BACKGROUND
there is an increasing realisation that copy-number variation, and in particular the loss of one copy of a gene from a diploid cell or organism, can have a significant phenotypic impact.
moreover, the importance of gene dosage in tumourigenesis is becoming increasingly recognised  <cit> , and the widespread aneuploidy and copy-number variations  that are the hallmarks of cancer cells are coming to be seen as potentially causative, rather than simply symptomatic  <cit> .
identifying causal cnv-related phenotypes for mammalian genes, however, is hampered by the difficulty in constructing deletion mutants in the higher eukaryotes, and the fact that homozygous knockout  phenotypes do not necessarily correlate with those elicited by the loss of a single copy of a gene  <cit> .
in the model eukaryote saccharomyces cerevisiae, in contrast, high-throughput screens on whole-genome libraries have facilitated the identification of yeast genes for which cnv has a significant effect on cell proliferation.
a screen of >  <dig>  deletion mutants, each heterozygous for a different protein-encoding gene in the s.cerevisiae genome, revealed that over 18% of these heterozygotes displayed a significantly reduced growth rate  <cit> .
this haploinsufficient phenotype was displayed even under conditions where there are no external constraints  on cell growth  <cit> .
for a smaller set  of ‘haploproficient’ genes, however, heterozygous deletion of the gene elicits significantly faster growth than the wild type.
using these yeast phenotypes, we have previously made correct predictions of a gene-dosage-related phenotype for their orthologous human genes  <cit> , and verified these predictions by controlled rnai knockdown in human cell lines  <cit> .
the existence of haploproficient genes in the yeast genome indicates that the organism has not evolved to maximize its rate of growth, even when sugars became abundantly available with the emergence of flowering plants approximately 80– <dig> million years ago  <cit> .
the persistence of these genes in the yeast genome therefore suggests that there must be some major selective advantage that outweighs their growth-rate disadvantages.
we demonstrate, here, that this advantage could be the maintenance of genome integrity, which is compromised when the dosage of these genes is reduced.
for hp yeast genes, the altered gene dosage not only increases growth rate but, as we will show, copy-number reduction, as opposed to complete gene deletion, is sufficient to result in abnormal progression through the cell cycle, increased accumulation of dna damage, and altered rates of apoptosis.
this set of phenotypes is strongly reminiscent of cancer in mammalian cells.
these results complement the recent conclusions from a study of aneuploid yeast strains  <cit>  that the detriment to genome stability is driven by gene-dosage effects , rather than simply by the presence of extra dna .
this emphasises the value of using model organisms to predict which human genes may impact on cancer in a dosage-dependent manner.
the screening of a library of yeast heterozygous deletion mutants for haploinsufficient or -proficient phenotypes is a high-throughput approach to determine what effects quantitative changes in the concentrations of gene products have on phenotype.
a related approach is the search for drug-induced haploinsufficiency – in which heterozygous deletion strains exhibit altered sensitivity to a compound as a result of the decrease in the dosage of the target gene.
in a pioneering study,  <cit>  exposed a pool of  <dig> heterozygous mutants to sub-lethal compound concentrations, and later work has successfully elucidated the mode-of-action of novel compounds such as the anti-tumour agent dihydromotuporamine c  <cit> , demonstrating the utility of such genome-wide yeast screens.
in the light of our findings on haploproficiency in yeast, we combined both approaches and carried out a screen of anti-cancer agents against a set of s. cerevisiae mutants heterozygous for hp genes involved in the dna damage response-pathway in order to search for altered sensitivities relative to both the wt and the corresponding homozygous  deletion mutant.
this screen might inform both the appropriate treatment of tumour cells that carry cnvs of the candidate cancer-related genes, and also suggest novel combinations of specific inhibitors which may prove more effect than either drug in isolation.
RESULTS
yeast haploproficient genes are involved in the maintenance of genome integrity and are orthologs of human cancer genes
the existence of haploproficient genes, and the inference that the yeast genome has not been optimised for maximal growth rate, does not appear to be an accident, nor unique to s. cerevisiae.
by examining orthology relationships across the ascomycetes, we find that haploproficient genes are more highly conserved than the genome average across the lineage  .
thus, selective pressure has existed toward the retention of hp genes for several hundred million years , including through a period of strong selective pressure toward maximizing growth rate that occurred at the time of the whole-genome duplication in the saccharomyces lineage .
congruent with our hypothesis of a trade-off between genome stability and growth rate optimisation, we find that haploproficient genes are overrepresented amongst those involved in the maintenance of genome integrity.
a gene ontology term-enrichment revealed that the hp set is enriched for genes involved in the mitotic cell cycle , and, in particular, the response to and repair of dna damage  .
given their integral role in maintaining genome stability, it is unsurprising that yeast hp genes are very much more likely to be orthologous to cancer genes than the s. cerevisaie genome average .
we selected the  <dig> hp genes  involved in the processes of dna damage repair and sister chromatid segregation as the most relevant candidates in which to examine the relationship between varying gene dosage and cancer-related phenotypes.
this ‘hp genome integrity’  set is even more highly conserved than the hp set as a whole ; and more likely  to be orthologous to a cancer gene .
nineteen genes in the set have a unique human ortholog  and, of these,  <dig>  are orthologous to cancer genes as annotated in the cancer gene index .
table  <dig> lists the cancer-specific omim disease associations of the orthologs of members of the hpgi set.
orthologs of haploproficient genes exhibit cnv in tumour cells
the haploproficiency phenotype is, by definition, linked to a reduction in gene copy number.
our hypothesis posits that it is copy number variation  of the orthologs of these genes that is relevant to human cancer.
to determine the rate of incidence of cnv in orthologs of our hp cell cycle set, we queried our list against the nci’s cancer genome atlas database .
no less than 17/ <dig> of the human orthologs of the hpgi set have a copy number log <dig> ratio of magnitude > <dig>  across eight types of cancer.
