Native Chromatin Proteomics Reveals a Role for Specific Nucleoporins in Heterochromatin Organization and Maintenance.

Spatially and functionally distinct domains of heterochromatin and euchromatin play important roles in the maintenance of chromosome stability and regulation of gene expression, but a comprehensive knowledge of their composition is lacking. Here, we develop a strategy for the isolation of native Schizosaccharomyces pombe heterochromatin and euchromatin fragments and analyze their composition by using quantitative mass spectrometry. The shared and euchromatin-specific proteomes contain proteins involved in DNA and chromatin metabolism and in transcription, respectively. The heterochromatin-specific proteome includes all proteins with known roles in heterochromatin formation and, in addition, is enriched for subsets of nucleoporins and inner nuclear membrane (INM) proteins, which associate with different chromatin domains. While the INM proteins are required for the integrity of the nucleolus, containing ribosomal DNA repeats, the nucleoporins are required for aggregation of heterochromatic foci and epigenetic inheritance. The results provide a comprehensive picture of heterochromatin-associated proteins and suggest a role for specific nucleoporins in heterochromatin function.

Interactions, structural aspects and evolutionary perspectives of the yeast 'START'-regulatory network.

Molecular signal transduction networks, which conduct transcription at the G1 to S phase transition of the eukaryotic cell division cycle have been identified in diverse taxa from mammals to baker's yeast with analogous functional organization. However, regarding some network components, such as the transcriptional regulators STB1 and WHI5, only few orthologs exist, which are confined to individual Saccharomycotina species. While Whi5 has been characterized as yeast analog of human Rb protein, in the particular case of Stb1 (Sin three binding protein 1) identification of functional analogs emerges as difficult because to date its exact functionality still remains obscured. By aiming to resolve Stb1's enigmatic role this Perspective article especially surveys works covering relations between Cyclin/CDKs, the heteromeric transcription factor complexes SBF (Swi4/Swi6) and MBF (Mbp1/Swi6), as well as additional coregulators (Whi5, Sin3, Rpd3, Nrm1) which are collectively associated with the orderly transcription at 'Start' of the Saccharomyces cerevisiae cell cycle. In this context, interaction capacities of the Sin3-scaffold protein are widely surveyed because its four PAH domains (Paired Amphiphatic Helix) represent a 'recruitment-code' for gene-specific targeting of repressive histone deacetylase activity (Rpd3) via different transcription factors. Here, Stb1 plays a role in Sin3's action on transcription at the G1/S-boundary. Through bioinformatic analyses a potential Sin3-interaction domain (SID) was detected in Stb1, and beyond that, connections within the G1/S-regulatory network are discussed in structural and evolutionary context thereby providing conceptual perspectives.

H3K9 methylation extends across natural boundaries of heterochromatin in the absence of an HP1 protein.

Proteins of the conserved HP1 family are elementary components of heterochromatin and are generally assumed to play a central role in the creation of a rigid, densely packed heterochromatic network that is inaccessible to the transcription machinery. Here, we demonstrate that the fission yeast HP1 protein Swi6 exists as a single highly dynamic population that rapidly exchanges in cis and in trans between different heterochromatic regions. Binding to methylated H3K9 or to heterochromatic RNA decelerates Swi6 mobility. We further show that Swi6 is largely dispensable to the maintenance of heterochromatin domains. In the absence of Swi6, H3K9 methylation levels are maintained by a mechanism that depends on polymeric self-association properties of Tas3, a subunit of the RNA-induced transcriptional silencing complex. Our results disclose a surprising role for Swi6 dimerization in demarcating constitutive heterochromatin from neighboring euchromatin. Thus, rather than promoting maintenance and spreading of heterochromatin, Swi6 appears to limit these processes and appropriately confine heterochromatin.

Induction of triacylglycerol synthesis in yeast by cell cycle arrest.

