in differentiated adipocytes, as shown by RNA-seq analysis and qPCR, suggesting a role in the growth of emerging lipid droplets. Conclusion In this research, 185 CDK genes were identified and grouped into eight distinct clades in Bovidae, showing extensively homology. Global expression analysis of different bovine tissues and specific expression analysis during adipocytes differentiation revealed CDK4, CDK7, CDK8, CDK9 and CDK14 may be involved in bovine adipocyte differentiation. The results provide a basis for further study to determine the roles of CDK gene family in regulating adipocyte differentiation, which is beneficial for beef quality improvement.

Download Full-text

Genome-wide identification and expression analysis of the Brassica oleracea L. chitin-binding genes and response to pathogens infections

Planta ◽

10.1007/s00425-021-03596-2 ◽

2021 ◽

Vol 253 (4) ◽

Author(s):

Mingzhao Zhu ◽

Shujin Lu ◽

Mu Zhuang ◽

Yangyong Zhang ◽

Honghao Lv ◽

...

Keyword(s):

Powdery Mildew ◽

Gene Family ◽

Brassica Oleracea ◽

Downy Mildew ◽

Fusarium Wilt ◽

Black Spot ◽

Housekeeping Genes ◽

Chitinase Gene ◽

Genome Wide ◽

A Genome

Abstract Main conclusion Chitinase family genes were involved in the response of Brassica oleracea to Fusarium wilt, powdery mildew, black spot and downy mildew. Abstract Abstract Chitinase, a category of pathogenesis-related proteins, is believed to play an important role in defending against external stress in plants. However, a comprehensive analysis of the chitin-binding gene family has not been reported to date in cabbage (Brassica oleracea L.), especially regarding the roles that chitinases play in response to various diseases. In this study, a total of 20 chitinase genes were identified using a genome-wide search method. Phylogenetic analysis was employed to classify these genes into two groups. The genes were distributed unevenly across six chromosomes in cabbage, and all of them contained few introns (≤ 2). The results of collinear analysis showed that the cabbage genome contained 1–5 copies of each chitinase gene (excluding Bol035470) identified in Arabidopsis. The heatmap of the chitinase gene family showed that these genes were expressed in various tissues and organs. Two genes (Bol023322 and Bol041024) were relatively highly expressed in all of the investigated tissues under normal conditions, exhibiting the expression characteristics of housekeeping genes. In addition, under four different stresses, namely, Fusarium wilt, powdery mildew, black spot and downy mildew, we detected 9, 5, 8 and 8 genes with different expression levels in different treatments, respectively. Our results may help to elucidate the roles played by chitinases in the responses of host plants to various diseases.

Download Full-text

Genome- Wide Identification, Structure Characterization and Expression Pattern Profiling of the Aquaporin Gene Family in Betula pendula

International Journal of Molecular Sciences ◽

10.3390/ijms22147269 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7269

Author(s):

Jean-Stéphane Venisse ◽

Eele Õunapuu-Pikas ◽

Maxime Dupont ◽

Aurélie Gousset-Dupont ◽

Mouadh Saadaoui ◽

...

Keyword(s):

Cold Stress ◽

Gene Family ◽

Betula Pendula ◽

Large Family ◽

Biochemical Properties ◽

Silver Birch ◽

Structure Characterization ◽

Transcriptional Expression ◽

Genome Wide ◽

A Genome

Aquaporin water channels (AQPs) constitute a large family of transmembrane proteins present throughout all kingdoms of life. They play key roles in the flux of water and many solutes across the membranes. The AQP diversity, protein features, and biological functions of silver birch are still unknown. A genome analysis of Betula pendula identified 33 putative genes encoding full-length AQP sequences (BpeAQPs). They are grouped into five subfamilies, representing ten plasma membrane intrinsic proteins (PIPs), eight tonoplast intrinsic proteins (TIPs), eight NOD26-like intrinsic proteins (NIPs), four X intrinsic proteins (XIPs), and three small basic intrinsic proteins (SIPs). The BpeAQP gene structure is conserved within each subfamily, with exon numbers ranging from one to five. The predictions of the aromatic/arginine selectivity filter (ar/R), Froger’s positions, specificity-determining positions, and 2D and 3D biochemical properties indicate noticeable transport specificities to various non-aqueous substrates between members and/or subfamilies. Nevertheless, overall, the BpePIPs display mostly hydrophilic ar/R selective filter and lining-pore residues, whereas the BpeTIP, BpeNIP, BpeSIP, and BpeXIP subfamilies mostly contain hydrophobic permeation signatures. Transcriptional expression analyses indicate that 23 BpeAQP genes are transcribed, including five organ-related expressions. Surprisingly, no significant transcriptional expression is monitored in leaves in response to cold stress (6 °C), although interesting trends can be distinguished and will be discussed, notably in relation to the plasticity of this pioneer species, B. pendula. The current study presents the first detailed genome-wide analysis of the AQP gene family in a Betulaceae species, and our results lay a foundation for a better understanding of the specific functions of the BpeAQP genes in the responses of the silver birch trees to cold stress.

