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.

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.

In this study, we experimentally validated the chromosome number in M. slooffiae and M. globosa by PFGE analysis. We sequenced and assembled the genomes of M. slooffiae, M. globosa, and M. furfur and compared each one with the genome of M. sympodialis in order to understand the karyotype differences observed in members of the Malassezia species complex. These species represent each of the three major clades of Malassezia species with chromosome numbers ranging from seven to nine. Because centromere loss or gain directly influences the chromosome number of a given species, we experimentally identified the centromeres of these representative species to understand the mechanisms of karyotype diversity. Kinetochore proteins are useful tools in identifying the centromeres of an organism. The localization of the evolutionarily conserved kinetochore protein Mtw1 suggested that kinetochores are clustered throughout the cell cycle in M. sympodialis. ChIP-sequencing analysis identified short regional (<5 kb long) centromeres in M. sympodialis that are depleted of histone H3, but enriched with an AT-rich sequence motif. The identification of centromeres in M. slooffiae, M. globosa, and M. furfur further suggested that centromere properties are conserved across these Malassezia species. By predicting putative centromeres in five other species and by considering four species with experimentally mapped centromeres described above across three clades of Malassezia, we concluded that an AT-rich centromere core of <1 kb in length enriched with the 12-bp sequence motif is a potential signature of centromeres in the nine Malassezia species analyzed in this study. Comparative genomics analysis revealed two major mechanisms of centromere inactivation resulting in karyotype change. The presence of a nine-chromosome state in each of the three clades, along with conserved centromere features and conserved gene synteny, helped us infer that the ancestral Malassezia species had nine chromosomes that had short regional centromeres with an AT-rich core enriched with the 12-bp sequence motif.

Centromeres in the Malassezia species complex represent the first example of short regional centromeres in basidiomycetes. Centromeres reported in other basidiomycetes, such as those in Cryptococcus and Ustilago species, are of the large regional type (Yadav et al., 2018b). The Malassezia species analyzed in this study have a significantly smaller genome (<9 Mb) than other basidiomycetes and lack RNAi machinery. The occurrence of short regional centromeres in RNAi-deficient Malassezia species is in line with a previous finding showing a reduction in centromere size in RNAi-deficient basidiomycete species as compared to their RNAi-proficient relatives (Yadav et al., 2018b). With the presence of clustered kinetochores across cell cycle stages, and the absence of key genes encoding the RNAi machinery, these Malassezia species resemble ascomycetes such as many of the CTG clade species with short regional centromeres (Sanyal et al., 2004; Nakayashiki et al., 2006; Padmanabhan et al., 2008; Kapoor et al., 2015; Chatterjee et al., 2016; Yadav et al., 2018a), rather than the basidiomycetes with large regional centromeres. By combining these features, we conclude that the genome size and the presence of complete RNAi machinery could determine the centromere type of a species, irrespective of the phylum to which it belongs.

Based on the binding patterns of the kinetochore protein across the M. sympodialis genome, the 3–5-kb long region can be divided into two domains: (a) an AT-rich CEN core that maps to the intergenic region containing the GC trough, which shows maximum kinetochore binding (<1 kb); and (b) the regions flanking the core, which show basal levels of kinetochore protein binding. We observed conservation of the 12-bp AT-rich motif in the centromere core across the nine Malassezia species. It should also be noted that the 12-bp motif is significantly enriched at the centromeres but is not exclusive to the centromeres as it is detected across the chromosomes. We did not observe any orientation bias for these motifs. The functional significance of the frequent occurrence of this motif at centromeres is unknown. It will be intriguing to test the roles played by this motif, the core, and the flanking sequences in centromere function. Testing these domains for centromere function in vivo by making centromeric plasmids in various Malassezia species is challenging at present because of technical limitations. Other than M. sympodialis, M. pachydermatis, and M. furfur, no other Malassezia species have been successfully transformed (Ianiri et al., 2016; Celis et al., 2017; Ianiri et al., 2017a). Moreover, in all of these cases, the genetic manipulations are performed by Agrobacterium-mediated transconjugation, which cannot be used for the introduction of circular plasmids. Hence, the functional significance of the 12-bp motif remains unknown and remains an important question to be addressed in the future to provide an understanding of centromere function in these species.

