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Chromatin abnormalities are common hallmarks of cancer cells, which exhibit alterations in DNA methylation profiles that can silence tumor suppressor genes. These epigenetic patterns are partly established and maintained by UHRF1 (ubiquitin-like PHD and RING finger domain-containing protein 1), which senses existing methylation states through multiple reader domains, and reinforces the modifications through recruitment of DNA methyltransferases. Small molecule inhibitors of UHRF1 would be important tools to illuminate molecular functions, yet no compounds capable of blocking UHRF1-histone binding in the context of the full-length protein exist. Here, we report the discovery and mechanism of action of compounds that selectively inhibit the UHRF1-histone interaction with low micromolar potency. Biochemical analyses reveal that these molecules are the first inhibitors to target the PHD finger of UHRF1, specifically disrupting histone H3 arginine 2 interactions with the PHD finger. Importantly, this unique inhibition mechanism is sufficient to displace binding of full-length UHRF1 with histones in vitro and in cells. Together, our study provides insight into the critical role of the PHD finger in driving histone interactions, and demonstrates that targeting this domain through a specific binding pocket is a tractable strategy for UHRF1-histone inhibition.

Ubiquitin-like containing PHD and ring finger (UHRF)1 and UHRF2 are multidomain epigenetic proteins that play a critical role in bridging crosstalk between histone modifications and DNA methylation. Both proteins contain two histone reader domains, called tandem Tudor domain (TTD) and plant homeodomain (PHD), which read the modification status on histone H3 to regulate DNA methylation and gene expression. To shed light on the mechanism of histone binding by UHRF2, we have undergone a detailed molecular investigation with the TTD, PHD and TTD-PHD domains and compared the binding activity to its UHRF1 counterpart. We found that unlike UHRF1 where the PHD is the primary binding contributor, the TTD of UHRF2 has modestly higher affinity toward the H3 tail, while the PHD has a weaker binding interaction. We also demonstrated that like UHRF1, the aromatic amino acids within the TTD are important for binding to H3K9me3 and a conserved aspartic acid within the PHD forms an ionic interaction with R2 of H3. However, while the aromatic amino acids in the TTD of UHRF1 contribute to selectivity, the analogous residues in UHRF2 contribute to both selectivity and affinity. We also discovered that the PHD of UHRF2 contains a distinct asparagine in the H3R2 binding pocket that lowers the binding affinity of the PHD by reducing a potential electrostatic interaction with the H3 tail. Furthermore, we demonstrate the PHD and TTD of UHRF2 cooperate to interact with the H3 tail and that dual domain engagement with the H3 tail relies on specific amino acids. Lastly, our data indicate that the unique stretch region in the TTD of UHRF2 can decrease the melting temperature of the TTD-PHD and represents a disordered region. Thus, these subtle but important mechanistic differences are potential avenues for selectively targeting the histone binding interactions of UHRF1 and UHRF2 with small molecules., (© 2021 Wiley Periodicals LLC.)

