Selective ploidy ablation, a high-throughput plasmid transfer protocol, identifies new genes affecting topoisomerase I–induced DNA damage

Reid, Robert J.D.; González-Barrera, Sergio; Šunjevarić, Ivana; Álvaro, David; Ciccone, Samantha; Wagner, Marisa; Rothstein, Rodney

doi:10.1101/gr.109033.110

Cited by 84 publications

(134 citation statements)

References 53 publications

Supporting

Mentioning

130

Contrasting

Order By: Relevance

“…We transferred the MTW1-GBP plasmid and, separately, the two controls, MTW1 and GBP, into the GFP collection of strains using selective ploidy ablation (SPA) (32) (Fig. S1A), which produces arrays of haploid GFPtagged strains, each containing one of the three plasmid constructs.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Synthetic physical interactions map kinetochore regulators and regions sensitive to constitutive Cdc14 localization

Ólafsson

Thorpe

2015

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The location of proteins within eukaryotic cells is often critical for their function and relocation of proteins forms the mainstay of regulatory pathways. To assess the importance of protein location to cellular homeostasis, we have developed a methodology to systematically create binary physical interactions between a query protein and most other members of the proteome. This method allows us to rapidly assess which of the thousands of possible protein interactions modify a phenotype. As proof of principle we studied the kinetochore, a multiprotein assembly that links centromeres to the microtubules of the spindle during cell division. In budding yeast, the kinetochores from the 16 chromosomes cluster together to a single location within the nucleus. The many proteins that make up the kinetochore are regulated through ubiquitylation and phosphorylation. By systematically associating members of the proteome to the kinetochore, we determine which fusions affect its normal function. We identify a number of candidate kinetochore regulators, including the phosphatase Cdc14. We examine where within the kinetochore Cdc14 can act and show that the effect is limited to regions that correlate with known phosphorylation sites, demonstrating the importance of serine phospho-regulation for normal kinetochore homeostasis.T he relocation of proteins between cellular compartments underlies many regulatory pathways. For example, protein relocation underpins most cell-signaling pathways (1) and the establishment of cell polarity and asymmetric cell division both require highly specialized relocation of proteins within the cell (2). Furthermore, the aberrant localization of proteins underlies the pathology of a number of diseases (3). However, our understanding of the effect of relocalizing members of the proteome is limited to specific studies typically concerning individual proteins, and only a select few studies have globally monitored protein relocation (4, 5).One highly localized structure within the cell is the kinetochore, which in budding yeast forms a single megadalton complex (6-8). The kinetochore attaches chromosomes to the spindle microtubules to drive accurate chromosome segregation. The kinetochore consists of at least 60 unique gene products present in multiple copies that stretch from the specialized H3 histone subunit (Cse4, CENP-A in mammals) to the microtubule binding proteins of the Dam1 complex (9). Kinetochores normally assemble hierarchically from the centromere-bound proteins (10). Once assembled, the structural homeostasis of the kinetochore is regulated by proteins that are recruited to kinetochores. For example, the histone subunit Cse4 is tightly regulated by the E3 ubiquitin ligase Psh1, which is localized at centromeres. Psh1 prevents excessive Cse4 centromere loading via ubiquitylation-dependent degradation of Cse4 (and at noncentromeric regions) (11). Cse4 is also phosphorylated by Ipl1, likely to destabilize aberrant microtubule interactions and ensure correct sister chromosome biorientation (12...

show abstract

Section: Resultsmentioning

confidence: 99%

“…Yeast plasmids are listed in Table S2. Plasmids were transferred into the GFP strains using selective ploidy ablation (32) and the resulting colony growth assessed using ScreenMill software (33). Other methods are described in SI Text.…”

Section: Methodsmentioning

confidence: 99%

Synthetic physical interactions map kinetochore regulators and regions sensitive to constitutive Cdc14 localization

Ólafsson

Thorpe

2015

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…This was of particular interest for our study, as Ent3 is also involved in SNARE (Vti1, Pep12 or Snc1) binding and trafficking. Surprisingly, the BTN3 gene was identified in a screen for deletion mutants sensitive to the overexpression of the DNA topoisomerase I top1-T 722 A mutant, and was thus named TDA3 (topoisomerase I damage affected) (Reid et al, 2011). Interestingly, the largest class of mutants identified in this screen affects the endosomal trafficking, including ESCRT subunits or the Fab1 lipid kinase.…”