In this study, we found that cell cycle arrest induced by alpha-factor mating pheromone (G1), hydroxyurea (S) or nocodazole (G2/M) was associated to increased lipid droplet (LD) content. To identify novel cell cycle genes involved in LD homeostasis, we screened a deletion library for strains with altered LD levels. Among the mutants related to mitotic cell cycle, we found 24 hits that displayed a significantly higher LD content. Ontology mapping showed that neither a biological process nor a specific cell cycle phase was enriched among the hits. We decided to further study the role of SWI4 on LD homeostasis as it is involved in G1/S transition, a stage where lipolysis is active. The high LD content of swi4Δ mutant was not due to inhibition of lipolysis, but due to an increase in triacylglycerol (TAG) synthesis. In addition, deletion of the AMP kinase gene SNF1 or inhibition of TORC1 activity, both known regulators of LD homeostasis, further increased the LD content of a swi4Δ mutant. These findings highlight a role of the cell cycle regulator SWI4 in the coordination of lipid metabolism which is independent of the TORC1 and SNF1/AMPK pathways.

An RNA aptamer to HP1/Swi6 facilitates heterochromatin formation at an ectopic locus in S.pombe.

In the fission yeast Schizosaccharomyces pombe (S.pombe), heterochromatin domains are established and maintained by protein complexes that contain numerous RNA binding domains among their components. The fission yeast HP1 protein Swi6 is one such component and contains an unstructured RNA-binding hinge, which is important for the integrity and silencing of heterochromatin. In this study, we have used an RNA aptamer that likely binds to the Swi6 hinge with high affinity, as a tool to perturb the natural interactions mediated by this domain. When the hinge is blocked by the aptamer RNA, Swi6 appears to become less restricted to the pericentromeres and is enriched at specific euchromatic loci. This suggests a role for the Swi6 hinge, along with the chromoshadow domain (previously shown) in controlling the spread of heterochromatin in S.pombe. The study also highlights the potential of using a synthetic aptamer RNA as a tool to perturb nucleic acid - protein interaction in vivo with the objective of understanding the functional relevance of such an interaction.

Contribution of the Slt2-regulated transcription factors to echinocandin tolerance in Candida glabrata.

Echinocandin-class antifungals, including micafungin, are considered as the first-line treatment for Candida glabrata infections. However, recent epidemiological surveys have revealed an increasing number of C. glabrata isolates exhibiting decreased echinocandin susceptibilities. The Slt2 mitogen-activated protein kinase pathway is important for maintenance of cell wall integrity in fungi. Rlm1 and Swi4-Swi6 cell cycle box binding factor (SBF) are transcription factors downstream of Slt2. While Slt2 and Rlm1 play important roles in response to cell wall stresses, such as micafungin exposure, little is known about SBF in C. glabrata. Here, we generated C. glabrata strains lacking or overexpressing SWI4 and SWI6 and evaluated their susceptibilities to micafungin. Micafungin tolerance considerably decreased in the ∆swi4 strain, whereas it increased in the strains overexpressing SWI4. On the other hand, deletion of SWI6 slightly impaired micafungin tolerance, but overexpression of SWI6 had no effect. These results suggest that, although Swi4 and Swi6 form a protein complex, Swi4 is involved in micafungin tolerance more predominantly than Swi6 in C. glabrata. Furthermore, the overexpression of RLM1 induced increased micafungin tolerance in the wild-type background but not in the ∆swi4 and ∆swi6 strains, suggesting that Rlm1 and SBF function interdependently in response to micafungin exposure.

The APSES transcription factor CfSwi6 is required for growth, cell wall integrity, and pathogenicity of Ceratocystis fimbriata.

Cell wall integrity (CWI) is crucial for the growth, development, and host invasion of pathogenic fungi. The APSES transcription factor Swi6 in fungi plays a role in mediating cell wall integrity through the mitogen-activated protein kinase (MAPK) signaling pathway. Ceratocystis fimbriata is a notorious pathogenic fungus responsible for causing black rot in sweet potatoes. In this study, an orthologous APSES transcription factor Swi6 (CfSwi6) downstream of the CWI regulatory pathway in C. fimbriata was characterized. Deletion of CfSWI6 leads to impaired hyphal development, conidiation, and compromised cell wall integrity, resulting in a significant reduction in virulence. Transcriptome analysis revealed the involvement of CfSWI6 in various pathways, including the MAPK pathway, DNA synthesis and stress response. ChIP-seq data provided predictions of potential target genes regulated by CfSwi6. Through yeast one-hybrid, we confirmed the direct binding of CfSwi6 to the promoter of the chitin synthetase gene. In summary, these findings indicated that CfSwi6 plays an important role in the growth, development, and pathogenicity of C. fimbriata. This study provides new insights into the pathogenic mechanism of C. fimbriata in sweet potato and inspires potential strategies to control sweet potato black rot.

