Histone deacetylase inhibitor activity in royal jellymight facilitate caste switching in beesAstrid Spannhoff1, Yong Kee Kim1, Noel J.-M. Raynal2, Vazganush Gharibyan2, Ming-Bo Su3, Yue-Yang Zhou3,Jia Li3, Sabrina Castellano4, Gianluca Sbardella4, Jean-Pierre J. Issa2 & Mark T. Bedford1+

1Department of Molecular Carcinogenesis, University of Texas MD Anderson Cancer Center, Smithville, 2Department of Leukemia,

University of Texas MD Anderson Cancer Center, Houston, Texas, USA, 3National Center for Drug Screening, State Key Laboratory of

Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China, and 4Dipartimento di Scienze

Farmaceutiche, Epigenetic Med. Chem. Lab, Universita di Salerno, Fisciano, Italy

Worker and queen bees are genetically indistinguishable. How-ever, queen bees are fertile, larger and have a longer lifespan thantheir female worker counterparts. Differential feeding of larvaewith royal jelly controls this caste switching. There is emergingevidence that the queen-bee phenotype is driven by epigeneticmechanisms. In this study, we show that royal jelly—thesecretion produced by the hypopharyngeal and mandibularglands of worker bees—has histone deacetylase inhibitor (HDACi)activity. A fatty acid, (E)-10-hydroxy-2-decenoic acid (10HDA),which accounts for up to 5% of royal jelly, harbours this HDACiactivity. Furthermore, 10HDA can reactivate the expression ofepigenetically silenced genes in mammalian cells. Thus, theepigenetic regulation of queen-bee development is probablydriven, in part, by HDACi activity in royal jelly.Keywords: Apis mellifera; epigenetic; HDAC inhibitor;phenotypic plasticity; royal jellyEMBO reports advance online publication 18 February 2011; doi:10.1038/embor.2011.9

INTRODUCTIONThe honey-bee (Apis mellifera) exemplifies a phenomenonin nature called environmentally driven phenotypic plasticity(Maleszka, 2008) which describes how diet dictates the ability ofdifferent phenotypes to arise from one genome. In social insectssuch as bees, it leads to the establishment of castes, with adifferentiation between reproduction (queen) and labour (workers;

Page Jr, 1991). This phenotypic difference is determined at an earlylarvae stage, and the imprint is established by distinct feedingregimens for queen and worker larvae (Page Jr & Peng, 2001). Forthe first 3 days of life, all larvae are fed royal jelly, a substancecomposed of a mixture of hypopharyngeal and mandibular-glandsecretions that is made by nurse bees. Worker larvae are thenswitched to a diet that contains predominantly honey and pollen(worker jelly; Haydak, 1970). The queen larva, however, remainson a royal-jelly diet and is fed it in abundance. This diet results in aqueen bee that is morphologically, behaviourally and physiologi-cally distinct from her worker-bee counterparts, and also has aprolonged lifespan; at least 20 times longer than a regular worker-bee (Winston, 1987). This dietary input has pleiotropic effects:activating the insulin signalling cascade, affecting brain-releasinghormones and regulating global epigenetic changes (Maleszka,2008). In other social insects such as ants, termites and somewasps, it is less clear how a selective diet or the environment leadsto caste determination (Schwander et al, 2010).

In mammals, nutrition has a direct influence on geneexpression and development, by affecting the epigenetic state ofthe genome (Dolinoy et al, 2007). The enzymes that maintain aspecific epigenetic state include DNA methyltransferases, histoneacetylases and deacetylases, and histone methyltransferases anddemethylases. Any of these enzymes could be targeted bynutritional factors. What we can learn about the mechanismsregulating phenotypic plasticity in insects will probably berelevant to our understanding of the way in which diet andnutrition regulate fetal programming and disease in humans.Recent reports show that honey-bees have a fully operationalDNA-methylation system (Wang et al, 2006), as opposed to flies,which do not have CpG methylation. The comparison betweenDNA-methylation patterns in the brains of queen and worker beesshows that there is no difference in the number of CpGs that aremethylated (Lyko et al, 2010). However, there are distinct loci thatare differentially methylated, especially in exonic regions, whichaffect alternative splicing. Importantly, small interfering RNA(siRNA)-induced knockdown of Dnmt3 levels in newly emerged

