Histone phosphorylation is often a direct outcome of activated intracellular signaling pathways, and functions to translate extracellular signals into appropriate biological outputs such as changes in gene expression. Growth factors and cellular stress trigger rapid and transient expression of immediate-early genes (such as c-fos, c-jun) in mammalian cells, and their induction strongly correlates with a transient phosphorylation of S10 and S28 on histone H3. While many signaling cascades that lead to H3 phosphorylation have been mapped out, mechanistic details of the downstream events and how H3 phosphorylation contributes to transcriptional activation are still poorly defined.
To investigate the direct effects of H3 phosphorylation on transcription, we targeted the H3 kinase MSK1 to endogenous c-fos promoter, and found that this is sufficient to activate its expression. Moreover, targeting MSK1 to the tissue-specific -globin gene induces H3S28 phosphorylation and reactivates expression of this polycomb-silenced gene. Mechanistically, H3S28 phosphorylation not only disrupts binding of polycomb repressive complexes, but also induces a methyl-acetylation switch of the adjacent K27 residue. This provides the first indication that H3 phosphorylation is involved in antagonizing polycomb silencing.
To further identify post-translational modifications (PTMs) that function together with MSK1-mediated H3 phosphorylation, I developed a novel nucleosome purification approach called Biotinylation-assisted Isolation of CO-modified Nucleosomes (BICON). This technique combines in vivo biotinylation by BirA and H3 phosphorylation by MSK1, allowing enrichment of phosphorylated nucleosomes using streptavidin. I found that MSK1-phosphorylated nucleosomes are hyper-acetylated on H3 and H4, and importantly, I identified a trans-tail crosstalk between H3 phosphorylation and H4 acetylation on K12. This proof-of-principle study demonstrates that BICON can be further adapted to study PTMs and crosstalks associated with other histone-modifying enzymes.
Taken together, work described in this thesis shows that histone H3 phosphorylation can initiate additional PTM changes on other residues within the nucleosome, and such crosstalk plays an important role in regulating gene expression.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/35875 |
| Date | 08 August 2013 |
| Creators | Lau, Nga Ieng |
| Contributors | Cheung, Peter |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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