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Characterization of chromatin assembly in murine embryos

University of British Columbia

During differentiation, changes in chromatin proteins lead to the establishment and maintenance of gene expression patterns. Histone H3 trimethylated at lysine 4 (H3K4me3) by the trithorax group (trxG) gene family Mixed lineage leukemia (MLL) is associated with active genes. H3K27me3 is trimethylated by the Polycomb group (PcG) Enhancer of Zeste (EZH2) at repressed genes. In Drosophila embryos, trxG and PcG proteins but not H3K4me3 or H3K27me3 are stable to DNA replication. In contrast, methylated histones are detected on nascent DNA in Drosophila and murine cell lines. Therefore some aspect of chromatin assembly or histone trimethylation must differ in different cells. My first aim was to determine if there is a change in the abundance of methylated histones at the replication fork in undifferentiated versus differentiated murine ES cells using two novel in vivo assays. Most undifferentiated ES cells lack early H3K4me3 and H3K27me3, but after 4 days of differentiation, most cells have early trimethylation of H3K4 and H3K27. I propose that the change in kinetics of histone methylation correlates with differentiation. To test this hypothesis, I carried out similar experiments on cells dissociated from day 9.5 (E9.5) and 14.5 (E14.5) murine embryos. In E9.5 cells there are two populations of cells, one that lacks methylated histones and the other contains methylated histones on nascent DNA. By E14.5 most cells exhibit H3K4me3 and H3K27me3 on nascent DNA. To determine if the presence of histone methyltransferases could account for the changes in histone methylation, I tested MLL1 and a subunit of the EZH2 complex, Su(z)12. Both are present continuously on nascent DNA, suggesting that their activity is regulated. Methylation and acetylation antagonize each other at the same residue. However I showed that the presence of acetylated H3K27 is not anticorrelated with H3K27me3 in most murine embryos cells. My results using inhibitors of the appropriate histone acetyltransferase were not conclusive owing to toxicity of the inhibitors. Overall, my results support the hypothesis that trimethylation of H3K4 and H3K27 on nascent DNA is developmentally regulated.

Text

2013-08-12

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/44768

Zoology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/