The centromeres in M. sympodialis contain transcribed ORFs, which have also been documented in the centromeres of rice, maize, and Zymoseptoria tritici (Nagaki et al., 2004; Wang et al., 2014). In contrast to these cases, our read count analysis did not reveal any significant difference in the transcription (RPKM values) of centromere-associated ORFs and ORFs elsewhere in the genome of M. sympodialis. We posit that the 12-bp AT-rich motif sequences could facilitate the transcription of these genes by recruiting transcription factors that have a possible role in kinetochore assembly. Binding of Cbf1 at CDEI in S. cerevisiae centromeres and of Ams2 at the central core sequences of S. pombe centromeres are classic examples of transcription factors facilitating kinetochore stability in fungal systems (Hemmerich et al., 2000; Chen et al., 2003). A fine regulation of transcription by Cbf1, Ste12, and Htz1, and the resulting low-level cenRNAs, have been implicated in proper chromosome segregation in budding yeast (Ohkuni and Kitagawa, 2011; Ling and Yuen, 2019). These studies reinforce the role of transcription in centromere function irrespective of the centromere structure.

In this study, we report three high-quality chromosome-level genome assemblies and identified centromeres in nine Malassezia species, representing all of the three Malassezia clades with differing numbers of chromosomes. This will serve as a rich resource for comparative genomics in the context of niche adaptation and speciation. Analysis of gene synteny conservation across centromeres using these genomes revealed breakage at the centromere as one of the mechanisms that results in a karyotype change between closely related species — those with nine chromosomes, such as M. slooffiae and M. globosa, and those with eight chromosomes, such as M. sympodialis. Gene synteny breakpoints adjacent to the centromeres have been reported in C. tropicalis, which has seven chromosomes, one less than C. albicans (Chatterjee et al., 2016). Centromere loss by breakage was proposed to have reduced the Ashbya gossypii karyotype by one when compared to the pre-whole genome duplication ancestor (Gordon et al., 2011). Breakpoints of conserved gene synteny between mammalian and chicken chromosomes were also mapped to the centromeres (International Chicken Genome Sequencing Consortium, 2004). Similar consequences in the karyotype have been reported in cases where centromeres were experimentally excised. Besides neocentromere formation, survival by fusion of acentric chromosome arms has been shown in S. pombe (Ishii et al., 2008). Such fusions are detected upon deletion of centromeres in another basidiomycete, C. deuterogattii (Schotanus and Heitman, 2019). By comparing the ancestral state (M. slooffiae) and other Malassezia species with either the same number or fewer chromosomes, we observed gene synteny breaks that were adjacent to centromeres (indicated by partial synteny conservation), in addition to the break observed at MgCEN2 or MslCEN5. Does this suggest that Malassezia centromeres are fragile in nature? We advance the following hypothesis to explain the observed breaks at centromeres.

Studies of the common fragile sites in the human genome suggest different forms of replication stress as a major source of instability and subsequent breakage at these sites (Helmrich et al., 2011; Letessier et al., 2011; Ozeri-Galai et al., 2011). The resolution of the resulting replication fork stall has been shown to be critical for the stability of these fragile sites (Schwartz et al., 2005). Studies of the human fragile site FRA16D show that the AT-rich DNA (Flex1) results in fork stalling as a consequence of cruciform or secondary structure formation (Zhang and Freudenreich, 2007). Centromeres are natural replication fork stall sites in the genome (Greenfeder and Newlon, 1992; Smith et al., 1995; Mitra et al., 2014). The AT-rich core centromere sequence in M. globosa is also predicted to form secondary structures (Figure 7—figure supplement 4), which can be facilitated by the inherent replication fork stall at the centromeres. Whenever these secondary structures are unresolved and the fork restart fails, double strand breaks (DSBs) can occur at the centromeres. Chromosomal breakage and aneuploidy resulting from such defects are known to occur in cancers (Kops et al., 2005). In mammals, centromeric DSBs are repaired efficiently compared to regions elsewhere in the genome, largely because of the presence of several homology tracts in the form of repetitive DNA sequences and the stiffness provided by the inherent heterochromatic state, which facilitates ligation (Rief and Löbrich, 2002). Malassezia species are haploid in nature and lack typical pericentric heterochromatin marks. Although the efficiency of centromeric DSB repair in the absence of long tracts of homologous sequences is not known in this species complex, we propose that the AT-rich core sequences, by virtue of secondary structure formation during DNA replication, could occasionally undergo DNA breakage at the centromere in Malassezia species.

