Histone deacetylation regulates de novo shoot regeneration.

During de novo plant organ regeneration, auxin induction mediates the formation of a pluripotent cell mass called callus, which regenerates shoots upon cytokinin induction. However, molecular mechanisms underlying transdifferentiation remain unknown. Here, we showed that the loss of HDA19, a histone deacetylase (HDAC) family gene, suppresses shoot regeneration. Treatment with an HDAC inhibitor revealed that the activity of this gene is essential for shoot regeneration. Further, we identified target genes whose expression was regulated through HDA19-mediated histone deacetylation during shoot induction and found that ENHANCER OF SHOOT REGENERATION 1 and CUP-SHAPED COTYLEDON 2 play important roles in shoot apical meristem formation. Histones at the loci of these genes were hyperacetylated and markedly upregulated in hda19. Transient ESR1 or CUC2 overexpression impaired shoot regeneration, as observed in hda19. Therefore, HDA19 mediates direct histone deacetylation of CUC2 and ESR1 loci to prevent their overexpression at the early stages of shoot regeneration.

Improved development of mouse somatic cell nuclear transfer embryos by chlamydocin analogues, class I and IIa histone deacetylase inhibitors†.

In mammalian cloning by somatic cell nuclear transfer (SCNT), the treatment of reconstructed embryos with histone deacetylase (HDAC) inhibitors improves efficiency. So far, most of those used for SCNT are hydroxamic acid derivatives-such as trichostatin A-characterized by their broad inhibitory spectrum. Here, we examined whether mouse SCNT efficiency could be improved using chlamydocin analogues, a family of newly designed agents that specifically inhibit class I and IIa HDACs. Development of SCNT-derived embryos in vitro and in vivo revealed that four out of five chlamydocin analogues tested could promote the development of cloned embryos. The highest pup rates (7.1-7.2%) were obtained with Ky-9, similar to those achieved with trichostatin A (7.2-7.3%). Thus, inhibition of class I and/or IIa HDACs in SCNT-derived embryos is enough for significant improvements in full-term development. In mouse SCNT, the exposure of reconstructed oocytes to HDAC inhibitors is limited to 8-10 h because longer inhibition with class I inhibitors causes a two-cell developmental block. Therefore, we used Ky-29, with higher selectivity for class IIa than class I HDACs for longer treatment of SCNT-derived embryos. As expected, 24-h treatment with Ky-29 up to the two-cell stage did not induce a developmental block, but the pup rate was not improved. This suggests that the one-cell stage is a critical period for improving SCNT cloning using HDAC inhibitors. Thus, chlamydocin analogues appear promising for understanding and improving the epigenetic status of mammalian SCNT-derived embryos through their specific inhibitory effects on HDACs.

Transcriptomic analysis of Arabidopsis thaliana plants treated with the Ky-9 and Ky-72 histone deacetylase inhibitors.

Histone acetylation plays a pivotal role in plant growth and development, and is regulated by the antagonistic relationship between histone acetyltransferase (HAT) and histone deacetylase (HDAC). We previously revealed that some HDAC inhibitors confer high-salinity stress tolerance in plants. In this study, we identified two HDAC inhibitors, namely Ky-9 and Ky-72, which enhanced the high-salinity stress tolerance of Arabidopsis thaliana. Ky-9 and Ky-72 are structurally similar chlamydocin analogs. However, the in vitro inhibitory activity of Ky-9 against mammalian HDAC is greater than that of Ky-72. A western blot indicated that Ky-9 and Ky-72 increased the acetylation levels of histone H3, suggesting they exhibit HDAC inhibitory activities in plants. We conducted a transcriptomic analysis to investigate how Ky-9 and Ky-72 enhance high-salinity stress tolerance. Although Ky-9 upregulated the expression of more genes than Ky-72, similar gene expression patterns were induced by both HDAC inhibitors. Additionally, the expression of high-salinity stress tolerance-related genes, such as anthocyanin-related genes and a small peptide-encoding gene, increased by Ky-9 and Ky-72. These data suggest that slight structural differences in chemical side chain between HDAC inhibitors can alter inhibitory effect on HDAC protein leading to influence gene expression, thereby enhancing high-salinity stress tolerance in different extent.

The Distinct Roles of Class I and II RPD3-Like Histone Deacetylases in Salinity Stress Response.

