Zinc finger transcription factors displaced SREBP proteins as the major Sterol regulators during Saccharomycotina evolution

Abstract

In most eukaryotes, including the majority of fungi, expression of sterol biosynthesis genes is regulated by Sterol-Regulatory Element Binding Proteins (SREBPs), which are basic helix-loop-helix transcription activators. However, in yeasts such as Saccharomyces cerevisiae and Candida albicans sterol synthesis is instead regulated by Upc2, an unrelated transcription factor with a Gal4-type zinc finger. The SREBPs in S. cerevisiae (Hms1) and C. albicans (Cph2) have lost a domain, are not major regulators of sterol synthesis, and instead regulate filamentous growth. We report here that rewiring of the sterol regulon, with Upc2 taking over from SREBP, likely occurred in the common ancestor of all Saccharomycotina. Yarrowia lipolytica, a deep-branching species, is the only genome known to contain intact and full-length orthologs of both SREBP (Sre1) and Upc2. Deleting YlUPC2, but not YlSRE1, confers susceptibility to azole drugs. Sterol levels are significantly reduced in the YlUPC2 deletion. RNA-seq analysis shows that hypoxic regulation of sterol synthesis genes in Y. lipolytica is predominantly mediated by Upc2. However, YlSre1 still retains a role in hypoxic regulation; growth of Y. lipolytica in hypoxic conditions is reduced in a Ylupc2 deletion and is abolished in a Ylsre1/Ylupc2 double deletion, and YlSre1 regulates sterol gene expression during hypoxia adaptation. We show that YlSRE1, and to a lesser extent YlUPC2, are required for switching from yeast to filamentous growth in hypoxia. Sre1 appears to have an ancestral role in the regulation of filamentation, which became decoupled from its role in sterol gene regulation by the arrival of Upc2 in the Saccharomycotina.

Figure 1. Conservation of sterol regulatory proteins in fungi.

(A) Schematic phylogenetic tree of Saccharomycotina species and outgroups. Two domains from the SREBP proteins are shown diagrammatically as red (bHLH) and purple (DUF2014) boxes. Transmembrane domains are indicated by wavy lines; probabilities of <40% are not shown (details in Figure S1). The asterisk indicates a partially conserved DUF2014 domain in M. guilliermondii. The presence of Upc2-like proteins is indicated by + (one ortholog) or ++ (two orthologs). The presence/absence of Scap proteins is indicated with +/−. CTG = CTG clade, S = Saccharomycetaceae, WGD = Whole Genome Duplication clade. (B) Alignment of the bHLH domain from SREBP proteins from mammals (yellow), Basidiomycetes (green) and Ascomycetes (orange, blue, pink, white). The alignment was generated using MUSCLE implemented in SeaView . The conserved Tyr residue is indicated with an asterisk. Clades are colored as in (C). (C) Unrooted phylogenetic tree of fungal SREBP-like proteins and mammalian SREBPF1/2. The tree was constructed from an alignment of bHLH regions (133 amino acid sites) by a maximum likelihood method. All sequences contain the atypical Tyr residue shown in (B). See Supplementary Figure S6 for taxon names and branch support values.

Figure 2. Upc2 has no clear orthologs outside the Saccharomycotina.

This tree was constructed from sequences of Upc2 (and its ohnolog Ecm22 in WGD species); Lys14; and all Zn₂Cys₆ zinc finger protein genes in the genomes of two representative outgroup species, Aspergillus nidulans (magenta branches) and Komagataella pastoris (cyan branches) that have significant BLASTP hits (E<1e-6) when Upc2 proteins are used as a query. Also included are top-scoring BLASTP hits from other Pezizomycotina species (black branches). The tree was drawn by the neighbor-joining method without correction for multiple hits due to the high extent of sequence divergence. Bootstrap support for two key branches is indicated. NCBI gene identifier (gi) numbers are shown for the three sequences closest to the yellow Upc2/Ecm22 clade, but there is no evidence that these proteins are Aspergillus orthologs of Upc2. Scale bar represents 0.2 amino acid substitutions per site.

