Deregulation of histone lysine methyltransferases contributes to oncogenic transformation of human bronchoepithelial cells

Watanabe, Hideo; Soejima, Kenzo; Yasuda, Hiroyuki; Kawada, Ichiro; Nakachi, Ichiro; Yoda, Satoshi; Naoki, Katsuhiko; Ishizaka, Akitoshi

doi:10.1186/1475-2867-8-15

Cited by 137 publications

(120 citation statements)

References 45 publications

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“…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.…”

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confidence: 99%

Inhibition of histone H3K9 methyltransferases by gliotoxin and related epipolythiodioxopiperazines

et al. 2012

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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 protein 2 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. 7-9 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 Suv39h1 11 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. 12-15 MATERIALS AND METHODS MaterialsAnti-mono-and di-methylated histone H3K9 antibodies were purchased from Merck Millipore (Billerica, MA, USA). pGEX4T-1-mG9a (706Àstop amino acid (a.a.)), pGEX4T-3-histone H3 (1À57 a.a.) and pGEX4T-3-mSuv39h1-H320R (74À412 a.a.) were previously described. 2 The pET-28a(+)-Set7/9 was kindly provided by Dr Kenichi Nishioka (National Institute of Genetics). Bacterial protein expression and purificationpGEX4T-1-mG9a (706Àstop a.a.), pGEX4T-3-mSuv39h1-H320R (74À412 a.a.), pET-28a(+)-Set7/9, or pGEX4T-3-histone H3 (1À57 a.a.) was introduced into Escherichia coli BL21 (DE3). Expression of each recombinant protein was induced by 0.1 mM isopropyl-b-D-galactopyranosid at 18 1C for 24 h. Purification of GST-and (His) 6 -fused proteins were carried out by either glutathioneaffinity (GE Healthcare UK Ltd, Little Chalfont, UK) or nickel (Ni 2+ ) affinity (GE Healthcare UK Ltd). In vitro HMT assayEach purified recombinant HMT was pretreated in the presence or absence of a given compound in HMT buffer (50 mM Tris-HCl (pH 8.5), 10 mM MgCl 2 , 20 mM KCl, 10 mM 2-mercaptoethanol and 250 mM sucrose) containing 3 mg ml À1 of BSA for 1 h. Next, 10 mg ml À1 of SAM and 20 mg ml À1 of GST-fused histone H3 (1À57 a.a.) were added into the reaction mixture and further incubated for 1 h at 37 1C. To detect histone methylation on western blots, samples were transferred onto Immobilon membranes (Merck Millipore), probed using appropriate primary antibodies, and visualized using horseradish peroxidase-linked secondary antibodies.In or...

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Inhibition of histone H3K9 methyltransferases by gliotoxin and related epipolythiodioxopiperazines

et al. 2012

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“…It is upregulated in hepatocellular carcinoma, 9 B cell acute lymphoblastic leukemia, 10 and lung cancers. 11 In addition, elevated expression of G9a in aggressive lung cancer correlates with poor prognosis, while its knockdown in highly invasive lung cancer cells suppressed metastasis in an in vivo mouse model. 12 In prostate cancer cells (PC3), G9a knockdown caused significant morphological changes and inhibition of cell growth.…”

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confidence: 99%

Discovery and Development of Potent and Selective Inhibitors of Histone Methyltransferase G9a

Sweis

Pliushchev

Brown

et al. 2014

ACS Med. Chem. Lett.