in particular, 12/ <dig> have a copy number reduction  in >25% of patients in one or more of the eight cancer types.
this is a significant overrepresentation compared with 7750/~ <dig> of all genes in tcga , and with the set of non-hp cell-cycle/dna repair orthologs .
for serous cystadenocarcinoma, kidney renal clear cell, and lung squamous cell carcinomas, there is a significant overrepresentation of orthologs of the hpgi set amongst genes exhibiting frequent copy-number reduction .
moreover, across all eight cancer types, orthologs of the hpgi set are overrepresented with a p-value of  <dig>  .
cnv of haploproficient genes is sufficient to elicit cancer-like phenotypic responses in yeast
cell cycle phasing
as their growth rate is higher than the previously observed maximum, heterozygous deletion mutants of hp genes must a priori progress faster through the cell cycle.
either progress through all the phases of the cycle may be accelerated, or one or more phases must be relatively shorter than in a diploid cell with two copies of the gene.
since the products of hpgi genes are involved in checkpoint controls of the cell cycle, we reasoned that the faster cycle time in the heterozygotes could be the result of cells skipping a compromised checkpoint and therefore progressing faster through that phase of the cycle.
this would manifest itself in an altered population distribution between the different cell cycle phases.
this same cell cycle dysregulation, arising from compromised tumour suppressor gene function, is a mechanism of oncogenesis in mammalian cells  <cit> .
previously, we found strong support for the hypothesis that changes in the copy number of the high flux control  genes alter cell cycle progression in a combined modelling/experimental study of a set of genes involved in the g2/m transition in yeast  <cit> .
this intrinsic link between haploproficiency and cell cycling is also borne out in our finding that copy-number variation of hp genes has no significant effect on yeast chronological lifespan; that is, survival in the extended non-cycling g <dig> state of stationary phase .
using flow cytometry, we analysed the cell cycle progression of heterozygous deletion mutants of the hpgi set, comparing them with the cell cycle profiles of the wt and of the non-cell cycle, non-hp controls his3/his <dig> and ho/ho, non-hp, cell-cycle hsl1/hsl <dig>  clb2/clb <dig>  clb5/clb <dig>  clb6/clb <dig>  cln2/cln <dig> and cln3/cln <dig> and the non-cell cycle genes pnp <dig>  met <dig>  hrk <dig> and tpo <dig>  to distinguish between the effects of the complete absence of a given gene from those of reducing its copy number from two to one, we also compared the cycle profiles of the heterozygous deletant against that of the diploid homozygous deletants of the same gene.
complete cell cycle profile data is given in additional file 4: table s <dig>
we find that correct dosage of the hpgi genes is essential for normal cell cycling: heterozygous deletion is indeed sufficient to significantly alter the cell cycle profile relative to the wt/non-cell cycle control genes for  <dig> of the  <dig> hpgi genes .
typically, the cell cycle perturbation for the heterozygote concurs with that previously reported for haploid null deletants of the gene , however bub <dig> heterozygous deletion confers the opposite phenotype  to that of both the null diploid and haploid.
apoptosis rate
in mammalian cells, compromising the link between the dna damage response and apoptotic pathways can diminish the apoptotic response  <cit> , which is a necessary step en route to cancer  <cit> .
therefore, we carried out tests to determine whether compromising the dna damage response in yeast by heterozygous deletion of hpgi genes affected the rate of apoptosis .
the degree of apoptosis occurring in the deletion strain populations, in response to treatment with methyl methanesulfonate  or tert-butyl hydroperoxide .
was measured by simultaneous annexin v  and propidium iodide  staining  <cit>  to distinguish between apoptotic and necrotic cells.
for  <dig> of the  <dig> hpgi genes, the heterozygous deletion mutant exhibited a degree of apoptosis significantly different  from than the wt – that is, partial knockdown of the gene expression is sufficient to disrupt the normal apoptotic response of the cell.
whilst exploring the specific mechanisms behind this disruption for each gene is beyond the scope of this paper, it is clear that deviating from the wt dosage of these particular genes has a significant effect on programmed cell death in response to genotoxic agents.
phenotypes for the mutants are described in table 4; apoptosis rates in his3/his3Δ and ho/hoΔ control strains are comparable to wt.
the phenotypes observed for the hpgi heterozygous deletants were compared with those reported in the literature for the rnai-mediated knockdown or pharmacological inhibition of their mammalian orthologs.
the congruence between the yeast and mammalian phenotypes is summarized in table  <dig>  for 2/ <dig> cases of increased apoptosis upon copy-number reduction, the phenotype is also observed in human cells, however for mlh1/mlh <dig> and ubx4/aspscr <dig> the relationship between gene dosage and apoptosis in human cells is acknowledged, but unclear  <cit> .
similarly, for 4/ <dig> genes prompting reduced apoptosis upon copy-number reduction, knockdown of the human ortholog has been reported to cause a similar phenotype; we could find no report for the orthologs of a further  <dig> of the  <dig>  for smc3/smc <dig> and dnl4/lig <dig>  however, copy-number reduction in yeast reduced apoptosis, whilst knockdown in mammalian systems has been reported to increase its occurrence  <cit> .
however, both of these reports involve a complete deletion of the gene, rather than the heterozygous deletion in which we observed the yeast phenotype.
this suggests that further investigation in human cells of the effects of varying gene dosage of these genes, in particular, would be worthwhile.
haploproficiency & cancer drug sensitivity
given the strong connection that we have observed between yeast haploproficiency and human cancer, it is unsurprising that the human orthologs of many of the hpgi genes are the targets of various anti-cancer compounds .
however, in the light of the increased growth upon reduction of hp gene dosage, and the dosage-specific phenotypes reported above, it is possible that inhibitor treatment of a tumour cell could elicit the opposite to the desired response – i.e.
increased proliferation rather than cell death – if complete ablation of the hp target function is not achieved.
