Mechanistic study of histone H3 lysine 79 methyltransferase Dot1 in Saccharomyces cerevisiae

Abstract

Dot1p is an evolutionarily conserved histone H3 lysine 79 (H3K79) methyltransferase and is associated with the maintenance of heterochromatin boundary, DNA damage response, and transcriptional regulation. It is well known that histone H2B mono-ubiquitylation (H2Bub1) by Rad6p/Bre1p is required for H3K79 di- and tri-methylation (H3K79me2 and H3K79me3) by Dot1p; however, the mechanism how Dot1p recognizes the mono-ubiquitin moiety of H2B has remained elusive. In this work, we generated a mono-ubiquitylated H2B through a one-step enzymatic reaction to reconstitute a nucleosome array containing H2Bub1. We found that ubiquitylated H2B could directly enhance the H3K79 di- and tri-methylation activity of Dot1p without aid of any accessory protein. In addition, we identified a lysine rich domain of Dot1p that spans amino acids (aa) of 101-140 that is responsible for the enhancement. Further analysis revealed the domain binds to ubiquitin, core histones and nucleosomes. An internal deletion mutant of the ubiquitin binding domain (Δ101-140) showed a defect in H3K79me3 both in vitro and in vivo, and in subtelomeric silencing as a consequence. Interestingly, Dot1p stimulated the sliding activity of remodeling complexes including Chd1, ISW, and RSC complex, via the nucleosome binding activity of the lysine rich domain rather than the methyltransferase activity or H4 tail binding activity of Dot1p. In addition, we found that H4 lysine 16 acetylation (H4K16ac) positively regulates H3K79 methylation in manner independent of H2Bub1. In the absence of Sas2p or H4K16ac defective mutant, a significant global decrease in H3K79me3 was observed. H3K79me1 was also shown to decrease in subtelomeric region in sas2Δ mutant. Despite of the decrease of H3K79me1 in subtelomeric region is partially due to ectopic Sir complex spreading, the direct connection between Sas2p and H3K79me also plays a part in subtelomeric silencing. Although further experiments will be required to dissect the mec...