Received 19 August 2010; revised 16 January 2011; accepted 19 January 2011;published online 18 February 2011

+Corresponding author. Tel: þ 1 512 237 9539; Fax: þ 1 512 237 2475;E-mail: mtbedford@mdanderson.org

1Department of Molecular Carcinogenesis, University of Texas MD Anderson CancerCenter, Park Road 1C, Smithville, Texas 78957, USA2Department of Leukemia, University of Texas MD Anderson Cancer Center,Houston, Texas 77030, USA3National Center for Drug Screening, State Key Laboratory of Drug Research,Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai201203, China4Dipartimento di Scienze Farmaceutiche, Epigenetic Med. Chem. Lab, Universitadi Salerno, Via Ponte Don Melillo, Fisciano (SA) I-84084, Italy

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larvae results in the development of queen bees with fullydeveloped ovaries (Kucharski et al, 2008). Thus, injection ofhoney-bee larvae with Dnmt3 siRNA can replace royal jellyconsumption and influence caste development. These dataindicate that a component of royal jelly has the ability to controlepigenetic pathways.

To investigate whether royal jelly harbours epigenetic regula-tory activity, we used the K-ras-transformed NIH 3T3 cell reportersystem. It has been shown previously that in these cells theactivated Ras pathway epigenetically silences expression of theproapoptotic Fas gene (Fenton et al, 1998; Peli et al, 1999).Furthermore, this system has been subjected to a genome-wideRNA-interference screen to identify factors required for Ras-mediated epigenetic silencing (Gazin et al, 2007). This screenidentified several transcriptional repressors including Dnmt1,Hdac9, Ezh2 and Sirt6. We therefore reasoned that this epigeneticreporter system is suited for the screening of potential royal jellyepigenetic regulatory activity. Indeed, when K-ras-transformedNIH 3T3 cells were treated with this hypopharyngeal glandsecretion, the Fas gene was reactivated. We have identified theactive ingredient as (E)-10-hydroxy-2-decenoic acid (10HDA) anddemonstrate that this compound has histone deacetylase inhibitor(HDACi) activity.

RESULTSRoyal jelly reactivates an epigenetically silenced locusAs siRNA-induced knockdown of Dnmt3 levels in bee larvaeresulted in the development of queen bees (Kucharski et al, 2008),we theorized that royal jelly harbours activity that can regulateDNA methylation. We used the K-ras-transformed NIH 3T3 cellreporter system to measure the presence of epigenetic regulatoryactivity in royal jelly (supplementary Fig S1A online). These cellswere treated with two concentrations of royal jelly (0.5% and 1%)for up to 3 days. By day 2, we observed re-expression of the Fasgene (Fig 1A). Furthermore, the regained expression was similar tothat obtained with 5-aza-20-deoxycytidine (5-Aza) treatment, awell-characterized inhibitor of DNA methylation (Stresemann &Lyko, 2008). Next, we fractionated royal jelly using an ultra-filtration membrane with a 3 kDa cut-off. The flow-through, whichcontains the low-molecular-weight fraction, harboured the epi-genetic regulatory activity (Fig 1B). This result—together with thefact that proteinase K treatment of royal jelly did not reduce itsactivity in this assay (data not shown)—indicates that theactive component of royal jelly is probably a small molecule ofnon-proteinaceous origin.

