Kaustuv Sanyal

Genes transcribed by the RNA polymerase (Pol) III of yeast are mostly found at sites of high transcription, genomic fragility, and replication fork stalling. Transcription terminator–dependent facilitated recycling of Pol III on naked DNA templates boosts transcription in vitro; its operability in vivo is not known. Recycling on the same template links transcriptional output directly to the reinitiation frequency. Using recycling-deficient Pol III or terminator-deficient templates, we found that terminator is essential to couple the facilitated Pol III recycling and chromatin remodeling for achieving high transcriptional activation. Mechanism of the full activation process constitutes three linked steps, antirepression, chromatin remodeling, and Pol III recycling. Normally high transcriptional demands are met by the terminator-facilitated Pol III recycling on genes, while different recycling frequencies result in different Pol III occupancies and gene-specific transcript yields. Abnormal increase in recycling frequency may cause the genomic sites fragility in normal cells or highly elevated Pol III transcription in cancerous cells or other related disorders.

Overexpression of pre-replication complex (pre-RC) components is frequently associated with poor prognosis in many types of cancer, yet the mechanisms underlying their pathological effects remain unclear. Using the model yeast Candida albicans, we investigated the consequences of elevated Mcm7 levels, a conserved pre-RC subunit previously linked to chromosome instability (CIN). Unlike other pre-RC components, Mcm7 overexpression severely compromises nuclear integrity, resulting in chromatin fragmentation, DNA double-strand breaks, and cell death. Excess Mcm7 disrupts chromosome segregation by impairing microtubule function, disrupting centromere organization, generating abnormal spindle structures, and causing defective nuclear migration, collectively leading to mitotic failure. We further discovered ectopic accumulation of Mcm7 within the nucleolus, accompanied by widespread transcriptional disruption of nucleolus-associated genes. Supporting the relevance of these findings to human disease, cancer transcriptomic datasets reveal a strong association between MCM7 expression and nucleolar gene expression programs, while human cancer cell lines with higher MCM7 expression exhibit increased nucleolar number and size. Together, our findings identify Mcm7 as a previously unrecognized regulator of cytoskeletal and nucleolar homeostasis and uncover an unexpected mechanism by which replication factor dysregulation promotes CIN.

Candida auris, a World Health Organisation–listed critical priority fungal pathogen, causes frequent multidrug-resistant outbreaks worldwide. While point mutations underlying antifungal resistance are well characterised, the contribution of structural genomic variation to antifungal responses remains poorly defined. Here, we integrate whole-genome sequencing, a genome-wide copy number variation (CNV) screen, electrophoretic karyotyping, and mutation-accumulation analyses to investigate the role of structural variation in antifungal susceptibility across a clinical cohort of C. auris isolates. We identify recurrent CNV hotspots, with segmental duplications representing a predominant mode of genome variation. Small duplications encompassing ERG11 arise at high frequency and frequently co-occur with drug resistance-associated ERG11 mutations, collectively enhancing azole resistance. In addition, large centromere-inclusive duplications of chromosome 1 generate supernumerary chromosomes, leading to paradoxical growth and reduced susceptibility to caspofungin, an echinocandin. At the population level, structural variants frequently arise in parallel to FKS1 mutations, suggesting multiple genetic mechanisms underlying reduced drug susceptibility. Together, our findings establish segmental duplication as a major, non-mutational driver of antifungal resistance, highlighting the need to consider structural genomic variation in both resistance surveillance and clinical susceptibility testing.

Fungal centromeres are clustered near microtubule organizing centers to help adopt the Rabl chromosomal organization. The role of centromere clustering in driving large-scale changes in structural and functional chromatin assembly remains unclear. Here, using Hi-C and superresolution microscopy, we show that cell cycle-dependent centromere declustering and clustering states in Cryptococcus neoformans drive global changes in the 3D genome architecture. Centromeres and telomeres are scattered around the nuclear periphery at interphase ^G1 , and this arrangement constrains the interarm interactions within a chromosome, providing a unique interphase ^G1 chromosome organization. Moreover, centromeres and telomeres are organized as separate compartments, segregating them from active euchromatic regions. Polymer modeling reveals that the transition from the unclustered to clustered centromere state during the cell cycle involves changes from a globular to an elongated chromosome architecture. Strikingly, while clustered centromeric regions replicate early in most yeasts, C. neoformans centromeres replicate late in S-phase, hinting at a possible link between centromere clustering dynamics and CEN DNA replication timing. Overall, our study uncovers several unique organizational principles governing the dynamic genome architecture in an evolutionarily diverged basidiomycete yeast.

Faithful chromosome segregation is facilitated by the centromeres, specialized genomic loci, which connect chromosomes to microtubules in every cell cycle by recruiting the kinetochore complex. However, a single conserved code does not govern the formation and maintenance of centromeres, as we begin to realize that enormous diversity exists in molecular mechanisms dictating centromere homeostasis across species. The fungal kingdom is a vast resource to study and appreciate the divergent nature of the conserved phenomenon of chromosome segregation. Studies in the fungal kingdom enable researchers to view the evolution of centromeres at the molecular level. While some organisms, such as Saccharomyces cerevisiae, rely on a strict genetically determined centromere establishment, most fungi adopt epigenetically driven mechanisms of centromere propagation. This epigenomic regulation ranges from modifications on the underlying DNA to histones forming the centric and pericentric regions. The centromere DNA sequence, arrangement of sequence elements, its transcription state, and the replication timing, as well as its spatial position in the nucleus, play a major role in determining centromere stability and its function. In this review, we aim to highlight the spectrum of centromere regulatory mechanisms observed in fungi and discuss the gaps in the research that can provide new perspectives on centromere biology.

