The Activity of HDAC8 Depends on Local and Distal Sequences of Its Peptide Substrates†

ABSTRACT: This paper introduces a flexible assay for characterizing the activities of the histone deacetylase enzymes. The approach combines mass spectrometry with self-assembled monolayers that present acetylated peptides and enables a label-free and one-step assay of this biochemical activity. The assay was used to characterize the activity of HDAC8 toward peptides taken from the N-terminal tail of the H4 histone and reveals that a distal region of the peptide substrate interacts with the deacetylase at an exosite and contributes to the activity of the substrate. Specifically, a peptide corresponding to residues 8-19 of H4 and having lysine 12 acetylated is an active substrate, but removal of the KRHR (residues 16-19) sequence abolishes activity. Mutation of glycine 11 to arginine in the peptide lacking the KRHR sequence restores activity, demonstrating that both local and distal sequences act synergistically to regulate the activity of the HDAC. Assays with peptides bearing multiply acetylated residues, but in which each acetyl group is isotopically labeled, permit studies of the processive deacetylation of peptides. Peptide substrates having an extended sequence that includes K20 were used to demonstrate that methylation of this residue directly affects HDAC8 activity at K12. This work provides a mechanistic basis for the regulation of HDAC activities by distal sequences and may contribute to studies aimed at evaluating the role of the histone code in regulating gene expression

Peptide Arrays Identify Isoform-Selective Substrates for Profiling Endogenous Lysine Deacetylase Activity

A cetylation of lysine is a post-translational modifi-cation involved inmany eukaryotic cellular pro-cesses (1). Though most commonly associated with histone proteins (2), a recent proteomic study by Mann and colleagues identified nearly 2,000 proteins in acetylated form and showed that this modification is used globally in regulating cell function (3). This recogni-tion hasmotivated a renaming of the histone acetyltrans-ferase (HAT) and histone deacetylase (HDAC) enzymes to reflect their broader roles [now the lysine acetyltrans-ferases (KATs) and the lysine deacetylases (KDACs) (4)] and has highlighted our limited understanding of the functions of the individual enzymes responsible for achieving andmaintaining acetylation states. A significant effort is now directed at understanding the differential roles that the 18 human KDAC isoforms play in regulating cell behavior (5−8). These studies re

Methylation of histone H3 lysine 9 occurs during translation

Histone post-translational modifications are key contributors to chromatin structure and function, and participate in the maintenance of genome stability. Understanding the establishment and maintenance of these marks, along with their misregulation in pathologies is thus a major focus in the field. While we have learned a great deal about the enzymes regulating histone modifications on nucleosomal histones, much less is known about the mechanisms establishing modifications on soluble newly synthesized histones. This includes methylation of lysine 9 on histone H3 (H3K9),a mark that primes the formation of heterochromatin, a critical chromatin landmark for genome stability. Here, we report that H3K9 mono- and dimethylation is imposed during translation by the methyltransferase SetDB1. We discuss the importance of these results in the context of heterochromatin establishment and maintenance and new therapeutic opportunities in pathologies where heterochromatin is perturbed

Methylation of histone H3 lysine 9 occurs during translation

Histone post-translational modifications are key contributors to chromatin structure and function, and participate in the maintenance of genome stability. Understanding the establishment and maintenance of these marks, along with their misregulation in pathologies is thus a major focus in the field. While we have learned a great deal about the enzymes regulating histone modifications on nucleosomal histones, much less is known about the mechanisms establishing modifications on soluble newly synthesized histones. This includes methylation of lysine 9 on histone H3 (H3K9),a mark that primes the formation of heterochromatin, a critical chromatin landmark for genome stability. Here, we report that H3K9 mono- and dimethylation is imposed during translation by the methyltransferase SetDB1. We discuss the importance of these results in the context of heterochromatin establishment and maintenance and new therapeutic opportunities in pathologies where heterochromatin is perturbed

