•EGR2 is a novel acetylated protein.•p300 and CBP act as acetyltransferases of EGR2.•HDAC6, HDAC10, and SIRT1 act as deacetylases of EGR2.EGR2 is a zinc finger transcription factor that regulates myelination in the peripheral nervous system and T cell anergy. The transcriptional activity of EGR2 is known to be regulated by its co-activators and/or co-repressors. Although the activity of transcription factors is generally regulated not only by interactions with co-regulators but also posttranslational modifications including acetylation, little is known about posttranslational modifications of EGR2. Here we show that EGR2 is a novel acetylated protein. Through immunoblotting analyses using an antibody that specifically recognizes the acetylated form of EGR2, CBP and p300 were identified as acetyltransferases, while HDAC6, 10 and SIRT1 were identified as deacetylases of EGR2. Although the NuRD complex containing HDAC1 and HDAC2 is known to associate with EGR2, the present study suggests that acetylation of EGR2 is regulated independently of NuRD.

SET domain containing lysine methyltransferase 7/9 (Set7/9), a histone lysine methyltransferase (HMT), also methylates non-histone proteins including estrogen receptor (ER) α. ERα methylation by Set7/9 stabilizes ERα and activates its transcriptional activities, which are involved in the carcinogenesis of breast cancer. We identified cyproheptadine, a clinically approved antiallergy drug, as a Set7/9 inhibitor in a high-throughput screen using a fluorogenic substrate-based HMT assay. Kinetic and X-ray crystallographic analyses revealed that cyproheptadine binds in the substrate-binding pocket of Set7/9 and inhibits its enzymatic activity by competing with the methyl group acceptor. Treatment of human breast cancer cells (MCF7 cells) with cyproheptadine decreased the expression and transcriptional activity of ERα, thereby inhibiting estrogen-dependent cell growth. Our findings suggest that cyproheptadine can be repurposed for breast cancer treatment or used as a starting point for the discovery of an anti-hormone breast cancer drug through lead optimization.

Conjugation of small ubiquitin-like modifier (SUMO) to protein (SUMOylation) regulates multiple biological systems by changing the functions and fates of a large number of proteins. Consequently, abnormalities in SUMOylation have been linked to multiple diseases, including breast cancer. Using an in situ cell-based screening system, we have identified spectomycin B1 and related natural products as novel SUMOylation inhibitors. Unlike known SUMOylation inhibitors such as ginkgolic acid, spectomycin B1 directly binds to E2 (Ubc9) and selectively blocks the formation of the E2-SUMO intermediate; that is, Ubc9 is the direct target of spectomycin B1. Importantly, either spectomycin B1 treatment or Ubc9 knockdown inhibited estrogen-dependent proliferation of MCF7 human breast-cancer cells. Our findings suggest that Ubc9 inhibitors such as spectomycin B1 have potential as therapeutic agents against hormone-dependent breast cancers.

The combination of histone posttranslational modifications occurring in nucleosomal histones determines the epigenetic code. Histone modifications such as acetylation are dynamically controlled in response to a variety of signals during the cell cycle and differentiation, but they are paradoxically maintained through cell division to impart tissue specific gene expression patterns to progeny. The dynamics of histone modifications in living cells are poorly understood, because of the lack of experimental tools to monitor them in a real-time fashion. Recently, FRET-based imaging probes for histone H4 acetylation have been developed, which enabled monitoring of changes in histone acetylation during the cell cycle and drug treatment. Further development of this type of fluorescent probes for other modifications will make it possible to visualize complicated epigenetic regulation in living cells.