The condensation of HP1α/Swi6 imparts nuclear stiffness.

Biomolecular condensates have emerged as major drivers of cellular organization. It remains largely unexplored, however, whether these condensates can impart mechanical function(s) to the cell. The heterochromatin protein HP1α (Swi6 in Schizosaccharomyces pombe) crosslinks histone H3K9 methylated nucleosomes and has been proposed to undergo condensation to drive the liquid-like clustering of heterochromatin domains. Here, we leverage the genetically tractable S. pombe model and a separation-of-function allele to elucidate a mechanical function imparted by Swi6 condensation. Using single-molecule imaging, force spectroscopy, and high-resolution live-cell imaging, we show that Swi6 is critical for nuclear resistance to external force. Strikingly, it is the condensed yet dynamic pool of Swi6, rather than the chromatin-bound molecules, that is essential to imparting mechanical stiffness. Our findings suggest that Swi6 condensates embedded in the chromatin meshwork establish the emergent mechanical behavior of the nucleus as a whole, revealing that biomolecular condensation can influence organelle and cell mechanics.

Heat stress-induced activation of MAPK pathway attenuates Atf1-dependent epigenetic inheritance of heterochromatin in fission yeast.

Eukaryotic cells are constantly exposed to various environmental stimuli. It remains largely unexplored how environmental cues bring about epigenetic fluctuations and affect heterochromatin stability. In the fission yeast Schizosaccharomyces pombe, heterochromatic silencing is quite stable at pericentromeres but unstable at the mating-type (mat) locus under chronic heat stress, although both loci are within the major constitutive heterochromatin regions. Here, we found that the compromised gene silencing at the mat locus at elevated temperature is linked to the phosphorylation status of Atf1, a member of the ATF/CREB superfamily. Constitutive activation of mitogen-activated protein kinase (MAPK) signaling disrupts epigenetic maintenance of heterochromatin at the mat locus even under normal temperature. Mechanistically, phosphorylation of Atf1 impairs its interaction with heterochromatin protein Swi6HP1, resulting in lower site-specific Swi6HP1 enrichment. Expression of non-phosphorylatable Atf1, tethering Swi6HP1 to the mat3M-flanking site or absence of the anti-silencing factor Epe1 can largely or partially rescue heat stress-induced defective heterochromatic maintenance at the mat locus.

Fdo1, Fkh1, Fkh2 and the Swi6-Mbp1 MBF complex regulate Mcd1 levels to impact eco1 rad61 cell growth in Saccharomyces cerevisiae.

Cohesins promote proper chromosome segregation, gene transcription, genomic architecture, DNA condensation, and DNA damage repair. Mutations in either cohesin subunits or regulatory genes can give rise to severe developmental abnormalities (such as Robert Syndrome and Cornelia de Lange Syndrome) and also are highly correlated with cancer. Despite this, little is known about cohesin regulation. Eco1 (ESCO2/EFO2 in humans) and Rad61 (WAPL in humans) represent two such regulators but perform opposing roles. Eco1 acetylation of cohesin during S phase, for instance, stabilizes cohesin-DNA binding to promote sister chromatid cohesion. On the other hand, Rad61 promotes the dissociation of cohesin from DNA. While Eco1 is essential, ECO1 and RAD61 co-deletion results in yeast cell viability, but only within a limited temperature range. Here, we report that eco1 rad61 cell lethality is due to reduced levels of the cohesin subunit Mcd1. Results from a suppressor screen further reveals that FDO1 deletion rescues the temperature sensitive (ts) growth defects exhibited by eco1 rad61 double mutant cells by increasing Mcd1 levels. Regulation of MCD1 expression, however, appears more complex. Elevated expression of MBP1, which encodes a subunit of the MBF transcription complex, also rescues eco1 rad61 cell growth defects. Elevated expression of SWI6, however, which encodes the Mbp1-binding partner of MBF, exacerbates eco1 rad61 cell growth and also abrogates the Mpb1-dependent rescue. Finally, we identify two additional transcription factors, Fkh1 and Fkh2, that impact MCD1 expression. In combination, these findings provide new insights into the nuanced and multi-faceted transcriptional pathways that impact MCD1 expression.

