Author response: Loss of centromere function drives karyotype evolution in closely related Malassezia species (2020)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Genomic rearrangements associated with speciation often result in variation in chromosome number among closely related species. Malassezia species show variable karyotypes ranging between six and nine chromosomes. Here, we experimentally identified all eight centromeres in M. sympodialis as 3–5-kb long kinetochore-bound regions that span an AT-rich core and are depleted of the canonical histone H3. Centromeres of similar sequence features were identified as CENP-A-rich regions in Malassezia furfur, which has seven chromosomes, and histone H3 depleted regions in Malassezia slooffiae and Malassezia globosa with nine chromosomes each. Analysis of synteny conservation across centromeres with newly generated chromosome-level genome assemblies suggests two distinct mechanisms of chromosome number reduction from an inferred nine-chromosome ancestral state: (a) chromosome breakage followed by loss of centromere DNA and (b) centromere inactivation accompanied by changes in DNA sequence following chromosome–chromosome fusion. We propose that AT-rich centromeres drive karyotype diversity in the Malassezia species complex through breakage and inactivation. eLife digest Millions of yeast, bacteria and other microbes live in or on the human body. A type of yeast known as Malassezia is one of the most abundantmicrobes living on our skin. Generally, Malassezia do not cause symptoms in humans but are associated with dandruff, dermatitis and other skin conditions in susceptible individuals. They have also been found in the human gut, where they exacerbate Crohn’s disease and pancreatic cancer. There are 18 closely related species of Malassezia and all have an unusually small amount of genetic material compared with other types of yeast. In yeast, like in humans, the genetic material is divided among several chromosomes. The number of chromosomes in different Malassezia species varies between six and nine. A region of each chromosome known as the centromere is responsible for ensuring that the equal numbers of chromosomes are passed on to their offspring. This means that any defects in centromeres can lead to the daughter yeast cells inheriting unequal numbers of chromosomes. Changes in chromosome number can drive the evolution of new species, but it remains unclear if and how centromere loss may have contributed to the evolution of Malassezia species. Sankaranarayanan et al. have now used biochemical, molecular genetic, and comparative genomic approaches to study the chromosomes of Malassezia species. The experiments revealed that nine Malassezia species had centromeres that shared common features such as being rich in adenine and thymine nucleotides, two of the building blocks of DNA. Sankaranarayanan et al. propose that these adenines and thymines make the centromeres more fragile leading to occasional breaks. This may have contributed to the loss of centromeres in some Malassezia cells and helped new species to evolve with fewer chromosomes. A better understanding of how Malassezia organize their genetic material should enable in-depth studies of how these yeasts interact with their human hosts and how they contribute to skin disease, cancer, Crohn’s disease and other health conditions. More broadly, these findings may help scientists to better understand how changes in chromosomes cause new species to evolve. Introduction Centromeres are the genomic loci on which the kinetochore, a multi-subunit complex, assembles to facilitate high-fidelity chromosome segregation. The centromere-specific histone H3 variant CENP-A is the epigenetic hallmark of centromeres, as it replaces canonical histone H3 in the nucleosomes to make specialized centromeric chromatin that acts as the foundation to recruit other kinetochore proteins. A remarkable diversity in the organization of centromere DNA sequences has been observed to accomplish this conserved role (Roy and Sanyal, 2011; Yadav et al., 2018b). The smallest known centromeres are the point centromeres present in budding yeasts of the family Saccharomycetaceae that span <200 bp in length (Clarke and Carbon, 1980; Gordon et al., 2011; Kobayashi et al., 2015). These centromeres are organized into conserved DNA elements I, II, and III that are recognized by a cognate kinetochore protein complex called the CBF3 complex, making them genetically defined centromeres. Small regional centromeres, identified in several Candida species, form the second category (Sanyal et al., 2004; Padmanabhan et al., 2008; Kapoor et al., 2015; Chatterjee et al., 2016) and have a 2–5-kb region enriched by kinetochore proteins. These centromeres can either have unique DNA sequences or a homogenized core that is flanked by inverted repeats. The third type of centromere structure is the large regional centromere, which is often repetitive in sequence and spans more than 15 kb. Large regional centromeres can be transposon-enriched, as in Cryptococcus species, or organized into repeat