therefore, we examined the drug sensitivity of wild-type yeast, and the heterozygous and homozygous deletion mutants for each of the non-essential hpgi genes.
we also included the deletion mutants for an additional five yeast genes , which are hp and whose products are orthologous to specific cancer-drug targets.
where the human ortholog of a yeast hpgi gene product is a cancer-drug target  we included in our screen either the commercial drug  or alternative inhibitors .
in total,  <dig> drugs were screened : two of these were specific alternative inhibitors;  <dig> were compounds either approved for human cancer treatment or shown to be effective in cell culture; and one control treatment of clotrimazole, a fungicide not known to have anti-cancer properties.
drug sensitivity profiles were clustered  by agglomerative hierarchical clustering  <cit> .
the profile elicited by a compound can reveal its mode of action, and thus similarities between drug profiles may indicate the targeting of the same pathways in yeast.
reassuringly, known modes of action are reflected in our profiles – for example, the sensitivity of cell wall integrity/morphogenesis deletion mutants to phleomycin , which causes cell wall lesions  <cit> .
control  clotrimazole treatments are reasonably distinct from the cancer drug treatments.
aminopterin and methotrexate  pd <dig>  cycloheximide and mitoxantrone cluster together, driven largely by the resistant phenotypes of deletion mutants of genes involved in chromosome condensation and segregation .
a second cluster is formed by bay <dig>  cantharidin and vorinostat , all of which act as inhibitors of the nf-κb pathway in mammalian cells  <cit> .
this pathway is absent in s.cerevisiae <cit>  however, the clustering of the compounds suggests that their modes of action, though distinct from the mammalian-cell pathway, may also be related in yeast.
rad54/rad <dig> mutants are particularly resistant to all three drugs, where, in general, these mutants are sensitive to all other treatments.
vorinostat has been demonstrated to induce dsbs in acute myeloid leukemia cells  <cit> , suggesting that the dsb-repair function of rad54p could potentially be a target.
indeed, the rad <dig> ortholog, rad54l, is strongly down-regulated in lncap  cells treated with vorinostat  <cit> .
this is also the case for the mammalian orthologs of chk <dig> and top <dig>  which are also resistant  to the vorinostat /bay11/cantharidin cluster.
we have since demonstrated  <cit>  that the magnitude of these drug-response phenotypes in yeast  is sufficient to translate into observable phenotypes in tumour cell lines.
targeting the cnv profile of tumour cells
where a copy of a gene is frequently lost in tumour cells, as is the case for orthologs of the hpgi set, the phenotype of the heterozygous deletion mutant of the yeast ortholog might better reflect the tumour cell’s response to drug treatment .
for a significant number of homozygous/heterozygous deletant pairs  the drug sensitivity phenotypes are quantitatively dissimilar  .
for these genes, therefore, gene dosage may be highly relevant to pharmaceutical efficacy.
these eight genes  have, on average, a greater number of protein-protein interactors  than the average for our hpgi set as a whole .
this suggests that gene dosage balance may be an important contributor to phenotype for genes which are more central in interaction networks.
we have explored the relationship between gene copy number and drug response further in  <cit> .
previously, we reported the striking result that treatment of wild-type yeast with low concentrations of inhibitors targeting hp genes can induce the same increased proliferative phenotype as is observed upon reducing the copy number of the hp target, a phenomenon we termed hypertolerance <cit> .
we observe this phenotype again for treatment with cantharidin, which inhibits the pp2a complex, the subunits of which are strongly haploproficient , but not for any of the general cytotoxic compounds in the drug screen, reinforcing our previously-established link between the specific inhibition of a haploproficient target and drug hypertolerance.
a number of heterozygous strains also exhibited hypertolerance in solid-media screens, which was subsequently confirmed by titrating across a range of drug concentrations in liquid medium .
for tumour cells bearing the orthologous cnvs, this would suggest a contraindication for treatment with the particular drug.
in particular, the nbp2/nbp <dig> heterozygote is multiply drug-hypertolerant, of note since its ortholog sh3gl <dig> frequently has reduced copy-number in serous cystadenocarcinoma.
conversely, for pnp <dig>  the heterozygotes are mitoxantrone/methotrexate/aminopterin and vorinostat/bay11/cantharidin sensitive, and the homozygotes resistant.
this phenotype may be a instance of ‘obligate haploinsufficiency’  <cit> , whereby fitness is compromised as the gene dosage is reduced below some threshold value, but the phenotype is subsequently rescued through the switching on of a compensatory pathway.
discussion
the data presented here for the s.cerevisiae hpgi set point to the striking result that copy-number variation , rather than complete gene loss, is sufficient to elicit deleterious phenotypes, in particular those reminiscent of cancer in mammalian cells.
this concurs with the increasing appreciation of the significance of copy-number variation to the genesis and progression of disease.
in particular, there is a general lack of significant correlation of phenotypes between the heterozygous and homozygous  deletion mutants of a given gene.
nor are the phenotypes of the null mutants necessarily more extreme than those of the heterozygous deletants, as might naively be expected from a linear ‘dosage hypothesis’  <cit> .
this emphasises the need to systematically investigate the phenotypic impact of gene dosage, rather than relying on knockout, or total-knockdown, studies.
a mechanism by which heterozygous deletion of a gene could be just as disruptive as a complete knockout is via the disruption of the stoichiometric ratios of the sub-units of protein complexes.
this has been proposed as a mechanism for haploinsufficiency in the 'balance hypothesis' of papp et al.
