Histone H4 Dosage Effects Figure 7. CGH analysis of a hhf22-hht2D/hhf22-hht2D HHF1/hhf1D (DAY1072) small colony (A) and a spontaneous large colony suppressor (B). Genomic DNA was purified from both a DAY1072 small colony and large colony and compared to genomic DNA from SC5314 (DAY963). The DAY1072 small colony is monosomic for Chromosome 3 while the DAY1072 large colony is trisomic for Chromosome 1. Both isolates have a short segmental aneuploidy on Chromosome 5R, which is present in the strain background [12,48]. doi:10.1371/journal.pone.0010629.g007 with no obvious effects on growth or colony morphology [74]. Therefore, the alterations in colony size observed in both histone H4 mutants could be attributed to the gain and loss of a segment or an entire copy of Chromosome R or Chromosome 1. Histone H4 deficit causes colony morphology changes When growing on agar plates at 30uC, C. albicans normally forms smooth, round, cream-colored colonies composed of yeast cells. Wrinkly colonies are generally composed of a higher percentage of cells that are filamenting, a fact that explains why it is possible to exacerbate some colony morphology phenotypic differences at 37uC. Altered C. albicans colony morphologies have been detected in strains isolated from infected patients, from studies in mouse models, and can also be induced in the laboratory through UV irradiation and genetic manipulation [4,14,88–93]. The changes in colony morphology are a manifestation of underlying genomic changes that can involve a group of genes (like in the white-opaque switching) or major karyotypic rearrangements [9]. Thus, alterations in diverse factors can lead to changes in colony morphology. When small, smooth colonies that carry only one H4 allele are sub-cultured onto a rich medium plate they give rise to colonies that have different morphologies (Figure 5A). The change in colony morphologies of small colonies was generally penetrant, although they also gave rise to colonies with other types of morphologies (Figure 5B). On the contrary, the large and smooth suppressor colonies did not produce colonies with altered morphology when they were sub-cultured, indicating that their colony morphology phenotype is stable (Figure 5C). One exception to this statement is the already mentioned formation of small colonies from re-streaking of the large colonies, which most likely arise by the loss of the duplicated chromosome R. One explanation for the formation of semi-penetrant morpho- logical variants in the histone mutants is karyotypic rearrange- ments. As previously mentioned, altered colony morphologies have been associated with altered karyotypes or with loss of heterozygozity [4,9,94]. Imbalances in histone dosage can lead to chromosome missegregation [28–31]. Further, the histone mutants grow slowly, a condition that might be more permissive to the References accumulation and tolerance of aneuploidies [4]. This latter idea is supported by the presence of a monosomic chromosome 3 in a hhf22-hht2D/D HHF1/hhf1D small colony (Figure 7). Thus, karyotypic rearrangements favored by the combination of the slow growth and the nucleosomal deficit of the histone H4 mutants might be one mechanism behind the formation of colony variants. Candida albicans is the most important human fungal pathogen, causing serious infections in immunosuppressed individuals. C. albicans has a diploid genome with an unexpectedly high level of heterozygozity, given the primarily clonal reproductive style of this organism [95]. The genome of C. albicans has a remarkably high tolerance for genomic rearrangements. The ability to thrive with an altered karyotype may provide a profound advantage to this organism, because it represents a potential source of genetic variation [5]. Karyotypic rearrangements and aneuploidies in C. albicans are associated with pathogenesis: they affect cellular and colonial morphology, increase metabolic diversity, are required for mating, and, importantly, constitute a mechanism of antifungal resistance. Histone modifying enzymes and chromatin remodeling proteins are also required for pathogenesis in C. albicans [16–22,96]. The study of chromatin dynamics and structure in this fungus therefore is critical for understanding the nature of C. albicans pathogenicity and, furthermore, it may uncover potential targets for antifungal therapies. In this study, we have generated and characterized strains that can be used for future analysis of specific histone H4 mutant alleles, in order to begin to dissect the function and impact of epigenetic regulation in C. albicans lifestyle and pathogenesis. Acknowledgments We are grateful to members of the Davis lab for helpful discussions and critical review of the manuscript. We are indebted to Tim Leonard in the UMN Department of Microbiology with photographic assistance. Author Contributions Conceived and designed the experiments: LFZ DD. Performed the experiments: LFZ AMS. Analyzed the data: LFZ AMS DD. Contributed reagents/materials/analysis tools: AMS JB. Wrote the paper: LFZ DD. 1. Pfaller MA, Diekema DJ, Jones RN, Sader HS, Fluit AC, et al. (2001) International Surveillance of Bloodstream Infections Due to Candida Species: Frequency of Occurrence and In Vitro Susceptibilities to Fluconazole, Ravuconazole, and Voriconazole of Isolates Collected from 1997 through PLoS ONE | www.plosone.org 10 May 2010 | Volume 5 | Issue 5 | e10629