structures around a central core, as in Schizosaccharomyces pombe (Chikashige et al., 1989; Clarke and Baum, 1990; Sun et al., 2017; Yadav et al., 2018b). Although the organization of DNA elements is variable, a majority of known centromeres share AT-richness as a common feature. Examples include the CDEII of point centromeres, central core sequences in S. pombe, and centromeres of Neurospora crassa, Magnaporthe oryzae, Plasmodium falciparum, and diatoms (Fitzgerald-Hayes et al., 1982; Iwanaga et al., 2010; Rhind et al., 2011; Kapoor et al., 2015; Diner et al., 2017; Yadav et al., 2019). Even the recently described mosaic centromere structure observed in Mucor circinelloides that has lost CENP-A comprises an AT-rich kinetochore-bound core region (Navarro-Mendoza et al., 2019). Although suppression of recombination around centromeres has been correlated with reduced GC content (Lynch et al., 2010), the genetic underpinning that determines how an AT-rich DNA region favors kinetochore assembly remains unclear. Ironically, AT-rich sequences have been shown to be fragile sites within a chromosome (Zhang and Freudenreich, 2007). Several lines of evidence suggest that centromeres are species-specific and are among the most rapidly evolving genomic regions, showing variation even between closely related species (Bensasson et al., 2008; Padmanabhan et al., 2008; Rhind et al., 2011; Roy and Sanyal, 2011). This evolution is accompanied by the concomitant evolution of CENP-A and the associated kinetochore proteins (Talbert et al., 2004). Functional incompatibilities between centromeres result in uniparental genome elimination in interspecies hybrids (Ravi and Chan, 2010; Sanei et al., 2011). The divergent nature of centromeres is proposed to be a driving force for speciation (Henikoff et al., 2001; Malik and Henikoff, 2009). Asexual organisms, by virtue of inter- and intra-chromosomal rearrangements, diversify into species clusters that are distinct in genotype and morphology (Barraclough et al., 2003). These genotypic differences include changes in both chromosomal organization and number. Centromere function is directly related to karyotype stabilization following a change in chromosome number. Rearrangements in the form of telomere–telomere fusions and nested chromosome insertions (NCIs), wherein an entire donor chromosome is ‘inserted’ into or near the centromere of a non-homologous recipient chromosome, are among the major sources of chromosome number reduction (Lysak, 2014). Such events often result in the formation of dicentric chromosomes that are subsequently stabilized by breakage-fusion-bridge cycles (McClintock, 1941) or via inactivation of one centromere through different mechanisms (Han et al., 2009; Sato et al., 2012). Well-known examples of telomere–telomere fusions include the formation of extant human chromosome 2 by fusion of two ancestral chromosomes (Yunis and Prakash, 1982; IJdo et al., 1991), the reduction in karyotype observed within members of the Saccharomycotina such as Candida glabrata, Vanderwaltozyma polyspora, Kluyveromyces lactis, and Zygosaccharomyces rouxii (Gordon et al., 2011), and the exchange of chromosomal arms seen in plants and fungi (Schubert and Lysak, 2011; Sun et al., 2017). NCIs have predominantly shaped karyotype evolution in grasses (Murat et al., 2010). Reduction of chromosome number by centromere loss has also been reported (Gordon et al., 2011). To investigate whether centromere breakage can be a natural source of karyotype diversity in closely related species, we sought to identify centromeres in a group of Malassezia yeast species that exhibit variation in chromosome number. Malassezia species are lipid-dependent basidiomycetous fungi that are naturally found as part of the animal skin microbiome (Theelen et al., 2018). At present, the Malassezia genus includes 18 species divided into three clades: A, B, and C. These species also have unusually compact genomes of less than 9 Mb, organized into six to nine chromosomes as revealed by electrophoretic karyotyping of some of these species (Boekhout and Bosboom, 1994; Boekhout et al., 1998; Wu et al., 2015). Fungemia-associated species such as Malassezia furfur belong to Clade A. Clade B includes common inhabitants of human skin that are phylogenetically clustered into two subgroups, namely Clade B1 that contains Malassezia globosa and Malassezia restricta and Clade B2 that contains Malassezia sympodialis and related species. Clade C includes Malassezia slooffiae and Malassezia cuniculi, which diverged earlier from a Malassezia common ancestor (Wu et al., 2015; Lorch et al., 2018). Besides humans, Malassezia species have been detected on the skin of other animals. For example, M. slooffiae was isolated from cows and goats, M. equina from horses, M. brasiliensis and M. psittaci from parrots, and the cold-tolerant species M. vespertilionis from bats (Lorch et al., 2018; Theelen et al., 2018). In addition, culture-independent studies of fungi from environmental samples showed that Malassezia species that are closely related to those found on human skin were