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Abstract
The acetylation isoforms of histone H4 from butyrate-treated HeLa cells were separated by C(4) reverse-phase high pressure liquid chromatography and by polyacrylamide gel electrophoresis. Histone H4 bands were excised and digested in-gel with the endoprotease trypsin. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to characterize the level of acetylation, and nanoelectrospray tandem mass spectrometric analysis of the acetylated peptides was used to determine the exact sites of acetylation. Although there are 15 acetylation sites possible, only four acetylated peptide sequences were actually observed. The tetra-acetylated form is modified at lysines 5, 8, 12, and 16, the tri-acetylated form is modified at lysines 8, 12, and 16, and the di-acetylated form is modified at lysines 12 and 16. The only significant amount of the mono-acetylated form was found at position 16. These results are consistent with the hypothesis of a "zip" model whereby acetylation of histone H4 proceeds in the direction of from Lys-16 to Lys-5, and deacetylation proceeds in the reverse direction. Histone acetylation and deacetylation are coordinated processes leading to a non-random distribution of isoforms. Our results also revealed that lysine 20 is di-methylated in all modified isoforms, as well as the non-acetylated isoform of H4.
Highlights
The acetylation isoforms of histone H4 from butyratetreated HeLa cells were separated by C4 reverse-phase high pressure liquid chromatography and by polyacrylamide gel electrophoresis
We report here the use of mass spectrometric methods to identify the acetylation sites of histone H4 to clarify the complicated and somewhat contradictory picture regarding the relative abundances of H4 acetylation isoforms
The resulting tryptic peptides were analyzed by MALDI-TOF mass spectrometry to determine their individual mass values (Fig. 2)
Summary
The acetylation isoforms of histone H4 from butyratetreated HeLa cells were separated by C4 reverse-phase high pressure liquid chromatography and by polyacrylamide gel electrophoresis. The only significant amount of the mono-acetylated form was found at position 16. These results are consistent with the hypothesis of a “zip” model whereby acetylation of histone H4 proceeds in the direction of from Lys-16 to Lys-5, and deacetylation proceeds in the reverse direction. The nucleosomes are joined by linker DNA and histone H1 to form chromatin. The acetylation of histone H4 is restricted to lysine 5, 8, 12, and 16 [16]. The specific role of each of these forms will remain unclear until measurable differences in the chromatographic, electrophoretic, and mass spectrometric properties of these forms can be correlated with cellular events [19]. Histone acetylation is a very specific phenomenon with various isoforms playing distinct roles [13]. Acetylation is a dynamic phenomenon with the steady state mediated by the
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