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The hunt for dim mutants: DNA
Methylation in
Neurospora crassa
Calvin Summers
Mentor: Andy Klocko
Selker Lab
What is DNA methylation?
Cytosine
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5’ Methylcytosine
When DNA is methylated, the elongation of RNA
Polymerase II during transcription is blocked.
Methylated DNA = Silenced DNA
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)
Neurospora crassa:
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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.
DNA Methylation is associated with
heterochromatin, or condensed, silenced DNA
Honda S , Selker E U Mol. Cell. Biol. 2008;28:6044-6055
DIM-5
DIM-2
HP1
CUL4
DIM-7
1. DNA binding protein that
recruits DIM-5 to A:T rich
DNA?
Dual Reporter Strain to find dim
(defective in methylation) mutants
Methylated
hyg gene
Methylated
bar gene
Grow on
Hygromycin and
Basta
+Basta
+Hygromycin
Growth=
A loss of
methylation
(dim mutant)
Southern Blot of xAM157 probed at 8:A6
W.T.
Me-
xAM 157-5
xAM 157
-1 -4 -5
DNA digested
with methylation
sensitive
restriction
endonuclease
AvaII
Wild type Me
Mutant Me (loss)
When there is a
loss of
methylation the
Ava II cuts and
you get a low
band.
Forced Heterokaryon Testing
known dim KO tester
dim mutant
dim z
his
hph
Δbar
Δam
Δinl
Δtrp -2
Δ dim x
Needs tryptophan to grow
Needs histidine, alanine, inositol to grow
Allow to grow on minimal media
dim ko
mutant
Cells form
multinucleate cell that
meets growth
requirements
Southern Blot of Forced Heterokaryons for xAM157-5
Wild type Me
Mutant Me (loss)
Methylation is restored
for all of these tester
strains=
NOVEL MUTANT
Now what?
cul 4
hpo
hda
dim 9
dim 8
dim 7
dim 5
dim 2
N2977
N3409 Me-
dim tester strains
Bulk Segregant Analysis
Oakridge
Mauriceville (N51)
Cross Strains
(Germinate on
Basta)
Look for mutants
Accrue Progeny and
extract DNA
Pool DNA of
Mutant progeny
and sequence
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:
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5x Basta Control
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6x Basta
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10x Basta
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3x Basta + 1x Hygromycin
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6x Basta + 1x Hygromycin
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10x Basta + 1x Hygromycin
Check to see
-How many progeny are able to grow (If at all)
-What proportion of progeny are dim mutants?
N2977
N3409 MeN51
N51 Me-
xAM 157-5xN51 progeny grown on drug tester plates
6x+Hyg
10x Basta
3x +Hyg
10x+Hyg
40 mutant progeny are present on this gel!
Bulk Segregant Analysis
Oakridge
Mauriceville (N51)
Cross Strains
(Germinate on
Basta)
Look for mutants
Accrue Progeny and
extract DNA
Pool DNA of
Mutant progeny
and sequence
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
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Andy Klocko
Eric Selker
Peter O Day
Mike Rountree
Paula Gisrafii
Jordan Gessman
Kirsty Jamieson
Robert Parrish
Michael Freitag’s Lab
NICHD UO R25