10HDA is the active component of royal jellyRoyal jelly is a complex mixture consisting of amino acids, sugars,proteins, fatty acids and mineral salts. A main component of royaljelly is 10HDA, which can compose 2–6% of royal jelly; thistranslates into a 100–300 mM concentration in royal jelly. Wefocussed on 10HDA because of its abundance, and publishedevidence that it is biologically active in mammalian cells, forwhich a mechanism has yet to be attributed. Again, by using thesame K-ras-transformed NIH 3T3 cell reporter system, we showthat a 5 mM solution of 10HDA restores Fas expression (Fig 1C).This demonstrates that a single component of royal jelly—10HDA—has the ability to activate gene expression, possiblythrough epigenetic reprogramming.

To establish that 10HDA functions broadly and that thereactivation of Fas expression is not idiosyncratic to the NIH 3T3K-ras cell reporter system, we tested 10HDA in a second epigeneticreporter system, which is based on the reactivation of a silencedgreen fluorescent protein (GFP) locus (supplementary Fig S1Bonline). This ear fibroblast cell line (GF2–4) harbours a GFP genethat is heavily CpG methylated, as determined by bisulphitegenomic sequencing (Kang et al, 2007). Treatment of GF2–4 cellswith 5-Aza results in the reactivation of the GFP locus (Kang et al,2007). Furthermore, a synergistic effect of a DNA demethylatingagent and an HDACi on the expression of GFP was reported. Wetreated GF2–4 cells with either 10HDA alone or in combinationwith 5-Aza and observed that 10HDA functions synergistically withthe 5-Aza (Fig 2A,B and supplementary Fig S2 online). 10HDAalone is unable to activate the silenced GFP locus. In this assay,10HDA behaves in a similar manner to sodium butyrate, a widelydocumented HDACi. These data indicate that 10HDA inhibits apathway involved in transcription repression that runs parallel to theDNA methylation pathway, and does not function as a DNAdemethylating agent per se.

10HDA does not inhibit DNA methylationWe studied a third model system—SW48 cells (a human coloncancer cell line) containing a hypermethylated cytomegalovirus

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Fig 1 | Royal jelly and 10HDA can reactivate the expression of an

epigenetically silenced Fas locus. (A) Western blot analysis showing that

a 1% and 0.5% royal jelly (RJ) suspension can restore Fas expression in

NIH 3T3 K-ras cells. Cells were cultured in RJ for 1–3 days. 5-Aza-2-

deoxycytidine (5-Aza) treatment acts as a control for reactivated Fas

expression. (B) The fraction of RJ that is smaller than 3 kDa in size

harbours this epigenetic regulatory activity. (C) A 5 mM solution of

10HDA, a main component of RJ, is able to reactivate the expression

of Fas in NIH 3T3 K-ras cells. Actin acts as a loading control. 10HDA,

(E)-10-hydroxy-2-decenoic acid.

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(CMV) immediate early promoter driving expression of GFP(supplementary Fig S1C online; Si et al, 2010). As observed in theear fibroblast cell line, 10HDA alone had no effect, but markedlypotentiated the ability of 5-Aza to activate GFP (supplementary FigS3D online). In the same cell line, both royal jelly and 10HDA canreactivate epigenetically silenced p21 expression (supplementaryFig S3A,B online). To test directly whether 10HDA can block DNAmethylation, we performed a pyrosequencing assay for LINE-1 (longinterspersed nuclear element) repeats, as a measure of globalhypomethylation in these cells. Although 5-Aza treatment reducesDNA methylation by 50% in this assay, 10HDA was not able toreduce the levels of LINE-1 DNA methylation alone, or to potentiatethe effect of 5-Aza in sequential combination treatment (Fig 2C).Importantly, royal-jelly treatment (0.4% and 4%, v/v) did not haveany effect on DNA methylation levels at LINE-1 elements either(supplementary Fig S3C online). We also evaluated the methylationlevel of the CMV promoter (by pyrosequencing) after 10HDAtreatment and observed the same result—10HDA does not reducethe DNA methylation level at this specific promoter (data notshown). Thus, 10HDA treatment of SW48 cells does not reducethe DNA methylation levels at LINE-1 repeat elements or at aspecific promoter that can be regulated by this compound. 10HDAis thus not a DNA methyltransferase inhibitor. It is possible that10HDA is not a direct regulator of epigenetic factors, but insteadinterferes with the extracellular signal-regulated kinase (ERK) orphosphoinositide 3-kinase (PI3K) signalling pathways, which linkK-ras to the epigenetic regulators (supplementary Fig S4A online).To test this possibility we treated cells with 10HDA, U0126