Histone acetylation is an essential process in the epigenetic regulation of diverse biological processes, including environmental stress responses in plants. Previously, our research group identified a histone deacetylase (HDAC) inhibitor (HDI) that confers salt tolerance in Arabidopsis (Arabidopsis thaliana). In this study, we demonstrate that class I HDAC (HDA19) and class II HDACs (HDA5/14/15/18) control responses to salt stress through different pathways. The screening of 12 different selective HDIs indicated that seven newly reported HDIs enhance salt tolerance. Genetic analysis, based on a pharmacological study, identified which HDACs function in salinity stress tolerance. In the wild-type Columbia-0 background, hda19 plants exhibit tolerance to high-salinity stress, while hda5/14/15/18 plants exhibit hypersensitivity to salt stress. Transcriptome analysis revealed that the effect of HDA19 deficiency on the response to salinity stress is distinct from that of HDA5/14/15/18 deficiencies. In hda19 plants, the expression levels of stress tolerance-related genes, late embryogenesis abundant proteins that prevent protein aggregation and positive regulators such as ABI5 and NAC019 in abscisic acid signaling, were induced strongly relative to the wild type. Neither of these elements was up-regulated in the hda5/14/15/18 plants. The mutagenesis of HDA19 by genome editing in the hda5/14/15/18 plants enhanced salt tolerance, suggesting that suppression of HDA19 masks the phenotype caused by the suppression of class II HDACs in the salinity stress response. Collectively, our results demonstrate that HDIs that inhibit class I HDACs allow the rescue of plants from salinity stress regardless of their selectivity, and they provide insight into the hierarchal regulation of environmental stress responses through HDAC isoforms.

Live imaging of H3K9 acetylation in plant cells.

Proper regulation of histone acetylation is important in development and cellular responses to environmental stimuli. However, the dynamics of histone acetylation at the single-cell level remains poorly understood. Here we established a transgenic plant cell line to track histone H3 lysine 9 acetylation (H3K9ac) with a modification-specific intracellular antibody (mintbody). The H3K9ac-specific mintbody fused to the enhanced green fluorescent protein (H3K9ac-mintbody-GFP) was introduced into tobacco BY-2 cells. We successfully demonstrated that H3K9ac-mintbody-GFP interacted with H3K9ac in vivo. The ratio of nuclear/cytoplasmic H3K9ac-mintbody-GFP detected in quantitative analysis reflected the endogenous H3K9ac levels. Under chemically induced hyperacetylation conditions with histone deacetylase inhibitors including trichostatin A, Ky-2 and Ky-14, significant enhancement of H3K9ac was detected by H3K9ac-mintbody-GFP dependent on the strength of inhibitors. Conversely, treatment with a histone acetyltransferase inhibitor, C646 caused a reduction in the nuclear to cytoplasmic ratio of H3K9ac-mintbody-GFP. Using this system, we assessed the environmental responses of H3K9ac and found that cold and salt stresses enhanced H3K9ac in tobacco BY-2 cells. In addition, a combination of H3K9ac-mintbody-GFP with 5-ethynyl-2'-deoxyuridine labelling confirmed that H3K9ac level is constant during interphase.

Identification of Cyproheptadine as an Inhibitor of SET Domain Containing Lysine Methyltransferase 7/9 (Set7/9) That Regulates Estrogen-Dependent Transcription.

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.

Ky-2, a Histone Deacetylase Inhibitor, Enhances High-Salinity Stress Tolerance in Arabidopsis thaliana.

Adaptation to environmental stress requires genome-wide changes in gene expression. Histone modifications are involved in gene regulation, but the role of histone modifications under environmental stress is not well understood. To reveal the relationship between histone modification and environmental stress, we assessed the effects of inhibitors of histone modification enzymes during salinity stress. Treatment with Ky-2, a histone deacetylase inhibitor, enhanced high-salinity stress tolerance in Arabidopsis. We confirmed that Ky-2 possessed inhibition activity towards histone deacetylases by immunoblot analysis. To investigate how Ky-2 improved salt stress tolerance, we performed transcriptome and metabolome analysis. These data showed that the expression of salt-responsive genes and salt stress-related metabolites were increased by Ky-2 treatment under salinity stress. A mutant deficient in AtSOS1(Arabidopis thaliana SALT OVERLY SENSITIVE 1), which encodes an Na(+)/H(+)antiporter and was among the up-regulated genes, lost the salinity stress tolerance conferred by Ky-2. We confirmed that acetylation of histone H4 at AtSOS1 was increased by Ky-2 treatment. Moreover, Ky-2 treatment decreased the intracellular Na(+)accumulation under salinity stress, suggesting that enhancement of SOS1-dependent Na(+)efflux contributes to increased high-salinity stress tolerance caused by Ky-2 treatment.

