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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM035690-28
Application #
8435319
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Carter, Anthony D
Project Start
1985-12-01
Project End
2017-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
28
Fiscal Year
2013
Total Cost
$558,530
Indirect Cost
$165,583
Name
University of Oregon
Department
Biochemistry
Type
Organized Research Units
DUNS #
948117312
City
Eugene
State
OR
Country
United States
Zip Code
97403
Wilinski, Daniel; Buter, Natascha; Klocko, Andrew D et al. (2017) Recurrent rewiring and emergence of RNA regulatory networks. Proc Natl Acad Sci U S A 114:E2816-E2825
Gessaman, Jordan D; Selker, Eric U (2017) Induction of H3K9me3 and DNA methylation by tethered heterochromatin factors in Neurospora crassa. Proc Natl Acad Sci U S A 114:E9598-E9607
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
Honda, Shinji; Bicocca, Vincent T; Gessaman, Jordan D et al. (2016) Dual chromatin recognition by the histone deacetylase complex HCHC is required for proper DNA methylation in Neurospora crassa. Proc Natl Acad Sci U S A 113:E6135-E6144
Klocko, Andrew D; Ormsby, Tereza; Galazka, Jonathan M et al. (2016) Normal chromosome conformation depends on subtelomeric facultative heterochromatin in Neurospora crassa. Proc Natl Acad Sci U S A 113:15048-15053
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 ? is required for normal heterochromatic formation and DNA methylation. PLoS Genet 11:e1005083
Aramayo, Rodolfo; Selker, Eric U (2013) Neurospora crassa, a model system for epigenetics research. Cold Spring Harb Perspect Biol 5:a017921
Qi, Xiaodong; Li, Yang; Honda, Shinji et al. (2013) The common ancestral core of vertebrate and fungal telomerase RNAs. Nucleic Acids Res 41:450-62

Showing the most recent 10 out of 70 publications