Biology subjects

Sanyal, K.

Publications and source records attributed to Sanyal, K..

27 records · Page 2Linked to original sources

bioRxiv2019

Identification of bridgin, an unconventional linker, connects the outer kinetochore to centromeric chromatin

The microtubule-binding outer kinetochore is linked to centromeric chromatin through the inner kinetochore CENP-CMif2, CENP-TCnn1, and CENP-UAme1 pathways. These are the only known kinetochore linker proteins across eukaryotes. Linker proteins are structurally less conserved than their outer kinetochore counterparts. Here, we demonstrate the recurrent loss of most inner kinetochore CCAN, including certain linker proteins during evolution in the fungal phylum of Basidiomycota. By studying the kinetochore interactome, a previously undescribed linker protein, bridgin was identified in the basidiomycete Cryptococcus neoformans, a human fungal pathogen. In vivo and in vitro functional analyses of bridgin reveal that it binds to the outer kinetochore and centromere chromatin simultaneously to ensure accurate kinetochore-microtubule attachments. Unlike known linker proteins, bridgin is recruited by the outer kinetochore. Homologs of bridgin were identified outside fungi. These results showcase a divergent strategy, with a more ancient origin than fungi, to link the outer kinetochore to centromeric chromatin.

cell biology↗

bioRxiv2019

Long transposon-rich centromeres in an oomycete reveal divergence of centromere features in Stramenopila-Alveolata-Rhizaria lineages

Centromeres are chromosomal regions that serve as platforms for kinetochore assembly and spindle attachments, ensuring accurate chromosome segregation during cell division. Despite functional conservation, centromere DNA sequences are diverse and often repetitive, making them challenging to assemble and identify. Here, we describe centromeres in an oomycete Phytophthora sojae by combining long-read sequencing-based genome assembly and chromatin immunoprecipitation for the centromeric histone CENP-A followed by high-throughput sequencing (ChIP-seq). P. sojae centromeres cluster at a single focus at different life stages and during nuclear division. We report an improved genome assembly of the P. sojae reference strain, which enabled identification of 15 enriched CENP-A binding regions as putative centromeres. By focusing on a subset of these regions, we demonstrate that centromeres in P. sojae are regional, spanning 211 to 356 kb. Most of these regions are transposon-rich, poorly transcribed, and lack the histone modification H3K4me2 but are embedded within regions with the heterochromatin marks H3K9me3 and H3K27me3. Strikingly, we discovered a Copia-like transposon (CoLT) that is highly enriched in the CENP-A chromatin. Similar clustered elements are also found in oomycete relatives of P. sojae, and may be applied as a criterion for prediction of oomycete centromeres. This work reveals a divergence of centromere features in oomycetes as compared to other organisms in the Stramenopila-Alveolata-Rhizaria (SAR) supergroup including diatoms and Plasmodium falciparum that have relatively short and simple regional centromeres. Identification of P. sojae centromeres in turn also advances the genome assembly. Author summaryOomycetes are fungal-like microorganisms that belong to the stramenopiles within the Stramenopila-Alveolata-Rhizaria (SAR) supergroup. The Phytophthora oomycetes are infamous as plant killers, threatening crop production worldwide. Because of the highly repetitive nature of their genomes, assembly of oomycete genomes presents challenges that impede identification of centromeres, which are chromosomal sites mediating faithful chromosome segregation. We report long-read sequencing-based genome assembly of the Phytophthora sojae reference strain, which facilitated the discovery of centromeres. P. sojae harbors large regional centromeres fully embedded in heterochromatin, and enriched for a Copia-like transposon that is also found in discrete clusters in other oomycetes. This study provides insight into the oomycete genome organization, broadens our knowledge of the centromere structure, function and evolution in eukaryotes, and may help elucidate the high frequency of aneuploidy during oomycete reproduction.

microbiology↗

bioRxiv2019

Early diverging fungus Mucor circinelloides lacks centromeric histone CENP-A and displays a mosaic of point and regional centromeres

Centromeres are rapidly evolving across eukaryotes, despite performing a conserved function to ensure high fidelity chromosome segregation. CENP-A chromatin is a hallmark of a functional centromere in most organisms. Due to its critical role in kinetochore architecture, the loss of CENP-A is tolerated in only a few organisms, many of which possess holocentric chromosomes. Here, we characterize the consequence of the loss of CENP-A in the fungal kingdom. Mucor circinelloides, an opportunistic human pathogen, lacks CENP-A along with the evolutionarily conserved CENP-C, but assembles a monocentric chromosome with a localized kinetochore complex throughout the cell cycle. Mis12 and Dsn1, two conserved kinetochore proteins were found to bind nine short overlapping regions, each comprising an [~]200-bp AT-rich sequence followed by a centromere-specific conserved motif that echoes the structure of budding yeast point centromeres. Resembling fungal regional centromeres, these core centromere regions are embedded in large genomic expanses devoid of genes yet marked by Grem-LINE1s, a novel retrotransposable element silenced by the Dicer-dependent RNAi pathway. Our results suggest that these hybrid features of point and regional centromeres arose from the absence of CENP-A, thus defining novel mosaic centromeres in this early-diverging fungus.