The second mechanism of chromosome number reduction suggested by our analyses comparing the M. furfur genome with the genomes of M. slooffiae or M. globosa involves the inactivation of centromeres in the process of transition from a nine-chromosome state to a seven-chromosome state. Centromere inactivation occurs in cases involving the fusion of centric chromosomal fragments, stabilizing the fusion product and generating a functionally monocentric chromosome, as seen during the origin of human Chr2 from the ancestor shared with the great apes (Yunis and Prakash, 1982; IJdo et al., 1991). A larger proportion of known centromere inactivation events were shown to be mediated by epigenetic modifications, in which inactivated centromeres are enriched with marks such as H3K9me2/3, H3K27me2/3, or DNA methylation, emphasizing the role of heterochromatin in this process (Zhang et al., 2010; Koo et al., 2011; Sato et al., 2012). Deletion of the centromere sequence corresponding to kinetochore binding has also been reported as an alternate mechanism, albeit a less frequent mechanism, in both humans and in yeast (Stimpson et al., 2010; Gordon et al., 2011; Sato et al., 2012). In difference to what might be expected according to the two modes described above, we observed divergence in the sequences corresponding to the inactivated centromeres (CEN8 and CEN9 of both M. slooffiae and M. globosa) in the arms of M. furfur Chr3, resulting in the loss of AT-richness of these centromere core regions (Figure 7C). This is also suggestive of a functional role for AT-rich DNA in centromere function in these species.

A change in chromosome number between two closely related species such as C. albicans and C. tropicalis is associated with a change in centromere structure: centromeres in C. albicans are unique short regions that are epigenetically regulated, whereas those in C. tropicalis are associated with genetically defined homogenized inverted repeats (Chatterjee et al., 2016). Strikingly, in both the transitions described above for Malassezia species, we did not observe any change in the centromere structure. The emergence of evolutionarily new centromeres, as seen in primate evolution, was not detected in Malassezia species (Rocchi et al., 2009; Kalitsis and Choo, 2012). This is particularly striking in the absence of conservation of any specific centromere-exclusive DNA sequence. This suggests that a strong driving force helps to maintain the highly conserved centromere properties in closely related Malassezia species that descended from a common ancestor, even after extensive chromosomal rearrangements involving centromeres that might have driven speciation. Furthermore, centromere inactivation/loss of centromere function seems to be a conserved theme mediating variation in chromosome number from unicellular yeast species to metazoans, including primates.

The reagents, strains, plasmids, and primers used in this study are listed in the Key Resources Table below.

The Mtw1 ChIP sequencing reads reported in this paper have been deposited under NCBI BioProject (Accession number PRJNA509412). The genome sequence assemblies of M. globosa, M. slooffiae, and M. furfur have been deposited in GenBank with accession numbers SAMN10720087, SAMN10720088, and SAMN13341476 respectively.

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Acceptance summary:

In this manuscript the authors identify the centromeres of several Malassezia species to trace the events that led to karyotype development in this species complex. The authors mapped centromeres in species with 8 or 9 chromosomes by Mtw1 ChIP. They discovered a 12 bp AT-rich consensus that is unique for centromeres but whose functional significance remains to be determined. They show convincingly that the reduction of chromosome number was accompanied by a break of one chromosome at its centromere and subsequent fusion of the chromosome arms with other chromosomes to reduce the number of chromosomes in the karyotype by one.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your work entitled "Centromere-mediated chromosome break drives karyotype evolution in closely related Malassezia species" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Wolf-Dietrich Heyer as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Senior Editor.

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

As you can see from the reviews, there was a span of opinions, which converged at the end of our discussion. We all agreed that the manuscript is interesting and that the topic of karyotype evolution is important. If you elect to address the comments in a future submission to eLife, I would be happy to serve as a reviewing editor again.

Reviewer #1:

In this manuscript the authors identify the centromeres of several Malassezia species to identify the events that led to karyotype development in this species complex. The authors mapped centromeres in species with 8 or 9 chromosomes by Mtw1 ChIP and identified a 12 bp AT-rich consensus that is unique for centromeres. They show convincingly that the reduction of chromosome number was accompanied by a break of one chromosome at its centromere and subsequent fusion of the chromosome arms with other chromosomes to reduce the number of chromosomes in the karyotype by one.

1) The classic molecular marker for the centromere in CENP-A or CENP-C enrichment. The depletion of normal histone H3 is used here as an argument that the identified regions are centromeres, but the more classic criterion would be better.

As there is little doubt that the identified regions are centromeres, I am unsure, whether this experiment must be in the revision or whether it is simply a possible suggested addition.