<cit> .
it also suggests that dosage compensation, in which the impact of the heterozygous deletion of a gene is mitigated by the increased expression of the remaining copy, does not play a significant role amongst our hpgi gene set.
indeed,  <cit>  showed that such dosage compensation is, in general, rare in s.cerevisae, and very few hp genes are amongst those exhibiting significant compensation.
for cell cycle phasing, heterozygous gene deletion is often sufficient to perturb the cycle from the wt profile, which indicates that cell cycle proteins are required in yeast at greater than 50% of wt dosage .
similarly, heterozygous deletion of the yeast ortholog is, in general, both sufficient to alter the degree of apoptosis occurring in response to dna-damaging agents , and to elicit the same apoptotic phenotype  as is reported for complete protein-product knockdown in mammalian cells.
this general conservation of apoptotic phenotypes suggests that the dna damage-related pathway controlling apoptosis in s.cerevisiae are similar to those in mammals.
this could be investigated further by undertaking a global survey of the effect of gene deletion on apoptosis rates in yeast, particularly since the current data on the degree of apoptosis in s.cerevisiae is limited .
in four cases, an altered apoptosis rate was observed in the yeast deletion mutants but has not, so far, been reported for the rnai knock-downs of their human orthologs.
we would suggest that, based on the apparent predictive value of the yeast phenotype, these genes should become the focus of an rnai study in mammalian cells.
hp genes and the efficacy of anti-cancer drugs
this critical importance of considering gene dosage in the context of pharmaceutical intervention in cancer is further emphasised in the copy-number-dependence of drug sensitivity phenotypes that we observed with our yeast model.
the mechanisms by which copy-number variation may exert a significant effect on phenotype are summarised in figure  <dig>
firstly, products of the mammalian orthologs of several of the yeast hpgi set are the targets of specific drugs used, or proposed for use, in treating cancers.
our data suggest that, in a majority of such cases, complete inhibition of the activity of a target protein product is necessary to achieve a positive therapeutic outcome.
incomplete inhibition, analogous to heterozygous deletion of the gene encoding the target, prompts increased proliferation or hypertolerance – the opposite of the intended anti-cancer effect.
furthermore, even if a given gene is not the intended specific target of a chemotherapeutic treatment, the modes-of-action, and secondary impacts, of many cancer drugs have not been fully elucidated.
drug-induced haploinsufficiency  data from yeast can contribute to a better understanding of both drug mechanisms and the functional conservation of drug-metabolism pathways between yeast and humans.
for example, the clustering of mutant phenotypes in response to the mammalian-nf-κb inhibitors in this study suggests that all three compounds act through a common pathway in s.cerevisiae, which may be mediated by rad54p.
secondly, we observed that varying the copy number of the yeast ortholog can significantly alter the phenotypic response to drug treatment.
this is especially for genes whose human orthologs have a high likelihood of cnv in tumour cells, in particular, several deletion strains are either more resistant than the wt, or themselves hypertolerant in response to a specific drug treatment.
the impact of specific cnvs on drug sensitivity is becoming increasingly appreciated, and we believe our yeast mutant approach represents a high-throughput complement to the creation of drug-cnv profile ‘fingerprints’ for tumour cells , and a model for determining the most effective drug interventions for tumours with a particular cnv profile.
lastly, our data on the sensitivity/resistance of deletion mutants could potentially inform the design of combination therapies.
where a particular  deletion mutant is sensitive to a given drug, a combination therapy consisting of that drug plus an inhibitor of the product of the heterozygous gene could have a synergistic effect in reducing cell proliferation.
conversely, considering pairs of genes/drugs for which the deletion mutant is resistant to treatment could assist in identifying those drug combinations which are unlikely to succeed, reducing the search space involved in an otherwise expensive combinatorial problem  <cit> .
finally, the several instances in which we observed that the heterozygous deletion mutant is more sensitive to drug treatment than the homozygote provide evidence for the ‘obligate haploinsufficiency’ hypothesis of berger et al.
<cit> .
identifying such genes, and defining the protein product threshold below which their associated compensatory pathways are activated, could help to define the precise inhibitor dosage to achieve maximal therapeutic efficacy.
CONCLUSIONS
haploproficient  genes and are for which, when one copy is deleted from a diploid cell, the rate of cell proliferation is increased.
we have demonstrated that, for a subset of yeast hp genes, heterozygous deletion causes aberrant cell cycling and altered rates of apoptosis, which are phenotypes associated with cancer in mammalian cells.
a majority of these yeast genes are the orthologs of mammalian cancer genes, and hence our studies suggest that cnv of these oncogenic orthologs may be sufficient to lead to tumourigenesis in human cells.
moreover, where not already implicated, this cluster of cancer-like phenotypes in this model eukaryote may be predictive of the involvement in cancer of the mammalian orthologs of these yeast hp genes.
using the yeast set as a model, we have shown that the response of the heterozygotes to a range of anti-cancer drugs is strongly dependent on gene dosage, such that intermediate concentrations of the drugs can actually increase a mutant’s growth rate.
this suggests such compounds may need to achieve near-total inhibition of target activity if they are to be a beneficial, rather than a deleterious, therapy.