Gcn5 serves as the defining member of the Gcn5-related N-acetyltransferase (GNAT) superfamily of proteins that display a common structural fold and catalytic mechanism involving the transfer of the acyl-group, primarily acetyl-, from CoA to an acceptor nucleophile. In the case of Gcn5, the target is the ε-amino group of lysine primarily on histones. Over the years, studies on Gcn5 structure-function have often formed the basis by which we understand the complex activities and regulation of the entire protein acetyltransferase family. It is now appreciated that protein acetylation occurs on thousands of proteins and can reversibly regulate the function of many cellular processes. In this review, we provide an overview of our fundamental understanding of catalysis, regulation of activity and substrate selection, and inhibitor development for this archetypal acetyltransferase., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Many epigenetic proteins recognize the posttranslational modification state of chromatin through their histone-binding domains and thereby recruit nuclear complexes to specific loci within the genome. A number of these domains have been implicated in cancer and other diseases through aberrant binding of chromatin; therefore, identifying small molecules that disrupt histone binding could be a powerful mechanism for disease therapy. We have developed a high-throughput assay for the detection of histone peptide-domain interactions utilizing AlphaScreen technology. Here, we describe how the assay can be first optimized and then performed for high-throughput screening of small molecule-binding inhibitors. We also describe strategies for biochemical validation of small molecules identified., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Rtt109 is a yeast histone acetyltransferase (HAT) that associates with histone chaperones Asf1 and Vps75 to acetylate H3K56, H3K9, and H3K27 and is important in DNA replication and maintaining genomic integrity. Recently, mass spectrometry and structural studies of Rtt109 have shown that active site residue Lys-290 is acetylated. However, the functional role of this modification and how the acetyl group is added to Lys-290 was unclear. Here, we examined the mechanism of Lys-290 acetylation and found that Rtt109 catalyzes intramolecular autoacetylation of Lys-290 ∼200-times slower than H3 acetylation. Deacetylated Rtt109 was prepared by reacting with a sirtuin protein deacetylase, producing an enzyme with negligible HAT activity. Autoacetylation of Rtt109 restored full HAT activity, indicating that autoacetylation is necessary for HAT activity and is a fully reversible process. To dissect the mechanism of activation, biochemical, and kinetic analyses were performed with Lys-290 variants of the Rtt109-Vps75 complex. We found that autoacetylation of Lys-290 increases the binding affinity for acetyl-CoA and enhances the rate of acetyl-transfer onto histone substrates. This study represents the first detailed investigation of a HAT enzyme regulated by single-site intramolecular autoacetylation.

Post-translational modifications of histones elicit structural and functional changes within chromatin that regulate various epigenetic processes. Epigenetic mechanisms rely on enzymes whose activities are driven by coenzymes and metabolites from intermediary metabolism. Lysine acetyltransferases (KATs) catalyze the transfer of acetyl groups from acetyl-CoA to epsilon amino groups. Utilization of this critical metabolite suggests these enzymes are modulated by the metabolic status of the cell. This review highlights studies linking KATs to metabolism. We cover newly identified acyl modifications (propionylation and butyrylation), discuss the control of KAT activity by cellular acetyl-CoA levels, and provide insights into how acetylation regulates metabolic proteins. We conclude with a discussion of the current approaches to identifying novel KATs and their metabolic substrates., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Most histone acetyltransferases (HATs) function as multisubunit complexes in which accessory proteins regulate substrate specificity and catalytic efficiency. Rtt109 is a particularly interesting example of a HAT whose specificity and catalytic activity require association with either of two histone chaperones, Vps75 or Asf1. Here, we utilize biochemical, structural, and genetic analyses to provide the detailed molecular mechanism for activation of a HAT (Rtt109) by its activating subunit Vps75. The rate-determining step of the activated complex is the transfer of the acetyl group from acetyl CoA to the acceptor lysine residue. Vps75 stimulates catalysis (> 250-fold), not by contributing a catalytic base, but by stabilizing the catalytically active conformation of Rtt109. To provide structural insight into the functional complex, we produced a molecular model of Rtt109-Vps75 based on X-ray diffraction of crystals of the complex. This model reveals distinct negative electrostatic surfaces on an Rtt109 molecule that interface with complementary electropositive ends of a symmetrical Vps75 dimer. Rtt109 variants with interface point substitutions lack the ability to be fully activated by Vps75, and one such variant displayed impaired Vps75-dependent histone acetylation functions in yeast, yet these variants showed no adverse effect on Asf1-dependent Rtt109 activities in vitro and in vivo. Finally, we provide evidence for a molecular model in which a 12 complex of Rtt109-Vps75 acetylates a heterodimer of H3-H4. The activation mechanism of Rtt109-Vps75 provides a valuable framework for understanding the molecular regulation of HATs within multisubunit complexes.

