- This Master's applied project (MAP) will focus on building a histone methylation-associated reporter gene. The purpose of this is to test chromatin protein function. These synthetic chtomatin proteins will have been constructed to test varying attributes: amino acid sequence of the methyl-histone binding domain, binding domain copy number, relative positioning of tandem binding domains and activation domain strength. In order to test the amino acid sequence of the methyl-histone binding domain, polycomb chromodomains (PCDs) will be taken from samples of human, zebrafish and Drosophilia tissue.
- Amino acid sequences vary outside of the binding regions in PCDs, but have conserved 3-D structures and methyl-histone binding surfaces. Tandem PCDs will vary the PCD copy number; their relative positioning will be determined by short or long rigid alpha helix, or flexible peptide linkers. The expression of the synthetic chromatin protein's target gene will be varied by the use of various activation modules (such as VP64, SP1 and p65).
- The activity of these synthetic chromatin proteins will be determined by the reporter gene that is to be built during the course of this project. Each protein will be expressed in transgenic cells that carry the gene in question, which produces cyan fluorescent protein upon activation. This gene will then be placed into a chromosomal FRT site in U2OS cells by way of enzymatic Flp-mediated recombination. The reporter's promoter will create histone methylation, either by the Gal4-EED fusion protein system or the human histone methylation signal.
- A Hill equation will be used to determine reporter output: f(X) = X < sup > n < / sup > / (K < sup > n < / sup > + X < sup > n < / sup > )
- In the resultant plot, red fluorescent protein will indicate the chromatin protein concentration (X) compared against the cyan fluorescence from the reporter. β indicates the maximum reporter output, K is the activation coefficient and n is the Hill coefficient.
- This project will help to understand chromatin protein engagement in living cells. The protein binding dynamics of Polycomb will also be explored, which plays a major role in cancer and tissue development. Finally, the amino acid primary structure and activation domain strength will be investigated, as well as linked domains. These design principles will be defined and quantitatively described, which will provide us with more information about natural chromatin components, making the engineering of chromatin a possibility.
- This project is currently in progress.
- Click on the individual dates, beginning on 1.24.13, to see specific protocols used.