HDAC8 substrates: Histones and beyond

The lysine deacetylase family of enzymes (HDACs) was first demonstrated to catalyze deacetylation of acetyllysine residues on histones. In subsequent years, HDACs have been shown to recognize a large pool of acetylated nonhistone proteins as substrates. Recently, thousands of acetylated proteins have been discovered, yet in most cases, the HDAC that catalyzes deacetylation in vivo has not been identified. This gap has created the need for better in vivo, in vitro, and in silico approaches for determining HDAC substrates. While HDAC8 is the best kinetically and structurally characterized HDAC, few efficient substrates have yet been substantiated in vivo. In this review, we delineate factors that may be important for determining HDAC8 substrate recognition and catalytic activity, including structure, complex formation, and post‐translational modifications. This summary provides insight into the challenges of identifying in vivo substrates for HDAC8, and provides a good vantage point for understanding the variables important for predicting HDAC substrate recognition. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 112–126, 2013.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/94512/1/22135_ftp.pd

Rapid and Complete Surface Modification with Strain‐Promoted Oxidation‐Controlled Cyclooctyne‐1,2‐Quinone Cycloaddition (SPOCQ)

Strain‐promoted oxidation‐controlled cyclooctyne‐1,2‐quinone cycloaddition (SPOCQ) between functionalized bicyclo[6.1.0]non‐4‐yne (BCN) and surface‐bound quinones revealed an unprecedented 100 % conjugation efficiency. In addition, monitoring by direct analysis in real time mass spectrometry (DART‐MS) revealed the underlying kinetics and activation parameters of this immobilization process in dependence on its microenvironment

Profiling the selectivity of DNA ligases in an array format with mass spectrometry

This article describes a method for the global profiling of the substrate specificities of DNA ligases and illustrates examples using the Taq and T4 DNA ligases. The method combines oligonucleotide arrays, which offer the benefits of high throughput and multiplexed assays, with mass spectrometry to permit label-free assays of ligase activity. Arrays were prepared by immobilizing ternary biotin-tagged DNA substrates to a self-assembled monolayer presenting a layer of streptavidin protein. The array represented complexes having all possible matched and mismatched base pairs at the 3′ side of the nick site and also included a number of deletions and insertions at this site. The arrays were treated with ligases and adenosine triphosphate or analogs of the nucleotide triphosphate and then analyzed by matrix-assisted laser desorption-ionization mass spectrometry to determine the yields for both adenylation of the 5′-probe strand and joining of the two probe strands. The resulting activity profiles reveal the basis for specificity of the ligases and also point to strategies that use ATP analogs to improve specificity. This work introduces a method that can be applied to profile a broad range of enzymes that operate on nucleic acid substrates

Structural basis of nucleosome assembly by the Abo1 AAA+ ATPase histone chaperone

The fundamental unit of chromatin, the nucleosome, is an intricate structure that requires histone chaperones for assembly. ATAD2 AAA+???ATPases are a family of histone chaperones that regulate nucleosome density and chromatin dynamics. Here, we demonstrate that the fission yeast ATAD2 homolog, Abo1, deposits histone H3???H4 onto DNA in an ATP-hydrolysis-dependent manner by in vitro reconstitution and single-tethered DNA curtain assays. We present cryo-EM structures of an ATAD2 family ATPase to atomic resolution in three different nucleotide states, revealing unique structural features required for histone loading on DNA, and directly visualize the transitions of Abo1 from an asymmetric spiral (ATP-state) to a symmetric ring (ADP- and apo-states) using high-speed atomic force microscopy (HS-AFM). Furthermore, we find that the acidic pore of ATP-Abo1 binds a peptide substrate which is suggestive of a histone tail. Based on these results, we propose a model whereby Abo1 facilitates H3???H4 loading by utilizing ATP

Epigenetic and Transcriptional Variability Shape Phenotypic Plasticity.

Epigenetic and transcriptional variability contribute to the vast diversity of cellular and organismal phenotypes and are key in human health and disease. In this review, we describe different types, sources, and determinants of epigenetic and transcriptional variability, enabling cells and organisms to adapt and evolve to a changing environment. We highlight the latest research and hypotheses on how chromatin structure and the epigenome influence gene expression variability. Further, we provide an overview of challenges in the analysis of biological variability. An improved understanding of the molecular mechanisms underlying epigenetic and transcriptional variability, at both the intra- and inter-individual level, provides great opportunity for disease prevention, better therapeutic approaches, and personalized medicine