Exploring Chromodomain Genes in the Fungus Fusarium graminearum Through Targeted Genetic Manipulation
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1 Exploring Chromodomain Genes in the Fungus Fusarium graminearum Through Targeted Genetic Manipulation Alec Peters Dr. Michael Freitag Laboratory Dept of Biochemistry/Biophysics Oregon State University
2 DNA and Chromatin Genomic DNA exists as chromatin Nucleosomes Chromatin structure alters gene expression Heterochromatin is densely packed and inactive Euchromatin is loosely packed and active
3 Histones
4 Histone Modification Alters Chromatin Structure
5 Chromodomain Proteins CDPs Chromatin binding motif Pc and HP1 Nuclear Localization GFP strains
6 Fusarium graminearum Cause of Fusarium Head Blight (FHB) Crop Damages Food Contamination Model Organism H3K27me3 Find possible PRC1 homolog 1/1e/Wheat_scab.jpg
7 F. graminearum CDP Gene Candidates Sequence homology with CDPs Focus on those with unknown functions 8 Gene Candidates Gene KOs gene is absent GFP fusion localization Observe phenotypes to identify CDP genes and perhaps PRC1 homolog CDP Candidate Fusarium Gene ID N. crassa Gene ID Proposed Function FgCDP1 FGSG_01512 NCU08362 Centromere Regulation FgCDP2 FGSG_05030 NCU00738 H3K9me Regulation FgCDP3 FGSG_04328 NCU01522 Unclear Function FgCDP5 FGSG_11309 N/A Unclear Function FgCDP6 FGSG_14036 NCU06788 Histone Acetylation FgCDP8 FGSG_03473 N/A Unclear Function FgCDP9 FGSG_02144 N/A Unclear Function FgCHD3 FGSG_07346 NCU06696 Histone Deacetylation
8 5 and 3 flanking fragments Flank targeted gene candidate DNA Cassette Generation GFP-S tag-hph or neo fragments neo neomycin antibiotic resistance gene Confers resistance to G418 hph hygromycin resistance resistance gene Confers resistance to hygromygin Synthesized by PCR, gel-purified
9 DNA Cassette Generation 1. Gene 2. 5 Flank (1kb) 3 Flank (1kb) Gene 3. 5 Flank (1kb) Antibiotic Resistance 3 Flank (1kb) 4. 5 Flank (1kb) Antibiotic Resistance 3 Flank (1kb)
10 Transformation and Selection DNA cassette is integrated into protoplast genome at target site Selection: G418 for strains with neo gene Hygromycin for strains with hph gene Genomic DNA is prepared from strains Used to verify targeted gene insertion PCR and Southern Blots
11 Verification of Transformant Strains PCR Verification Primers: Amplify gene segment Amplify cassette Southern Blots Probes: Gene candidate specific Antibiotic gene specific
12 Photo taken by Lanelle Connolly, Freitag Lab Phenotypic Assays Changes in appearance Color, shape, etc. 7 days, YPD Growth rate changes Protein localization GFP tagged strains WT ΔKmt-6
13 Growth Assay/Westerns Growth Assay Conidia grown, single spores picked Linear growth on agar plate YPD (rich) and FMM (poor) media Western Blots Histone isolation Coomassie blue stain Western Antibodies specific for histone modifications
14 Verified Transformants Transformation Obtained Verified Strains ΔFgCdp1::neo 1 FgCdp1-GFP-S tag-hph 0 ΔFgCdp2::neo 2 FgCdp2-GFP-S tag-hph 1 ΔFgCdp3::neo 1 FgCdp3-GFP-S tag-hph 4 ΔFgCdp5::neo 1 ΔFgCdp6::neo 2 FgCdp6-GFP-S tag-hph 1 ΔFgCdp8::neo 0 ΔFgCdp9::neo 1 ΔFgChd3::neo 1
