Rme1

[Elisabet Schwartzkopf]

In haploid cells of Ogataea (Hansenula) polymorpha an environmental signal, nitrogen starvation, induces a reversible change in the structure of a chromosome. This process, mating-type switching, inverts a 19-kb DNA region to place either MATa or MATα genes under centromeric repression of transcription, depending on the orientation of the region. Here, we investigated the genetic pathway that controls switching. We characterized the transcriptomes of haploid and diploid O. polymorpha by RNAseq in rich and nitrogen-deficient media, and found that there are no constitutively a-specific or α-specific genes other than the MAT genes themselves. We mapped a switching defect in a sibling species (O. parapolymorpha strain DL-1) by interspecies bulk segregant analysis to a frameshift in the transcription factor EFG1, which in Candida albicans regulates filamentous growth and white-opaque switching. Gene knockout, overexpression and ChIPseq experiments show that EFG1 regulates RME1, which in turn regulates STE12, to achieve mating-type switching. All three genes are necessary both for switching and for mating. Overexpression of RME1 or STE12 is sufficient to induce switching without a nitrogen depletion signal. The homologous recombination genes RAD51 and RAD17 are also necessary for switching. The pathway controlling switching in O. polymorpha shares no components with the regulation of HO in S. cerevisiae, which does not involve any environmental signal, but it shares some components with mating-type switching in Kluyveromyces lactis and with white-opaque phenotypic switching in C. albicans.

The molecular mechanisms of self-fertility (homothallism) vary enormously among fungal species. We previously found that in the yeast Ogataea polymorpha, homothallism is achieved by a novel mating-type switching mechanism that exchanges the locations of MATa and MATα genes between expression and repression contexts. Switching in this species is induced by nitrogen depletion, unlike the analogous process in Saccharomyces cerevisiae. Here, we show that the upstream parts of the genetic pathway controlling the environmental induction of switching in O. polymorpha are the same as the environmental pathway that induces competence for mating in this species.

Copyright: 2017 Hanson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

In yeast species (unicellular fungi) that can reproduce sexually, the ability of a cell to mate with other cells is governed by which mating-type genes it expresses [1, 2]. In ascomycete yeasts, these genes are located at a single genomic site called the mating-type (MAT) locus. Mating generally occurs between two haploid cells with opposite genotypes (MATa and MATα) at this locus, to form a diploid zygote (MATa/α). In some ascomycete yeasts such as Saccharomyces cerevisiae, haploid cells are able to change their MAT genotypes by a process called mating-type switching [3, 4]. During this process, DNA at the MAT locus is physically replaced, exchanging a MATa allele for a MATα allele or vice versa. Mating-type switching is a form of secondary homothallism [5] because it enables a yeast strain to mate with any other strain of the same species, regardless of their initial mating types, by means of fusion between a-cells and α-cells [6, 7].

O. polymorpha chromosome 3 contains both a MATa locus and a MATα locus, approximately 19 kb apart (Fig 1B). The two MAT loci are beside two copies of an identical 2-kb DNA sequence that form an inverted repeat (IR) on the chromosome. During mating type switching, the two copies of the IR recombine, inverting the orientation of the 19-kb region relative to the rest of the chromosome. The centromere of chromosome 3 is located just to the left of the left copy of the IR (Fig 1B). The MAT locus proximal to the centromere is not transcribed, probably due to silencing by centromeric heterochromatin, whereas the distal MAT locus is transcribed. By inverting the 19-kb region, mating type switching swaps the locations of the MATa and MATα genes, repressing the MAT genes that were previously expressed, and expressing the ones that were previously repressed. Similar flip/flop mating type switching mechanisms are now known in three other Saccharomycotina species (Komagataella phaffii, Pachysolen tannophilus, and Ascoidea rubescens) [4, 11, 12].

Mating type switching in O. polymorpha is induced by an environmental signal, nitrogen depletion [10, 11]. In a culture transferred into media that contains no nitrogen, up to approximately 25% the cells in the culture switch their mating type (Fig 1C). This situation, in which an environmental signal reproducibly induces a DNA rearrangement at a specific chromosomal locus, is unusual in biology and we were motivated to investigate its mechanism. Our aim in the current study was to identify the pathway in O. polymorpha that detects the environmental signal and executes rearrangement of chromosome 3 in response. A priori, we know that the pathway in O. polymorpha must be quite different from the pathway that regulates mating-type switching in S. cerevisiae [3, 13], because switching in S. cerevisiae is not regulated by the environment and occurs even in rich media, and because O. polymorpha has no ortholog of the S. cerevisiae HO endonuclease gene. Therefore, both the upstream (nitrogen-sensing) and downstream (DNA inversion) parts of the pathway in O. polymorpha must be different from S. cerevisiae. Furthermore, since the DNA rearrangements that occur during switching in S. cerevisiae, O. polymorpha and Kluyveromyces lactis are all substantially different but are descendants of a common ancestral switching mechanism [4, 14, 15], we were interested to determine how the pathways that regulate these rearrangements have evolved.

