Summary How different cell types with the same genotype are formed and heritability maintained is a fundamental question in biology. We utilized white-opaque switching in Candida albicans as a system to study mechanisms of cell type formation and maintenance. Each cell type has tractable characters, which are maintained over many cell divisions. Cell type specification is under the control of interlocking transcriptional feedback loops, with Wor1 being the master regulator of the opaque cell type. Here we show that deletion of RTT109, encoding the acetyltransferase for histone H3K56, impairs stochastic and environmentally stimulated white-opaque switching. Ectopic expression of WOR1 mostly bypasses the requirement for RTT109, but opaque cells lacking RTT109 cannot be maintained. We have also discovered that nicotinamide induces opaque cell formation, and this activity of nicotinamide requires RTT109. Reducing the copy number of HST3, which encodes the H3K56 deacetylase, also leads to increased opaque formation. We further show that the Hst3 level is down regulated in the presence of genotoxins and ectopic expression of HST3 blocks genotoxin induced switching. This finding links genotoxin induced switching to Hst3 regulation. Together these findings suggest RTT109 and HST3 genes play an important role in the regulation of white-opaque switching in C. albicans.

CAF-1 and HIR are highly conserved histone chaperone protein complexes that function in the assembly of nucleosomes onto chromatin. CAF-1 is characterized as having replication-coupled nucleosome activity, whereas the HIR complex can assemble nucleosomes independent of replication. Histone H3K56 acetylation, controlled by the acetyltransferase Rtt109 and deacetylase Hst3, also plays a significant role in nucleosome assembly. In this study, we generated a set of deletion mutants to genetically characterize pathway-specific and overlapping functions of CAF-1 and HIR in C. albicans. Their roles in epigenetic maintenance of cell type were examined by using the white-opaque switching system in C. albicans. We show that CAF-1 and HIR play conserved roles in UV radiation recovery, repression of histone gene expression, correct chromosome segregation, and stress responses. Unique to C. albicans, the cac2⌬/⌬ mutant shows increased sensitivity to the Hst3 inhibitor nicotinamide, while the rtt109⌬/⌬ cac2⌬/⌬ and hir1⌬/⌬ cac2⌬/⌬ mutants are resistant to nicotinamide. CAF-1 plays a major role in maintaining cell types, as the cac2⌬/⌬ mutant exhibited increased switching frequencies in both directions and switched at a high frequency to opaque in response to nicotinamide. Like the rtt109⌬/⌬ mutant, the hir1⌬/⌬ cac2⌬/⌬ double mutant is defective in maintaining the opaque cell fate and blocks nicotinamide-induced opaque formation, and the defects are suppressed by ectopic expression of the master white-opaque regulator Wor1. Our data suggest an overlapping function of CAF-1 and HIR in epigenetic regulation of cell fate determination in an H3K56 acetylation-associated manner.

The glomerular capillary wall functions as both a size-selective and charge-selective barrier. Heparan sulfate is known to be an important component of the charge-selective barrier to filtration of polyanions. We studied the alterations in both the charge and size selectivity barriers in a model of experimental membranous nephropathy in the rabbit. The fractional clearance of both charged and uncharged dextrans compared to inulin was measured. Sulfate incorporation into glycosaminoglycans was measured and the glomerular heparan sulfate was isolated and biochemically characterized. Membranous nephropathy in the rabbit was induced with daily injections of cationic bovine serum albumin. After three weeks of injection animals had 735 +/- 196 mg/24 hours of protein excretion. There was no change in [35S] incorporation in 24 hours by experimental animals, 440 +/- 91 DPM/mg dry weight of glomeruli, N = 9 versus 410 +/- 98, N = 11 in controls. The percentage of [35S] incorporated into heparan sulfate versus chondroitin sulfate was decreased, 60% +/- 3 versus 79% +/- 2, P less than 0.001. Heparan sulfate from membranous nephropathy eluted from ion exchange chromatography in a lower molarity salt, indicating a lower effective charge. Fractional clearance of neutral dextrans was significantly increased in membranous nephropathy for dextrans greater than 48 A, while fractional clearance of dextran sulfates was significantly increased compared to controls for dextrans greater than 32 A. Thus, in membranous nephropathy there is loss of both charge selectivity and size selectivity. The loss of charge selectivity correlated with a change in the structure of the glomerular heparan sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)

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