Reviewer #2:

Changes of chromosome numbers are related to centromere inactivation events and/or chromosome fusions. Because karyotype changes seem to be related to speciation, it is important to characterize and compare centromere organization at genome level between closed species. In this paper, authors focused on Malassezia species, who belong to Basiodiomycota and have variable karyotypes. They experimentally defined 8 centromeres in Malassezia sympodialis by ChIP-seq with kinetochore protein Mtw1 and proposed a 12 bp consensus motif in centromeres. Furthermore, they examined synteny and predicted centromeres in other Malassezia species and found that some centromeres are located in a synteny breakpoint. Finally they proposed a model how karyotype diversity occurred through centromere breakage and inactivation during evolution.

Overall, subject itself is interesting and it important to understand karyotype evolution. However, to improve quality of the paper, some additional experiments are needed. My major concern is lack of functional analysis of the 12 bp sequence. Identification of this sequence is one of highlights in this paper, but there was no functional assay. Authors should present functional significance of this sequence. In addition, they also should confirm centromere sequence using ChIP experiments in other Malassezia species.

Reviewer #3:

This manuscript describes the identification of centromeres in Malassezia sympodialis using ChIP-seq of a GFP-tagged kinetochore protein (Mtw1). Centromeres are described as being 3-5 kb with an AT-rich core enriched with a 12 bp consensus motif. Using these features, the authors predict centromere locations in the genomes of several other Malassezia species, including M. globosa, M. slooffiae and M. restricta. These three species have 9 chromosomes, whilst M. sympodialis has 8. Mapping of synteny breaks between the genomes leads the authors to suggest that centromere loss has resulted in M. sympodialis having one less chromosome than the other species. The authors speculate that chromosome breakage due to merotelic centromere attachments drive karyotype diversity in Malessezia. The genomes of M. globosa and M. slooffiae are also assembled de novo.

The manuscript contains potentially interesting data on Malassezia centromeres and karyotype diversity. However, the merotely model presented is highly speculative and overplayed.

Merotely requires that a centromere/kinetochore is capable of binding at least two microtubules (in merotely a single kinetochore is bound to microtubules emanating from both poles). Point centromeres which bind only one microtubule are not capable of merotelic attachments. Large regional centromeres such as vertebrate centromeres bind multiple microtubules per centromere, as do the regional centromeres of S. pombe (2-4 microtubules bound per kinetochore). Smaller regional centromeres, such as those of Candida albicans (3-5 kb) have been reported to bind only one microtubule (e.g. Joglekar et al., 2008, Burrack et al., 2011). Thus, it is entirely possible, given the size of the centromeres in Malessezia (3-5 kb – or even smaller – see below) that each binds only one microtubule. If that were the case, merotelic attachments would not be possible. Is there any data available in these species to suggest that the kinetochores bind multiple microtubules? Have lagging chromosomes (typical of merotely) been observed in these species during anaphase? There is no discussion of these points in the manuscript.

Could other processes have led to rearrangements involving ancestral CEN2? Perhaps centromeres are for some reason fragile (i.e. due to AT-richness), leading to 'ordinary' rearrangements like those on chromosome arms, without invoking centromere-specific processes such as merotely?

Malesezzia centromeres are described as being 3-5 kb, but it looks as if the Mtw1 ChIP-seq reads cover approximately 1 kb. It is very hard to interpret the data because of the low resolution in Supplementary Figure 1A and the lack of scale in Supplementary Figure 1B. Zooming in further on the ChIP-seq and providing a diagram of the positions of features such as 12 bp motif and% GC would be helpful and informative.

Figure 3—figure supplement 2C and Supplementary Figure 1F. No indication of size/region covered. Figure 3—figure supplement 2 appears to show that there are several 12 bp motifs in a 500 bp window at centromeres, but these are not apparent in Figure 3—figure supplement 2C and Supplementary Figure 1F.

Some means of confirming centromere location in the other Malassezia species is needed. Whilst the difficulty in transforming different yeast species and therefore tagging proteins is understood, have the authors contemplated raising an antibody to Cse4/CENP-A or testing for cross-reactivity of an existing antibody? Failing that, other Malassezia centromeres would be expected to have a reduced level of histone H3 incorporated, where it is replaced by CENP-A, compared to chromosome arms (as in Supplementary Figure 1D).

Figure 2B: a negative control locus for GFP-Mtw1 should be included.