 there is an increasing realisation that copy-number variation, and in particular the loss of one copy of a gene from a diploid cell or organism, can have a significant phenotypic impact. moreover, the importance of gene dosage in tumourigenesis is becoming increasingly recognised, and the widespread aneuploidy and copy-number variations that are the hallmarks of cancer cells are coming to be seen as potentially causative, rather than simply symptomatic. identifying causal cnv-related phenotypes for mammalian genes, however, is hampered by the difficulty in constructing deletion mutants in the higher eukaryotes, and the fact that homozygous knockout phenotypes do not necessarily correlate with those elicited by the loss of a single copy of a gene. in the model eukaryote saccharomyces cerevisiae, in contrast, high-throughput screens on whole-genome libraries have facilitated the identification of yeast genes for which cnv has a significant effect on cell proliferation. a screen of >  deletion mutants, each heterozygous for a different protein-encoding gene in the s.cerevisiae genome, revealed that over 18% of these heterozygotes displayed a significantly reduced growth rate. this haploinsufficient phenotype was displayed even under conditions where there are no external constraints on cell growth. for a smaller set of ‘haploproficient’ genes, however, heterozygous deletion of the gene elicits significantly faster growth than the wild type. using these yeast phenotypes, we have previously made correct predictions of a gene-dosage-related phenotype for their orthologous human genes, and verified these predictions by controlled rnai knockdown in human cell lines. the existence of haploproficient genes in the yeast genome indicates that the organism has not evolved to maximize its rate of growth, even when sugars became abundantly available with the emergence of flowering plants approximately 80– million years ago. the persistence of these genes in the yeast genome therefore suggests that there must be some major selective advantage that outweighs their growth-rate disadvantages. we demonstrate, here, that this advantage could be the maintenance of genome integrity, which is compromised when the dosage of these genes is reduced. for hp yeast genes, the altered gene dosage not only increases growth rate but, as we will show, copy-number reduction, as opposed to complete gene deletion, is sufficient to result in abnormal progression through the cell cycle, increased accumulation of dna damage, and altered rates of apoptosis. this set of phenotypes is strongly reminiscent of cancer in mammalian cells. these results complement the recent conclusions from a study of aneuploid yeast strains that the detriment to genome stability is driven by gene-dosage effects, rather than simply by the presence of extra dna. this emphasises the value of using model organisms to predict which human genes may impact on cancer in a dosage-dependent manner. the screening of a library of yeast heterozygous deletion mutants for haploinsufficient or -proficient phenotypes is a high-throughput approach to determine what effects quantitative changes in the concentrations of gene products have on phenotype. a related approach is the search for drug-induced haploinsufficiency – in which heterozygous deletion strains exhibit altered sensitivity to a compound as a result of the decrease in the dosage of the target gene. in a pioneering study, exposed a pool of heterozygous mutants to sub-lethal compound concentrations, and later work has successfully elucidated the mode-of-action of novel compounds such as the anti-tumour agent dihydromotuporamine c, demonstrating the utility of such genome-wide yeast screens. in the light of our findings on haploproficiency in yeast, we combined both approaches and carried out a screen of anti-cancer agents against a set of s. cerevisiae mutants heterozygous for hp genes involved in the dna damage response-pathway in order to search for altered sensitivities relative to both the wt and the corresponding homozygous deletion mutant. this screen might inform both the appropriate treatment of tumour cells that carry cnvs of the candidate cancer-related genes, and also suggest novel combinations of specific inhibitors which may prove more effect than either drug in isolation.  yeast haploproficient genes are involved in the maintenance of genome integrity and are orthologs of human cancer genes the existence of haploproficient genes, and the inference that the yeast genome has not been optimised for maximal growth rate, does not appear to be an accident, nor unique to s. cerevisiae. by examining orthology relationships across the ascomycetes, we find that haploproficient genes are more highly conserved than the genome average across the lineage. thus, selective pressure has existed toward the retention of hp genes for several hundred million years, including through a period of strong selective pressure toward maximizing growth rate that occurred at the time of the whole-genome duplication in the saccharomyces lineage. congruent with our hypothesis of a trade-off between genome stability and growth rate optimisation, we find that haploproficient genes are overrepresented amongst those involved in the maintenance of genome integrity. a gene ontology term-enrichment revealed that the hp set is enriched for genes involved in the mitotic cell cycle, and, in particular, the response to and repair of dna damage. given their integral role in maintaining genome stability, it is unsurprising that yeast hp genes are very much more likely to be orthologous to cancer genes than the s. cerevisaie genome average. we selected the hp genes involved in the processes of dna damage repair and sister chromatid segregation as the most relevant candidates in which to examine the relationship between varying gene dosage and cancer-related phenotypes. this ‘hp genome integrity’ set is even more highly conserved than the hp set as a whole; and more likely to be orthologous to a cancer gene. nineteen genes in the set have a unique human ortholog and, of these, are orthologous to cancer genes as annotated in the cancer gene index. table  lists the cancer-specific omim disease associations of the orthologs of members of the hpgi set. orthologs of haploproficient genes exhibit cnv in tumour cells the haploproficiency phenotype is, by definition, linked to a reduction in gene copy number. our hypothesis posits that it is copy number variation of the orthologs of these genes that is relevant to human cancer. to determine the rate of incidence of cnv in orthologs of our hp cell cycle set, we queried our list against the nci’s cancer genome atlas database. no less than 17/ of the human orthologs of the hpgi set have a copy number log ratio of magnitude > across eight types of cancer. in particular, 12/ have a copy number reduction in >25% of patients in one or more of the eight cancer types. this is a significant overrepresentation compared with 7750/~ of all genes in tcga, and with the set of non-hp cell-cycle/dna repair orthologs. for serous cystadenocarcinoma, kidney renal clear cell, and lung squamous cell carcinomas, there is a significant overrepresentation of orthologs of the hpgi set amongst genes exhibiting frequent copy-number reduction. moreover, across all eight cancer types, orthologs of the hpgi set are overrepresented with a p-value