1. Neurospora: The Mystery of Methylation By: Kayla Garrett, Rochester Institute of Technology

2. Basic Biology Haploid genome Haploid genome Have multiple nuclei (can Have multiple nuclei (can be homokaryon or heterokaryon) Three main cell types: Three main cell types: Ascospore Ascospore Conidia Conidia Hyphae Hyphae DNA methylation is DNA methylation isdispensable Courtesy of http://www.fgsc.net/Neurospora/

3. Life Cycle Courtesy of http://www.fgsc.net/Neurospora/

4. DNA Methylation Methyl (CH3) groups are added by proteins at selected sites on DNA, which in turn alters its properties. 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 Methylation makes up 70%-80% of human CpG These changes can occur during one’s life and are heritable. These changes can occur during one’s life and are heritable. Much of it is still unknown Much of it is still unknown

5. Methylation Model

6. Part I: Insertional Mutants and UV mutants Methods: Spot tests were done on Insertional Mutants to identify a lack of DNA methylation. 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. Results are judged by a marked resistance to Hygromycin and Basta. Two mutants were identified. Two mutants were identified. Complementation Tests were done with both insertional mutants and UV mutants to determine if the mutation was novel: 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. Heterokaryons were made with the unknown mutant and a 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 identified Two UV mutants and Two Insertional Mutants were identified

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

8. Southern Blots: Cont. Southern Blots: Cont. Part 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.

9. Southern Blots: Cont. Southern Blots: Cont. Part 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.

10. Southern Blots: Cont. Southern Blots: Cont. Part 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.

11. Part 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 genome Visualization of bands after probe

12. 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

13. Part II Cont.: Growth of E2 Growth Phenotype of E2: Slow growth and poor conidiation Wild TypeE2 mutant Growth Rate: E2 grows significantly slower

14. Part II Cont. :Confirmation of KO location E2 mutant was knocked out by replacing the E2 gene with hph 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. 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. 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. The results confirm that the KO exists in the E2 gene. 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

15. 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 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 E2 mutant clearly shows a similar loss of methylation as dim-2 9:E1 Probe WT dim-2 E2 SD SD SD 2:B3 Probe WT dim-2 E2 SD SD SD WT dim-2 E2 SD SD SD WT dim-2 E2 SD SD SD WT dim-2 E2 SD SD SD 8:A6 Probe8:G3 Probe2:G9 Probe

16. Part II Cont. : Western Blot of E2c A Western blot with nuclear extracts was done to determine at which point E2 affects methylation. 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. Antibodies for Histone 3 and Histone 3 lysine 9 methylation were used. WT dim-5 (N/A) E2 H3 H3K9me3 38.4kDa 31.5kDa 18kDa 7kDa 38.4kDa 31.5kDa 18kDa 7kDa Histones are about 17 kDa in size.

17. Part III: Yellow Fluorescent Protein Yellow Fluorescent Protein (YFP) is a useful approach in determining protein to protein interactions. 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. A protein is attached to each half of the YFP. If an interaction occurs there is fluorescence. This approach was used for hpo This approach was used for hpo

18. Part III Cont. : YFP Methods Transformed E.coli Removed Hpo segment and inserted in pYFP GLOW Mixed samples GLOW Purified plasmids and transformed Neurospora

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

20. 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. 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 E2 plays an important role in DNA methylation for Neurospora 1. It affects the organisms growth and development 2. It’s required for the trimethylation of H3K9 YFP was successfully used with hpo YFP was successfully used with hpo

21. Special Thanks My mentors, Keyur Adhvaryu and Anthony Shiver. My mentors, Keyur Adhvaryu and Anthony Shiver. The Selker Lab The Selker Lab SPUR SPUR