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "Loss of centromere function drives karyotype evolution in closely related Malassezia species" for further consideration by eLife. Your revised article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Kevin Struhl as the Senior Editor.

The manuscript has been improved but there a few remaining issues that need to be addressed before acceptance, as outlined below:

Essential revisions:

1) The identification of the 12 bp AT-rich consensus that is unique for centromeres may suggest functional importance, but this is currently not experimentally shown. The manuscript should clearly state that the importance of the 12 bp consensus sequence is not known.

Reviewer #2:

Authors of this manuscript have submitted a previous manuscript on karyotype in Malassezia species, which was rejected by eLife ~9 months ago. Here, they added various new data and submit a new manuscript. Changes of chromosome numbers might be associated with centromere inactivation or centromere brake events followed by chromosome fusions. Although comparative genome analyses in close species suggest such possibilities, it is not well characterized in very closed species. Because karyotype changes seem to be related to speciation, it is important to characterize and compare centromere organization at genome level between closed species. In this manuscript, authors focused on Malassezia species and characterized whole genome sequences and centromere positions of several Malassezia species, which have different numbers of chromosomes. Based on comparison of genome synteny and chromosome numbers, authors conclude that ancestral Malassezia species has 9 chromosomes, but chromosome breakage events or centromere inactivation events independently occur in different lineages, and these events associated with chromosome fusion caused reduction of chromosome numbers in some of Malassezia species.

I think that the manuscript is substantially revised and data themselves have high quality. However, as I asked in previous review, it is important to clarify the 12 bp sequence for centromere function. It is not clear whether the centromere is specified by sequence dependent or independent. In their model, the centromere is not created in speciation in Malassezia after centromere loss. In general, after centromere loss, chromosome can fuse with another chromosome or acentric chromosome can get a neocentromere. As this species does not form new centromere, it is really important to address whether the centromere is specified by sequence dependent or not. Although it might be hard to do genetic manipulation in this species, it might be possible to deplete the 12 bp motif. If they can show the significance of the 12bp sequence in any methods, paper would be suitable for eLife.

In addition, they evaluated CENP-A accumulation as reduction of H3. Quantitative ChIP-seq is not easy. If they want to do that, they should normalize seq-reads using the Spike-in method.

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

Reviewer #1:

[…]

1) The classic molecular marker for the centromere in CENP-A or CENP-C enrichment. The depletion of normal histone H3 is used here as an argument that the identified regions are centromeres, but the more classic criterion would be better.

As there is little doubt that the identified regions are centromeres, I am unsure, whether this experiment must be in the revision or whether it is simply a possible suggested addition.

We thank reviewer 1 for the careful review and the positive feedback on the data set. We concur with the reviewer 1’s comment that CENP-A and CENP-C are the classical markers for centromeres. The proteins of the Mis12 complex are evolutionarily conserved across phyla and are shown to be enriched at the centromeres in various organisms (Goshima et al., 1999; Westerman et al., 2003; Goshima et al., 2003; Roy et al., 2011) and hence can be used as an authentic centromere marker. Also, the functionality of an epitope tagged CENP-A in a haploid organism remains uncertain. Taking these facts into consideration, we instead tagged the Mis12 homolog, Mtw1, with GFP to identify centromeres in M. sympodialis.

In centromeric nucleosomes, histone H3 is replaced by CENP-A. Reduced levels of histone H3 at the centromeres has been shown in Candida lusitaniae, in which centromeres are similar in length (Kapoor et al., 2015) to those observed in M. sympodialis. To confirm centromere identity of Mtw1-enriched regions in the M. sympodialis genome, we demonstrated reduced levels of histone H3 enrichment as it is expected that histone H3 molecules are replaced by centromere-specific histone H3 variant CENP-A. This further supports that the Mtw1-bound regions are indeed centromeres in M. sympodialis. This has been mentioned in the revised manuscript (Introduction: eighth paragraph; Results: subsection “Histone H3 is depleted at the core centromere with active genes at the pericentric regions in M. sympodialis”).

Reviewer #2:

[…]

Overall, subject itself is interesting and it important to understand karyotype evolution. However, to improve quality of the paper, some additional experiments are needed. My major concern is lack of functional analysis of the 12 bp sequence. Identification of this sequence is one of highlights in this paper, but there was no functional assay. Authors should present functional significance of this sequence.