of. cnv of haploproficient genes is sufficient to elicit cancer-like phenotypic responses in yeast cell cycle phasing as their growth rate is higher than the previously observed maximum, heterozygous deletion mutants of hp genes must a priori progress faster through the cell cycle. either progress through all the phases of the cycle may be accelerated, or one or more phases must be relatively shorter than in a diploid cell with two copies of the gene. since the products of hpgi genes are involved in checkpoint controls of the cell cycle, we reasoned that the faster cycle time in the heterozygotes could be the result of cells skipping a compromised checkpoint and therefore progressing faster through that phase of the cycle. this would manifest itself in an altered population distribution between the different cell cycle phases. this same cell cycle dysregulation, arising from compromised tumour suppressor gene function, is a mechanism of oncogenesis in mammalian cells. previously, we found strong support for the hypothesis that changes in the copy number of the high flux control genes alter cell cycle progression in a combined modelling/experimental study of a set of genes involved in the g2/m transition in yeast. this intrinsic link between haploproficiency and cell cycling is also borne out in our finding that copy-number variation of hp genes has no significant effect on yeast chronological lifespan; that is, survival in the extended non-cycling g state of stationary phase. using flow cytometry, we analysed the cell cycle progression of heterozygous deletion mutants of the hpgi set, comparing them with the cell cycle profiles of the wt and of the non-cell cycle, non-hp controls his3/his and ho/ho, non-hp, cell-cycle hsl1/hsl clb2/clb clb5/clb clb6/clb cln2/cln and cln3/cln and the non-cell cycle genes pnp met hrk and tpo to distinguish between the effects of the complete absence of a given gene from those of reducing its copy number from two to one, we also compared the cycle profiles of the heterozygous deletant against that of the diploid homozygous deletants of the same gene. complete cell cycle profile data is given in additional file 4: table s we find that correct dosage of the hpgi genes is essential for normal cell cycling: heterozygous deletion is indeed sufficient to significantly alter the cell cycle profile relative to the wt/non-cell cycle control genes for of the hpgi genes. typically, the cell cycle perturbation for the heterozygote concurs with that previously reported for haploid null deletants of the gene, however bub heterozygous deletion confers the opposite phenotype to that of both the null diploid and haploid. apoptosis rate in mammalian cells, compromising the link between the dna damage response and apoptotic pathways can diminish the apoptotic response, which is a necessary step en route to cancer. therefore, we carried out tests to determine whether compromising the dna damage response in yeast by heterozygous deletion of hpgi genes affected the rate of apoptosis. the degree of apoptosis occurring in the deletion strain populations, in response to treatment with methyl methanesulfonate or tert-butyl hydroperoxide. was measured by simultaneous annexin v and propidium iodide staining to distinguish between apoptotic and necrotic cells. for of the hpgi genes, the heterozygous deletion mutant exhibited a degree of apoptosis significantly different from than the wt – that is, partial knockdown of the gene expression is sufficient to disrupt the normal apoptotic response of the cell. whilst exploring the specific mechanisms behind this disruption for each gene is beyond the scope of this paper, it is clear that deviating from the wt dosage of these particular genes has a significant effect on programmed cell death in response to genotoxic agents. phenotypes for the mutants are described in table 4; apoptosis rates in his3/his3Δ and ho/hoΔ control strains are comparable to wt. the phenotypes observed for the hpgi heterozygous deletants were compared with those reported in the literature for the rnai-mediated knockdown or pharmacological inhibition of their mammalian orthologs. the congruence between the yeast and mammalian phenotypes is summarized in table  for 2/ cases of increased apoptosis upon copy-number reduction, the phenotype is also observed in human cells, however for mlh1/mlh and ubx4/aspscr the relationship between gene dosage and apoptosis in human cells is acknowledged, but unclear. similarly, for 4/ genes prompting reduced apoptosis upon copy-number reduction, knockdown of the human ortholog has been reported to cause a similar phenotype; we could find no report for the orthologs of a further of the for smc3/smc and dnl4/lig however, copy-number reduction in yeast reduced apoptosis, whilst knockdown in mammalian systems has been reported to increase its occurrence. however, both of these reports involve a complete deletion of the gene, rather than the heterozygous deletion in which we observed the yeast phenotype. this suggests that further investigation in human cells of the effects of varying gene dosage of these genes, in particular, would be worthwhile. haploproficiency & cancer drug sensitivity given the strong connection that we have observed between yeast haploproficiency and human cancer, it is unsurprising that the human orthologs of many of the hpgi genes are the targets of various anti-cancer compounds. however, in the light of the increased growth upon reduction of hp gene dosage, and the dosage-specific phenotypes reported above, it is possible that inhibitor treatment of a tumour cell could elicit the opposite to the desired response – i.e. increased proliferation rather than cell death – if complete ablation of the hp target function is not achieved. therefore, we examined the drug sensitivity of wild-type yeast, and the heterozygous and homozygous deletion mutants for each of the non-essential hpgi genes. we also included the deletion mutants for an additional five yeast genes, which are hp and whose products are orthologous to specific cancer-drug targets. where the human ortholog of a yeast hpgi gene product is a cancer-drug target we included in our screen either the commercial drug or alternative inhibitors. in total, drugs were screened: two of these were specific alternative inhibitors; were compounds either approved for human cancer treatment or shown to be effective in cell culture; and one control treatment of clotrimazole, a fungicide not known to have anti-cancer properties. drug sensitivity profiles were clustered by agglomerative hierarchical clustering. the profile elicited by a compound can reveal its mode of action, and thus similarities between drug profiles may indicate the targeting of the same pathways in yeast. reassuringly, known modes of action are reflected in our profiles – for example, the sensitivity of cell wall integrity/morphogenesis deletion mutants to phleomycin, which causes cell wall lesions. control clotrimazole treatments are reasonably distinct from the cancer drug treatments. aminopterin and methotrexate pd cycloheximide and mitoxantrone cluster together, driven largely by the resistant phenotypes of deletion mutants of genes involved in chromosome condensation and segregation. a second cluster is formed by bay cantharidin and vorinostat, all of which act as inhibitors of the nf-κb pathway in mammalian cells. this pathway