The transcription cofactor FgSwi6 plays a role in growth and development, carbendazim sensitivity, cellulose utilization, lithium tolerance, deoxynivalenol production and virulence in the filamentous fungus Fusarium graminearum.

Fusarium head blight (FHB) is a worldwide devastating disease of wheat, barley and other small grain cereals and caused primarily by Fusarium graminearum. Carbendazim (MBC) is one of the fungicides widely used to control FHB in China. In the present study, we have identified the F. graminearum locus FGSG_04220 as the sequence homolog for Saccharomyces cerevisiae ScSWI6, named FgSWI6 hereafter. Deletion of FgSWI6 causes mycelium of F. graminearum become sensitive to MBC in liquid medium. In addition, deletion of FgSWI6 reduces mycelial growth as well as production and development of conidia. F. graminearum cells lacking FgSWI6 show reduced production efficiency and sizes of perithecia as well as a defect in the production of ascus and ascospore. FgSWI6 is required for the cellulose utilization, lithium tolerance and deoxynivalenol (DON) production of this pathogen. Furthermore, deletion of FgSWI6 significantly attenuates the virulence of F. graminearum on wheat. Therefore, FgSwi6p plays an important role in growth and development of the economically important fungal pathogen F. graminearum as well as its resistance to MBC.

The Mechanism of Transcription Factor Swi6 in Regulating Growth and Pathogenicity of Ceratocystis fimbriata: Insights from Non-Targeted Metabolomics.

Ceratocystis fimbriata (C. fimbriata) is a notorious pathogenic fungus that causes sweet potato black rot disease. The APSES transcription factor Swi6 in fungi is located downstream of the cell wall integrity (CWI)-mitogen-activated protein kinase (MAPK) signaling pathway and has been identified to be involved in cell wall integrity and virulence in several filamentous pathogenic fungi. However, the specific mechanisms by which Swi6 regulates the growth and pathogenicity of plant pathogenic fungi remain elusive. In this study, the SWI6 deletion mutants and complemented strains of C. fimbriata were generated. Deletion of Swi6 in C. fimbriata resulted in aberrant growth patterns. Pathogenicity assays on sweet potato storage roots revealed a significant decrease in virulence in the mutant. Non-targeted metabolomic analysis using LC-MS identified a total of 692 potential differentially accumulated metabolites (PDAMs) in the ∆Cfswi6 mutant compared to the wild type, and the results of KEGG enrichment analysis demonstrated significant enrichment of PDAMs within various metabolic pathways, including amino acid metabolism, lipid metabolism, nucleotide metabolism, GPI-anchored protein synthesis, and ABC transporter metabolism. These metabolic pathways were believed to play a crucial role in mediating the growth and pathogenicity of C. fimbriata through the regulation of CWI. Firstly, the deletion of the SWI6 gene led to abnormal amino acid and lipid metabolism, potentially exacerbating energy storage imbalance. Secondly, significant enrichment of metabolites related to GPI-anchored protein biosynthesis implied compromised cell wall integrity. Lastly, disruption of ABC transport protein metabolism may hinder intracellular transmembrane transport. Importantly, this study represents the first investigation into the potential regulatory mechanisms of SWI6 in plant filamentous pathogenic fungi from a metabolic perspective. The findings provide novel insights into the role of SWI6 in the growth and virulence of C. fimbriata, highlighting its potential as a target for controlling this pathogen.

Opposing Roles of FACT for Euchromatin and Heterochromatin in Yeast.