An efficient synthesis of SK-658 and its analogs as potent histone deacetylase inhibitors.

SK-658 is a potent histone deacetylase (HDAC) inhibitor that showed higher activity than SAHA due to the presence of extended hydrophobic group. We designed and synthesized thioester and SS-hybrid bearing SK-658 analogs as HDAC inhibitors. All the compounds were active in nano molar range and showed higher inhibitory activity than SAHA and SK-658. Among these, disulfide compounds showed the highest activity.

Bicyclic tetrapeptide histone deacetylase inhibitors with methoxymethyl ketone and boronic acid zinc-binding groups.

Histone deacetylase (HDAC) inhibitors are a class of potential therapeutics for the treatment of cancer. Bicyclic tetrapeptides equipped with methoxymethyl ketone and boronic acid as zinc-binding group were designed and synthesized. The inhibitory activities of these compounds were evaluated against HDAC enzymes. The cell-free and cell-based assay data showed that both potency and selectivity changed with the change in zinc-binding group. Boronic acid-based compound showed poor activity whereas methoxymethyl ketone-based compound displayed impressive activity in both cell-free and cell-based conditions.

Bicyclic tetrapeptides as potent HDAC inhibitors: effect of aliphatic loop position and hydrophobicity on inhibitory activity.

Several histone deacetylase (HDAC) inhibiting bicyclic tetrapeptides have been designed and synthesized through intramolecular ring-closing metathesis (RCM) reaction and peptide cyclization. We designed bicyclic tetrapeptides based on CHAP31, trapoxin B and HC-toxin I. The HDAC inhibitory and p21 promoter assay results showed that the aliphatic loop position as well as the hydrophobicity plays an important role toward the activity of the bicyclic tetrapeptide HDAC inhibitors.

Design and synthesis of CHAP31, trapoxin B and HC-toxin based bicyclic tetrapeptides disulfide as potent histone deacetylase inhibitors.

The naturally occurring cyclic depsipeptide, FK228 inhibits histone deacetylase (HDAC) enzymes after reductive cleavage of intra-molecular disulfide bond. One of the sulfhydryl groups produced in the reduction interacts with zinc atom that involved in the catalytic mechanism of type 1 and 2 HDACs such as HDAC1, HDAC4, and HDAC6. In the present study, we describe the development of CHAP31, trapoxin B and HC-toxin based cyclic tetrapeptides with intra-molecular disulfide bond as HDAC inhibitors. The bicyclic tetrapeptides disulfide showed potent HDAC1 and HDAC4 inhibition and p21 promoting activity.

Design and synthesis of mono and bicyclic tetrapeptides thioester as potent inhibitor of histone deacetylases.

Inhibitors of histone deacetylases (HDACs) are a promising class of anticancer agents that have an effect on gene regulation. The naturally occurring cyclic depsipeptide FK228 containing disulfide and Largazole possessing thioester functionalities act as pro-drugs and share the same HDAC inhibition mechanism in cell. Inspired from these facts, we have reported bicyclic tetrapeptide disulfide HDAC inhibitors resembling FK228 with potent activity and enhanced selectivity. In the present study, we report the design and synthesis of several mono and bicyclic tetrapeptide thioester HDAC inhibitors that share the inhibition mechanism similar to Largazole. Most of the compounds showed HDAC1 and HDAC4 inhibition and p21 promoting activity in nanomolar ranges. Among these the monocyclic peptides 1, 2 and bicyclic peptide, 4 are notable demanding more advanced research to be promising anticancer drug candidates.

A convenient preparation of N (ε)-methyl-L-lysine derivatives and its application to the synthesis of histone tail peptides.

A convenient route is established for the preparation of N (α)-Fmoc-N (ε)-(Boc, methyl)-L-lysine and N (α)-Fmoc-N (ε)-dimethyl-L-lysine as building blocks to be used for the synthesis of methylated peptides. This methodology is based on the use of malonate derivatives and dibromobutane to produce key intermediates, L-2-amino-6-bromohexanoic acid derivatives, which could be modified to the required group at the ε-position. Fmoc-protection is accessible, so these compounds can be used in solution as well as in solid-phase peptide synthesis. Also the peptides containing these methylated lysines have been proved to resist the action of trypsin and lysyl endopeptidase. Thus, this new method could be considered as an improvement of the synthesis of N (ε)-methyl-L-lysine derivatives.