(an ERK inhibitor) and LY294002 (a PI3K inhibitor). 10HDA doesnot affect either of these signalling pathways (supplementaryFig S4B online), suggesting that it directly targets an epigeneticeffector molecule.

10HDA is an HDACiOn the basis of the fact that 10HDA (i) functions synergisticallywith DNA demethylating agents (Fig 2A,B), but does not inhibitDNA methylation itself (Fig 2C) and (ii) is structurally similar toknown HDACi compounds (Fig 3A), we investigated whether itwas an HDACi. First, NIH 3T3 K-ras cells were treated withsodium butyrate, 10HDA and a 5% royal jelly suspension for 3days. When core histones from these cells were analysed withpan-acetyl-lysine antibodies, acetylation levels were increased byall three treatments (Fig 3B). These data indicate that 10HDAblocks HDAC activity or promotes histone acetyltransferaseactivity. Next, we directly tested whether 10HDA and royal jellyinhibit HDAC activity using an in vitro assay. Both royal jelly and10HDA harbour HDACi activity (Fig 3C). Importantly, the royaljelly used in this experiment was composed of 2% 10HDA, whichat a 1% dilution translates into a 1 mM 10HDA solution. Thus,most of the HDACi activity in royal jelly can be attributed to10HDA. (E)-9-oxodec-2-enoic acid (90DA) is an analogue of10HDA that is produced by the queen bee (Plettner et al, 1996),and it also has similar HDACi activity (supplementary Fig S5online). This activity of 90DA acid might be important formaintaining the royal phenotype, if it is produced and consumedby the queen herself.

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Fig 2 | 10HDA can reactivate epigenetically silenced green fluorescent protein loci. (A) 10HDA (5 or 1 mM) can restore GFP-expression in pig-ear

fibroblasts when used in combination with 5-Aza (100 nM). (B) Quantification by western blot analysis shows a dose-dependent effect of 10HDA.

10HDA shows a stronger effect on GFP expression in pig-ear fibroblasts than sodium butyrate (SB), a known HDACi. Actin acts as a loading control.

(C) 10HDA does not alter the degree of DNA methylation at LINE-1 repeat elements. SW48 colon-cancer cells were treated with 10HDA or a

combination of 10HDA and 5-Aza (50 nM). 10HDA alone does not reduce the level of DNA methylation at LINE-1 repeat elements, as determined

by pyrosequencing. The experiment was performed twice, with three replicates each. 5-Aza, 5-Aza-2-deoxycytidine; GFP, green fluorescent protein;

HDACi, histone deacetylase inhibitor; LINE-1, long interspersed nucleotide elements; 10HDA, (E)-10-hydroxy-2-decenoic acid.

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Next, we revisited the K-ras-transformed NIH 3T3 cell reportersystem that we used initially (Fig 1) and compared the efficacy oftrichostatin A (TSA), sodium butyrate and 10HDA in reactivatingthe Fas gene over a 2-day period (Fig 3D). Sodium butyrate and10HDA behave similarly and show activity within 24 h, whereasTSA was able to reactivate the Fas gene within 12 h. Finally, weperformed individual inhibitor assays using recombinant HDAC1,3, 8, 10 and 11 (supplementary Fig S6 online). 10HDA inhibits allof these recombinant HDACs with and half-maximal inhibitoryconcentration (IC50), in the range of 5–8 mM.