We appreciate that the reviewer finds this study, and karyotype evolution in general, important and relevant. Functional analysis of the 12 bp motif identified by our study is challenged by technical limitations described below. Among the Malassezia species, only M. sympodialis, M. furfur, and M. pachydermatis have been successfully transformed in the laboratory. In all three cases, transformation was accomplished with Agrobacterium mediated trans-conjugation which is not amenable for episomal maintenance of plasmids. Any sequence provided within the T-DNA border elements is integrated into the genome upon transconjugation. This precludes us from performing functional assays such as stabilization of a plasmid containing the centromere or the associated 12 bp motif. This has been described in the Discussion section in the revised manuscript (third paragraph).

In addition, they also should confirm centromere sequence using ChIP experiments in other Malassezia species.

Following the reviewer’s recommendation, we have experimentally validated the centromeres in three other species: M. furfur, M. globosa, and M. slooffiae. These results are presented in Figure 4 of the revised manuscript (Results: subsection “Centromeres in M. furfur, M. slooffiae, and M. globosa map to chromosomal GC minima”).

1) For M. furfur, we generated strains functionally expressing CENP-A with a 3xFLAG at the carboxy terminus. All seven centromeres we predicted in M. furfur were validated by ChIP-qPCR using this strain (Figure 4B and C).

2) In the case of M. slooffiae and M. globosa, we used the reduction in histone H3 levels (as observed in M. sympodialis) to validate centromere identity. Histone H4 was used as a control for the presence of nucleosomes in both cases. The reduction in the relative abundance of H3/H4 at the centromeres as compared to a control region away from and unlinked to the centromere is included (Figure 4D, E). Furthermore, the enrichment profile of H3/H4 across one centromere and the flanking pericentric region in both M.

slooffiae and M. globosa has also been included (Figure 4F and G).

Reviewer #3:

This manuscript describes the identification of centromeres in Malassezia sympodialis using ChIP-seq of a GFP-tagged kinetochore protein (Mtw1). Centromeres are described as being 3-5 kb with an AT-rich core enriched with a 12 bp consensus motif. Using these features, the authors predict centromere locations in the genomes of several other Malassezia species, including M. globosa, M. slooffiae and M. restricta. These three species have 9 chromosomes, whilst M. sympodialis has 8. Mapping of synteny breaks between the genomes leads the authors to suggest that centromere loss has resulted in M. sympodialis having one less chromosome than the other species. The authors speculate that chromosome breakage due to merotelic centromere attachments drive karyotype diversity in Malessezia. The genomes of M. globosa and M. slooffiae are also assembled de novo.

The manuscript contains potentially interesting data on Malassezia centromeres and karyotype diversity. However, the merotely model presented is highly speculative and overplayed.

Merotely requires that a centromere/kinetochore is capable of binding at least two microtubules (in merotely a single kinetochore is bound to microtubules emanating from both poles). Point centromeres which bind only one microtubule are not capable of merotelic attachments. Large regional centromeres such as vertebrate centromeres bind multiple microtubules per centromere, as do the regional centromeres of S. pombe (2-4 microtubules bound per kinetochore). Smaller regional centromeres, such as those of Candida albicans (3-5 kb) have been reported to bind only one microtubule (e.g. Joglekar et al., 2008, Burrack et al., 2011). Thus, it is entirely possible, given the size of the centromeres in Malessezia (3-5 kb – or even smaller – see below) that each binds only one microtubule. If that were the case, merotelic attachments would not be possible. Is there any data available in these species to suggest that the kinetochores bind multiple microtubules? Have lagging chromosomes (typical of merotely) been observed in these species during anaphase? There is no discussion of these points in the manuscript.

Could other processes have led to rearrangements involving ancestral CEN2? Perhaps centromeres are for some reason fragile (i.e. due to AT-richness), leading to 'ordinary' rearrangements like those on chromosome arms, without invoking centromere-specific processes such as merotely?