With the prevalence of immunocompromising infections, the emergence of new pathogenic species, rising multidrug resistance, hospital outbreaks, and high mortality rates, once neglected fungal pathogens are now taking over the global healthcare arena, necessitating breakthroughs in diagnostics and therapeutics. The World Health Organization (WHO) recently published a list of critical and high-priority fungal pathogens, underlining the need and significance of broadening the focus to include fungal infections, among other life-threatening infectious diseases. This is also a wake-up call to the scientific community to facilitate a better understanding of the emergence, life cycle, spread, virulence, and drug resistance mechanisms in fungal pathogens. The strategies that fungal pathogens adopt to escape the immune system, establish an infection, and combat antifungal drugs are diverse. In this review, we focus on the pathogenic lifestyles of WHO critical and high priority fungal pathogens, with a brief introduction to the epidemiology and infection statistics.

Fungal centromeres are clustered near microtubule organizing centers to help adopt the Rabl chromosomal organization. The role of centromere clustering in driving large-scale changes in structural and functional chromatin assembly remains unclear. Here, using Hi-C and super-resolution microscopy, we show that cell cycle-dependent centromere declustering and clustering states in Cryptococcus neoformans drive global changes in the 3D genome architecture. Centromeres and telomeres are scattered around the nuclear periphery at interphase ^G1 , and this arrangement constrains the inter-arm interactions within a chromosome, providing a unique interphase ^G1 chromosome organization. Moreover, centromeres and telomeres are organized as compartments, segregating them from active euchromatic regions. Polymer modeling reveals that the transition from the unclustered to clustered centromere state during the cell cycle involves changes from a globular to an elongated chromosome architecture. Strikingly, while clustered centromeres replicate early in most yeasts, C. neoformans centromeres replicate late in S-phase, hinting a possible link between centromere clustering dynamics and CEN DNA replication timing. Overall, our study uncovers several unique organizational principles governing the dynamic genome architecture in an evolutionarily diverged basidiomycete yeast.

Candida albicans is an opportunistic pathogen associated with healthy humans. It is the major causative agent for superficial and invasive candidiasis in immunocompromised individuals globally. Lack of awareness toward fungal infections, poor disease management, and increasing drug resistance have contributed to the burden of C. albicans-associated diseases. C. albicans displays polymorphism, assisting in host interaction, virulence, and sustenance of the pathogen. Well-known polymorphic forms of C. albicans include yeast, hyphal, pseudohyphal, white, opaque, gray, GUT, and chlamydospores. While the predominant mode of propagation is via mitosis, the organism also displays a parasexual cycle. The pathogen's parasexual mating and genome plasticity introduce variants in the population, leading to adaptability and drug resistance. This organism can adopt robust pathogenic modules capable of destroying host tissues and forming drug-resistant biofilms. On the other hand, its ability to maintain an amicable interaction with the host immune system, coexisting microbiome, and adaptability to the host microenvironment makes it a successful commensal. Over the years, C. albicans has emerged as a useful model for understanding basic cell biology and drug resistance in fungal pathogens. Advancements in genetic manipulation tools have accelerated our understanding of these biological processes and eased the drug development process. This review provides comprehensive information about C. albicans and the tools available to investigate it. Studying its life cycle, disease epidemiology, mechanisms of infection, and host immune responses is crucial for developing effective therapeutic interventions.

Clinical isolates of Candida auris show a high prevalence of resistance to Amphotericin B (AmB)—an uncommon trait in most Candida species. Alterations in ergosterol biosynthesis can contribute to acquired AmB resistance in C. auris laboratory strains but are rarely seen in clinical isolates. In this study, we experimentally evolved two drug‐susceptible Clade II isolates of C. auris to develop AmB resistance. The evolved strains displayed a four to eight fold increase in MIC₅₀ compared to the parental cells. We analyzed changes in their karyotype, genome, lipidome, and transcriptome associated with this acquired resistance. In one lineage, AOX2 was upregulated, and its deletion reversed the AmB resistance phenotype. The aox2Δ mutant also failed to evolve AmB resistance under experimental conditions. In the same lineage, restoring the UPC2^S332R and RTG3^S101T mutations to the wild‐type allele restored AmB susceptibility. In another lineage, the ergosterol and sphingolipid pathways were observed to play a critical role, and upregulation of the ERG genes elevated the total sterol content, while significant downregulation of HSX11 (glucosylceramide synthase) resulted in lower levels of glucosylceramides. To our knowledge, this study is the first to show that AmB resistance in C. auris can be acquired through mechanisms both dependent on or independent of sterol content modulation, highlighting Aox2 and Upc2 as key regulators of amphotericin resistance.