Quantitative Proteomic Analysis of Post-translational Modifications of Human Histones

Abstract

Eukaryote DNA is wrapped around nucleosomes that are comprised of octamers of core histones (two heterodimers of histones H2A and H2B and a heterotetramer of histones H3 and H4). Nucleosomes are joined by linker DNA and histone H1 to form chromatin. A central mechanism for regulating chromatin activity is the (reversible) covalent modification of histones by enzymes. A complex interplay between posttranslational modifications (PTMs) 1 either represses or activates transcription in a site-specific and cooperative manner (1). The NH 2 -and COOH-terminal tails of the core histones are subjected to multisite modifications by methylation, acetylation, phosphorylation, ADP-ribosylation, ubiquitination (2, 3), and sumoylation (4). A "histone code" model has been hypothesized where the cell uses a combinatorial system of (clustered) covalent modifications to regulate specific genomic functions (5-7). The current hypothesis is that the pattern and types of modifications modulate protein-protein and protein-DNA interactions, e.g. for recruitment of transcription factors, as a function of the cellular state and environment (8).Acetylation of lysine residues is a major mediator of the histone code. Hyperacetylation of histone tails is usually a sign of high transcriptional activity, whereas hypoacetylation indicates transcriptional silencing (9). Reversible acetylation/ deacetylation of lysine residues is a dynamic balance between the activities of histone acyltransferases and histone deacetylases (HDACs). Inhibition of HDAC activity results in hyperacetylation of lysine residues in the NH 2 tails of histones H3 and H4 (10), and it leads to the expression of genes that induce growth arrest, cell differentiation, and apoptotic cell death in cultured tumor cells (11,12). Non-toxic levels of several HDACis have shown promising pharmacological properties by inhibiting tumor growth in animal models (13)(14)(15). Consequently there is a great interest in developing HDACis as anticancer drugs, and some of these compounds are currently being evaluated in clinical trails (16,17).Mass spectrometry is rapidly becoming a key analytical technology in molecular cell biology and proteomics. Mass spectrometry is particularly suited for the examination of protein primary structure and for determining post-translational modifications of proteins as it reveals covalent modifications From the ‡Danish Technological Institute,

Mammalian Protein Arginine Methyltransferase 7 (PRMT7) Specifically Targets RXR Sites in Lysine- and Arginine-rich Regions

“…The very high methyl-accepting activity of H2B in vitro suggests that it might be a physiological substrate for PRMT7. Previous studies identified multiple post-translational modifications on human H2B, including acetylation on Lys-5, Lys-15, Lys-16, and Lys-20 (43,44), phosphorylation on Ser-14 (45), and methylation on Lys-46, Lys-57, and Lys-108 (43). However, arginine methylation on HBR has not been reported to our knowledge.…”

Analyses of Histone Proteoforms Using Front-end Electron Transfer Dissociation-enabled Orbitrap Instruments

“…They are less abundant and are more difficult to characterize because their signals are split among multiple acetylated forms (ϩ1-3 lysine-acetyl confirmed by MS/MS). The acetylations are shared among multiple sites, including Lys-11, Lys-12, Lys-15, Lys-16, and Lys-20, which are all sites that were confirmed by MS/MS in the intact form and that have previously been identified by MS in an earlier study (35). Truncations derived from two additional H2B isoforms (possibly H2B.2-E, Q16778, and H2B.1-K, O60814) were also present.…”

Maspin: The New Frontier

“…Recently, quantitative proteomic approach has revealed that treatment of human cell cultures with histone deacetylase inhibitor PXD101 leads to the expression of genes regulating growth arrest, differentiation, and apoptosis (21). In line with these studies, treatment of maspin-deficient breast cancer cell lines with trichostatin A (a histone deacetylase inhibitor) induced maspin expression in breast and bladder cancer cell lines (22,23), thus indicating that histone deacetylation may suppress maspin gene activation in these cancers.…”

“…The ''histone code'' (constituting a combination of modifications, including acetylation, methylation, and ubiquitination) dictates chromatin structure and function during development, growth, differentiation, and homeostasis of cells (21). Recently, quantitative proteomic approach has revealed that treatment of human cell cultures with histone deacetylase inhibitor PXD101 leads to the expression of genes regulating growth arrest, differentiation, and apoptosis (21).…”