We exploit the fungus Neurospora crassa to elucidate the control and function of DNA methylation, an pigenetic process required for X chromosome-inactivation, genomic imprinting and normal development in mammals. In humans, loss of a DNA methyltransferase (MTase) causes ICF syndrome, loss of the methyl- DNA binding protein MeCP2 causes Rett syndrome and abnormal methylation is associatedwith cancer. . Isolation of Neurospora mutants defective in DNA methylation (dim) has led to insights into the control and function of methylation in eukaryotes. For example, our identification of DIM-5 as a histone H3 MTase demonstrated for the first time that histone modifications can control DNA methylation. Our work is helped by valuable resources including: 1. the sequence of the Neurospora genome, 2. efficient methods to knock out genes in Neurospora, S.epitope tagging systems for localization and biochemical studies, 4. antibodies specific for modified histones and other relevant epitopes and 5. powerful analytical methods (e.g., mass spectrometry, chromatin immunoprecipitation and microarray analysis). Most of our effort will go towards identifying and characterizing gene products involved in DNA methylation. We will identify mutants defective in silencing methylated DNA using both forward and reverse genetics. The latter will take advantage of the genome knock-out project, which will disrupt most Neurospora genes within the next few years. We will test all knock-outs for de-repression of our silenced bar gene. We will also employ a biochemical approach to identify protein(s) that trigger de novo methylation based on our identification of sequences that trigger DNA methylation in vivo. In other work, we will elucidate the function, mechanism and interrelationships of histone modifications associated with methylated DNA. We will determine which gene/protein is responsible for each observed histone modification in this fungus and test the phenotypes of mutants unable to perform the modifications. We also will determine the extent to which the histone modifications depend on each other. In addition, we will characterize the Neurospora 'epigenome'in wildtype and mutants using a 'ChlP-on-chip' approach with stock and custom microarrays and will use proteomics to investigate whether critical histone MTases are in protein complexes. Finally, we will follow up our observations suggesting that histone demethylases influence DNA methylation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM035690-27
Application #
8197759
Study Section
Special Emphasis Panel (NSS)
Program Officer
Carter, Anthony D
Project Start
1985-12-01
Project End
2013-03-31
Budget Start
2011-12-01
Budget End
2013-03-31
Support Year
27
Fiscal Year
2012
Total Cost
$572,014
Indirect Cost
$185,518
Name
University of Oregon
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
948117312
City
Eugene
State
OR
Country
United States
Zip Code
97403
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
Selker, Eric U (2011) Neurospora. Curr Biol 21:R139-40
Xiong, Lei; Adhvaryu, Keyur K; Selker, Eric U et al. (2010) Mapping of lysine methylation and acetylation in core histones of Neurospora crassa. Biochemistry 49:5236-43
Kothe, Gregory O; Kitamura, Maki; Masutani, Mitsuko et al. (2010) PARP is involved in replicative aging in Neurospora crassa. Fungal Genet Biol 47:297-309
Lee, Heng-Chi; Li, Liande; Gu, Weifeng et al. (2010) Diverse pathways generate microRNA-like RNAs and Dicer-independent small interfering RNAs in fungi. Mol Cell 38:803-14
Honda, Shinji; Lewis, Zachary A; Huarte, Maite et al. (2010) The DMM complex prevents spreading of DNA methylation from transposons to nearby genes in Neurospora crassa. Genes Dev 24:443-54
Lewis, Zachary A; Adhvaryu, Keyur K; Honda, Shinji et al. (2010) Identification of DIM-7, a protein required to target the DIM-5 H3 methyltransferase to chromatin. Proc Natl Acad Sci U S A 107:8310-5
Rountree, M R; Selker, E U (2010) DNA methylation and the formation of heterochromatin in Neurospora crassa. Heredity (Edinb) 105:38-44
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
Lee, Dong W; Freitag, Michael; Selker, Eric U et al. (2008) A cytosine methyltransferase homologue is essential for sexual development in Aspergillus nidulans. PLoS One 3:e2531

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