Design and synthesis of peptide-MCA substrates for a novel assay of histone methyltransferases and their inhibitors.

Histone methyltransferases (HMTs) play an important role in controlling gene expression through site-specific methylation of lysines in core and linker histones within chromatin. As the typical HMTs, G9a and Set7/9 have been intensively studied that G9a is specific to the methylation at H3K9 and H3K27 and represses transcription, while Set7/9 methylates at H3K4. In this report we prepared various peptide-MCAs (4-methylcoumaryl-7-amides) related to histone tail and protein-substrates such as p53 and estrogen receptor-α. The fluorogenic substrates are applied for the assay of HMTs and an inhibitor, for example. The most sensitive and specific MCA-substrates to G9a and Set7/9 are discovered. The peptide-MCAs corresponding to the methylation sequences are promising for screening of HMT inhibitors.

Discovery of potent HDAC inhibitors based on chlamydocin with inhibitory effects on cell migration.

The histone deacetylase (HDAC) family is a promising drug target class owing to the importance of these enzymes in a variety of cellular processes. Docking studies were conducted to identify novel HDAC inhibitors. Subtle modifications in the recognition domain were introduced into a series of chlamydocin analogues, and the resulting scaffolds were combined with various zinc binding domains. Remarkably, cyclo(L-Asu(NHOH)-L-A3mc6c-L-Phe-D-Pro, compound 1 b), with a methyl group at positions 3 or 5 on the aliphatic ring, exhibited better antiproliferative effects than trichostatin A (TSA) against MCF-7 and K562 cell lines. In addition to cell-cycle arrest and apoptosis, cell migration inhibition was observed in cells treated with compound 1 b. Subsequent western blot analysis revealed that the balance between matrix metalloproteinase 2 (MMP2) and tissue inhibitors of metalloproteinase 1 (TIMP1) determines the degree of metalloproteinase activity in MCF-7 cells, thereby regulating cell migration. The improved inhibitory activity imparted by altering the hydrophobic substitution pattern at the bulky cap group is a valuable approach in the development of novel HDAC inhibitors.

Cyclic tetrapeptides with -SS- bridging between amino acid side chains for potent histone deacetylases' inhibition.

Cyclic depsipeptide FK228 with an intramolecular disulfide bond is a potent inhibitor of histone deacetylases (HDAC). FK228 is stable in blood because of its prodrug function, whose -SS- bond is reduced within the cell. Here, cyclic peptides with -SS- bridges between a variety of amino acids were synthesized and assayed for HDAC inhibition. Cyclic peptide 3, cyclo(-L-amino acid-L-amino acid-L-Val-D-Pro-), with an -SS- bridge between the first and second amino acids, was found to be a potent HDAC inhibitor. Cyclic peptide 7, cyclo(-L-amino acid-D-amino acid-L-Val-D-Pro-), with an -SS- bridge between the first and second amino acids, was also a potent HDAC inhibitor.

Dynamics and regulation of lysine-acetylation during one-cell stage mouse embryos.

Previous studies show that treatment of zygotes with trichostatin A (TSA), a histone deacetylase inhibitor (HDACi), impacts the subsequent development to a blastocyst as well as full-term development. To reveal the dynamics of protein acetylation, with and without TSA treatment during one-cell stage, we examined oocytes and zygotes by immunofluorescence and Western Blot analyses using anti-acetylated lysine and acetylated α-tubulin antibodies. In unfertilized oocytes, lysine acetylation level was extremely low over all but faintly detected in the spindle. Once oocyte activation occurs, a dramatic increase of lysine acetylation signal was observed mostly in the pronuclei and a fiber-like structure, the so called midbody, suggesting activation coupled up-regulation of lysine acetylation presumably in histones and α-tubulin. TSA treatment resulted in significantly more hyperacetylation not only in the midbody structure and pronuclei but also in the whole cytoplasm. Consistently, Western Blot analysis revealed that acetylation of proteins about 53 kDa and 11 kDa in size, corresponding to α-tubulin and histone H4 sizes respectively, were increased mainly after oocyte activation and exclusively enhanced by TSA treatment in zygotes. To confirm this behavior of acetylated nonhistone proteins, acetylated α-tubulin was examined and found to be faintly detected in the spindle of MII oocytes but later in whole in the cell of zygotes including the midbody, which was enhanced by TSA treatment. To elucidate the mechanism underlying up-regulation of lysine acetylation following oocyte activation, we assayed the HDAC activity, and found significant reduction of HDAC activity from MII to zygotic stages. Taken together, our data indicate that HDACs play an important role in maintaining low acetylated status in a MII oocyte. However, once an oocyte has been activated, histone and nonhistone proteins including α-tubulin are hyperacetylated partly due to a reduction of HDAC activity. TSA treatment of zygotes enhances their acetylation, which could affect subsequent embryonic development.

