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Bridgin Connects the Outer Kinetochore to Centromeric Chromatin ✉ ✉ Shreyas Sridhar 1,4, Tetsuya Hori2, Reiko Nakagawa 3, Tatsuo Fukagawa 2 & Kaustuv Sanyal 1,2

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Bridgin Connects the Outer Kinetochore to Centromeric Chromatin ✉ ✉ Shreyas Sridhar 1,4, Tetsuya Hori2, Reiko Nakagawa 3, Tatsuo Fukagawa 2 & Kaustuv Sanyal 1,2 ARTICLE https://doi.org/10.1038/s41467-020-20161-9 OPEN Bridgin connects the outer kinetochore to centromeric chromatin ✉ ✉ Shreyas Sridhar 1,4, Tetsuya Hori2, Reiko Nakagawa 3, Tatsuo Fukagawa 2 & Kaustuv Sanyal 1,2 The microtubule-binding outer kinetochore is coupled to centromeric chromatin through CENP-CMif2, CENP-TCnn1, and CENP-UAme1 linker pathways originating from the constitutive centromere associated network (CCAN) of the inner kinetochore. Here, we demonstrate the 1234567890():,; recurrent loss of most CCAN components, including certain kinetochore linkers during the evolution of the fungal phylum of Basidiomycota. By kinetochore interactome analyses in a model basidiomycete and human pathogen Cryptococcus neoformans, a forkhead-associated domain containing protein “bridgin” was identified as a kinetochore component along with other predicted kinetochore proteins. In vivo and in vitro functional analyses of bridgin reveal its ability to connect the outer kinetochore with centromeric chromatin to ensure accurate chromosome segregation. Unlike established CCAN-based linkers, bridgin is recruited at the outer kinetochore establishing its role as a distinct family of kinetochore proteins. Presence of bridgin homologs in non-fungal lineages suggests an ancient divergent strategy exists to bridge the outer kinetochore with centromeric chromatin. 1 Molecular Mycology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Center for Advanced Scientific Research (JNCASR), Bangalore, India 560064. 2 Laboratory of Chromosome Biology, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan. 3 Laboratory for Phyloinformatics, RIKEN Center for Biosystems Dynamics Research (BDR), Kobe, Japan. 4Present address: Graduate School of Frontier ✉ Biosciences, Osaka University, Suita, Osaka 565-0871, Japan. email: [email protected]; [email protected] NATURE COMMUNICATIONS | (2021) 12:146 | https://doi.org/10.1038/s41467-020-20161-9 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-20161-9 ccurate chromosome segregation ensures faithful trans- prediction. Species representing 31 fungal orders across the three Amission of the genetic material to progeny. The kine- subphyla, Pucciniomycotina, Ustilagomycotina, and Agar- tochore is a multicomplex protein structure assembled on icomycotina, were considered. Additionally, representative spe- centromere DNA of each eukaryotic chromosome1–4 and is cies across 7 fungal phyla were included48. CENP-ACse4, the 16- attached to the spindle microtubules for accurate chromosome member CCAN, and the 10-member KMN network were chosen segregation5. Components involved in error correction mechan- for this study (Fig. 1a and Supplementary Data 1). Our analysis, isms, including the spindle assembly checkpoint (SAC), are in accordance with the previous reports25,26,29, indicates the recruited at kinetochores to ensure the biorientation of robust conservation of CENP-ACse4 and the KMN network sister chromatids in mitosis6–8. The inner kinetochore is com- proteins across basidiomycetes. On the other hand, we observed posed of centromeric histone H3 variant CENP-ACse4 9–11 and recurrent loss of most CCAN proteins across 23 of the 31 basi- the 16-member constitutive centromere-associated network diomycete orders (Fig. 1a). In the subphylum of Agar- (CCAN)12–16. The outer kinetochore members of the KMN icomycotina, to which C. neoformans belongs, the loss event may (Knl1, Mis12, and Ndc80 complexes) network1,17 are recruited to have occurred early at the time of divergence of Wallemiales from CCAN to form the kinetochore ensemble across eukaryotes, other orders. Retention of CCAN proteins in a few discrete orders including budding yeast and vertebrates18. Additional compo- in the subphyla of Pucciniomycotina and Ustilogamycotina sug- nents such as the 10-member Dam1 complex (Dam1C) in fungi gests the occurrence of multiple independent loss events of most and the three-member Ska complex in vertebrates localize to the CCAN subunits (Fig. 1a). The kinetochore linker CENP-CMif2 outer kinetochore to ensure accurate kinetochore–microtubule was the only conserved CCAN component across basidiomycetes. interactions19–24. Other known linker proteins, CENP-TCnn1 and CENP-UAme1, Genome sequencing data reveal that while the KMN network were often lost together. Although the primary protein sequence components are highly conserved25–27, the inner kinetochore conservation is low among CENP-TCnn1 homologs, they share a composition is variable across eukaryotes25–29. The Ndc80 typical