To identify components of the switching pathway in O. polymorpha, we used several strategies including transcriptomic analysis, candidate gene approaches, and mapping the defective gene in a naturally-occurring mutant that is unable to switch mating types. We identified five genes that are required for switching. Although we were unable to deduce all the steps that lead from nitrogen depletion to mating type switching, we infer that O. polymorpha senses nitrogen depletion using the Protein Kinase A (PKA) pathway, which then transmits a signal via Ste12 to induce mating and/or mating type switching, and that recombination between the IRs is mediated by the homologous recombination pathway for DNA repair. We compare the roles of genes in the O. polymorpha pathway to the roles of their orthologs in other species.

To examine the transcriptional response induced by nitrogen depletion, we used mRNAseq to compare the transcriptomes of O. polymorpha cells 2 h after transfer from a YPD pre-induction culture into NaKG, to parallel cultures transferred into fresh YPD. Furthermore, because we expect that switching occurs only in haploid cells, we conducted this experiment in parallel on haploid (MATa and MATα isogenic strains) and diploid (MATa/α) cells.

Growth of all three cell types in NaKG resulted in a robust transcriptional response to nitrogen depletion, with a large number of genes significantly up- or down-regulated relative to YPD (S1 Fig; S1 Table). Regardless of cell type, homologs of S. cerevisiae genes for nitrogen starvation responses were induced, such as transporters of amino acids (DIP5, GAP1), urea (DUR3), and allantoate (SEO1), and amidases for the release of amide groups from urea (DUR1,2), pyrimidines (PYD3), or other substrates (AMD2). Ribosomal protein genes were strongly repressed, as expected because of the reduced growth rate in NaKG (S1 Table). However, orthologs of S. cerevisiae genes with mating or sporulation functions were not induced by these nitrogen depletion conditions alone, even though mating (of haploids) and sporulation (of diploids) can be induced by plating cells onto similar nitrogen-depleted solid media [17]. Among the genes strongly upregulated in NaKG were two transcription factors, RME1 and CZF1-like3 (one of three O. polymorpha co-orthologs of C. albicans CZF1, which is a singleton zinc finger gene with no S. cerevisiae ortholog [18]). Both of these genes were uniformly induced in all three cell types (MATa, MATα and MATa/α), with CZF1-like3 upregulated 69- to 93-fold, and RME1 upregulated 26- to 79-fold, upon transfer into NaKG (S1 Table).

In S. cerevisiae, defined sets of a- and α-specific genes that allow haploid cells to identify and respond to the presence of a mating partner are well established [19]. These genes are constitutively expressed in S. cerevisiae cells of the appropriate mating type. Surprisingly, comparison of gene expression between haploid O. polymorpha a-cells and α-cells in either NaKG or YPD media revealed that there are essentially no constitutive a- or α-specific genes in this species, apart from the MAT genes themselves (S1 Fig; S2 Fig). All haploid cells of O. polymorpha contain four MAT genes (MAT α1, α2, a1, and a2), and the orientation of the 19-kb region specifies whether the α1 and α2 genes, or the a1 and a2 genes, are placed at the expression site (Fig 1B). In NaKG, transcription of α1 and α2 was respectively 53-fold and 39-fold higher in α-cells than in a-cells; a2 was 31-fold lower, and a1 was just 2-fold lower. In YPD, α1 and α2 were 4-fold and 9-fold higher, a1 was 6-fold lower, and a1 showed no difference. KAR4, which in S. cerevisiae is a general pheromone-induced gene [20] required for fusion of the haploid nuclei after mating, showed moderately higher expression in a-cells than in α-cells (2 to 3-fold; S2 Fig). No other genes showed more than a 2-fold difference in transcription between a- and α-cells, in either of the two media (S2 Fig; S2 Table). This result contrasts sharply with S. cerevisiae, where for example several a-specific genes such as MFA2, STE2 and BAR1 have more than 10-fold higher expression in MATa than MATα cells in YPD [19]. It is also consistent with previous observations that expression of the pheromone receptors STE2 and STE3 in haploid O. polymorpha is independent of cell type [10].

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