Centromeres form the foundation of kinetochores, the attachment points for spindle microtubules that transport chromosomes into daughter nuclei during nuclear division. Defective centromeres result in faulty chromosome segregation and aneuploidy, implicated as one cause of cancer. A conserved centromere-specific histone variant (CenH3), repeated DNA and posttranslational histone modifications are universally required for centromere function, but mechanisms for centromere assembly and maintenance remain unresolved. The relative impact of DNA composition vs. epigenetic modifications is difficult to separate in most species. Here, two filamentous fungi, Neurospora crassa and Fusarium graminearum, are used as powerful systems to test the importance of DNA sequence and heterochromatin for centromere function. Both fungi lack tandem repeats, making the centromeric DNA amenable to high-throughput sequencing analyses. Most characteristics of human centromeres are found in these species, making them excellent reference organisms. All planned genetic studies are straightforward with these fungi but difficult to carry out in mammals. This project draws on exciting results from our work with Neurospora that suggest that current models for centromere maintenance are inadequate. Long-term goals are to determine how centromeres assemble and how they are maintained in filamentous fungi, an important - but in this respect still poorly characterized - group of human, animal and plant pathogens. The two major hypotheses are that maintenance of Neurospora centromeres relies on interactions of centromere-specific nucleosomes with heterochromatic histone modifications, and that incorporation of CenH3 during meiosis is controlled by a novel mechanism mediated via CenH3 mRNA.
Specific aims will test these hypotheses by: (1) characterizing critical features of centromere components (2) determining why heterochromatin is essential for maintenance of Neurospora centromeres, and (3) deciphering mechanisms of CenH3 regulation. To accomplish these aims, centromeric DNA will be tested for the propensity to nucleate centromeric chromatin in vivo and a novel suppressor screen for mutants that bypass the requirement for heterochromatin will be carried out. Biochemical methods (chromatin immunoprecipitation, chromosome conformation capture, affinity purification of centromere proteins) will complement genetic and cytological approaches. Large amounts of supporting preliminary data have been accumulated, most materials and methods to address underlying mechanisms are at hand, and currently no other lab is working on this fundamental problem with filamentous fungi. The proposed experiments will not only provide much needed key knowledge eventually to be used to guide development of new antifungal drugs, but will also lead to a better understanding of epigenetic determinants for the regulation of centromere assembly and maintenance.
During cell division, faulty chromosome segregation can occur, which has been implicated as one root cause of cancer and several inherited diseases. It is not well understood how centromeres assemble, but much of what we have learned about these essential components of chromosomes stems from studies with simple model systems, such as filamentous fungi. Our long-term goal is to shed light on mechanisms of centromere assembly and inheritance in filamentous fungi, a currently ill-characterized group of human pathogens. One translational goal of this project is to guide the design of drugs that interfere with chromosome segregation, which can be used for both cancer research and for the treatment of invasive fungal infections.
|Galazka, Jonathan M; Klocko, Andrew D; Uesaka, Miki et al. (2016) Neurospora chromosomes are organized by blocks of importin alpha-dependent heterochromatin that are largely independent of H3K9me3. Genome Res 26:1069-80|
|Studt, Lena; RÃ¶sler, Sarah M; Burkhardt, Immo et al. (2016) Knock-down of the methyltransferase Kmt6 relieves H3K27me3 and results in induction of cryptic and otherwise silent secondary metabolite gene clusters in Fusarium fujikuroi. Environ Microbiol 18:4037-4054|
|Freitag, Michael (2016) The kinetochore interaction network (KIN) of ascomycetes. Mycologia 108:485-505|
|Soyer, Jessica L; MÃ¶ller, Mareike; Schotanus, Klaas et al. (2015) Chromatin analyses of Zymoseptoria tritici: Methods for chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). Fungal Genet Biol 79:63-70|
|Schotanus, Klaas; Soyer, Jessica L; Connolly, Lanelle R et al. (2015) Histone modifications rather than the novel regional centromeres of Zymoseptoria tritici distinguish core and accessory chromosomes. Epigenetics Chromatin 8:41|
|Sasaki, Takahiko; Lynch, Kelsey L; Mueller, Caitlin V et al. (2014) Heterochromatin controls Î³H2A localization in Neurospora crassa. Eukaryot Cell 13:990-1000|
|Galazka, Jonathan M; Freitag, Michael (2014) Variability of chromosome structure in pathogenic fungi--of 'ends and odds'. Curr Opin Microbiol 20:19-26|
|Karimi-Aghcheh, Razieh; Bok, Jin Woo; Phatale, Pallavi A et al. (2013) Functional analyses of Trichoderma reesei LAE1 reveal conserved and contrasting roles of this regulator. G3 (Bethesda) 3:369-78|
|Connolly, Lanelle R; Smith, Kristina M; Freitag, Michael (2013) The Fusarium graminearum histone H3 K27 methyltransferase KMT6 regulates development and expression of secondary metabolite gene clusters. PLoS Genet 9:e1003916|
|Niehaus, Eva-Maria; Kleigrewe, Karin; Wiemann, Philipp et al. (2013) Genetic manipulation of the Fusarium fujikuroi fusarin gene cluster yields insight into the complex regulation and fusarin biosynthetic pathway. Chem Biol 20:1055-66|
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