Neurospora: The Mystery of Neurospora: The Mystery of MethylationMethylation

By: Kayla Garrett, Rochester Institute of Technology

Basic BiologyBasic Biology Haploid genomeHaploid genome Have multiple nuclei (can Have multiple nuclei (can

be homokaryon or be homokaryon or

heterokaryon)heterokaryon) Three main cell types: Three main cell types:

AscosporeAscospore Conidia Conidia HyphaeHyphae

DNA methylation is DNA methylation is

dispensabledispensable

Courtesy of http://www.fgsc.net/Neurospora/

Life CycleLife Cycle

Courtesy of http://www.fgsc.net/Neurospora/

DNA MethylationDNA Methylation

Methyl (CH3) groups are added by proteins at Methyl (CH3) groups are added by proteins at selected sites on DNA, which in turn alters its selected sites on DNA, which in turn alters its properties. properties.

Methylation makes up 70%-80% of human CpG Methylation makes up 70%-80% of human CpG These changes can occur during one’s life and are These changes can occur during one’s life and are

heritable. heritable. Much of it is still unknownMuch of it is still unknown

Methylation ModelMethylation Model

Part I: Insertional Mutants and UV mutantsPart I: Insertional Mutants and UV mutantsMethods:Methods: Spot tests were done on Insertional Mutants to identify a lack Spot tests were done on Insertional Mutants to identify a lack

of DNA methylation. of DNA methylation. Results are judged by a marked resistance to Hygromycin Results are judged by a marked resistance to Hygromycin

and Basta. and Basta. Two mutants were identified.Two mutants were identified.

Complementation Tests were done with both insertional Complementation Tests were done with both insertional mutants and UV mutants to determine if the mutation was mutants and UV mutants to determine if the mutation was novel:novel: Heterokaryons were made with the unknown mutant and a Heterokaryons were made with the unknown mutant and a

known mutant. known mutant. DNA was isolated and tested for DNA methylation. DNA was isolated and tested for DNA methylation. Two UV mutants and Two Insertional Mutants were Two UV mutants and Two Insertional Mutants were

identifiedidentified

Southern Blots: DNA was digested with AvaII and a non-Southern Blots: DNA was digested with AvaII and a non-radioactive probe was used for the region. radioactive probe was used for the region.

Part I: Insertional Mutants and UV mutantsPart I: Insertional Mutants and UV mutants

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.

Southern Blots: Cont. Southern Blots: Cont.

Part I: Insertional Mutants and UV mutantsPart I: Insertional Mutants and UV mutants

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.

Southern Blots: Cont. Southern Blots: Cont.

Part I: Insertional Mutants and UV mutantsPart I: Insertional Mutants and UV mutants

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.

Southern Blots: Cont. Southern Blots: Cont.

Part I: Insertional Mutants and UV mutantsPart I: Insertional Mutants and UV mutants

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 methylationPart II: Identifying E2 in DNA methylation 57 knockouts were tested for the presence of methylation with a Southern Blot. 57 knockouts were tested for the presence of methylation with a Southern Blot.

1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 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

3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 0 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

Southern Legend:

Lane 1: WT N150

Lane 2: dim-2

Lane 8: dim-7 (heterokaryon)

Lane 48: E2 Ubiquitin Ligase

Location of Probe on the genomeVisualization of bands after probe

Part II: Model of E2Part 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 E2Part II Cont.: Growth of E2Growth Phenotype of E2: Slow growth and poor conidiation

Wild Type E2 mutant

Growth Rate: E2 grows significantly slower

0

5

10

15

20

25

30

Hours

Centimeters

WT

E2

Part II Cont. :Confirmation of KO locationPart II Cont. :Confirmation of KO location E2E2 mutant was knocked out by replacing mutant was knocked out by replacing

the E2 gene with the E2 gene with hphhph The portion was amplified and digested The portion was amplified and digested

with XbaI in both the mutant and WT. with XbaI in both the mutant and WT. There is restriction site for XbaI in the There is restriction site for XbaI in the

E2 gene which should be missing in the E2 gene which should be missing in the mutant. mutant.

