Within the cell nucleus, there are regions of highly condensed, transcriptionally silent chromatin called heterochromatin. Heterochromatin plays an important role in both chromosomal stability and gene regulation within the cell. Heterochromatin assembly is mediated by Heterochromatin Protein 1 (HP1) binding to epigenetically marked histone tails, most notably methylated H3K9. HP1 is post-translationally phosphorylated at serine and threonine residues, and this phosphorylation has been shown to increase HP1’s binding affinity for methylated H3K9 and heterochromatin formation. To study the effect of phosphorylation on heterochromatin assembly and HP1 localization within the nucleus, the unicellular protozoan Tetrahymena thermophila was used. Tetrahymena is an ideal model for this work because cells have a dynamic chromatin environment. Tetrahymena have an HP1-like protein, tHP1, which localizes to transcriptionally silent chromatin bodies within the otherwise transcriptionally active macronucleus. tHP1 is known to be phosphorylated at threonine-64 (site one) and at either serine-102 or threonine-103 (site two). Previous work shows that when phosphorylation at both sites is prevented, tHP1 exhibits decreased localization to chromatin bodies. In order to determine which site of phosphorylation accounts for tHP1’s localization to regions of heterochromatin, mutant proteins that allow phosphorylation at only one of the two sites were generated. The efforts to engineer a mutant protein that cannot be phosphorylated at site two and to visualize the protein’s localization throughout cell development are discussed. When phosphorylation is prevented at site two, tHP1 localization to regions of heterochromatin remains intact. These results suggest that phosphorylation at site one, not site two, may be responsible for tHP1 localization to macronuclear chromatin bodies. A mechanism by which site one phosphorylation influences tHP1 targeting to regions of heterochromatin is proposed. Furthermore, bioinformatics techniques are employed to identify other tHP1-like proteins within Tetrahymena. Characterization of these proteins will likely contribute to a more complete model of how heterochromatin is assembled in Tetrahymena.
| Identifer | oai:union.ndltd.org:CLAREMONT/oai:scholarship.claremont.edu:scripps_theses-1149 |
| Date | 01 January 2013 |
| Creators | Bulley, Emily, Wiley, Emily |
| Publisher | Scholarship @ Claremont |
| Source Sets | Claremont Colleges |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Scripps Senior Theses |
| Rights | © 2012 Emily Bulley and Emily Wiley, default |
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