The complete spectrum of yeast chromosome instability genes identifies candidate CIN cancer genes and functional roles for ASTRA complex components

Abstract

Chromosome instability (CIN) is observed in most solid tumors and is linked to somatic mutations in genome integrity maintenance genes. The spectrum of mutations that cause CIN is only partly known and it is not possible to predict a priori all pathways whose disruption might lead to CIN. To address this issue, we generated a catalogue of CIN genes and pathways by screening ∼ 2,000 reduction-of-function alleles for 90% of essential genes in Saccharomyces cerevisiae. Integrating this with published CIN phenotypes for other yeast genes generated a systematic CIN gene dataset comprised of 692 genes. Enriched gene ontology terms defined cellular CIN pathways that, together with sequence orthologs, created a list of human CIN candidate genes, which we cross-referenced to published somatic mutation databases revealing hundreds of mutated CIN candidate genes. Characterization of some poorly characterized CIN genes revealed short telomeres in mutants of the ASTRA/TTT components TTI1 and ASA1. High-throughput phenotypic profiling links ASA1 to TTT (Tel2-Tti1-Tti2) complex function and to TORC1 signaling via Tor1p stability, consistent with the role of TTT in PI3-kinase related kinase biogenesis. The comprehensive CIN gene list presented here in principle comprises all conserved eukaryotic genome integrity pathways. Deriving human CIN candidate genes from the list allows direct cross-referencing with tumor mutational data and thus candidate mutations potentially driving CIN in tumors. Overall, the CIN gene spectrum reveals new chromosome biology and will help us to understand CIN phenotypes in human disease.

Figure 1. The spectrum of chromosome instability genes in yeast.

(A) Schematic of genome integrity screens showing the cellular phenotype measured (top box – i.e. pigmentation for CTF, capacity to mate for ALF, acquired 5′FOA and canavanine resistance for GCR), the chromosomal context of the CIN marker (lower box; blue line indicates chromosome, black oval indicates the centromere, other features are described below) and the type of alleles screened by each assay (bottom). For CTF (left), red pigment accumulates due to a block in adenine production (ade2-101) that is relieved in the presence of SUP11 (black rectangle) carried on a non-essential fragment of chromosome III or VII (CF) (Blue line). Strains carrying the CF will be unpigmented while those that lose the CF will be red. Colonies of mutants with whole chromosome instability will grow with red sectors in an unpigmented colony. For ALF (center), loss of the MATα locus leads to dedifferentiation to an a-mating phenotype. α-mating type mutant strains are mated to a MATα test strain and growth of diploid mated progeny is assessed on selective media, where the ability to mate and form diploids reflects loss, deletion or inactivation of the MATα locus. For GCR (right), URA3 is integrated near the CAN1 gene on the left arm of chromosome V where the distal portion of the chromosome arm is non-essential. GCR assay strains are patched onto double counter-selection media where only ura3, can1 strains can survive thus revealing increased rates of terminal chromosome arm deletion and non-reciprocal rearrangements. (B) Overview of CIN gene list. (i) CIN mutations in essential, non-essential and overexpressed genes. (ii) Overlap among five high-throughput CIN phenotypes links 683 of the CIN genes (overlap of 36 LOH mutants (dotted lines) is not shown for clarity). (iii) CIN phenotypic strength in nuclear localized versus other genes. Bars colored according to 1C. (C) Cellular schematic of 692 CIN genes grouped according to function (Table S1). The large dotted circle represents the nucleus. Colors indicate a strong phenotype (red) or a weak phenotype (blue) where darker represents CIN in multiple assays and lighter represents CIN in a single assay. Yellow boxes and bold labels indicate groups, and dotted circles and italic labels indicate subgroups or protein complexes. Subgroup abbreviations; IMP – inosine monophosphate synthesis; SRP – signal recognition particle; GPI – glycophosphatidylinositol synthesis; NUP – nucleoporins; PFD – prefoldin complex; FeS – Iron Sulfur cluster synthesis; m-ribosome – mitochondrial ribosomal subunit.

Figure 2. A constellation of GO-terms define CIN pathways and human CIN candidates.

(A) Enriched GO terms representing cellular CIN pathways are plotted in the context of the GO-hierarchy. Blue coloration and node size increase with higher fold enrichment. Highlighted sections of the network are marked with roman numerals. (B) CIN gene orthologs and CIN-GO terms generate a partly overlapping candidate gene space. (C) Cross-referencing CIN candidate genes with the COSMIC database and the cancer gene census (CC) databases. Asterisk indicates significantly more mutated CIN-GO genes in COSMIC and CC than the population (Z-test of two proportions).

Figure 3. A screen for altered telomeres among poorly characterized essential CIN genes identifies components of ASTRA/TTT.

(A) Southern blots of telomere length alterations in indicated mutants. 11 strains are shown, other strains screened had WT telomere length (data not shown). Telomeres are bright bands under 1.5 kb, control bands are seen at the top and bottom of the gel. Triangles indicate short (filled) or long (open) telomeres. (B) Conservation of ASTRA components from yeast to humans. (C) Temperature-sensitive growth of TTT subunit and ASA1 mutant alleles in spots of 10-fold serially diluted yeast cultures. (D) Southern blot of TTT-Asa1 telomere length as in (A). (E) Western analysis of 2xFLAG-Tel1p in the TTT-Asa1 ts-alleles including α-Pgk1p blots as a loading control.

Figure 4. High-resolution phenotypic profiles link TTT and ASA1 to TORC1 function.

(A) Hierarchical clustering of genome-wide SGA profiles for TTT and ASA1 ts-alleles with the global genetic interaction network . Queries are grey and TORC1 components are yellow. (B) Dilution series of TTT-Asa1 ts-allele growth +/− the TORC1 inhibitor rapamycin. (C) Growth curve analysis of hypomorphic tti1-1 and asa1-1 alleles in congenic strains +/− TIP41. (D) Localization of Gat1-GFP in TTT-Asa1 mutants. Below the sample images a graph shows the mean proportion of cells with nuclear GFP +/− SEM (*  =  p<0.01 compared to WT; tti2-1 not done). (E) Western blot analysis of 3xHA-Tor1p in the TTT-Asa1 ts-alleles including α-Pgk1p blots as a loading control.