also detected in diverse niches, such as deep-sea vents, soil invertebrates, hydrothermal vents, corals, and Antarctic soils (Amend, 2014). More than ten Malassezia species have been detected as a part of the human skin microbiome (Findley and Grice, 2014). The human skin commensals such as M. globosa, M. restricta, and M. sympodialis have been associated with dermatological conditions such as dandruff/seborrheic dermatitis, atopic dermatitis, and folliculitis (Theelen et al., 2018). Recent reports implicate M. restricta in conditions such as Crohn’s disease and M. globosa in the progression of pancreatic cancer (pancreatic ductal adenocarcinoma) (Aykut et al., 2019; Limon et al., 2019). Elevated levels of Malassezia species and the resulting inflammatory host response have been implicated in both of these disease states. The nature of genomic rearrangements in each species may influence its ability to adapt and cause disease in a specific host niche. Thus, studying the mechanisms of karyotype evolution is an important step towards understanding the evolution of the Malassezia species complex. Kinetochore proteins serve as useful tools in the identification of centromeres because of their centromere-exclusive localization. CENP-A replaces histone H3 in the centromeric nucleosomes. This has been shown as a reduction in histone H3 levels at the centromeres in Candida lusitaniae (Kapoor et al., 2015) and in a human neocentromere (Lo et al., 2001). These CENP-A nucleosomes act as a foundation to recruit CENP-C, the KMN (KNL1C-MIS12C-NDC80C) network, and other kinetochore proteins (Musacchio and Desai, 2017). In this study, we experimentally validated all of the eight centromeres of M. sympodialis using the Mtw1 protein (Mis12 in S. pombe), a subunit of the KMN complex, as the kinetochore marker. The Mis12 complex proteins are evolutionarily conserved outer kinetochore proteins that link the chromatin-associated inner kinetochore proteins to the microtubule-associated outer kinetochore proteins. Members of the Mis12 complex localize to centromeres in other organisms (Goshima et al., 1999; Goshima et al., 2003; Westermann et al., 2003; Roy et al., 2011). Recent studies suggest that the protein domains associated with the Mis12 complex members are exclusive to kinetochore proteins and are not detected in any other proteins, making them attractive tools for identifying centromere sequences (Tromer et al., 2019). Using the features of centromeres of M. sympodialis and newly generated chromosome-level genome assemblies, we predicted centromeres in related Malassezia species carrying seven, eight, or nine chromosomes, and experimentally validated the centromere identity in representative species of each karyotype, each belonging to a different Malassezia clade. We employed gene synteny conservation across these centromeres to understand their transitions from an inferred ancestral state of nine chromosomes. On the basis of our results, we propose that centromere loss by two distinct mechanisms drives karyotype diversity. Results Chromosome number varies in the Malassezia species complex Previous reports that are based on pulsed-field gel electrophoresis (PFGE) have suggested that chromosome number varies within the Malassezia species complex. The early diverged species M. slooffiae of Clade C was reported to have nine chromosomes (Boekhout et al., 1998). Clade B Malassezia species are reported to have nine (M. globosa and M. restricta), eight (M. sympodialis), or six chromosomes (M. pachydermatis). Among the Clade A species, M. obtusa and M. furfur CBS14141 were both reported to have seven chromosomes (Boekhout and Bosboom, 1994; Zhu et al., 2017). A high-quality reference genome is a prerequisite to understanding the rearrangements associated with chromosome number variation. In addition, such a reference genome will also assist in resolving ambiguities in PFGE-based estimates of chromosome number when similar-sized chromosomes are present. Complete genome assemblies were not available for many of the species with reported numbers of chromosomes. To obtain better-assembled reference genomes, we sequenced the genomes of M. slooffiae and M. globosa as representatives of the nine-chromosome state, and of M. furfur as a representative of the seven-chromosome state, using PacBio SMRT sequencing technology (Figure 1, Figure 1—figure supplement 1). Figure 1 with 1 supplement see all Download asset Open asset Genome assembly and karyotype diversity in representative Malassezia species. The genomes of (A) M. globosa, (B) M. slooffiae, and (C) M. furfur were sequenced and assembled in this study, whereas the genome assembly of (D) M. sympodialis was reported earlier (Zhu et al., 2017) and is shown for comparison. In each panel, bar plots represent the assembled chromosomes of the indicated Malassezia species, with the telomeres and the ribosomal DNA (rDNA) marked as dark gray and blue bars, respectively. Telomere-repeat motifs are shown at the 5′- and 3′-ends of the Chr1 in each species. Electrophoretic karyotypes of M. globosa (Mg) and M. sloffiiae (Msl), are