is absent in s.cerevisiae however, the clustering of the compounds suggests that their modes of action, though distinct from the mammalian-cell pathway, may also be related in yeast. rad54/rad mutants are particularly resistant to all three drugs, where, in general, these mutants are sensitive to all other treatments. vorinostat has been demonstrated to induce dsbs in acute myeloid leukemia cells, suggesting that the dsb-repair function of rad54p could potentially be a target. indeed, the rad ortholog, rad54l, is strongly down-regulated in lncap cells treated with vorinostat. this is also the case for the mammalian orthologs of chk and top which are also resistant to the vorinostat /bay11/cantharidin cluster. we have since demonstrated that the magnitude of these drug-response phenotypes in yeast is sufficient to translate into observable phenotypes in tumour cell lines. targeting the cnv profile of tumour cells where a copy of a gene is frequently lost in tumour cells, as is the case for orthologs of the hpgi set, the phenotype of the heterozygous deletion mutant of the yeast ortholog might better reflect the tumour cell’s response to drug treatment. for a significant number of homozygous/heterozygous deletant pairs the drug sensitivity phenotypes are quantitatively dissimilar. for these genes, therefore, gene dosage may be highly relevant to pharmaceutical efficacy. these eight genes have, on average, a greater number of protein-protein interactors than the average for our hpgi set as a whole. this suggests that gene dosage balance may be an important contributor to phenotype for genes which are more central in interaction networks. we have explored the relationship between gene copy number and drug response further in. previously, we reported the striking result that treatment of wild-type yeast with low concentrations of inhibitors targeting hp genes can induce the same increased proliferative phenotype as is observed upon reducing the copy number of the hp target, a phenomenon we termed hypertolerance. we observe this phenotype again for treatment with cantharidin, which inhibits the pp2a complex, the subunits of which are strongly haploproficient, but not for any of the general cytotoxic compounds in the drug screen, reinforcing our previously-established link between the specific inhibition of a haploproficient target and drug hypertolerance. a number of heterozygous strains also exhibited hypertolerance in solid-media screens, which was subsequently confirmed by titrating across a range of drug concentrations in liquid medium. for tumour cells bearing the orthologous cnvs, this would suggest a contraindication for treatment with the particular drug. in particular, the nbp2/nbp heterozygote is multiply drug-hypertolerant, of note since its ortholog sh3gl frequently has reduced copy-number in serous cystadenocarcinoma. conversely, for pnp the heterozygotes are mitoxantrone/methotrexate/aminopterin and vorinostat/bay11/cantharidin sensitive, and the homozygotes resistant. this phenotype may be a instance of ‘obligate haploinsufficiency’, whereby fitness is compromised as the gene dosage is reduced below some threshold value, but the phenotype is subsequently rescued through the switching on of a compensatory pathway. discussion the data presented here for the s.cerevisiae hpgi set point to the striking result that copy-number variation, rather than complete gene loss, is sufficient to elicit deleterious phenotypes, in particular those reminiscent of cancer in mammalian cells. this concurs with the increasing appreciation of the significance of copy-number variation to the genesis and progression of disease. in particular, there is a general lack of significant correlation of phenotypes between the heterozygous and homozygous deletion mutants of a given gene. nor are the phenotypes of the null mutants necessarily more extreme than those of the heterozygous deletants, as might naively be expected from a linear ‘dosage hypothesis’. this emphasises the need to systematically investigate the phenotypic impact of gene dosage, rather than relying on knockout, or total-knockdown, studies. a mechanism by which heterozygous deletion of a gene could be just as disruptive as a complete knockout is via the disruption of the stoichiometric ratios of the sub-units of protein complexes. this has been proposed as a mechanism for haploinsufficiency in the'balance hypothesis' of papp et al.. it also suggests that dosage compensation, in which the impact of the heterozygous deletion of a gene is mitigated by the increased expression of the remaining copy, does not play a significant role amongst our hpgi gene set. indeed, showed that such dosage compensation is, in general, rare in s.cerevisae, and very few hp genes are amongst those exhibiting significant compensation. for cell cycle phasing, heterozygous gene deletion is often sufficient to perturb the cycle from the wt profile, which indicates that cell cycle proteins are required in yeast at greater than 50% of wt dosage. similarly, heterozygous deletion of the yeast ortholog is, in general, both sufficient to alter the degree of apoptosis occurring in response to dna-damaging agents, and to elicit the same apoptotic phenotype as is reported for complete protein-product knockdown in mammalian cells. this general conservation of apoptotic phenotypes suggests that the dna damage-related pathway controlling apoptosis in s.cerevisiae are similar to those in mammals. this could be investigated further by undertaking a global survey of the effect of gene deletion on apoptosis rates in yeast, particularly since the current data on the degree of apoptosis in s.cerevisiae is limited. in four cases, an altered apoptosis rate was observed in the yeast deletion mutants but has not, so far, been reported for the rnai knock-downs of their human orthologs. we would suggest that, based on the apparent predictive value of the yeast phenotype, these genes should become the focus of an rnai study in mammalian cells. hp genes and the efficacy of anti-cancer drugs this critical importance of considering gene dosage in the context of pharmaceutical intervention in cancer is further emphasised in the copy-number-dependence of drug sensitivity phenotypes that we observed with our yeast model. the mechanisms by which copy-number variation may exert a significant effect on phenotype are summarised in figure  firstly, products of the mammalian orthologs of several of the yeast hpgi set are the targets of specific drugs used, or proposed for use, in treating cancers. our data suggest that, in a majority of such cases, complete inhibition of the activity of a target protein product is necessary to achieve a positive therapeutic outcome. incomplete inhibition, analogous to heterozygous deletion of the gene encoding the target, prompts increased proliferation or hypertolerance – the opposite of the intended anti-cancer effect. furthermore, even if a given gene is not the intended specific target of a chemotherapeutic treatment, the modes-of-action, and secondary impacts, of many cancer drugs have not been fully elucidated. drug-induced haploinsufficiency data from yeast can contribute to a better understanding of both drug mechanisms and the functional conservation of drug-metabolism pathways between yeast and humans. for example, the clustering of mutant phenotypes in response to the mammalian-nf-κb