A modified "cross-talk" between histone H2B Lys-120 ubiquitination and H3 Lys-79 methylation.
Western blot analysis is currently the major method utilized for quantitatively assessing histone global modifications. However, there is a growing need to develop a highly specific, accurate, and multisite quantitative method. Herein, we report a liquid chromatography-tandem mass spectrometry-multiple reaction monitoring method to simultaneously quantify multisite modifications with unmatched specificity, sensitivity, and throughput. With one set of purification of histones by high pressure liquid chromatography or SDS-PAGE, nearly 20 modification sites including acetylation, propionylation, methylation, and ubiquitination were quantified within 2 h for two samples to be compared. Using this method, the relative levels of H2B ubiquitination and H3 Lys-79 methylation were quantified in the U937 human leukemia cell line, U937 derivative cell lines overexpressing anti-secretory factor 10 (AF10) and mutant AF10 with the deletion of the hDot1 binding domain OM-LZ. We found that H2B ubiquitination is inversely correlated with H3 Lys-79 methylation. Therefore, we propose that a catalytic and inhibitory loop mechanism may better describe the cross-talk relationship between H2B ubiquitination and H3 Lys-79 methylation.
Darwanto
Agus
A
Department of Basic Sciences, Biochemistry Division, School of Medicine, Loma Linda University, Loma Linda, California 92350, USA.
Curtis
Matthew P
MP
Schrag
Matthew
M
Kirsch
Wolff
W
Liu
Peng
P
Xu
Guoliang
G
Neidigh
Jonathan W
JW
Zhang
Kangling
K
D002853:Chromatography, Liquid / Q000379:methods; D019175:DNA Methylation; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D018922:HL-60 Cells; D006657:Histones / Q000737:chemistry*; D006801:Humans; D008239:Lysine / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D008954:Models, Biological; D009154:Mutation; D040901:Proteomics / Q000379:methods; D020298:U937 Cells; D025801:Ubiquitin / Q000737:chemistry*
D006657:Histones; D025801:Ubiquitin; D008239:Lysine