A Modified “Cross-talk” between Histone H2B Lys-120 Ubiquitination and H3 Lys-79 Methylation

Abstract

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 crosstalk relationship between H2B ubiquitination and H3 Lys-79 methylation.Acetylation, methylation, and ubiquitination are widely known modifications of lysine in histones (1). Histone acetylation normally plays a vital role in gene activation with an exception when acetylation is utilized for other functions such as protein-protein interactions (2, 3). The function of histone methylation depends on the site that is modified, i.e. H3 Lys-9, Lys-27 and H4 Lys-20 methylation are associated with heterochromatin where genes are predominantly in a silenced state, whereas H3 Lys-4 and Lys-79 methylation are linked to euchromatin where the majority of genes are in an active format (4). Methylation may also function oppositely, under two different conditions such that both H3 Lys-9 and Lys-36 methylation have a positive effect in the coding region and negative effect in the promotor region (5, 6). Histone H2B ubiquitination has been demonstrated in vitro to be required for histone H3 Lys-4 and Lys-79 di-and tri-methylation, indicating a cross-talk pathway exists between the two types of modifications in two histone subclasses in the process of regulating gene expression (7-9).Human leukemia cell line U937 typically contains reciprocal CALM-AF10 2 fusion genes resulting from the rearrangement of CALM and AF10 genes (10). These CALM-AF10 and/or AF10/CALM fusion proteins were identified in many leukemia patients with acute myeloid, acute lymphoblastic, and T cell acute lymphoblastic leukemia (11-13). The AF10 protein associates with the histone H3 methyltransferase hDot1L in the nucleus (14, 15). Literally, the fusion protein, CALM-AF10, might compete with the endogenous AF10 protein for hDot1L binding and bring the bound Dot1 out of the nucleus to the cytoplasm along with the shuttling vehicle of CALM. As a consequence, the net concentration of the hDot1L protein in the nucleus decreases, ...

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Total kinetic analysis reveals how combinatorial methylation patterns are established on lysines 27 and 36 of histone H3

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