165

141

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G9a is a histone lysine methyltransferase responsible for the methylation of histone H3 lysine 9. The discovery of A-366 arose from a unique diversity screening hit, which was optimized by incorporation of a propyl-pyrrolidine subunit to occupy the enzyme lysine channel. A-366 is a potent inhibitor of G9a (IC 50 : 3.3 nM) with greater than 1000-fold selectivity over 21 other methyltransferases.KEYWORDS: G9a, methyltransferase, A-366, methylation, epigenetics H istone methyltransferases (HMTs), a class of enzymatic "writers" of epigenetic marks, have recently emerged as targets of potential therapeutic value.1,2 They catalyze the methylation of histone lysines and arginines utilizing Sadenosyl-methionine (SAM) as the cofactor/methyl-source. This process can result in either the activation or repression of transcription.3,4 Dysregulation of methylation at specific histone sites (alterations in the "histone code") has been implicated in many cancers.5−7 Hence, targeting HMT activity has been the subject of much investigation in the field of oncology, even recently reaching human clinical trials. 8 Euchromatic histone methyltransferase 2 (EHMT2), also known as G9a, is an HMT that is primarily responsible for the dimethylation of lysine 9 on histone H3 (H3K9). Several reports have highlighted its link to a variety of cancers. It is upregulated in hepatocellular carcinoma, 9 B cell acute lymphoblastic leukemia, 10 and lung cancers. 11 In addition, elevated expression of G9a in aggressive lung cancer correlates with poor prognosis, while its knockdown in highly invasive lung cancer cells suppressed metastasis in an in vivo mouse model. 12 In prostate cancer cells (PC3), G9a knockdown caused significant morphological changes and inhibition of cell growth. 13While small molecule inhibitors of G9a (Figure 1) have been reported as early as 2005, 14 BIX01294 (1) was the first reported potent and selective inhibitor. 15 It was found to reduce H3K9 dimethyl levels in cells and notably was not a SAM-competitive inhibitor. Recently, a second G9a inhibitor, UNC0638 (2), was disclosed. Quinazoline 2, which incorporated a lysine-mimic via an n-propyl-pyrrolidine exhibited a >10-fold enhancement in potency over 1 along with selectivity over a panel of 17 other epigenetic targets. 16−19 Our efforts toward the identification of chemically distinct G9a inhibitors commenced with a chemical diversity subset screen of our compound collection. The assay format employed was a peptide-based AlphaLISA, measuring the levels of H3K9 dimethylation. Several clusters of related chemical matter were identified, but a singleton stood out: spiro[cyclobutane-1,3′-indol]-2′-amine (3) as having robust potency (IC 50 : 153 nM). The low molecular weight of this compound translated to a high binding efficiency index (BEI) value of 27. 20 Such a feature rendered 3 as an attractive starting point for chemistry efforts aimed at further potency optimization.The importance of the various subunits of 3 was initially interrogated by evaluation of a ...

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“…Enhancer of zeste (EZH)2, a methyltransferase component of polycomb repressive complex 2(PRC2) has been found deregulated in many sorts of cancers, including lymphoma [148,149], bladder [150][151][152][153], gastric [154,155], lung [156], breast cancers [157][158][159]. EZH2 catalyzes H3K27me3 in the presence of suppressor of zeste 12 (SUZ12) and embryonic ectoderm development (EED) which possess a carboxy-terminal domain specifically recognizes histone tails that carry trimethyl-lysine residues [160].…”

Section: Histone Associated Protein Deregulation In Cancer and Epigenmentioning

confidence: 99%

Chromatin Memory in the Development of Human Cancers

2014

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Cancer is a complex disease with acquired genomic and epigenomic alterations that affect cell proliferation, viability and invasiveness. Almost all the epigenetic mechanisms including cytosine methylation and hydroxymethylation, chromatin remodeling and non-coding RNAs have been found associate with carcinogenesis and cancer specific expression profile. Altered histone modification as an epigenetic hallmark is frequently found in tumors. Understanding the epigenetic alterations induced by carcinogens or infectious agents may help us understand early epigenetic changes prior to the development of cancer. In this review, we focus on chromatin remodeling and the associated histone modifiers in the development of cancer; the application of these modifiers as a cancer therapy target in different clinical trial phases is also discussed.

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Deregulation of histone lysine methyltransferases contributes to oncogenic transformation of human bronchoepithelial cells

Cited by 137 publications

References 45 publications

Inhibition of histone H3K9 methyltransferases by gliotoxin and related epipolythiodioxopiperazines

Inhibition of histone H3K9 methyltransferases by gliotoxin and related epipolythiodioxopiperazines

Discovery and Development of Potent and Selective Inhibitors of Histone Methyltransferase G9a

Chromatin Memory in the Development of Human Cancers

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