We thank the reviewer for the comprehensive review and raising this point and giving us the opportunity to improve our hypothesis. As mentioned by the reviewer, organisms with similar sized small regional centromeres (ranging from 3 to 5 kb) have been reported to have only one microtubule attachment per kinetochore, making merotelic attachments less likely in Malassezia species. Further, we do not have any direct evidence for merotelic attachments in any of these organisms. In the revised manuscript, we propose that the AT-rich centromeres could be fragile sites in the genome. Drawing parallels from our understanding of the instability caused by fragile sites in the human genome, we advance the following hypothesis. Instability of chromosomal fragile sites has been correlated with replication stress that results in replication fork stalling. Stable inheritance of these regions is directly related to the ability of cells to resolve the fork stall with the aid of DNA repair machinery. Centromeres are reported to be natural replication fork stall sites in several yeasts (Smith et al., 1995; Mitra et al., 2014). The presence of AT-rich or repetitive sequences at the fork stall sites can result in the formation of secondary structures. Such structures are usually repaired by the DNA repair machinery (Schwartz et al., 2005). However, failure of efficient repair may result in a double-strand break at the centromere. The outcome of repair of these broken ends depends on the availability of a homologous sequence. Absence of any DNA sequence homology can result in events such as translocations that we observed in Malassezia species. This is a likely possibility in the Malassezia species given the haploid nature of these organisms. We have discussed this model in the revised manuscript and removed the merotely model (Discussion section, sixth paragraph).

Malesezzia centromeres are described as being 3-5 kb, but it looks as if the Mtw1 ChIP-seq reads cover approximately 1 kb. It is very hard to interpret the data because of the low resolution in Supplementary Figure 1A and the lack of scale in Supplementary Figure 1B. Zooming in further on the ChIP-seq and providing a diagram of the positions of features such as 12 bp motif and% GC would be helpful and informative.

We thank the reviewer for pointing this out. The resolution of figure has been improved and this panel has been included in Figure 3 in the revised manuscript.

Figure 3—figure supplement 2C and Supplementary Figure 1F. No indication of size/region covered. Figure 3—figure supplement 2 appears to show that there are several 12 bp motifs in a 500 bp window at centromeres, but these are not apparent in Figure 3—figure supplement 2C and Supplementary Figure 1F.

For the dot-plot analysis, were used sequences of 5 kb in length flanking the Mtw1 enriched regions in M. sympodialis. We have included the coordinates of the sequences used for the dot-plot in the figure legend. We thank the reviewer for pointing this out. The motif we identified was developed from a position weight matrix. Hence the motifs represented in Figure 3—figure supplement 2 may not be identical in sequence thereby not being picked up in the dot-plot.

Some means of confirming centromere location in the other Malassezia species is needed. Whilst the difficulty in transforming different yeast species and therefore tagging proteins is understood, have the authors contemplated raising an antibody to Cse4/CENP-A or testing for cross-reactivity of an existing antibody? Failing that, other Malassezia centromeres would be expected to have a reduced level of histone H3 incorporated, where it is replaced by CENP-A, compared to chromosome arms (as in Supplementary Figure 1D).

We appreciate the reviewer’s understanding of the experimental limitations related to

Malassezia. As the reviewer suggested, we attempted to raise antibodies against the epitope ‘EANYASSASA’ in M. furfur CENP-A (75-84 aa), which we expected would cross-react with M. globosa (EENYDSSASA) and M. sympodialis (EANYDSSASA) CENP-A. The purified antisera could detect the peptide in ELISA at a titer of 1:64,000. However, we could not detect a protein corresponding to CENP-A by western blot at any of the dilutions tested (ranging between 1:200 and 1:2000). The results are included here (see Author response image 1).

Considering these results, we analyzed the depletion of histone H3 as an alternative approach to validate our centromere predictions in the two species with 9 chromosomes: M. slooffiae and M. globosa. In addition, we also generated M. furfur strains that functionally express CENP-A with a 3xFLAG tag at the carboxy terminus. We used this strain to experimentally to conduct ChIP-qPCR analysis of the candidate centromere regions, and thereby validate that the approaches employed for centromere prediction in M. furfur and related species are accurate. This has been further described in our response to a point raised by reviewer 2.

Author response image 1

Download asset Open asset

Figure 2B: a negative control locus for GFP-Mtw1 should be included.

We would like to point out that the graph was plotted after normalizing with a non-centromeric locus. This has been mentioned in the revised axis label and legend (Figure 3—figure supplement 1B in the revised manuscript).

[Editors’ note: what follows is the authors’ response to the second round of review.]

Essential revisions:

1) The identification of the 12 bp AT-rich consensus that is unique for centromeres may suggest functional importance, but this is currently not experimentally shown. The manuscript should clearly state that the importance of the 12 bp consensus sequence is not known.

We have revised the manuscript, as requested, to clearly state in the Discussion section (third paragraph) that for technical reasons, the function of the motif remains unknown. However, we would like to point out that this has already been mentioned in the aforementioned paragraph.