Histone lysine methyltransferases (HMTs) regulate transcriptional activity by writing epigenetic marks. Methylation of histone H3K9, a hallmark of silent chromatin,1 is mainly regulated by two subgroups of HMTs, G9a/G9a-like protein2 and Suv39h.3 G9a and G9a-like protein induce mono- and di-methylation of histone H3K9 (H3K9me1 and H3K9me2) in the euchromatin region,4 whereas Suv39h contributes to tri-methylation of histone H3K9 (H3K9me3) in the heterochromatin.3 As methylation at histone H3K9, increased DNA methylation and reduced levels of activating chromatin modifications (e.g., histone acetylation) have been detected at promoter regions of aberrantly silenced tumor suppressor genes in cancer cells,5, 6 HMTs responsible for histone H3K9 methylation may represent promising targets for drug discovery. Indeed, overexpression of G9a is associated with several types of cancers and downregulation of G9a by RNAi inhibits tumor cell proliferation.7, 8, 9 Chaetocin and BIX-01294 are small molecules that inhibit histone H3K9 HMTs. Chaetocin, which inhibit both G9a and Suv39h activities, is a member of the epipolythiodioxopiperazine (ETP) class of fungal metabolites.10 On the other hand, BIX-01294 is a synthetic compound that selectively inhibits G9a but not Suv39h111 Based on a co-crystallization analysis of G9a with BIX-01294, BIX-01294-related molecules have been developed; these novel compounds are both more potent inhibitors and more membrane permeable than the parent compound.12, 13, 14, 15In order to identify small molecules that affect the function of histone H3K9 methyltransferases, we used enzyme-linked immunosorbent assay to screen a chemical library derived from microorganisms.16 From this screen, we identified gliotoxin, an ETP secondary metabolite produced by fungal pathogens, as a G9a inhibitor (Figure 1a). To examine the inhibitory activity of gliotoxin against G9a in detail, we performed in vitro HMT assay by western blotting. In this assay, methylation of recombinant GST-fused histone H3 (1−57 a.a.) by G9a was measured using specific antibodies against methylated histone H3. As shown in Figure 1e, in the presence of recombinant GST-fused G9a together with SAM, G9a exhibited HMTase activity, producing mono- and di-methylated histone H3K9. However, the enzymatic activity of G9a was inhibited by gliotoxin in a dose-dependent manner. The IC50 value of gliotoxin for di-methylation of histone H3 was 0.53 μM (Table 1). Moreover, in almost the same concentration range, gliotoxin inhibited the HMTase activity of Suv39h1H320R (active form3) (Figure 1f and Table 1). On the other hand, gliotoxin did not inhibit the HMTase activity of Set7/9, another methyltransferase that methylates histone H3K4 in vitro17 (Figure 1g and Table 1). These results suggested that gliotoxin selectively inhibits histone H3K9 methyltransferases.In further analysis of the inhibitory activity of related ETP compounds, we selected chetomin (Figure 1b), 11-11′-dideoxyverticillin A (Figure 1c) and bisdethiobis-acetylgliotoxin (Figure 1d), and tested these compounds for their activity to inhibit histone H3K9 methyltransferases in vitro. Indeed, chetomin and 11-11′-dideoxyverticillin A could inhibit the HMTase activities of both G9a and Suv39h1H320R (Table 1). The capacity of chetomin to inhibit Suv39h1H320R was relatively strong (IC50: 0.066 μM). Importantly, however, bisdethiobis-acetylgliotoxin, which does not contain the disulfide bridge, exhibited no inhibitory activity up to 10 μM (Table 1). This observation suggests that the disulfide group is essential for the inhibition of HMTs by ETPs. This is also consistent with the previous report of the activity of synthetic analogs of chaetocin lacking disulfide.16 Because G9a and Suv39h1, but not Set7/9, possess pre- and post-SET domains, which bind zinc atoms with their cysteine-rich regions,2 ETPs might interact with these domains through their own sulfur-containing functional groups.Gliotoxin and the other ETPs inhibit several other proteins, including creatine kinase, NF-κB, adenine nucleotide transporter and farnesyltransferase;18 these compounds also exhibit antitumor and immunomodulatory activities. However, the structure−activity relationship of ETPs is poorly understood. In this study, we demonstrated that gliotoxin, chetomin and 11-11′-dideoxyverticillin A strongly inhibit H3K9 HMTs (Table 1) probably through their disulfide bonds, suggesting that the inhibition of H3K9 HMTs is a common activity of ETPs. Because accumulating evidence suggests that G9a and Suv39h1 are potential targets for cancer therapy, ETPs may serve as a drug seed. In particular, gliotoxin is one of the structurally simplest ETPs; derivatives of this compound may provide information that will be useful for development of anticancer small molecules that specifically inhibit HMTs but not other proteins.This work was supported in part by the Chemical Genomics Research Project, RIKEN ASI, the CREST Research Project, the Japan Science and Technology Corporation, the Project for Development of Innovative Research Cancer Therapeutics and a Grant-in-Aid for Scientific Research on Priority Area ‘Cancer’ from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Histone acetylation constitutes an epigenetic mark for transcriptional regulation. Here we developed a fluorescent probe to visualize acetylation of histone H4 Lys12 (H4K12) in living cells using fluorescence resonance energy transfer (FRET) and the binding of the BRD2 bromodomain to acetylated H4K12. Using this probe designated as Histac-K12, we demonstrated that histone H4K12 acetylation is retained in mitosis and that some histone deacetylase (HDAC) inhibitors continue to inhibit cellular HDAC activity even after their removal from the culture. In addition, a small molecule that interferes with ability of the bromodomain to bind to acetylated H4K12 could be assessed using Histac-K12 in cells. Thus, Histac-K12 will serve as a powerful tool not only to understand the dynamics of H4K12-specific acetylation but also to characterize small molecules that modulate the acetylation or interaction status of histones.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (249 K)Download as PowerPoint slideHighlights► Development of a FRET-based probe for visualizing histone H4K12 acetylation ► Real-time quantification of H4K12 acetylation dynamics during mitosis ► Description of H4K12 acetylation dynamics in living cells using HDAC inhibitors ► Identification of a compound that inhibits BRD2 association with H4K12 acetylation

Protein modification by small ubiquitin-related modifier proteins (SUMOs) controls diverse cellular functions. Dysregulation of SUMOylation or deSUMOylation processes has been implicated in the development of cancer and neurodegenerative diseases. However, no small-molecule inhibiting protein SUMOylation has been reported so far. Here, we report inhibition of SUMOylation by ginkgolic acid and its analog, anacardic acid. Ginkgolic acid and anacardic acid inhibit protein SUMOylation both in vitro and in vivo without affecting in vivo ubiquitination. Binding assays with a fluorescently labeled probe showed that ginkgolic acid directly binds E1 and inhibits the formation of the E1-SUMO intermediate. These studies will provide not only a useful tool for investigating the roles of SUMO conjugations in a variety of pathways in cells, but also a basis for the development of drugs targeted against diseases involving aberrant SUMOylation.

Eukaryotic translation initiation factor 5A (eIF5A) is a protein subject to hypusination, which is essential for its function. eIF5A is also acetylated, but the role of that modification is unknown. Here, we report that acetylation regulates the subcellular localization of eIF5A. We identified PCAF as the major cellular acetyltransferase of eIF5A, and HDAC6 and SIRT2 as its major deacetylases. Inhibition of the deacetylases or impaired hypusination increased acetylation of eIF5A, leading to nuclear accumulation. As eIF5A is constitutively hypusinated under physiological conditions, we suggest that reversible acetylation plays a major role in controlling the subcellular localization of eIF5A.Highlights► Acetylation regulates subcellular localization of eIF5A. ► PCAF is the cellular acetylatrasferase of eIF5A. ► HDAC6 and SIRT2 are major cellular deacetylases of eIF5A. ► eIF5A hypusination prevents its acetylation.