The hunt for dim mutants: DNA Methylation in Neurospora crassa Calvin Summers Mentor: Andy Klocko Selker Lab

What is DNA methylation? When DNA is methylated, the elongation of RNA Polymerase II during transcription is blocked. Methylated DNA = Silenced DNA Cytosine5’ Methylcytosine

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 , ISSN ,S )

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!

Basic model for control of DNA methylation in Neurospora. Honda S, Selker E U Mol. Cell. Biol. 2008;28: DNA Methylation is associated with heterochromatin, or condensed, silenced DNA

DIM-5 DIM-2 HP1 CUL4 DIM-7 1. DNA binding protein that recruits DIM-5 to A:T rich DNA?

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)

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 W.T. Me DNA digested with methylation sensitive restriction endonuclease AvaII

Forced Heterokaryon Testing known dim KO tester Δ dim x Δtrp -2 dim z his hph Δbar Δam Δinl 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

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?

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

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.

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?

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!

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

Bulk Segregant Analysis O M Maps Single Nucleotide Polymorphisms (SNP’s) 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

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.

Thank You  Andy Klocko  Eric Selker  Peter O Day  Mike Rountree  Paula Gisrafii  Jordan Gessman  Kirsty Jamieson  Robert Parrish  Michael Freitag’s Lab  NICHD UO R25