genetics↗

bioRxiv2019

MTOC clustering and nuclear division through proper spindle positioning in yeast: a microtubule grow-and-catch model

The dynamic process of mitotic spindle assembly depends on multitudes of inter-dependent interactions involving kinetochores (KTs), microtubules (MTs), spindle pole bodies (SPBs), and molecular motors. Before forming the mitotic spindle, multiple visible microtubule organizing centers (MTOCs) coalesce into a single focus to serve as a SPB in the pathogenic budding yeast, Cryptococcus neoformans. To explain this unusual phenomenon in the fungal kingdom, we propose a search and capture model, in which cytoplasmic MTs (cMTs) nucleated by MTOCs grow and capture each other to promote MTOC clustering. Our quantitative modeling identifies multiple redundant mechanisms mediated by a combination of cMT-cell cortex interactions and inter-cMT coupling to facilitate MTOC clustering within the physiological time limit as determined by time-lapse live-cell microscopy. Besides, we screen various possible mechanisms by computational modeling and propose optimal conditions that favor proper spindle positioning - a critical determinant for timely chromosome segregation. These analyses also reveal that a combined effect of MT buckling, dynein pull, and cortical push maintains spatiotemporal spindle localization.

biophysics↗

bioRxiv2019

Aurora kinase Ipl1 facilitates bilobed distribution of clustered kinetochores to ensure error-free chromosome segregation in Candida albicans

Candida albicans, an ascomycete, has an ability to switch to diverse morphological forms. While C. albicans is predominatly diploid, it can tolerate aneuploidy as a survival strategy under stress. Aurora kinase B homolog Ipl1 is a critical ploidy regulator that controls microtubule dynamics and chromosome segregation in Saccharomyces cerevisiae. In this study, we show that Ipl1 in C. albicans has a longer activation loop than that of the well-studied ascomycete S. cerevisiae. Ipl1 localizes to the kinetochores during the G1/S phase and associates with the spindle during mitosis. Ipl1 regulates cell morphogenesis and is required for cell viability. Ipl1 monitors microtubule dynamics which is mediated by separation of spindle pole bodies. While Ipl1 is dispensable for maintaining structural integrity and clustering of kinetochores in C. albicans, it is required for the maintenance of kinetochore geometry to form bilobed structures along the mitotic spindle, a feature of Ipl1 that was not observed in other yeasts. Depletion of Ipl1 results in erroneous kinetochore-microtubule attachments leading to aneuploidy-associated resistance to fluconazole, the most common anti-fungal drug used to treat Candida infections. Taking together, we suggest that Ipl1 spatiotemporally ensures kinetochore geometry to facilitate bipolar spindle assembly crucial for ploidy maintenance in C. albicans.

cell biology↗

bioRxiv2019

Centromere-mediated chromosome break drives karyotype evolution in closely related Malassezia species

Intra-chromosomal or inter-chromosomal genomic rearrangements often lead to speciation (1). Loss or gain of a centromere leads to alterations in chromosome number in closely related species. Thus, centromeres can enable tracing the path of evolution from the ancestral to a derived state (2). The Malassezia species complex of the phylum Basiodiomycota shows remarkable diversity in chromosome number ranging between six and nine chromosomes (3-5). To understand these transitions, we experimentally identified all eight centromeres as binding sites of an evolutionarily conserved outer kinetochore protein Mis12/Mtw1 in M. sympodialis. The 3 to 5 kb centromere regions share an AT-rich, poorly transcribed core region enriched with a 12 bp consensus motif. We also mapped nine such AT-rich centromeres in M. globosa and the related species Malassezia restricta and Malassezia slooffiae. While eight predicted centromeres were found within conserved synteny blocks between these species and M. sympodialis, the remaining centromere in M. globosa (MgCEN2) or its orthologous centromere in M. slooffiae (MslCEN4) and M. restricta (MreCEN8) mapped to a synteny breakpoint compared with M. sympodialis. Taken together, we provide evidence that breakage and loss of a centromere (CEN2) in an ancestral Malassezia species possessing nine chromosomes resulted in fewer chromosomes in M. sympodialis. Strikingly, the predicted centromeres of all closely related Malassezia species map to an AT-rich core on each chromosome that also shows enrichment of the 12 bp sequence motif. We propose that centromeres are fragile AT-rich sites driving karyotype diversity through breakage and inactivation in these and other species. Significance statementThe number of chromosomes can vary between closely related species. Centromere loss destabilizes chromosomes and results in reduced number of chromosomes to drive speciation. A series of evidence from studies on various cancers suggest that an imbalance in kinetochore-microtubule attachments results in breaks at the centromeres. To understand if such events can cause chromosome number changes in nature, we studied six species of Malassezia, of which three possess eight chromosomes and others have nine chromosomes each. We find signatures of chromosome breakage at the centromeres in organisms having nine chromosomes. We propose that the break at the centromere followed by fusions of acentric chromosomes to other chromosomes could be a plausible mechanism shaping the karyotype of Malassezia and related organisms. ClassificationBiological sciences, Genetics