DNA is stored in the nucleus of a cell in a folded state; however, only the necessary genetic information is extracted from the required group of genes. The key to extracting genetic information is chromatin ambivalence. Depending on the chromosomal region, chromatin is characterized into low-density "euchromatin" and high-density "heterochromatin", with various factors being involved in its regulation. Here, we focus on chromatin regulation and gene expression by the yeast FACT complex, which functions in both euchromatin and heterochromatin. FACT is known as a histone H2A/H2B chaperone and was initially reported as an elongation factor associated with RNA polymerase II. In budding yeast, FACT activates promoter chromatin by interacting with the transcriptional activators SBF/MBF via the regulation of G1/S cell cycle genes. In fission yeast, FACT plays an important role in the formation of higher-order chromatin structures and transcriptional repression by binding to Swi6, an HP1 family protein, at heterochromatin. This FACT property, which refers to the alternate chromatin-regulation depending on the binding partner, is an interesting phenomenon. Further analysis of nucleosome regulation within heterochromatin is expected in future studies.

Exportin Crm1 is important for Swi6 nuclear shuttling and MBF transcription activation in Saccharomyces cerevisiae.

BACKGROUND: Swi6 acts as a transcription factor in budding yeast, functioning in two different heterodimeric complexes, SBF and MBF, that activate the expression of distinct but overlapping sets of genes. Swi6 undergoes regulated changes in nucleocytoplasmic localization throughout the cell cycle that correlate with changes in gene expression. This study investigates how nucleocytoplasmic transport by multiple transport factors may influence specific Swi6 activities. RESULTS: Here we show that the exportin Crm1 is important for Swi6 nuclear export and activity. Loss of a putative Crm1 NES or inhibition of Crm1 activity results in changes in nucleocytoplasmic Swi6 localization. Alteration of the Crm1 NES in Swi6 results in decreased MBF-mediated gene expression, but does not affect SBF reporter expression, suggesting that export of Swi6 by Crm1 regulates a subset of Swi6 transcription activation activity. Finally, alteration of the putative Crm1 NES in Swi6 results in cells that are larger than wild type, and this increase in cell size is exacerbated by deletion of Msn5. CONCLUSIONS: These data provide evidence that Swi6 has at least two different exportins, Crm1 and Msn5, each of which interacts with a distinct nuclear export signal. We identify a putative nuclear export signal for Crm1 within Swi6, and observe that export by Crm1 or Msn5 independently influences Swi6-regulated expression of a different subset of Swi6-controlled genes. These findings provide new insights into the complex regulation of Swi6 transcription activation activity and the role of nucleocytoplasmic shuttling in regulated gene expression.

The regional sequestration of heterochromatin structural proteins is critical to form and maintain silent chromatin.

Budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe are good models for heterochromatin study. In S. pombe, H3K9 methylation and Swi6, an ortholog of mammalian HP1, lead to heterochromatin formation. However, S. cerevisiae does not have known epigenetic silencing markers and instead has Sir proteins to regulate silent chromatin formation. Although S. cerevisiae and S. pombe form and maintain heterochromatin via mechanisms that appear to be fundamentally different, they share important common features in the heterochromatin structural proteins. Heterochromatin loci are localized at the nuclear periphery by binding to perinuclear membrane proteins, thereby producing distinct heterochromatin foci, which sequester heterochromatin structural proteins. In this review, we discuss the nuclear peripheral anchoring of heterochromatin foci and its functional relevance to heterochromatin formation and maintenance.

Two secured FACT recruitment mechanisms are essential for heterochromatin maintenance.

FACT (facilitate chromatin transcription) is involved in heterochromatic silencing, but its mechanisms and function remain unclear. We reveal that the Spt16 recruitment mechanism operates in two distinct ways in heterochromatin. First, Pob3 mediates Spt16 recruitment onto the heterochromatin through its Spt16 dimerization and tandem PH domains. Without Pob3, Spt16 recruitment is partially reduced, exhibiting a silencing defect and impaired H2A/H2B organization. Second, heterochromatin protein 1 (HP1)/Swi6 mediates Spt16 recruitment onto the heterochromatin by physical interaction of the Swi6 chromo-shadow domain (CSD) and Spt16 peptidase-like domains. Several CSD mutants are tested for Spt16 binding activity, and the charged loop connecting ß1 and ß2 is critical for Spt16 binding and heterochromatic silencing. Loss of these pathways causes a severe defect in H3K9 methylation and HP1/Swi6 localization in the pericentromeric region, exhibiting transcriptional silencing defects and disordered heterochromatin. Our findings suggest that FACT and HP1/Swi6 work intimately to regulate heterochromatin organization.