Section: Identification Of Btn3 As a New Direct Ent3 And Ent5 Interactormentioning

confidence: 99%

Btn3 regulates the endosomal sorting function of the yeast Ent3 epsin, an adaptor for SNARE proteins

Morvan

Craene

Rinaldi

et al. 2014

Journal of Cell Science

View full text Add to dashboard Cite

Ent3 and Ent5 are yeast epsin N-terminal homology (ENTH) domain-containing proteins involved in protein trafficking between the Golgi and late endosomes. They interact with clathrin, clathrin adaptors at the Golgi (AP-1 and GGA) and different SNAREs (Vti1, Snc1, Pep12 and Syn8) required for vesicular transport at the Golgi and endosomes. To better understand the role of these epsins in membrane trafficking, we performed a protein-protein interaction screen. We identified Btn3 (also known as Tda3), a putative oxidoreductase, as a new partner of both Ent3 and Ent5. Btn3 is a negative regulator of the Batten-disease-linked protein Btn2 involved in the retrieval of specific SNAREs (Vti1, Snc1, Tlg1 and Tlg2) from the late endosome to the Golgi. We show that Btn3 endosomal localization depends on the epsins Ent3 and Ent5. We demonstrated that in btn3D mutant cells, endosomal sorting of ubiquitylated cargos and endosomal recycling of the Snc1 SNARE are delayed. We thus propose that Btn3 regulates the sorting function of two adaptors for SNARE proteins, the epsin Ent3 and the Batten-disease-linked protein Btn2.

show abstract

“…Although SDL has been used to identify protein targets of enzymes (82,100,101) and to identify specific subsets of genes within chromosome segregation mutants (29), it is underused to uncover genetic contexts that could selectively target cancer cells with elevated levels of a specific gene (26,27,102). SDL screens therefore represent a powerful approach for fast and easy identification of candidate chemotherapeutic drug targets within the context of dCIN gene overexpression that could enable targeted elimination of these cells.…”

Section: Sdl Screens As a Platform To Identify Therapeutic Targets Inmentioning

confidence: 99%

Overexpression screens identify conserved dosage chromosome instability genes in yeast and human cancer

Duffy

Fam

Wang

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Somatic copy number amplification and gene overexpression are common features of many cancers. To determine the role of gene overexpression on chromosome instability (CIN), we performed genome-wide screens in the budding yeast for yeast genes that cause CIN when overexpressed, a phenotype we refer to as dosage CIN (dCIN), and identified 245 dCIN genes. This catalog of genes reveals human orthologs known to be recurrently overexpressed and/or amplified in tumors. We show that two genes, TDP1, a tyrosyl-DNA-phosphdiesterase, and TAF12, an RNA polymerase II TATA-box binding factor, cause CIN when overexpressed in human cells. Rhabdomyosarcoma lines with elevated human Tdp1 levels also exhibit CIN that can be partially rescued by siRNA-mediated knockdown of TDP1. Overexpression of dCIN genes represents a genetic vulnerability that could be leveraged for selective killing of cancer cells through targeting of an unlinked synthetic dosage lethal (SDL) partner. Using SDL screens in yeast, we identified a set of genes that when deleted specifically kill cells with high levels of Tdp1. One gene was the histone deacetylase RPD3, for which there are known inhibitors. Both HT1080 cells overexpressing hTDP1 and rhabdomyosarcoma cells with elevated levels of hTdp1 were more sensitive to histone deacetylase inhibitors valproic acid (VPA) and trichostatin A (TSA), recapitulating the SDL interaction in human cells and suggesting VPA and TSA as potential therapeutic agents for tumors with elevated levels of hTdp1. The catalog of dCIN genes presented here provides a candidate list to identify genes that cause CIN when overexpressed in cancer, which can then be leveraged through SDL to selectively target tumors.dosage chromosome instability | overexpression | synthetic dosage lethality | TDP1 | rhabdomyosarcoma

show abstract

Selective ploidy ablation, a high-throughput plasmid transfer protocol, identifies new genes affecting topoisomerase I–induced DNA damage

Cited by 84 publications

References 53 publications

Synthetic physical interactions map kinetochore regulators and regions sensitive to constitutive Cdc14 localization

Synthetic physical interactions map kinetochore regulators and regions sensitive to constitutive Cdc14 localization

Btn3 regulates the endosomal sorting function of the yeast Ent3 epsin, an adaptor for SNARE proteins

Overexpression screens identify conserved dosage chromosome instability genes in yeast and human cancer

Contact Info

Product

Resources

About