shown in (A) and (B), respectively, with chromosome sizes estimated from the genome assembly. Chromosomes of Saccharomyces cerevisiae (Sc) served as size markers. The chromosome containing the rDNA (marked with an ‘r’), in M. globosa, co-migrates with Chr3. This was assessed by chromoblot hybridization using unique sequences from Chr3, Chr4, Chr5, and Chr6 as probes (regions indicated by red arrowheads). Chromosomes of similar size (denoted in red) migrate together in the gel and appear as a doublet band (i.e. MgChr3–MgChr5, MslChr2–MslChr3, and MslChr5–MslChr6). The M. globosa genome was completely assembled into nine contigs with telomeres on both ends (BioSample accession SAMN10720087). We validated these contigs by matching each band on the pulsed-field gel with the contig sizes from the genome assembly, and further confirmed these by chromoblot analysis following PFGE. This analysis shows that chromosome 5 contains the rDNA locus and migrates further than the expected size of 902 kb, as a diffuse ensemble of different sizes along with chromosome 3 (Figure 1A). The assembled genome of M. slooffiae has 14 contigs of which nine contigs have telomeres on both ends, indicative of nine chromosomes (BioSample accession SAMN10720088). Each of the nine contigs could be assigned to the bands observed in the pulsed-field gel (Figure 1B). For M. furfur, the final genome assembly consisted of seven contigs with telomeres on both ends and matched the expected chromosome sizes obtained from an earlier PFGE analysis of CBS14141 (Figure 1C). The complete genome assembly of M. sympodialis reported earlier is distributed into eight chromosomes with telomere repeats on both ends (Figure 1D), and serves as a representative of an eight-chromosome state in this study. Changes in chromosome number are always associated with the birth or loss of centromeres, which stabilizes the karyotype in organisms with monocentric chromosomes. To understand the transitions between these different karyotypic states observed in the Malassezia species complex, we sought to validate centromeres in species representative of each karyotype experimentally. Kinetochores cluster and localize to the nuclear periphery in M. sympodialis Organisms that have point centromeres possess Ndc10, Cep3, and Ctf13 of the CBF3 complex, a cognate protein complex that is specific to point centromeres. None of these point-centromere-specific proteins could be detected in M. sympodialis. However, we could detect homologs of CENP-A, CENP-C, and most of the outer kinetochore proteins in the genome of M. sympodialis (Figure 2A and Figure 2—figure supplement 1). We functionally expressed an N-terminally GFP-tagged Mtw1 protein (Protein ID: SHO76526.1) from its native promoter, and the expression of the fusion protein was confirmed by western blotting (Figure 2B). Upon staining with anti-GFP antibodies and DAPI (4′,6-diamidino-2-phenylindole), we were able to detect punctate localization of Mtw1 at the nuclear periphery (Figure 2C), consistent with the clustered kinetochore localization observed in other yeasts (Goshima et al., 1999; Euskirchen, 2002; Roy et al., 2011). Live-cell images of MSY001 (GFP-MTW1) cells revealed that the kinetochores (GFP-Mtw1) remained clustered throughout the cell cycle, starting from unbudded G1 cells in interphase to large-budded cells in mitosis (Figure 2D). Figure 2 with 1 supplement see all Download asset Open asset Kinetochores cluster and localize at the nuclear periphery in M. sympodialis. (A) Schematic of the kinetochore organization of M. sympodialis. Gray boxes indicate proteins absent in M. sympodialis. The outer kinetochore protein Mtw1 (a component of Mis12C) served as the kinetochore marker in the present study. (B) Line diagram representation of Mtw1 tagged with GFP at the N- terminus. Immunoblot analysis of whole-cell lysates prepared from the untagged M. sympodialis strain (ATCC42132) and from GFP-Mtw1 expressing cells (MSY001) probed with anti-GFP antibodies and anti-PSTAIRE antibodies. PSTAIRE served as a loading control. Relative intensity values normalized to PSTAIRE are indicated below each lane. (C) Logarithmically grown MSY001 cells expressing GFP-Mtw1 were fixed and stained with DAPI (blue) and anti-GFP antibodies (pseudo-colored in red). Scale bar, 2.5 µm. (D) Cell cycle stage-specific localization dynamics of GFP-Mtw1. Scale bar, 2.5 µm. Mtw1 is localized to a single region at the GC minima of each M. sympodialis chromosome Having identified Mtw1 as an authentic kinetochore protein, we performed ChIP-sequencing using the GFP-Mtw1 expressing strain of M. sympodialis (MSY001). Mapping the reads to the reference genome of M. sympodialis strain ATCC42132 (Zhu et al., 2017) identified one significantly enriched locus on each of the eight chromosomes (Figure 3A). The lengths of the Mtw1-enriched centromere regions identified from the ChIP-seq analysis ranged from 3167 bp to 5143 bp with an average length of 4165 bp (Table 1). However, the region of maximum Mtw1 enrichment on each chromosome (based on the number of sequenced reads aligned) mapped to the