DISCUSSIONRoyal jelly has been bestowed with mythical qualities because ofits long-recognized ability to induce the differentiation of beelarvae into queens. As the queen bee is fertile, longer-lived and

more substantial than her worker counterparts, royal jelly isconsumed in large quantities all over the world as a healthsupplement. The consequences of human consumption of royaljelly are unknown, although several health benefits have beenclaimed, which lack scientific evidence.

Royal jelly is a complex mixture produced in the hypo-pharyngeal and mandibular glands of nurse bees and used to feedthe queen throughout her life (Haydak, 1970). It containsapproximately 65% water, 12% crude protein and 10% mono-saccharides. The remainder of the royal jelly is composed of anether-soluble fraction of fatty acids. This fatty acid component iscomprised primarily of a single compound—10HDA (Barker et al,1959). 10HDA contributes 2–6% of the wet weight of royal jelly,depending on the source, making it the dominant small moleculein this secretion. If we start with the lower 10HDA (molecular

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Fig 3 | 10HDA is an HDAC inhibitor. (A) 10HDA, also referred to as royal jelly (RJ) acid, has a few structural similarities to the known HDACis,

sodium butyrate (SB) and SAHA (suberoylanilide hydroxamic acid). (B) NIH 3T3 K-ras cells were treated with SB (5 mM), 10HDA (5 mM) or a

5% RJ suspension for 72 h. Core histones were then isolated and western blot analysis was performed with a pan-acetyl-lysine antibody. Increased

histone acetylation levels were observed in the treated samples. (C) 10HDA and diluted RJ inhibit HDAC activity in vitro. The data were generated

using an HDAC colorimetric activity assay kit. Error bars represent s.d. calculated from three independent experiments. (D) Trichostatin A (TSA), SB

and 10HDA are able to reactivate the expression of Fas in NIH 3T3 K-ras cells. Fas expression at three different time points was assayed. Actin acts as

a loading control. HDAC, histone deacetylase; HDACi, histone deacetylase inhibitor; 10HDA, (E)-10-hydroxy-2-decenoic acid.

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weight¼ 186) concentration (2%), then this fatty acid is present ata level of 107 mM in royal jelly, which is thus the minimumconcentration that the larvae are exposed too. In tissue culture, wesee epigenetic regulatory effects of 10HDA at 0.5–1 mM;concentrations more than 100-fold lower than the developinglarvae are soaked in. Thus, although 10HDA is not a potentHDACi (IC50 approximately 5 mM), it is present at concentrationsthat will elicit in vivo effects.

10HDA has several pharmacological activities, includingantitumour activity (Townsend et al, 1959) and inhibitory effectson vascular endothelial growth factor-induced angiogenesis (Izutaet al, 2009). Interestingly, valproic acid (a known HDACi) is aninhibitor of angiogenesis (Hrebackova et al, 2010) and severalHDACis are used in cancer treatment (Beumer & Tawbi, 2010).Furthermore, neural stem-cell differentiation into neurons isstimulated by 10HDA (Hattori et al, 2007), a phenomenonthat is also observed using TSA (Balasubramaniyan et al, 2006),sodium butyrate (Yao et al, 2010) and valproic acid (Yu et al,2009). Thus, many of the reported pharmacological activities of10HDA might be due to its ability to inhibit HDACs.

There are four annotated histone deacetylases encoded in thehoney-bee (A. mellifera) genome, with orthologues in both class Iand class II. 10HDA inhibits class I and class II HDACs, asdetermined by using a HDAC colorimetric assay, specific in vitroassays using a panel of recombinant HDACs, and western blotanalysis with pan-acetyllysine antibodies. Most HDACis isfunction by chelating a crucial zinc atom in the active site ofthe enzyme (Finnin et al, 1999; Bertrand, 2010). Carboxylic acidsare a subset of these, and 10HDA can be classed within this group,which includes valproic acid and butyric acid (Bertrand, 2010).Thus, similarly to the other members of this group, 10HDA hasproven to be a broad-spectrum class I and class II inhibitor, whichcauses an increase in lysine acetylation levels and generates atranscriptionally permissive state.

