1. Use of Genetic analysis to study the nature of the smyd1b gene in Cardiac and Skeletal Muscular Systems of Zebra Fish By, Prajwal Keranahalli Poolesville High School

2. Objectives We want to observe the localization of Smyd1 genes and proteins in zebra-fish through a developmental study This will tell us what muscle structures this gene is active in and give us clues as to its function We will look for co-localizations with Hsp90 to support the idea that Smyd is a protein methyltransferase 3. Basic Unit of Muscle 4. Proposed Role of SMYD1b 5. Protein Methyltransferase Methylation on lysine can regulate a Hsp90 activation and interaction with other proteins Titin Myomesin Actin 6. The Smyd1b Protein The same gene codes for 2 mRNA strands due to alternative splicing Smyd1b-tv1, extra 13 amino acids Smyd1b-tv2 7. Ribbon diagram of the SmyD1 structure. The location of SET domain, MYND domain, and C- terminal (TPR) domain are represented by the orange, green, and gray color, respectively. 8. Purpose Knockdown of both TV1 and TV2 genes caused death in zebra-fish embryos 1 of every 5,600 to 7,700 males 5 through 24 years of age has genetic Muscular Dystrophy 9. Zebra Fish as Model Organisms Fully sequenced genome Easily observable, fast and testable developmental behaviors Robust, large and transparent embryos Similar to Mammalian Models Short Life Cycle Large Clutch Sizes Express trans-genes 10. Humans with Smyd Mutations Living people with Smyd mutations are most likely hypomorphic. If the gene is totally knocked out they would be dead. Active Site 11. GFP Tagged Smyd Promoter: Day1 12. Observations At this stage most of the promoter expression is in the trunk of the embryo Surrounding the notochord is a lot of skeletal muscles essential for movement Just like in humans we see that motor limbs develop early on in the fish's life cycle This supports the idea that these proteins are needed for sarcomere assembly 13. GFP Tagged Smyd Promoter: Day 8 14. GFP Tagged Smyd Promoter: Day 8 15. Observations After 8 days there is a lot more development in the head region of the embryos Especially near the heart, eyes, and mouth Cardiac muscle was another predicted site of function for this gene It is still important to observe protein localization 16. 1 2 3 4 5 6 7 8 0 10 20 30 40 50 60 70 80 Day vs Gene Expression Line 1 Set 1 Line 1 Set 2 Line 3 Set 1 Line 3 Set 2 Day PercentGeneExpression 17. Day Vs Percent Gene Expression Line 1 Set 1 Line 1 Set 2 Line 3 Set 1 Line 3 Set 2 19.34 24.07 31.84 33.55 27.85 30.23 33.84 36.56 45.52 44.13 43.08 38.75 44.80 47.95 38.32 47.30 31.30 48.55 49.19 37.88 32.03 44.68 48.40 47.78 54.69 51.71 43.61 45.90 61.00 40.21 63.33 75.31 6 2 3 4 5 1 7 8 18. Smyd1b-TV1 protein Localization Smyd1b-TV1 Day 4 Day 5 19. Smyd1b-TV2 protein Localization Smyd1b-TV2 Day 5 Day 5 20. Myomesin Localization 21. Myomesin and Smyd Co- Localization 1.046 pixels 1.146 pixels 22. Myomesin and Smyd Co-Localization Box Dimensions 0.5 W 1 L Myomesin BOP1 1.134 1.069 0.934 1.113 1.2 1 1.2 1.3 1.12 0.91 1 1.133 0.933 1.001 1.034 1 1 1.1 1.33 1.21 1.167 0.969 1.001 1.113 0.901 1 1.2 1.3 1.33 1.14 1.100 1.057 0.956 1.076 1.045 1.000 1.133 1.233 1.260 1.087 Avg. Pixel Width 1.047 Avg. Pixel Width 1.143 23. Conclusion Not only is the Smyd1b gene expressed in skeletal and cardiac muscles but its proteins also have important functions in the M-Line's of sarcomeres. The TV1 strain of the protein seems to be the more strongly expressed version of this gene because of this extra 13 amino acids This gene is co-localized with proteins like myomesin so it is logical to conclude that the Smyd gene works to regulate the structures of such proteins 24. Future work It would be useful to see if this protein is co- localized with any other sarcomere proteins. We also need to properly characterize its molecular mechanism of function so that research with this gene can have more practical applications 25. Thank You for Listening