Download Full-text

Genome-wide mapping of unexplored essential regions in the Saccharomyces cerevisiae genome: evidence for hidden synthetic lethal combinations in a genetic interaction network

Nucleic Acids Research ◽

10.1093/nar/gku576 ◽

2014 ◽

Vol 42 (15) ◽

pp. 9838-9853 ◽

Cited By ~ 6

Author(s):

Saeed Kaboli ◽

Takuya Yamakawa ◽

Keisuke Sunada ◽

Tao Takagaki ◽

Yu Sasano ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Genetic Interaction ◽

Interaction Network ◽

Essential Genes ◽

Genetic Interactions ◽

Lethal Gene ◽

Synthetic Lethal ◽

Genome Wide ◽

A Genome ◽

Wide Scale

Abstract Despite systematic approaches to mapping networks of genetic interactions in Saccharomyces cerevisiae, exploration of genetic interactions on a genome-wide scale has been limited. The S. cerevisiae haploid genome has 110 regions that are longer than 10 kb but harbor only non-essential genes. Here, we attempted to delete these regions by PCR-mediated chromosomal deletion technology (PCD), which enables chromosomal segments to be deleted by a one-step transformation. Thirty-three of the 110 regions could be deleted, but the remaining 77 regions could not. To determine whether the 77 undeletable regions are essential, we successfully converted 67 of them to mini-chromosomes marked with URA3 using PCR-mediated chromosome splitting technology and conducted a mitotic loss assay of the mini-chromosomes. Fifty-six of the 67 regions were found to be essential for cell growth, and 49 of these carried co-lethal gene pair(s) that were not previously been detected by synthetic genetic array analysis. This result implies that regions harboring only non-essential genes contain unidentified synthetic lethal combinations at an unexpectedly high frequency, revealing a novel landscape of genetic interactions in the S. cerevisiae genome. Furthermore, this study indicates that segmental deletion might be exploited for not only revealing genome function but also breeding stress-tolerant strains.

Download Full-text

Transcriptome analysis ofSchistosoma mansonilarval development using serial analysis of gene expression (SAGE)

Parasitology ◽

10.1017/s0031182009005733 ◽

2009 ◽

Vol 136 (5) ◽

pp. 469-485 ◽

Cited By ~ 22

Author(s):

A. S. TAFT ◽

J. J. VERMEIRE ◽

J. BERNIER ◽

S. R. BIRKELAND ◽

M. J. CIPRIANO ◽

...

Keyword(s):

Gene Expression ◽

Sequence Data ◽

Subsequent Development ◽

Differentially Expressed ◽

Cdna Libraries ◽

Genome Wide ◽

A Genome ◽

Genome Wide Expression ◽

Cell Conditioned Medium

SUMMARYInfection of the snail,Biomphalaria glabrata, by the free-swimming miracidial stage of the human blood fluke,Schistosoma mansoni, and its subsequent development to the parasitic sporocyst stage is critical to establishment of viable infections and continued human transmission. We performed a genome-wide expression analysis of theS. mansonimiracidia and developing sporocyst using Long Serial Analysis of Gene Expression (LongSAGE). Five cDNA libraries were constructed from miracidia andin vitrocultured 6- and 20-day-old sporocysts maintained in sporocyst medium (SM) or in SM conditioned by previous cultivation with cells of theB. glabrataembryonic (Bge) cell line. We generated 21 440 SAGE tags and mapped 13 381 to theS. mansonigene predictions (v4.0e) either by estimating theoretical 3′ UTR lengths or using existing 3′ EST sequence data. Overall, 432 transcripts were found to be differentially expressed amongst all 5 libraries. In total, 172 tags were differentially expressed between miracidia and 6-day conditioned sporocysts and 152 were differentially expressed between miracidia and 6-day unconditioned sporocysts. In addition, 53 and 45 tags, respectively, were differentially expressed in 6-day and 20-day cultured sporocysts, due to the effects of exposure to Bge cell-conditioned medium.

Download Full-text

Genome-wide identification and analysis of cystatin family genes in Sorghum (Sorghum bicolor (L.) Moench)

PeerJ ◽

10.7717/peerj.10617 ◽

2021 ◽

Vol 9 ◽

pp. e10617

Author(s):

Jie Li ◽

Xinhao Liu ◽

Qingmei Wang ◽

Junyan Sun ◽

Dexian He

Keyword(s):

Gene Family ◽

Seed Development ◽

Stress Responses ◽

Abiotic Stresses ◽

Expression Profiles ◽

Seed Formation ◽

Aphid Infestation ◽

Genome Wide ◽

A Genome ◽

Cystatin Family

To set a systematic study of the Sorghum cystatins (SbCys) gene family, a genome-wide analysis of the SbCys family genes was performed by bioinformatics-based methods. In total, 18 SbCys genes were identified in Sorghum, which were distributed unevenly on chromosomes, and two genes were involved in a tandem duplication event. All SbCys genes had similar exon/intron structure and motifs, indicating their high evolutionary conservation. Transcriptome analysis showed that 16 SbCys genes were expressed in different tissues, and most genes displayed higher expression levels in reproductive tissues than in vegetative tissues, indicating that the SbCys genes participated in the regulation of seed formation. Furthermore, the expression profiles of the SbCys genes revealed that seven cystatin family genes were induced during Bipolaris sorghicola infection and only two genes were responsive to aphid infestation. In addition, quantitative real-time polymerase chain reaction (qRT-PCR) confirmed that 17 SbCys genes were induced by one or two abiotic stresses (dehydration, salt, and ABA stresses). The interaction network indicated that SbCys proteins were associated with several biological processes, including seed development and stress responses. Notably, the expression of SbCys4 was up-regulated under biotic and abiotic stresses, suggesting its potential roles in mediating the responses of Sorghum to adverse environmental impact. Our results provide new insights into the structural and functional characteristics of the SbCys gene family, which lay the foundation for better understanding the roles and regulatory mechanism of Sorghum cystatins in seed development and responses to different stress conditions.

Download Full-text