The results confirm that the KO exists in The results confirm that the KO exists in the E2 gene. the E2 gene.

Digestion with XbaI

1 2 3 4

Lane 1: WT undigestedLane 2: E2 mutant undigestedLane 3: WT digestedLane 4: E2 mutant digested

Part II Cont. : Extent of loss of MethylationPart II Cont. : Extent of loss of MethylationSouthern blot with methylation sensitive (Sau3AI) and methylation Southern blot with methylation sensitive (Sau3AI) and methylation

insensitive (DpnII) restriction enzymesinsensitive (DpnII) restriction enzymes 5 regions known to have DNA methylation were probed to 5 regions known to have DNA methylation were probed to

understand the extent of DNA methylation lossunderstand the extent of DNA methylation lossEtBr Stain

WT dim-2 E2SD SD SD

E2 mutant clearly shows a similar loss of methylation as dim-2

9:E1 ProbeWT dim-2 E2SD SD SD

2:B3 ProbeWT dim-2 E2SD SD SD

WT dim-2 E2SD SD SD

WT dim-2 E2SD SD SD

WT dim-2 E2SD SD SD

8:A6 Probe 8:G3 Probe 2:G9 Probe

Part II Cont. : Western Blot of E2cPart II Cont. : Western Blot of E2c A Western blot with nuclear extracts was done to determine at which A Western blot with nuclear extracts was done to determine at which

point E2 affects methylation.point E2 affects methylation. Antibodies for Histone 3 and Histone 3 lysine 9 methylation were Antibodies for Histone 3 and Histone 3 lysine 9 methylation were

used. used.

WT dim-5 (N/A) E2

H3

H3K9me3

38.4kDa31.5kDa

18kDa 7kDa

38.4kDa31.5kDa

18kDa 7kDa

• Histones are about 17 kDa in size.

Part III: Yellow Fluorescent ProteinPart III: Yellow Fluorescent Protein Yellow Fluorescent Protein (YFP) is a useful approach in Yellow Fluorescent Protein (YFP) is a useful approach in

determining protein to protein interactions. determining protein to protein interactions.

A protein is attached to each half of the YFP. If an interaction A protein is attached to each half of the YFP. If an interaction occurs there is fluorescence.occurs there is fluorescence.

This approach was used for This approach was used for hpohpo

Part III Cont. : YFP MethodsPart III Cont. : YFP Methods

Transformed E.coli

Removed Hpo segment and

inserted in pYFP

GLOWMixed

samples

GLOW

Purified plasmids and transformed Neurospora

Part III Cont.: YFP was successfulPart III Cont.: YFP was successful

Above is a microscope image taken of one of the heterokaryon Above is a microscope image taken of one of the heterokaryon macroconidia samples. The fluorescence is localized to the macroconidia samples. The fluorescence is localized to the nuclei of the macroconidianuclei of the macroconidia

ConclusionsConclusions

The Insertional mutants were not methylation The Insertional mutants were not methylation mutants and the UV mutants were already mutants and the UV mutants were already known mutants (known mutants (raf-1raf-1 and and hda-1hda-1), both ), both shown by Southern blotting. shown by Southern blotting.

E2 plays an important role in DNA E2 plays an important role in DNA methylation for methylation for NeurosporaNeurospora

1.1. It affects the organisms growth and developmentIt affects the organisms growth and development

2.2. It’s required for the trimethylation of H3K9It’s required for the trimethylation of H3K9 YFP was successfully used with YFP was successfully used with hpo hpo

Special ThanksSpecial Thanks My mentors, Keyur Adhvaryu and Anthony My mentors, Keyur Adhvaryu and Anthony

Shiver. Shiver. The Selker LabThe Selker Lab SPURSPUR