Mechanisms of G1 cell cycle arrest and apoptosis in myeloma cells induced by hybrid-compound histone deacetylase inhibitor.

OBJECTIVES: Histone deacetylase (HDAC) inhibitors are new therapeutic agents, used to treat various types of malignant cancers. In the present study, we investigated the effects of Ky-2, a hybrid-compound HDAC inhibitor, on the growth of mouse myeloma cells. MATERIALS AND METHODS: Myeloma cells, HS-72, P3U1, and mouse normal cells were used in this study. Effect of HDAC inhibitors on cell viability was determined by WST-assay and trypan blue assay. Cell cycle was analyzed using flow cytometer. The expression of cell cycle regulatory and the apoptosis associated proteins were examined by Western blot analysis. Hoechst's staining was used to detect apoptotic cells. RESULTS: Our findings showed that Ky-2 decreased the levels of HDACs, while it enhanced acetylation of histone H3. Myeloma cell proliferation was inhibited by Ky-2 treatment. Interestingly, Ky-2 had no cytotoxic effects on mouse normal cells. Ky-2 treatment induced G1-phase cell cycle arrest and accumulation of a sub-G1 phase population, while Western blotting analysis revealed that expressions of the cell cycle-associated proteins were up-regulated. Also, Ky-2 enhanced the cleavage of caspase-9 and -3 in myeloma cells, followed by DNA fragmentation. In addition, Ky-2 was not found to induce apoptosis in bcl-2 overexpressing myeloma cells. CONCLUSION: These findings suggest that Ky-2 induces apoptosis via a caspase-dependent cascade and Bcl-2-inhibitable mechanism in myeloma cells.

Cyclic tetrapeptides with thioacetate tails or intramolecular disulfide bridge as potent inhibitors of histone deacetylases.

Two thioacetate tails were introduced to the chlamydocin- and CHAP31-related cyclic tetrapeptides. An intramolecular disulfide bridge could be formed in the CHAP31-related cyclic peptides. Both the thioacetate-tailed and disulfide-bridged peptides were potent histone deacetylase inhibitors in the presence of sulfhydryl compound. Potent p21 promoter inducing activity was also observed in vivo.

Identification of novel molecular targets regulated by tumor suppressive miR-375 induced by histone acetylation in esophageal squamous cell carcinoma.

The aim of this study was to determine whether histone acetylation regulates tumor suppressive microRNAs (miRNAs) in esophageal squamous cell carcinoma (ESCC) and to identify genes which are regulated by these miRNAs. We identified a miRNA that was highly upregulated in an ESCC cell line by cyclic hydroxamic acid-containing peptide 31 (CHAP31), one of the histone deacetylase inhibitors (HDACIs), using a miRNA array analysis. miR-375 was strongly upregulated by CHAP31 treatment in an ESCC cell line. The expression levels of the most upregulated miRNA, miR-375 were analyzed by quantitative real-time PCR in human ESCC specimens. The tumor suppressive function of miR-375 was revealed by restoration of miR-375 in ESCC cell lines. We performed a microarray analysis to identify target genes of miR-375. The mRNA and protein expression levels of these genes were verified in ESCC clinical specimens. LDHB and AEG-1/MTDH were detected as miR­375-targeted genes. The restoration of miR-375 suppressed the expression of LDHB and AEG-1/MTDH. The ESCC clinical specimens exhibited a high level of LDHB expression at both the mRNA and protein levels. A loss-of-function assay using a siRNA analysis was performed to examine the oncogenic function of the gene. Knockdown of LDHB by RNAi showed a tumor suppressive function in the ESCC cells. The correlation between gene expression and clinicopathological features was investigated by immunohistochemistry for 94 cases of ESCC. The positive staining of LDHB correlated significantly with lymph node metastasis and tumor stage. It also had a tendency to be associated with a poor prognosis. Our results indicate that HDACIs upregulate miRNAs, at least some of which act as tumor suppressors. LDHB, which is regulated by the tumor suppressive miR-375, may therefore act as an oncogene in ESCC.