Heterochromatin structure: Lessons from the budding yeast

Bi, Xin

doi:10.1002/iub.1322

Cited by 19 publications

(12 citation statements)

References 75 publications

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“…APEX2‐Htb1 tagging led to the isolation of hundreds of proteins with roles in chromatin organization, transcription, DNA replication, or histone modification that were absent in the cytosolic APEX2 list (Table S1). Even very low abundant proteins were identified upon APEX2‐Htb1 PL including the silencing factors Sir3 and Sir4 that form a stoichiometric complex with the histone deacetylase Sir2 in subtelomeric regions , as well as other proteins with roles at telomers, centromers, or in DNA repair. Conversely, both control biotin tagging reactions via cytosolic APEX2 resulted in the enrichment of hundreds of cytosolic proteins or proteins facing the cytosolic side of organelles that were absent in MM‐APEX2 and APEX2‐Htb1 lists (Table S1).…”

Section: Resultsmentioning

confidence: 99%

APEX2‐mediated proximity labeling resolves protein networks in Saccharomyces cerevisiae cells

et al. 2019

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Enzyme‐catalyzed proximity labeling (PL) with the engineered ascorbate peroxidase APEX2 is a novel approach to map organelle compartmentalization and protein networks in living cells. Current procedures developed for mammalian cells do not allow delivery of the cosubstrate, biotin‐phenol, into living yeast cells. Here, we present a new method based on semipermeabilized yeast cells. Combined with stable isotope labeling by amino acids in cell culture (SILAC), we demonstrate proteomic mapping of a membrane‐enclosed and a semiopen compartment, the mitochondrial matrix and the nucleus. APEX2 PL revealed nuclear proteins that were previously not identified by conventional techniques. One of these, the Yer156C protein, is highly conserved but of unknown function. Its human ortholog, melanocyte proliferating gene 1, is linked to developmental processes and dermatological diseases. A first characterization of the Yer156C neighborhood reveals an array of proteins linked to proteostasis and RNA binding. Thus, our approach establishes APEX2 PL as another powerful tool that complements the methods palette for the model system yeast.

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Section: Resultsmentioning

confidence: 99%

APEX2‐mediated proximity labeling resolves protein networks in Saccharomyces cerevisiae cells

et al. 2019

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“…These results indicated the presence of basal transcription originating from the region from Ϫ1000 to Ϫ500 of STL1 under both nutrient-replete (YPD) and nutrientpoor (GluϪ) conditions, corresponding to the region of Cac2 binding by ChIP. However, under the nutrient-rich condition, a subpopulation of WT cells displayed suppression using episomal constructs which replicate autonomously, which could suggest a requirement for chromatin-dependent promoter-binding factor(s) for more effective CAC2-dependent chromatin silencing (39). Thus, linear PCR fragments containing the indicated promoter, STL1 coding region, and selection marker were transformed into WT and cac2Δ strains as integrated fragments, demonstrated comigration with genomic DNA on Southern blot analysis.…”

Section: Resultsmentioning

confidence: 99%

Transcriptional Profiling of Patient Isolates Identifies a Novel TOR/Starvation Regulatory Pathway in Cryptococcal Virulence

Park

Jarvis

et al. 2018

mBio

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Human infection with Cryptococcus causes up to a quarter of a million AIDS-related deaths annually and is the most common cause of nonviral meningitis in the United States. As an opportunistic fungal pathogen, Cryptococcus neoformans is distinguished by its ability to adapt to diverse host environments, including plants, amoebae, and mammals. In the present study, comparative transcriptomics of the fungus within human cerebrospinal fluid identified expression profiles representative of low-nutrient adaptive responses. Transcriptomics of fungal isolates from a cohort of HIV/AIDS patients identified high expression levels of an alternative carbon nutrient transporter gene, STL1, to be associated with poor early fungicidal activity, an important clinical prognostic marker. Mouse modeling and pathway analysis demonstrated a role for STL1 in mammalian pathogenesis and revealed that STL1 expression is regulated by a novel multigene regulatory mechanism involving the CAC2 subunit of the chromatin assembly complex 1, CAF-1. In this pathway, the global regulator of virulence gene VAD1 was found to transcriptionally regulate a cryptococcal homolog of a cytosolic protein, Ecm15, in turn required for nuclear transport of the Cac2 protein. Derepression of STL1 by the CAC2-containing CAF-1 complex was mediated by Cac2 and modulated binding and suppression of the STL1 enhancer element. Derepression of STL1 resulted in enhanced survival and growth of the fungus in the presence of low-nutrient, alternative carbon sources, facilitating virulence in mice. This study underscores the utility of ex vivo expression profiling of fungal clinical isolates and provides fundamental genetic understanding of saprophyte adaption to the human host. IMPORTANCE Cryptococcus is a fungal pathogen that kills an estimated quarter of a million individuals yearly and is the most common cause of nonviral meningitis in the United States. The fungus is carried in about 10% of the adult population and, after reactivation, causes disease in a wide variety of immunosuppressed individuals, including the HIV infected and patients receiving transplant conditioning, cancer therapy, or corticosteroid therapy for autoimmune diseases. The fungus is widely carried in the soil but can also cause infections in plants and mammals. However, the mechanisms for this widespread ability to infect a variety of hosts are poorly understood. The present study identified adaptation to low nutrients as a key property that allows the fungus to inhabit these diverse environments. Further studies identified a nutrient transporter gene, STL1, to be upregulated under low nutrients and to be associated with early fungicidal activity, a marker of poor clinical outcome in a cohort of HIV/AIDS patients. Understanding molecular mechanisms involved in adaptation to the human host may help to design better methods of control and treatment of widely dispersed fungal pathogens such as Cryptococcus.

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“…The CRISPR/Cas9 system prefers to target the lae1 or clr2 locus rather than the vib1 locus. The gene vib1 may be located in a region of low transcriptional activity [ 30 ], which complicates the process of gRNA directing Cas9 to the vib1 locus. The increase in the molar concentration ratio of gRNA- vib1 (the gRNA targeting vib1 ) during co-transformation resulted in an increase in frequency for double recombination from 16 to 45% ( Supplementary Figure S3 ).…”

Section: Discussionmentioning

confidence: 99%

Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system

et al. 2015

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Filamentous fungi have wide applications in biotechnology. The CRISPR/Cas9 system is a powerful genome-editing method that facilitates genetic alterations of genomes in a variety of organisms. However, a genome-editing approach has not been reported in filamentous fungi. Here, we demonstrated the establishment of a CRISPR/Cas9 system in the filamentous fungus Trichoderma reesei by specific codon optimization and in vitro RNA transcription. It was shown that the CRISPR/Cas9 system was controllable and conditional through inducible Cas9 expression. This system generated site-specific mutations in target genes through efficient homologous recombination, even using short homology arms. This system also provided an applicable and promising approach to targeting multiple genes simultaneously. Our results illustrate that the CRISPR/Cas9 system is a powerful genome-manipulating tool for T. reesei and most likely for other filamentous fungal species, which may accelerate studies on functional genomics and strain improvement in these filamentous fungi.

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Heterochromatin structure: Lessons from the budding yeast

Cited by 19 publications

References 75 publications

APEX2‐mediated proximity labeling resolves protein networks in Saccharomyces cerevisiae cells

APEX2‐mediated proximity labeling resolves protein networks in Saccharomyces cerevisiae cells

Transcriptional Profiling of Patient Isolates Identifies a Novel TOR/Starvation Regulatory Pathway in Cryptococcal Virulence

Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system

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