Figure 3. Role of YlUpc2 and YlSre1 in hypoxic growth, drug resistance and iron uptake.

(A) YlUPC2 controls susceptibility to azole drugs. YlUPC2 and YlSRE1 were deleted as described in Figure S2, and the deletion and re-constituted strains were plated as serial dilutions on YPD or synthetic complete (SC) media with additions as noted, and incubated in normoxic or hypoxic (1% O₂) conditions. Deleting YlUPC2 reduces growth in the presence of ketoconazole on YPD and SC media. Deleting YlUPC2 or YlSRE1 reduces growth in hypoxia, particularly on SC media. The strains shown are (in order): JMY2900, SMY2, SMY6, SMY5, SMY7 and SMY4. (B) Deleting YlUPC2 reduces sterol content. The strains were grown in defined synthetic media and sterol levels were measured by absorbance as described in methods. The strains are the same as in (A), except that two independent deletions of YlSRE1 were tested. The difference between the Ylupc2 deletion and the wild type is statistically significant (P-value<0.05). (C) YlUPC2 is required for iron uptake. The strains listed in (A) were spotted as serial dilutions on YPD, YPD +BPS and YPD+BPS+ additional iron.

Figure 4. YlSre1 and YlUpc2 regulate filamentation.

(A) Wild type (JMY2900), Ylupc2 deletion (SMY2) and UPC2 re-integration (SMY6), Ylsre1 deletion (SMY5) and YlSRE1 re-integration (SMY7), and ylupc2/Ylsre1 double deletion (SMY4) strains were grown overnight in liquid YPD or liquid SC media containing methionine, in normoxic or hypoxic (1% O₂) conditions. Tween80 (1%) was added where indicated. The cells are stained with Calcofluor White. (B) The strains listed in (A) were grown on solid YPD or SC media in hypoxic or normoxic conditions for 2 days. Cells were removed from individual colonies, stained with Calcofluor White, and photographed.

Figure 5. YlUpc2 regulates expression of ergosterol genes.

(A) Hierarchical clustering of wild type (JMY2900), Ylupc2Δ (SMY2), and Ylsre1Δ (SMY5 and SMY8), all compared in hypoxia vs normoxia. Yellow indicates decreased expression in hypoxic growth. One cluster is shown in more detail. The full heatmap with gene names and descriptions is available in Figure S7. (B) Overlap in genes differentially expressed in hypoxia in wild type (JMY2900), Ylupc2Δ (SMY2) and Ylsre1Δ (SMY5 and SMY8) backgrounds. (C) Illustration of the sterol synthesis pathway in fungi. Changes in expression of indicated genes in wild type cells during growth in hypoxia compared to normoxia (Hyp) or in Ylupc2 deletion strains compared to wild type cells both grown in hypoxia (upc2Δ) are indicated. Blue color indicates decreased expression and black color indicates increased expression. No changes in expression of genes in gray were identified.

Figure 6. YlUPC2 and YlSRE1 regulate expression of ergosterol genes during hypoxic adaptation.

Expression of the indicated ERG pathway genes was tested using qRT-PCR. All strains were grown to an A₆₀₀ of 1.0 in SC media at 28°C under normoxic conditions (21% O₂) and then switched to an hypoxic environment (1% O₂) for 2 hours. Expression was normalized against actin, and is shown relative to the wild type strain grown in normoxia. Standard error of three replicates is shown. ** P<0.05, *** P<0.005.

Figure 7. Model of sterol regulon evolution in Saccharomycotina.

The hypothesized ancestral state, with sterol synthesis (ERG) genes and filamentation genes both being regulated by SREBP and Scap, is shown on the left. The DUF2014 domain of SREBP is shown in blue. SREBP replaced Upc2 in the promoters of ergosterol genes in the Saccharomycotina, but retains some role ergosterol metabolism in Y. lipolytica. Upc2 also regulates filamentation in Y. lipolytica (not shown). SREBP is known to have a role in regulation of filamentation in the three species shown. SREBP may have a similar function in A. fumigatus, where the deletion affects hyphal branching . Loss of Scap is also indicated.