Histone modifications play key roles in the regulation of gene expression. In embryonic stem cells, promoters of developmental genes simultaneously carry histone H3 lysine 4 trimethylation (H3K4me3), which is usually found at active promoters, and H3K27me3, a hallmark of repressive chromatin. These so-called bivalent domains are thought to poise genes for timely induction during development, but it remains unclear whether bivalent domains are required for proper differentiation and if so, how their associated marks regulate gene expression. In addition to the two defining methylation marks, additional modifications are found at bivalent domains, but have received much less attention. Moreover, bivalent domains feature the histone variants H2A.Z and H3.3; however, it remains unclear how their presence might contribute to establishing a poised state at bivalent promoters.
In this project, we will perform nucleosome pulldown assays with recombinant nucleosome templates carrying bivalent modifications and histone variants to determine how presence of histone variants affects recruitment of activating and repressive factors to bivalent domains. Given the close proximity of H3K27 to H3-3-specific residues, binders of the repressive H3K27me3 mark might be especially sensitive to incorporation of H3.3. These assays will reveal potential histone variant-specific binders of the bivalent state. Using CRISPR-based knockout studies, we will deplete embryonic stem (ES) cells for H3.3 and/or H2A.Z and determine how this will affect recruitment of bivalent and repressive factors to bivalent domains and expression of bivalent genes in ES cells. These studies will add to our list of candidate proteins for mediating poising at bivalent domains and expand our knowledge on how combinatorial readout of histone modifications might shape their function. We will then further characterise candidates for interpreting the bivalent state identified here or previously in the lab, to determine their interaction partners, the mechanisms through which they bind to bivalent domains, and their functional importance in maintenance of pluripotency and differentiation through CRISPR-based genome editing techniques and stem cell differentiation approaches.
This project will allow to gain extensive knowledge in chromatin biology and epigenetics. It will provide excellent training opportunities in chromatin biochemistry and state-of-the-art quantitative mass spectrometry approaches. Moreover, it will involve genome-wide approaches such as ChIP-seq and RNA-seq and associated bioinformatics approaches required for data analysis. It will provide training opportunities in CRISPR-based genome editing and stem cell biology.
Please visit the lab website for more information: http://www.wcb.ed.ac.uk/research/philipp-voigt
. A variety of funding sources are available, including for UK, EU, and international students. Informal enquiries should be made to Dr. Philipp Voigt: [email protected]
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Voigt, P., Tee, W.-W., & Reinberg, D. (2013). A double take on bivalent promoters. Genes Dev 27, 1318-1338.
Voigt, P., LeRoy, G., Drury III, W. J., Zee, B. M., Son, J., Beck, D. B., Young, N. L., Garcia, B. A., & Reinberg, D. (2012). Asymmetrically Modified Nucleosomes. Cell 151, 181–193.