protein architecture: an N-terminal α-helix composed of complex (Ndc80C) of the KMN network directly binds to spindle conserved hydrophobic residues, observed to occur within the microtubules30–32. CCAN proteins, CENP-CMif2 and CENP- PITH domain in Ustilaginales, and the CENP-TCnn1 motif at the TCnn1, have been shown to independently link centromeric C terminus (Fig. 1b). As a result, the CENP-CMif2 pathway chromatin with the KMN network33–38. In addition, CENP- remains the only known linker pathway among 23 of the 31 UAme1 functions as a linker in budding yeast39,40. CENP-CMif2 basidiomycete orders investigated. These observations suggest and CENP-TCnn1, through their amino(N)-termini, interact with that additional factors may exist to compensate for the recurrent the KMN network, while their carboxy(C)-termini interact with loss of most CCAN proteins in Basidiomycota. centromeric chromatin41–43. CENP-UAme1 cooperatively with CENP-CMif2 also binds to Mis12C, to ensure a linker function in budding yeast39,40. These three critical kinetochore linker pro- Identification of the kinetochore interactome in C. neoformans. teins, CENP-CMif2, CENP-TCnn1, and CENP-UAme1, are often In search of such unknown kinetochore proteins, we attempted to lost or significantly diverged during evolution. Despite the analyze the kinetochore composition in the fungal phylum of observed loss or functional divergence of linker proteins, the Basidiomycota using a relatively well-studied model basidiomy- existence of other molecular innovations to bridge centromeric cetous yeast C. neoformans (Fig. 1a). To comprehensively deter- chromatin with the outer kinetochore to ensure accurate chro- mine the constitution of the kinetochore and its interactome mosome segregation remains largely unknown21,28,44. Although in vivo, C. neoformans strains expressing 3×FLAG-tagged CENP- each of the kinetochore linker pathways has been shown to play CMif2, Dsn1 (Mis12C), and Spc25 (Ndc80C) under control of the important roles in chromosome segregation, the architectural native promoter were generated. The functionality of the dependence on a specific pathway varies across species34,35,37. 3×FLAG-tagged proteins was validated by chromatin immuno- Cryptococcus neoformans is a ubiquitous environmental fungus precipitation (ChIP) and co-immunoprecipitation (co-IP) assays and an opportunistic pathogen causing fatal cryptococcal (Supplementary Fig. 1a–c). meningitis45,46. Our previous studies using this model basidio- Mass-spectrometry (MS) analyses were performed after FLAG- mycete suggested a stepwise kinetochore assembly on a long IP for CENP-CMif2, Dsn1, and Spc25 from metaphase-enriched repetitive regional centromere24,47. (mitotic index > 90%) cell population (Fig. 1c and Supplementary In this work, by analyzing a number of fungal genomes, we Data 2). Nearly all predicted inner kinetochore proteins (CENP- strikingly find the recurrent absence of most CCAN components, ACse4 and CENP-CMif2) and outer kinetochore KMN network including certain conventional linker proteins, CENP-TCnn1 and components were identified from each of the FLAG-IPs (Table 1 CENP-UAme1, in the phylum Basidiomycota. We identify “brid- and Supplementary Data 2). Except for CENP-CMif2,no gin”, a previously undescribed protein, in the kinetochore inter- components of the CCAN were identified in the IP–MS (Table 1 actome of C. neoformans. We predict the existence of bridgin and Supplementary Table. 2). Identified KMN network compo- homologs in eukaryotic lineages outside the fungal kingdom as nents included evolutionarily conserved but previously unanno- well. Based on in vivo and in vitro functional analyses, we tated ORFs coding for subunits of Mis12C (CNAG_04300Nsl1, demonstrate that bridgin connects the outer kinetochore with CNAG_04479Nnf1) and Knl1C (CNAG_03715Sos7) (Table 1 and centromeric chromatin revealing the existence of a distinct Supplementary Data 2). CNAG_03715Sos7 (Knl1C) was used as a strategy to bridge the outer kinetochore and centromeric chro- candidate to authenticate the predicted ORFs as bona fide matin across eukaryotes. kinetochore proteins. CNAG_03715Sos7 was bioinformatically identified as the Sos7 homolog in C. neoformans using Schizosaccharomyces pombe Sos749 (Supplementary Fig. 1d, e). Results Microscopic observations of GFP-CnSos7 suggested a cell-cycle- Recurrent independent loss events of CCAN proteins in Basi- dependent kinetochore-exclusive localization in C. neoformans diomycota. To have a comprehensive understanding of the (Fig. 1d and Supplementary Fig. 1f). Further analysis confirmed kinetochore composition in Basidiomycota, we analyzed putative that while Sos7 was dispensable for cell viability, yet was essential kinetochore homologs using high-confidence protein homology for accurate chromosome segregation in C. neoformans (Supple- searches combined with secondary and tertiary structure
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