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Use of Genetic analysis to study the nature of the Smyd1b gene in Cardiac and Skeletal muscular
- 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
- 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
Editor's Notes
- The relationship between the proteins and the regions of the sarcomere are as follows: Actin filaments, the thin filaments, are the major component of the I-band and extend into the A-band. Myosin filaments, the thick filaments, are bipolar and extend throughout the A-band. They are crosslinked at the centre by the M-band. The giant protein titin (connectin) extends from the Z-line of the sarcomere, where it binds to the thick filament (myosin) system, to the M-band, where it is thought to interact with the thick filaments. Titin (and its splice isoforms) is the biggest single highly elasticated protein found in nature. It provides binding sites for numerous proteins and is thought to play an important role as sarcomeric ruler and as blueprint for the assembly of the sarcomere. Another giant protein, nebulin, is hypothesised to extend along the thin filaments and the entire I-Band. Similar to titin, it is thought to act as a molecular ruler along for thin filament assembly. Several proteins important for the stability of the sarcomeric structure are found in the Z-line as well as in the M-band of the sarcomere. Actin filaments and titin molecules are cross-linked in the Z-disc via the Z-line protein alpha-actinin. The M-band proteins myomesin as well as C-protein crosslink the thick filament system (myosins) and the M-band part of titin (the elastic filaments). The interaction between actin and myosin filaments in the A-band of the sarcomere is responsible for the muscle contraction (sliding filament model).
- The MYND domain (named after myeloid translocation protein 8, Nervy, and DEAF-1) is a conserved zinc binding domain. It is defined by seven conserved cysteine residues and a single histidine residue that are arranged in a C4-C2HC consensus. The SET domain appears generally as one part of a larger multidomain protein, and recently there were described three structures of very different proteins with distinct domain compositions: Neurospora crassa DIM-5, a member of the Su(var) family of HKMTs which methylate histone H3 on lysine 9,human SET7 (also called SET9), which methylates H3 on lysine 4 and garden pea Rubisco LSMT, an enzyme that does not modify histones, but instead methylates lysine 14 in the flexible tail of the large subunit of the enzyme Rubisco. The 13 aa insertion is coded for by the exon 5 on the Smyd1b-tv1 isoform
- Muscular dystrophy is a group of genetic diseases in which muscle fibers are unusually susceptible to damage. These damaged muscles become progressively weaker. Most people who have muscular dystrophy will eventually need to use a wheelchair. There are many different kinds of muscular dystrophy. Symptoms of the most common variety begin in childhood, primarily in boys. Other types of muscular dystrophy don't surface until adulthood. People who have muscular dystrophy may have trouble breathing or swallowing. Their limbs may also draw inward and become fixed in that position — a problem called contracture. Some varieties of the disease can also affect the heart and other organs. While there is no cure for muscular dystrophy, medications and therapy can slow the course of the disease.