15 Verification of Transformants via PCR ΔFgCdp6::neo KO * 7 8 FgCdp6 10 kb 1 kb 10 kb ** WT Primers StartF GlyR WT 2.0 kb KO 0 kb 1 kb
16 Verification of Transformants via Southern Blot FgCdp6 FgCdp6 FgCdp6 KO Digested with MluI and BglII restriction enzymes Gene Probe WT control 5.4 kb KO strains 0 kb GFP-hph strains 7.9 kb Antibiotic Probe WT control (both probes) 0 kb KO strains (neo) 5.0 kb GFP-hph strains (hph) 7.9 kb FgCdp6-GFP
17 Verification of Transformants via Southern Blot ΔFgCdp6::neo KO C neo * 5 6 7** FgCdp6-GFP-hph C hph kb 5.4 kb 5.4 kb FgCdp6 gene probe WT control 5.4 kb KO strains 0 kb GFP-hph strains 7.9 kb
18 Verification of Transformants via Southern Blot ΔFgCdp6::neo KO FgCdp6-GFP-hph C neo * 5 6 7** C hph kb 3.6kb 7.9kb - 3.0kb - Antibiotic gene probe WT control (both probes) 0 kb KO strains (neo) 5.0 kb GFP-hph strains (hph) 7.9 kb
19 Verified Transformants Transformation Obtained Verified Strains ΔFgCdp1::neo 1 FgCdp1-GFP-S tag-hph 0 ΔFgCdp2::neo 2 FgCdp2-GFP-S tag-hph 1 ΔFgCdp3::neo 1 FgCdp3-GFP-S tag-hph 4 ΔFgCdp5::neo 1 ΔFgCdp6::neo 2 FgCdp6-GFP-S tag-hph 1 ΔFgCdp8::neo 0 ΔFgCdp9::neo 1 ΔFgChd3::neo 1
20 Phenotypic Assays Most strains had no difference in phenotype ΔFgCdp1::neo ΔFgCdp2::neo ΔFgCdp3::neo WT ΔFgCdp5::neo ΔFgChd3::neo ΔFgCdp9::neo
21 FgCdp6 Transformant Strain Phenotypes YPD, 3 days of growth GFP tagged strains, conidia ΔFgCdp6 KO ΔKmt-6 KO FgCdp1-GFP FgCdp2-GFP FgCdp6-GFP-hph WT FgCdp3-GFP FgCdp6-GFP
22 FgCdp6 GFP
23 Growth Assay YPD, 7 days of growth WT ΔFgCdp6 KO FMM, 7 days of growth WT ΔFgCdp6 KO
24 Growth Assay Quantification
25 Growth Assay Quantification
26 Coomassie Stain Calf Thymus WT FgCdp6 KO FgCdp6-GFP
27 Western Blot Calf Thymus WT FgCdp6 KO FgCdp6-GFP H3K4me2 H3 Acetylation H3K36me3 H3K27Ac
28 Gene Candidates With WT Phenotype Different Possibilities Pseudogenes Redundant Function Further Studies Multi-KO and gene reintroduction Transformation with no colonies Lethal phenotype Unsuccessful transformation Transformation Obtained Verified Strains ΔFgCdp1::neo 1 FgCdp1_GFP-S tag-hph 0 ΔFgCdp2::neo 2 FgCdp2_GFP-S tag-hph 1 ΔFgCdp3::neo 1 FgCdp3_GFP-S tag-hph 4 ΔFgCdp5::neo 1 ΔFgCdp6::neo 2 FgCdp6_GFP-S tag-hph 1 ΔFgCdp8::neo 0 ΔFgCdp9::neo 1 ΔFgChd3::neo 1
29 FgCdp6 Functional CDP gene Necessary for normal growth Nuclear localization (GFP) Eaf3 homolog Sequence homology Western Results H3K36me3 NuA4 complex, Rpd3S recruitment Histone Acetylation, Transcriptional Elongation (H3K36me3)
30 Further Studies FgCdp6 Re-introduce gene Reversion to wildtype More evidence for gene existence Western blots H3K36me3 quantification Complex pull-down/ms analysis Identify other protein in complex with tagged protein (GFP) Chromatin Immunoprecipitation DNA segments influenced by gene product
31 Acknowledgements Lanelle Connolly Michael Freitag Jonathan Galazka David Hendrix Kate Field Kevin Ahern Freitag Lab Oregon State University Honors College, OSU Department of Biochemistry/Biophysics, Howard Hughed Medical Institute (Summer 2011 Scholarship)
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