genomics↗

bioRxiv2018

Cellular dynamics and genomic identity of centromeres in Rice Blast

A series of well-synchronized events mediated by kinetochore-microtubule interactions ensure faithful chromosome segregation in eukaryotes. Centromeres scaffold kinetochore assembly and are among the fastest evolving chromosomal loci in terms of the DNA sequence, length, and organization of intrinsic elements. Neither the centromere structure nor the kinetochore dynamics is well studied in plant pathogenic fungi. Here, we sought to understand the process of chromosome segregation in the rice blast fungus, Magnaporthe oryzae. High-resolution confocal imaging of GFP-tagged inner kinetochore proteins, CenpA and CenpC, revealed an unusual albeit transient declustering of centromeres just before anaphase separation in M. oryzae. Strikingly, the declustered centromeres positioned randomly at the spindle midzone without an apparent metaphase plate per se. Using chromatin immunoprecipitation followed by deep sequencing, all seven centromeres were identified as CenpA-rich regions in the wild-type Guy11 strain of M. oryzae. The centromeres in M. oryzae are regional and span 57 to 109 kb transcriptionally poor regions. No centromere-specific DNA sequence motif or repetitive elements could be identified in these regions suggesting an epigenetic specification of centromere function in M. oryzae. Highly AT-rich and heavily methylated DNA sequences were the only common defining features of all the centromeres in the Rice Blast. Detailed gene synteny analyses helped identify and compare the centromere regions in distinct isolates of M. oryzae and its related species Magnaporthe poae. Overall, this study identifies unusual centromere dynamics and maps the centromere DNA sequences in the top model fungal pathogen M. oryzae, which causes severe losses in global rice production.\n\nSignificanceMagnaporthe oryzae is an important fungal pathogen that causes an annual loss of 10-30% rice crop due to the devastating blast disease. In most organisms, kinetochores are arranged either in the metaphase plate or are clustered together to facilitate synchronized anaphase separation of chromosomes. In this study, we show that the initially clustered kinetochores separate and position randomly prior to anaphase in M. oryzae. Centromeres, identified as the site of kinetochore assembly, are regional type without any shared sequence motifs in M. oryzae. Together, this study reveals atypical kinetochore dynamics and identifies functional centromeres in M. oryzae, thus paving the way to define heterochromatin boundaries and understand the process of kinetochore assembly on epigenetically specified centromere loci.

genetics↗

bioRxiv2018

DNA replication initiator proteins facilitate CENPA loading on early replicating compact chromatin

Spatiotemporal regulation in DNA replication maintains kinetochore stability. The epigenetically regulated centromeres (CENs) in the budding yeast Candida albicans have unique DNA sequences, replicate early and are clustered throughout the cell cycle. In this study, the genome-wide occupancy of replication initiation protein Orc4 reveals its abundance at all CENs in C. albicans. Orc4 associates with four different DNA motifs, one of which coincides with tRNA genes. Hi-C combined with genome-wide replication timing analyses identify enriched interactions among early or late replicating Orc4-bound regions. A simulated polymer model of chromosomes reveals that early replicating and strongly enriched Orc4-bound sites localize towards the kinetochores. Orc4 is constitutively localized to CENs, and both Orc4 and Mcm2 stabilize CENPA. CENPA chaperone Scm3 localizes at the kinetochore during anaphase, coinciding with the loading time of CENPA. We propose that this spatiotemporal nuclear localization of Orc4, with Mcm2 and Scm3, recruits CENPA and stabilizes centromeric chromatin.

molecular biology↗

bioRxiv2018

Identification and in silico analysis of the origin recognition complex in the human fungal pathogen Candida albicans

DNA replication in eukaryotes is initiated by the orchestrated assembly and association of initiator proteins (heterohexameric Origin Recognition Complex, ORC) on the replication origins. These functionally conserved proteins play significant roles in diverse cellular processes besides their central role in ignition of DNA replication at origins. While Candida albicans, a major human fungal pathogen, is an ascomycetous, asexual, diploid budding yeast but it is significantly diverged from a much better studied model organism Saccharomyces cerevisiae. The components of the DNA replication machinery in C. albicans remain largely uncharacterized. Identification of factors required for DNA replication is essential for understanding the evolution of the DNA replication machinery. We identified the putative ORC homologs in C. albicans and determined their relatedness with those of other eukaryotes including several yeast species. Our extensive in silico studies demonstrate that the domain architecture of CaORC proteins share similarities with the ORC proteins of S. cerevisiae. We dissect the domain organization of ORC (trans-acting factors) proteins that seem to associate with DNA replication origins in C. albicans. We present a model of the 3D structure of CaORC4 to gain further insights of this proteins function.

bioinformatics↗