inhibitors in this study suggests that all three compounds act through a common pathway in s.cerevisiae, which may be mediated by rad54p. secondly, we observed that varying the copy number of the yeast ortholog can significantly alter the phenotypic response to drug treatment. this is especially for genes whose human orthologs have a high likelihood of cnv in tumour cells, in particular, several deletion strains are either more resistant than the wt, or themselves hypertolerant in response to a specific drug treatment. the impact of specific cnvs on drug sensitivity is becoming increasingly appreciated, and we believe our yeast mutant approach represents a high-throughput complement to the creation of drug-cnv profile ‘fingerprints’ for tumour cells, and a model for determining the most effective drug interventions for tumours with a particular cnv profile. lastly, our data on the sensitivity/resistance of deletion mutants could potentially inform the design of combination therapies. where a particular deletion mutant is sensitive to a given drug, a combination therapy consisting of that drug plus an inhibitor of the product of the heterozygous gene could have a synergistic effect in reducing cell proliferation. conversely, considering pairs of genes/drugs for which the deletion mutant is resistant to treatment could assist in identifying those drug combinations which are unlikely to succeed, reducing the search space involved in an otherwise expensive combinatorial problem. finally, the several instances in which we observed that the heterozygous deletion mutant is more sensitive to drug treatment than the homozygote provide evidence for the ‘obligate haploinsufficiency’ hypothesis of berger et al.. identifying such genes, and defining the protein product threshold below which their associated compensatory pathways are activated, could help to define the precise inhibitor dosage to achieve maximal therapeutic efficacy.  haploproficient genes and are for which, when one copy is deleted from a diploid cell, the rate of cell proliferation is increased. we have demonstrated that, for a subset of yeast hp genes, heterozygous deletion causes aberrant cell cycling and altered rates of apoptosis, which are phenotypes associated with cancer in mammalian cells. a majority of these yeast genes are the orthologs of mammalian cancer genes, and hence our studies suggest that cnv of these oncogenic orthologs may be sufficient to lead to tumourigenesis in human cells. moreover, where not already implicated, this cluster of cancer-like phenotypes in this model eukaryote may be predictive of the involvement in cancer of the mammalian orthologs of these yeast hp genes. using the yeast set as a model, we have shown that the response of the heterozygotes to a range of anti-cancer drugs is strongly dependent on gene dosage, such that intermediate concentrations of the drugs can actually increase a mutant’s growth rate. this suggests such compounds may need to achieve near-total inhibition of target activity if they are to be a beneficial, rather than a deleterious, therapy. 
using the yeast set as a model, we show that the response to a range of anti-cancer drugs is strongly dependent on gene dosage, such that intermediate concentrations of the drugs can actually increase a mutant’s growth rate. our yeast data also suggest that the identification of cnvs in tumour cells may assist both the selection of anti-cancer drugs and the dosages at which they should be administered if they are to be a beneficial, rather than a deleterious, therapy. using the genome-wide deletion library for the model eukaryote, saccharomyces cerevisiae, we have identified genes which, when one copy is deleted from a diploid cell, result in an increased rate of proliferation. a majority of these yeast genes are the orthologs of mammalian cancer genes, and hence our studies suggest that cnv of these oncogenic orthologs may be sufficient to lead to tumourigenesis in human cells. copy-number variation, rather than complete loss of gene function, is increasingly implicated in human disease. we show that, for a subset of hp genes, heterozygous deletion is sufficient to cause aberrant cell cycling and altered rates of apoptosis, phenotypes associated with cancer in mammalian cells. moreover, where not already implicated, this cluster of cancer-like phenotypes in this model eukaryote may be predictive of the involvement in cancer of the mammalian orthologs of these yeast hp genes. however, the identification of cnv-related phenotypes for mammalian genes is a slow process, due to the technical difficulty of constructing deletion mutants. the exploitation of data on the phenotypic impact of heterozygosis in saccharomyces cerevisiae has permitted the prediction of cnvs affecting tumourigenesis in humans. since haploproficiency under nutrient-sufficient conditions is a novel phenotype, we sought here to characterise a subset of the yeast haploproficient genes which seem particularly relevant to human cancers. moreover, gene dosage is recognised as important in tumourigenesis, and there is an increasing realisation that cnvs may not be just symptomatic of the cancerous state but may, in fact, be causative.
 copy-number variation, rather than complete loss of gene function, is increasingly implicated in human disease. moreover, gene dosage is recognised as important in tumourigenesis, and there is an increasing realisation that cnvs may not be just symptomatic of the cancerous state but may, in fact, be causative. however, the identification of cnv-related phenotypes for mammalian genes is a slow process, due to the technical difficulty of constructing deletion mutants. using the genome-wide deletion library for the model eukaryote, saccharomyces cerevisiae, we have identified genes which, when one copy is deleted from a diploid cell, result in an increased rate of proliferation. since haploproficiency under nutrient-sufficient conditions is a novel phenotype, we sought here to characterise a subset of the yeast haploproficient genes which seem particularly relevant to human cancers.  we show that, for a subset of hp genes, heterozygous deletion is sufficient to cause aberrant cell cycling and altered rates of apoptosis, phenotypes associated with cancer in mammalian cells. a majority of these yeast genes are the orthologs of mammalian cancer genes, and hence our studies suggest that cnv of these oncogenic orthologs may be sufficient to lead to tumourigenesis in human cells. moreover, where not already implicated, this cluster of cancer-like phenotypes in this model eukaryote may be predictive of the involvement in cancer of the mammalian orthologs of these yeast hp genes. using the yeast set as a model, we show that the response to a range of anti-cancer drugs is strongly dependent on gene dosage, such that intermediate concentrations of the drugs can actually increase a mutant’s growth rate.  the exploitation of data on the phenotypic impact of heterozygosis in saccharomyces cerevisiae has permitted the prediction of cnvs affecting tumourigenesis in humans. our yeast data also suggest that the identification of cnvs in tumour cells may assist both the selection of anti-cancer drugs and the dosages at which they should be administered if they are to be a beneficial, rather than a deleterious, therapy. copy-number variationmodel organismyeastcancerhaploinsufficiency 