intergenic region harboring the GC trough (approximately 1 kb long), which was previously predicted to be the centromeres of M. sympodialis (Figure 3B) (Zhu et al., 2017). The regions of Mtw1 enrichment span beyond the core centromeres and include active genes located proximal to these troughs (Figure 3B, Figure 3—figure supplement 1A). However, these open reading frames (ORFs) do not show consensus features such as the orientation of transcription or functional classification. We validated this enrichment by ChIP-qPCR analysis with primers to the predicted centromeres compared to those to a region from the centromere (Figure 3—figure supplement 1B). Figure 3 with 3 see all Download asset Open asset of Mtw1 to centromeres on each of the eight chromosomes of M. sympodialis. (A) GFP-Mtw1 ChIP-seq reads were mapped to each The chromosomal and the reads from reads from Mtw1-enriched regions by reads from those of the values observed in both and on are from the rDNA (B) A of the Mtw1 enrichment as and in two different red) along with the GC content and regions of transcription in the in indicate chromosomal The for the are as reads Gray in each indicate predicted based on data with the of transcription of the also marked as and in the (C) in Mtw1 and histone H3 enrichment at as compared to a region kb on the of by Schematic of a region of with the core is below the lines indicate regions by to the GC and bp from the bp from the and a region kb from centromere in The regions across the probed by and the in the enrichment of Mtw1 and histone H3 as compared to the indicate from three each performed with three were used to the (D) of the consensus DNA sequence identified from M. sympodialis centromeres, with the size of the with the of Figure data 1 data for Figure for GFP-Mtw1 and H3 across Download 1 of centromeres and their GC content in M. sympodialis. and length of Mtw1-enriched regions in with those of the core centromeres in M. sympodialis. Chromosome Genome average GC content H3 is depleted at the core centromere with active genes at the regions in M. sympodialis The of CENP-A nucleosomes at the kinetochore should result in reduced histone H3 enrichment at the centromeres when compared to a To we performed with H3 antibodies and the DNA by compared to a region that is to the centromere kb from the regions the core centromere showed a reduction in histone H3 which was further reduced at the core, that to the GC trough with the enrichment of the kinetochore protein the core centromere region showing the maximum of histone H3 with the regions most enriched with Mtw1 further that histone in these regions, is by its centromere-specific variant CENP-A (Figure The regional centromeres of M. sympodialis are enriched with a AT-rich consensus sequence To understand the features of M. sympodialis centromeres, we the centromere DNA sequences for the of consensus motifs or structures such as inverted repeats. et al., predicted a AT-rich common to all of the centromere sequences of M. sympodialis (Figure We the from the across each chromosome of M. sympodialis and the number of in a by bp at a with of were as The is the natural of the of the of as a from the to the of it being In each the with the centromere (Figure 3—figure supplement In each chromosome, the centromere region shows between and whereas other shows more than three This suggests that the AT-rich is more enriched at the centromeres than at any other region in the M. sympodialis genome (Figure 3—figure supplement To that this is not an of the nature of the centromere, we the analysis with a by the of the of the the in the the the This showed more in the centromeres than are seen in the genomic but significantly fewer than are seen in the authentic centromeric sequences of most chromosomes (Figure 3—figure supplement 2B). A analysis was performed to detect the of any or structure associated with the centromeres in M. sympodialis. Analysis of all of the centromere sequences and sequences using confirmed the of repeat structures (Figure 3—figure supplement In the of any centromere-exclusive DNA the unique and features of centromere regions in M. sympodialis are an AT-rich core region of kb content of as compared to the genome average of that is enriched with the (Figure 3—figure supplement within a kinetochore region of kb. the kinetochore-bound region contains a reduced of histone H3. Centromeres in M. furfur, M. slooffiae, and M. globosa to chromosomal GC minima Using the unique centromere features identified in M. we predicted one centromere locus on each of the seven M. furfur chromosomes, and these all to chromosomal GC troughs (Figure supplement We also predicted the centromeres in M. slooffiae, M. globosa, and M. restricta, each of which contains nine chromosomes (Figure supplement Each of the predicted centromere regions is enriched with the AT-rich identified in M. sympodialis centromere sequences as compared to other regions in the genomes (Figure supplement 2 and GC content of the centromeres predicted in M. furfur, M. globosa, M. slooffiae, and M. GC furfur globosa slooffiae restricta