A charge-based interaction between histone H4 and Dot1 is required for H3K79 methylation and telomere silencing: identification of a new trans-histone pathway.
Saccharomyces cerevisiae cells lacking Dot1 exhibit a complete loss of H3K79 methylation and defects in heterochromatin-mediated silencing. To further understand the mechanism of Dot1-mediated methylation, the substrate requirement of Dot1 was determined. This analysis found that Dot1 requires histone H4 for in vitro methyltransferase activity and the histone H4 tail for Dot1-mediated methylation in yeast. Mutational analyses demonstrated that the basic patch residues (R(17)H(18)R(19)) of the histone H4 N-terminal tail are required for Dot1 methyltransferase activity in vitro as well as Dot1-mediated histone H3K79 methylation in vivo. In vitro binding assays show that Dot1 can interact with the H4 N-terminal tail via the basic patch residues. Furthermore, an acidic patch at the C terminus of Dot1 is required for histone H4 tail binding in vitro, histone H3K79 di- and trimethylation in vivo, and proper telomere silencing. Our data suggest a novel trans-histone regulatory pathway whereby charged residues of one histone are required for the modification of another histone. These findings not only provide key insights into the mechanism of Dot1 histone methylation but also illustrate how chromatin-modifying enzymes engage their nucleosomal substrates in vivo.
Department of Biochemistry and Purdue Cancer Center, Purdue University, West Lafayette, Indiana 47907, USA.
Ian M
IM
Fingerman
Hui-Chun
HC
Li
Scott D
SD
Briggs
D024361:Amino Acids, Basic / Q000737:chemistry; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002645:Chickens; D004563:Electrochemistry; D020868:Gene Silencing; D011495:Histone-Lysine N-Methyltransferase; D006657:Histones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D016615:Telomere / Q000235:genetics* / Q000378:metabolism*
D024361:Amino Acids, Basic; D006657:Histones; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae; D008780:Methyltransferases; C416235:Dot1 protein, S cerevisiae; D011495:Histone-Lysine N-Methyltransferase