Histone H4 N-Terminal Acetylation in Kasumi-1 Cells Treated with Depsipeptide Determined by Acetic Acid−Urea Polyacrylamide Gel Electrophoresis, Amino Acid Coded Mass Tagging, and Mass Spectrometry

Abstract

Disrupted patterns of acetylation and deacetylation of core histones play an important role in silencing transcription of hematopoietic important genes in acute myeloid leukemia (AML). A thorough investigation of these mechanisms and the response to pharmacologic modifiers will provide a better understanding of the role of histone acetylation in leukemogenesis. We describe here an analytical approach that combines acid urea polyacrylamide gel electrophoresis (AU-PAGE), amino acid coded mass tagging (AACM), and mass spectrometry (MS) for the investigation of histone acetylation patterns. The combined approach was used to follow the dynamics of H4 acetylation in Kasumi-1 cells harboring the fusion gene AML1/ETO shown to aberrantly recruit histone deacetylases (HDACs). The histones in Kasumi-1 cells were labeled by growing the cells in media in which lysine was replaced with stable isotope-labeled lysine (Lys-D 4 ). Labeled and unlabeled cells were treated with depsipeptide and analyzed at different time points (0,4,8,12,24, and 48 h). The cells were mixed, the histone was extracted, and acetylated H4 isoforms were separated using AU-PAGE before in-gel trypsin digestion. The digests were analyzed by MALDI-TOF MS. Peptides were identified by mass and isotope pattern. LC-MS/MS of Arg-C digests were also performed to verify the acetylation pattern for H4. The major pattern of acetylation was determined as follows: initial acetylation at K16, followed by acetylation at K12, and finally acetylation of either K8 and/or K5.

Global profiling of histone modifications in the polyomavirus BK virion minichromosome

“…After a careful examination of the sites, we determined that histone H4 was acetylated sequentially, first at Lys12 and then at Lys16, Lys8, and Lys5. This acetylation sequence accords with that determined in a human cell line (Zhang et al, 2007), providing evidence that the acetylation machinery for BKPyV histone H4 modification was completely adapted from the host cells because the original host PTM pattern was preserved. For those peptides including Lys-8 and Lys-12, the acetylation of Lys-12 but not Lys-8 was observed in the lower acetylated band of TAU gel.…”

Modelling DNA Methylation Dynamics

“…Even when the probability assigned to the three preferred states was lowered for a test set, the system preferred the other two states containing lysine acetylation. Such consistent results demonstrate the ability of our model to reproduce the presence of the modifications mentioned above, during transcription, (as reported, (Taplick, 1998;Zhang et al, 2007) in particular, during expression of oncogenes). For higher levels of DNA methylation (>0.85, Figure 6), the preference is more towards choosing methylated histone states leading to reduced transcription rate.…”

Validation of an LC‐MS based approach for profiling histones in chronic lymphocytic leukemia

“…Using additional approaches, we determined that the peak 14,017 Da was not mono-methylated H2AFC/D/I/N/P and 14,045 Da was not mono-acetylated H2AFC/D/I/N/P [28–30]. The identity of these species was inferred through use of double SILAC labeling and LC-MS/MS [24].…”