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The hunt for dim mutants: DNA Methylation in Neurospora crassa Calvin Summers Mentor: Andy Klocko Selker Lab Slide 2 Slide 3 What is DNA methylation? When DNA is methylated, the elongation of RNA Polymerase II during transcription is blocked. Methylated DNA = Silenced DNA Cytosine5 Methylcytosine Slide 4 Why is methylation important? Aberrant DNA methylation is highly correlated to cancer. Loss of DNA methylation in mice is either lethal or leads to improper development Masaki Okano, Daphne W Bell, Daniel A Haber, En Li, DNA Methyltransferases Dnmt3a and Dnmt3b Are Essential for De Novo Methylation and Mammalian Development, Cell, Volume 99, Issue 3, 29 October 1999, Pages 247-257, ISSN0092-8674,S0092867400816566) Slide 5 Neurospora crassa: Crosses Easily Highly methylated genome due to RIP Can form heterokaryotic cells Haploid, easy to identify recessive mutations Genome has been sequenced A loss of methylation mutant is still viable! Slide 6 Basic model for control of DNA methylation in Neurospora. Honda S, Selker E U Mol. Cell. Biol. 2008;28:6044-6055 DNA Methylation is associated with heterochromatin, or condensed, silenced DNA Slide 7 DIM-5 DIM-2 HP1 CUL4 DIM-7 1. DNA binding protein that recruits DIM-5 to A:T rich DNA? Slide 8 Methylated hyg gene Methylated bar gene Dual Reporter Strain to find dim (defective in methylation) mutants Grow on Hygromycin and Basta +Basta +Hygromycin Growth= A loss of methylation (dim mutant) Slide 9 Mutant Me (loss) Wild type Me When there is a loss of methylation the Ava II cuts and you get a low band. Southern Blot of xAM157 probed at 8:A6 xAM 157 xAM 157-5 W.T. Me- -1 -4 -5 DNA digested with methylation sensitive restriction endonuclease AvaII Slide 10 Forced Heterokaryon Testing known dim KO tester dim x trp -2 dim z his hph bar am inl Needs tryptophan to grow Needs histidine, alanine, inositol to grow dim mutant Allow to grow on minimal media dim komutant Cells form multinucleate cell that meets growth requirements Slide 11 Southern Blot of Forced Heterokaryons for xAM157-5 N2977 N3409 Me- dim 2dim 5dim 7dim 8dim 9hdahpo cul 4 dim tester strains Mutant Me (loss) Wild type Me Methylation is restored for all of these tester strains= NOVEL MUTANT Now what? Slide 12 Oakridge Mauriceville (N51) Bulk Segregant Analysis Cross Strains (Germinate on Basta) Accrue Progeny and extract DNA Pool DNA of Mutant progeny and sequence Look for mutants Slide 13 Issues with BSA: So far I have tested about 120 N51 cross progeny for DNA methylation. Of those 120, only about 30 look to have the genotype. There is a problem with this method, it takes too long to gather enough progeny for testing. We are getting false positives. There needs to be a faster way to obtain mutants from the cross to Mauriceville so these dim strains can be mapped. Slide 14 Is 5x Basta selecting for mutants powerfully enough? Test: Germinate xAM 157-5xN51 on plates of varying drug concentrations: 5x Basta Control 6x Basta 10x Basta 3x Basta + 1x Hygromycin 6x Basta + 1x Hygromycin 10x Basta + 1x Hygromycin Check to see -How many progeny are able to grow (If at all) -What proportion of progeny are dim mutants? Slide 15 xAM 157-5xN51 progeny grown on drug tester plates 10x Basta 3x +Hyg6x+Hyg10x+Hyg N2977 N3409 Me- N51 N51 Me- 40 mutant progeny are present on this gel! Slide 16 Oakridge Mauriceville (N51) Bulk Segregant Analysis Cross Strains (Germinate on Basta) Accrue Progeny and extract DNA Pool DNA of Mutant progeny and sequence Look for mutants Slide 17 Bulk Segregant Analysis O M Maps Single Nucleotide Polymorphisms (SNPs) for Oakridge vs. Mauriceville This assay enable us to focus in on the genic region causing the lost methylation phenotype. Once the region is narrowed down we can look for causative mutations Slide 18 Future Directions Sequence the pooled DNA of: xAM 157-5xN51, 12-1xN51, and 152-4xN51 Use Bulk Segregant Analysis to locate the region causing the loss of methylation. Pinpoint the mutation causing that loss of methylation. Characterize the protein involved. Carry out biochemical and biomolecular assays that determine the role of the protein in DNA methylation. Slide 19 Thank You Andy Klocko Eric Selker Peter O Day Mike Rountree Paula Gisrafii Jordan Gessman Kirsty Jamieson Robert Parrish Michael Freitags Lab NICHD UO R25