Rtt109 is a histone acetyltransferase (HAT) involved in promoting genomic stability, DNA repair, and transcriptional regulation. Rtt109 associates with the NAP1 family histone chaperone Vps75 and stimulates histone acetylation. Here we explore the mechanism of histone acetylation and report a detailed kinetic investigation of the Rtt109-Vps75 complex. Rtt109 and Vps75 form a stable complex with nanomolar binding affinity (K(d) = 10 +/- 2 nM). Steady-state kinetic analysis reveals evidence of a sequential kinetic mechanism whereby the Rtt109-Vps75 complex, AcCoA, and histone H3 substrates form a complex prior to chemical catalysis. Product inhibition studies demonstrate that CoA binds competitively with AcCoA, and equilibrium measurements reveal AcCoA or CoA binding is not stimulated in the presence of H3 substrate. Additionally, the Rtt109-Vps75 complex binds H3 substrates in the absence AcCoA. Pre-steady-state kinetic analysis suggests the chemical attack of substrate lysine on the bound AcCoA is the rate-limiting step of catalysis, while the pH profile of k(cat) reveals a critical ionization with a pK(a) of 8.5 that must be unprotonated for catalysis. Amino acid substitution at D287 and D288 did not substantially change the shape of the k(cat)-pH profile, suggesting these conserved residues do not function as base catalysts for histone acetylation. However, the D288N mutant revealed a dramatic 1000-fold decrease in k(cat)/K(m) for AcCoA, consistent with a role in AcCoA binding. Together, these data support a sequential mechanism in which AcCoA and H3 bind to the Rtt109-Vps75 complex without obligate order, followed by the direct attack of the unprotonated epsilon-amino group on AcCoA, transferring the acetyl group to H3 lysine residues.

Sirtuins are NAD+-dependent enzymes that have been implicated in a wide range of cellular processes, including pathways that affect diabetes, cancer, lifespan and Parkinson's disease. To understand their cellular function in these age-related diseases, identification of sirtuin targets and their subcellular localization is paramount. SIRT3 (sirtuin 3), a human homologue of Sir2 (silent information regulator 2), has been genetically linked to lifespan in the elderly. However, the function and localization of this enzyme has been keenly debated. A number of reports have indicated that SIRT3, upon proteolytic cleavage in the mitochondria, is an active protein deacetylase against a number of mitochondrial targets. In stark contrast, some reports have suggested that full-length SIRT3 exhibits nuclear localization and histone deacetylase activity. Recently, a report comparing SIRT3-/- and SIRT+/+ mice have provided compelling evidence that endogenous SIRT3 is mitochondrial and appears to be responsible for the majority of protein deacetylation in this organelle. In this issue of the Biochemical Journal, Cooper et al. present additional results that address the mitochondrial and nuclear localization of SIRT3. Utilizing fluorescence microscopy and cellular fractionation studies, Cooper et al. have shown that SIRT3 localizes to the mitochondria and is absent in the nucleus. Thus this study provides additional evidence to establish SIRT3 as a proteolytically modified, mitochondrial deacetylase.

Distinct catalytic mechanisms have been proposed for the Gcn5 and MYST histone acetyltransferase (HAT) families. Gcn5-like HATs utilize an ordered sequential mechanism involving direct nucleophilic attack of the N-epsilon-lysine on the enzyme-bound acetyl-CoA. Recently, MYST enzymes were reported to employ a ping-pong route of catalysis via an acetyl-cysteine intermediate. Here, using the prototypical MYST family member Esa1, and its physiological complex (piccolo NuA4), steady-state kinetic analyses revealed a kinetic mechanism that requires the formation of a ternary complex prior to catalysis, where acetyl-CoA binds first and CoA is the last product released. In the absence of histone acceptor, slow rates of enzyme auto-acetylation (7 x 10(-4) s(-1), or approximately 2500-fold slower than histone acetylation; kcat = 1.6 s(-1)) and of CoA formation (0.0021 s(-1)) were inconsistent with a kinetically competent acetyl-enzyme intermediate. Previously, Cys-304 of Esa1 was the proposed nucleophile that forms an acetyl-cysteine intermediate. Here, mutation of this cysteine (C304A) in Esa1 or within the piccolo NuA4 complex yielded an enzyme that was catalytically indistinguishable from the wild type. Similarly, a pH rate (kcat) analysis of the wild type and C304A revealed an ionization (pKa = 7.6-7.8) that must be unprotonated. Mutation of a conserved active-site glutamate (E338Q) reduced kcat approximately 200-fold at pH 7.5; however, at higher pH, E338Q exhibited nearly wild-type activity. These data are consistent with Glu-338 (general base) activating the N-epsilon-lysine by deprotonation. Together, the results suggest that MYST family HATs utilize a direct-attack mechanism within an Esa1 x acetyl-CoA x histone ternary complex.