Histone H4 Dosage Effects Figure 4. Growth defect and phenotypic instability of mutants containing a single allele of histone H4. A small colony of a histone H4 mutant (DAY1072) was re-streaked on YPD medium and incubated 4 days at 30uC (A), close-up picture (B). Re-isolated small (C) and large (C, E) colonies of hhf22-hht2D/hhf22-hht2D HHF1/hhf1D (DAY1072), and small (D) and large (D, F) colonies of HHF22-HHT2/hhf22-hht2D hhf1D/hhf1D (DAY1074 and DAY1079) incubated on YPD for 48 hrs at 30uC. doi:10.1371/journal.pone.0010629.g004 HHT2/hhf22-hht2D hhf1D/D small colonies (data not shown), indicating that the suppressor phenotype may be reversible. These results suggest that the severe growth defect caused by the low dosage of both HHF1 and HHF22 confers a strong selective pressure for the generation of secondary suppressor mutations, which restore growth. Further, the differences observed in the emergence of small colonies during re-isolation of large colonies in both type of mutants is likely a consequence of the stability of the suppressors that arose in the mutants. Aneuploidy as a mechanism for H4 dosage compensation in C. albicans Two copies of histone H4 are necessary and sufficient for wild- type growth. Thus, we reasoned that the increased colony size and phenotypic stability observed in the large suppressor colonies reflected an increase in histone H4 copy number. Since aneuploi- dies are common in C. albicans strains [5,7,8,10,11,13,48,74–78], we hypothesized that the large suppressor colonies had duplicated the remaining histone H4 gene, thereby restoring the number of histone H4 alleles to two, which supports normal and phenotypically stable growth (Figure 3). The duplication of large DNA fragments, including whole chromosomes, as a mechanism to suppress a slow growth defect has also been described in S. cerevisiae [79]. In order to determine if the large colonies of the hhf22-hht2D/D HHF1/hhf1D and HHF22-HHT2/hhf22-hht2D hhf1D/D mutants had increased the number of wild-type H4 allele copies, we performed quantitative Southern blot analysis. Two probes, Probe D and Probe G, were designed to anneal equally to both the wild- type and the mutated versions of HHF1 or HHF22, respectively (Figure 1). Densitometry analysis was performed on Southern blots of small and large colonies isolated from the mutants to determine the ratio of mutated to wild-type histone H4 allele (Figure 6). We included the HHF22-HHT2/hhf22-hht2D HHF1/hhf1D heterozy- gous strain DAY1070 as a control, which had a 1:1.1 hhf22D/ HHF22 ratio by quantitative Southern blot (Figure 6B). Indeed, the small colonies of the histone mutants retained a 1:0.9–1.1 ratio of mutated to wild-type H4 alleles while the large colonies presented a 1:1.5–2.6 ratio of mutated to wild-type H4 alleles (Figure 6A and 6B). Thus, the large colonies of the hhf22-hht2D/D HHF1/hhf1D and HHF22-HHT2/hhf22-hht2D hhf1D/D mutants analyzed showed an increase in DNA associated with the wild-type histone H4 compared to the small colonies. Thus, the suppressor colonies arise by increasing the genomic dosage of histone H4. We reasoned that an increase in histone H4 copy number could involve either a segmental aneuploidy or a trisomy. Using comparative genome hybridization (CGH) arrays, we found that, as expected, a small colony of hhf22-hht2D/D HHF1/hhf1D had a diploid content of chromosome 1 (Figure 7). However, CGH analysis of a large suppressor colony derived from the hhf22-hht2D/ D HHF1/hhf1D small colony revealed a trisomy of chromosome 1. Thus, a reduction in histone H4 dosage causes a severe growth PLoS ONE | www.plosone.org 7 May 2010 | Volume 5 | Issue 5 | e10629