Centromere Stability: The Replication Connection.

The fission yeast centromere, which is similar to metazoan centromeres, contains highly repetitive pericentromere sequences that are assembled into heterochromatin. This is required for the recruitment of cohesin and proper chromosome segregation. Surprisingly, the pericentromere replicates early in the S phase. Loss of heterochromatin causes this domain to become very sensitive to replication fork defects, leading to gross chromosome rearrangements. This review examines the interplay between components of DNA replication, heterochromatin assembly, and cohesin dynamics that ensures maintenance of genome stability and proper chromosome segregation.

The Transcription Cofactor Swi6 of the Fusarium graminearum Is Involved in Fusarium Graminearum Virus 1 Infection-Induced Phenotypic Alterations.

The transcription cofactor Swi6 plays important roles in regulating vegetative growth and meiosis in Saccharomyces cerevisiae. Functions of Swi6 ortholog were also characterized in Fusarium graminearum which is one of the devastating plant pathogenic fungi. Here, we report possible role of FgSwi6 in the interaction between F. graminearum and Fusarium graminearum virus 1 (FgV1) strain DK21. FgV1 perturbs biological characteristics of host fungi such as vegetative growth, sporulation, pigmentation, and reduction of the virulence (hypovirulence) of its fungal host. To characterize function(s) of FgSWI6 gene during FgV1 infection, targeted deletion, over-expression, and complementation mutants were generated and further infected successfully with FgV1. Deletion of FgSwi6 led to severe reduction of vegetative growth even aerial mycelia while over-expression did not affect any remarkable alteration of phenotype in virus-free isolates. Virus-infected (VI) FgSWI6 deletion isolate exhibited completely delayed vegetative growth. However, VI FgSWI6 over-expression mutant grew faster than any other VI isolates. To verify whether these different growth patterns in VI isolates, viral RNA quantification was carried out using qRT-PCR. Surprisingly, viral RNA accumulations in VI isolates were similar regardless of introduced mutations. These results provide evidence that FgSWI6 might play important role(s) in FgV1 induced phenotype alteration such as delayed vegetative growth.

Aurora B kinase controls the separation of centromeric and telomeric heterochromatin.

The segregation of chromosomes is coordinated at multiple levels to prevent chromosome loss, a phenotype frequently observed in cancers. We recently described an essential role for telomeres in the physical separation of chromosomes and identified Aurora B kinase as a double agent involved in the separation of centromeric and telomeric heterochromatin.

Escape from Mitotic Arrest: An Unexpected Connection Between Microtubule Dynamics and Epigenetic Regulation of Centromeric Chromatin in Schizosaccharomyces pombe.

Accurate chromosome segregation is necessary to ensure genomic integrity. Segregation depends on the proper functioning of the centromere, kinetochore, and mitotic spindle microtubules and is monitored by the spindle assembly checkpoint (SAC). In the fission yeast Schizosaccharomyces pombe, defects in Dis1, a microtubule-associated protein that influences microtubule dynamics, lead to mitotic arrest as a result of an active SAC and consequent failure to grow at low temperature. In a mutant dis1 background (dis1-288), loss of function of Msc1, a fission yeast homolog of the KDM5 family of proteins, suppresses the growth defect and promotes normal mitosis. Genetic analysis implicates a histone deacetylase (HDAC)-linked pathway in suppression because HDAC mutants clr6-1, clr3∆, and sir2∆, though not hos2∆, also promote normal mitosis in the dis1-288 mutant. Suppression of the dis phenotype through loss of msc1 function requires the spindle checkpoint protein Mad2 and is limited by the presence of the heterochromatin-associated HP1 protein homolog Swi6. We speculate that alterations in histone acetylation promote a centromeric chromatin environment that compensates for compromised dis1 function by allowing for successful kinetochore-microtubule interactions that can satisfy the SAC. In cells arrested in mitosis by mutation of dis1, loss of function of epigenetic determinants such as Msc1 or specific HDACs can promote cell survival. Because the KDM5 family of proteins has been implicated in human cancers, an appreciation of the potential role of this family of proteins in chromosome segregation is warranted.