Reviewer #2:

[…]

I think that the manuscript is substantially revised and data themselves have high quality. However, as I asked in previous review, it is important to clarify the 12 bp sequence for centromere function. It is not clear whether the centromere is specified by sequence dependent or independent. In their model, the centromere is not created in speciation in Malassezia after centromere loss. In general, after centromere loss, chromosome can fuse with another chromosome or acentric chromosome can get a neocentromere. As this species does not form new centromere, it is really important to address whether the centromere is specified by sequence dependent or not. Although it might be hard to do genetic manipulation in this species, it might be possible to deplete the 12 bp motif. If they can show the significance of the 12bp sequence in any methods, paper would be suitable for eLife.

In addition, they evaluated CENP-A accumulation as reduction of H3. Quantitative ChIP-seq is not easy. If they want to do that, they should normalize Seq-reads using the Spike-in method.

We concur with the reviewer that it is important to understand the nature of centromere function (sequence dependent or sequence independent) and the role of the 12 bp motif. At present, we are unable to ascribe a function to this motif due to the technical limitations described in the manuscript (Discussion, third paragraph) that we hope to address in our future studies.

Author details

1. Sundar Ram Sankaranarayanan

   Molecular Mycology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, India

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing - original draft, Writing - review and editing, performed western blot, microscopy, synteny conservation analysis, ChIP-qPCR analysis, and prepared the samples for ChIP-sequencing

   Competing interests

   No competing interests declared

2. Giuseppe Ianiri

   Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, United States

   Present address

   Department of Agricultural, Environmental and Food Sciences, University of Molise, Campobasso, Italy

   Contribution

   Formal analysis, Investigation, Methodology, Writing - review and editing, Transformation of Malassezia strains and PCR confirmation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3278-8678

3. Marco A Coelho

   Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, United States

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - review and editing, performed genome comparisons, phylogenetic analysis, and prepared the figures

   Competing interests

   No competing interests declared

4. Md Hashim Reza

   Molecular Mycology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, India

   Contribution

   Formal analysis, Investigation, Writing - original draft, Writing - review and editing, generated the constructs for tagging MsyMtw1 and MfCENP-A, and synteny conservation analysis

   Competing interests

   No competing interests declared

5. Bhagya C Thimmappa

   Molecular Mycology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, India

   Present address

   Department of Biochemistry, Robert-Cedergren Centre for Bioinformatics and Genomics, University of Montreal, Montreal, Canada

   Contribution

   Formal analysis, performed synteny conservation analysis

   Competing interests

   No competing interests declared

6. Promit Ganguly

   Molecular Mycology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, India

   Contribution

   Formal analysis, analyzed the ChIP-seq data and RNA-seq data for M. sympodialis

   Competing interests

   No competing interests declared

7. Rakesh Netha Vadnala

   The Institute of Mathematical Sciences/HBNI, Chennai, India

   Contribution

   Formal analysis, Visualization, performed GC/GC3 analysis

   Competing interests

   No competing interests declared

8. Sheng Sun

   Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, United States

   Contribution

   Formal analysis, Investigation, Writing - review and editing, performed PFGE and chromoblot analysis of M. slooffiae and M. globosa

   Competing interests

   No competing interests declared

9. Rahul Siddharthan

   The Institute of Mathematical Sciences/HBNI, Chennai, India

   Contribution

   Software, Formal analysis, Visualization, Writing - review and editing, identified the 12 bp motif and performed motif scan analysis across Malassezia genomes

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2233-0954

10. Christian Tellgren-Roth

    National Genomics Infrastructure, Science for Life Laboratory, Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden

    Contribution

    Resources, Methodology, genome assemblies of M. globosa

    Competing interests

    No competing interests declared

11. Thomas L Dawson Jnr

    1. Skin Research Institute Singapore, Agency for Science, Technology and Research (A*STAR), Singapore, Singapore
    2. Department of Drug Discovery, Medical University of South Carolina, School of Pharmacy, Charleston, United States

    Contribution

    Resources, Writing - review and editing, genome assemblies of M. globosa, M. slooffiae, and M. furfur

    Competing interests

    No competing interests declared

12. Joseph Heitman

    Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, United States

    Contribution

    Conceptualization, Resources, Supervision, Funding acquisition, Project administration, Writing - review and editing

    For correspondence

    heitm001@duke.edu

    Competing interests

    No competing interests declared

13. Kaustuv Sanyal

    Molecular Mycology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bengaluru, India

    Contribution

    Conceptualization, Resources, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing

    For correspondence

    sanyal@jncasr.ac.in

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-6611-4073

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