Download DNA Methylation

Neurospora: The Mystery of
Methylation
By: Kayla Garrett, Rochester Institute of Technology
Basic Biology
Haploid genome
 Have multiple nuclei (can
be homokaryon or
heterokaryon)
 Three main cell types:
 Ascospore
 Conidia
 Hyphae
 DNA methylation is
dispensable

Courtesy of http://www.fgsc.net/Neurospora/
Life Cycle
Courtesy of http://www.fgsc.net/Neurospora/
DNA Methylation




Methyl (CH3) groups are added by proteins at
selected sites on DNA, which in turn alters its
properties.
Methylation makes up 70%-80% of human CpG
These changes can occur during one’s life and are
heritable.
Much of it is still unknown
Methylation Model
Part I: Insertional Mutants and UV mutants
Methods:
 Spot tests were done on Insertional Mutants to identify a lack
of DNA methylation.
 Results are judged by a marked resistance to Hygromycin
and Basta.
 Two mutants were identified.
 Complementation Tests were done with both insertional
mutants and UV mutants to determine if the mutation was
novel:
 Heterokaryons were made with the unknown mutant and a
known mutant.
 DNA was isolated and tested for DNA methylation.
 Two UV mutants and Two Insertional Mutants were
identified
Part I: Insertional Mutants and UV mutants

Southern Blots: DNA was digested with AvaII and a nonradioactive probe was used for the region.
Insertional Mutant 135-3B
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15
Methylation 
Loss of


Methylation
Lane 1 is the 135-3B mutant and Lane 2 is the WT control. Both show signs of
methylation, therefore 135-3B is not a methylation mutant.
Part I: Insertional Mutants and UV mutants

Southern Blots: Cont.
Insertional Mutant- 138-2E
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15
Methylation 
Loss of


Methylation
Lane 1 is the 138-2E mutant and Lane 2 is the WT control. Both show signs of
methylation, therefore 138-2B is not a methylation mutant.
Part I: Insertional Mutants and UV mutants

Southern Blots: Cont.
UV Mutant- UV154-4
1 2
3 4
5 6 7 8 9 10 11 12 13
Methylation 
Loss of


Methylation
Lane 1 is the UV154-4 mutant and Lane 4 is the raf-1 mutant (although not
visible, it’s assumed that the raf-1 mutant is not methylated.) In Lane 5 is the
resulting heterokaryon. Since the loss of methylation is maintained, UV154-4
has a the same mutation as raf-1.
Part I: Insertional Mutants and UV mutants

Southern Blots: Cont.
UV Mutant- UV201-1
1
2
3 4
5
6
7 8 9 10 11 12 13
Methylation 

Loss of

Methylation
Lane 1 is the UV201-1 mutant and Lane 2 is the hda-1 mutant. In Lane 3 is
the resulting heterokaryon. Since the loss of methylation is maintained,
UV201-1 has a the same mutation hda-1.
Part II: Identifying E2 in DNA methylation

57 knockouts were tested for the presence of methylation with a
Southern Blot.
Visualization of
bands after probe
Location of Probe on the genome
1 1 1 1 11 11 1 1 2 2 2 2 2 2 22 2 2
1 2 3 4 5 6 7 8 9 0 1
2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9
Southern Legend:
Lane 1: WT N150
Lane 2: dim-2
Lane 8: dim-7 (heterokaryon)
Lane 48: E2 Ubiquitin Ligase
3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4
0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8
4 5 5 5 5 5 5 5 5
9 0 1 2 3 4 5 6 7
Part II: Model of E2
E2’s role in eukaryotes is as a Ubiquitin ligase, which, in
conjugation with other enzymes, attaches a ubiquitin to a
substrate
Part II Cont.: Growth of E2
Growth Phenotype of E2: Slow growth and poor conidiation
Wild Type
E2 mutant
Growth Rate: E2 grows significantly slower
30
20
WT
15
E2
10
5
Hours
ur
s
60
ho
ur
s
48
ho
ur
s
36
ho
ur
s
ho
24
ho
ur
s
0
12
Centimeters
25
Part II Cont. :Confirmation of KO location
Digestion with XbaI
1
2
3
4




Lane 1: WT undigested
Lane 2: E2 mutant undigested
Lane 3: WT digested
Lane 4: E2 mutant digested
E2 mutant was knocked out by replacing
the E2 gene with hph
The portion was amplified and digested
with XbaI in both the mutant and WT.
There is restriction site for XbaI in the
E2 gene which should be missing in the
mutant.
The results confirm that the KO exists in
the E2 gene.
Part II Cont. : Extent of loss of Methylation
Southern blot with methylation sensitive (Sau3AI) and methylation
insensitive (DpnII) restriction enzymes

5 regions known to have DNA methylation were probed to
understand the extent of DNA methylation loss
EtBr Stain
WT dim-2 E2
SD SD SD
9:E1 Probe
WT dim-2 E2
SD SD SD
2:B3 Probe
WT dim-2 E2
SD SD SD
8:A6 Probe
WT dim-2 E2
SD SD SD
8:G3 Probe
WT dim-2 E2
SD SD SD
2:G9 Probe
WT dim-2 E2
SD SD SD
E2 mutant clearly shows a similar loss of methylation as dim-2
Part II Cont. : Western Blot of E2c


A Western blot with nuclear extracts was done to determine at
which point E2 affects methylation.
Antibodies for Histone 3 and Histone 3 lysine 9 methylation
were used.
WT dim-5 (N/A) E2
H3
38.4kDa
31.5kDa
18kDa
7kDa
38.4kDa
31.5kDa
H3K9me3
18kDa
7kDa
• Histones are about 17
kDa in size.
Part III: Yellow Fluorescent Protein

Yellow Fluorescent Protein (YFP) is a useful approach in
determining protein to protein interactions.

A protein is attached to each half of the YFP. If an interaction
occurs there is fluorescence.
This approach was used for hpo

Part III Cont. : YFP Methods
Removed Hpo
segment and
inserted in pYFP
Transformed E.coli
Purified plasmids and
transformed Neurospora
GLOW
Mixed
samples
GLOW
Part III Cont.: YFP was successful

Above is a microscope image taken of one of the heterokaryon
macroconidia samples. The fluorescence is localized to the
nuclei of the macroconidia
Conclusions


The Insertional mutants were not methylation
mutants and the UV mutants were already
known mutants (raf-1 and hda-1), both
shown by Southern blotting.
E2 plays an important role in DNA
methylation for Neurospora
1.
2.

It affects the organisms growth and development
It’s required for the trimethylation of H3K9
YFP was successfully used with hpo
Special Thanks



My mentors, Keyur Adhvaryu and Anthony
Shiver.
The Selker Lab
SPUR