DNA methylation is required for normal development of higher eukaryotes, presumably in part because of its role in genome defense, genomic imprinting and in other silencing processes such as X-chromosome inactivation. Abnormal DNA methylation has been implicated in tumorigenesis, imprinting disorders, Fragile-X syndrome, diabetes and neurologic disorders (e.g. bipolar syndrome, Alzheimer's, autism and schizophrenia). DNA methylation is dispensable in the fungus Neurospora crassa, facilitating genetic studies. The overall goal of the proposed research is to elucidate the mechanism of DNA methylation in eukaryotes by taking advantage of this outstanding model system. The work will be facilitated by valuable resources and tools including: 1. Efficient procedures for gene targeting and epitope-tagging;2. A collection of gene knockout strains covering ~80% of the genome;3. The mutagenic process, RIP (repeat-induced point mutation), for reverse-genetics;4. Efficient high throughput sequencing coupled with bisulfite methylation analysis, chromatin immunoprecipitation and DamID methodology;5. Sensitive proteomic (mass spectrometry) and immunological methods for identification of proteins and their modifications. An important goal of the proposed research is to identify all the components of the DNA methylation machinery and to elucidate their interrelationships. It is important to determine how sequences to be methylated are targeted and to expand our mechanistic understanding of the downstream steps required for heterochromatin formation and DNA methylation (Aims 1 &2). Another general goal of the research is to advance our understanding of the regulation of DNA methylation by identifying and characterizing the mechanisms that modulate methylation patterns (Aim 3). A final goal is to improve our understanding of mechanisms that work alongside DNA methylation to silence sequences (Aim 4).
Specific aims of the project are: 1. To exploit new and existing dim (defective in methylation) mutants to elucidate the mechanism of DNA methylation. 2. To test elements of heterochromatin for roles in the establishment and/or maintenance of silencing. 3. To characterize modifiers of DNA methylation and silencing. 4. To characterize silencing mutants that do not show DNA methylation defects.

Public Health Relevance

Methylation of DNA is a prominent feature of particular chromosomal regions of many eukaryotes including in mammals, plants and some fungi. In mammals, altered DNA methylation has been implicated in tumorigenesis, imprinting disorders, Fragile-X syndrome, diabetes, neurologic disorders and aging, but little is understood about how it is controlled. The fungus Neurospora crassa, which is the simplest model organism with DNA methylation, provides us with an exceptionally tractable system to advance our understanding of the control and function of this important process.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular Genetics B Study Section (MGB)
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Carter, Anthony D
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University of Oregon
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Jamieson, Kirsty; Wiles, Elizabeth T; McNaught, Kevin J et al. (2016) Loss of HP1 causes depletion of H3K27me3 from facultative heterochromatin and gain of H3K27me2 at constitutive heterochromatin. Genome Res 26:97-107
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
Adhvaryu, Keyur K; Gessaman, Jordan D; Honda, Shinji et al. (2015) The cullin-4 complex DCDC does not require E3 ubiquitin ligase elements to control heterochromatin in Neurospora crassa. Eukaryot Cell 14:25-8
Klocko, Andrew D; Rountree, Michael R; Grisafi, Paula L et al. (2015) Neurospora importin α is required for normal heterochromatic formation and DNA methylation. PLoS Genet 11:e1005083
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Honda, Shinji; Lewis, Zachary A; Shimada, Kenji et al. (2012) Heterochromatin protein 1 forms distinct complexes to direct histone deacetylation and DNA methylation. Nat Struct Mol Biol 19:471-7, S1
Adhvaryu, Keyur K; Berge, Emanuela; Tamaru, Hisashi et al. (2011) Substitutions in the amino-terminal tail of neurospora histone H3 have varied effects on DNA methylation. PLoS Genet 7:e1002423
Belden, William J; Lewis, Zachary A; Selker, Eric U et al. (2011) CHD1 remodels chromatin and influences transient DNA methylation at the clock gene frequency. PLoS Genet 7:e1002166
Anderson, D C; Green, George R; Smith, Kristina et al. (2010) Extensive and varied modifications in histone H2B of wild-type and histone deacetylase 1 mutant Neurospora crassa. Biochemistry 49:5244-57

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