Recent experiments in the Maleszka laboratory have beenpivotal in highlighting the importance of epigenetics in bee caste-switching (Kucharski et al, 2008). It is intriguing that no grosschanges in CpG methylation levels were observed when compar-ing DNA in the brains of queen and worker bees (Lyko et al,2010), which is consistent with the notion that royal jelly does notharbour activity that inhibits DNA methylation, but instead targetshistone-modifying enzymes, such as HDACs. Indeed, on its own,10HDA is sufficient to reactivate an epigenetically silenced locus,but does not inhibit DNA methylation. However, it is possible thatroyal jelly does harbour a compound that can selectively targetDNA methyltransferases at specific loci, which would functionsynergistically with 10HDA to promote the generation of thequeen bee. Alternatively, extended hyperacetylation might induceselective hypomethylation of DNA at specific loci—a phenom-enon that has been observed in mammalian cells (Wu et al, 2008).It is important to note that for the first 3 days of development, allbee larvae are fed royal jelly, and workers only get worker jelly onthe fourth day; thus, this induced hyperacetylated state might be ameans by which to reprogramme the epigenetic code in earlydevelopment.

METHODSAntibodies, chemicals and royal jelly. The NIH 3T3 K-ras cellsand the parental NIH 3T3 cells were obtained from the American

Type Culture Collection (ATCC; CRL-6361 and CRL-1658). Thepig-ear fibroblast cell line (GF2–4) was a gift from Dr Jeung-WhanHan (Kang et al, 2007). Royal jelly was obtained from GloryBeeFoods (Eugene, OR, USA; Cat. no. 12366). 5-aza-20-deoxycytidineand sodium butyrate were obtained from Sigma-Aldrich (St Louis,MO, USA). The following antibodies were used: Fas from MBL(Woburn, MA, USA; Cat. no. JM-3070-100), K-ras from Abcam(Cambridge, MA, USA; Cat. no. ab16795), actin from Sigma-Aldrich (Cat. no. A3853), histone H3 from Millipore (Billerica,MA, USA; Cat. no. 06-755) and pan-acetyllysine from CellSignalling (Danvers, MA, USA; Cat. no. 9441). 10HDA forpreliminary experiments was obtained from Matreya LLC (PleasantGap, PA, USA) and larger amounts of it, as well as of 90DA, wereprepared following a new, improved procedure (details for thesynthesis and characterization are provided as supplementaryinformation online). The fractionation of royal jelly was performedusing Amicon-Ultra spin columns (Millipore).Pyrosequencing. Pyrosequencing was performed as describedpreviously (Figueiredo et al, 2009).Histone extraction and HDACi assays. Histones are extractedusing the TCA-precipitation method (Butler et al, 1986). HDACactivity was determined using an HDAC colorimetric kit fromEnzo Lifesciences (Plymouth Meeting, PA, USA; Cat. no. ALX-850-294-KI01). This kit uses total HeLa cell nuclear extract as theenzyme source.Supplementary information is available at EMBO reports online(http://www.emboreports.org).

ACKNOWLEDGEMENTSM.T.B. is supported by an institutional grant from the National Instituteof Environmental Health Sciences (NIEHS ES007784). A.S. is supportedby a Fellowship from the German Research Foundation, SP 1262/1-1.G.S. is partially supported by the Ministero dell’Universita e della RicercaScientifica e Tecnologica—PRIN 2007 and by Universita di Salerno,Italy.

CONFLICT OF INTERESTThe authors declare that they have no conflict of interest.

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Social insect epigenetics and royal jelly

A. Spannhoff et al

EMBO reports &2011 EUROPEAN MOLECULAR BIOLOGY ORGANIZATION

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Supplemental Experimental Procedures Tissue culture, Western blot procedure and Fluorescent microscopy Mouse NIH3T3 cells and GF2-4 cells (pig ear fibroblasts) were cultured in DMEM with

10% newborn calf serum and 100 µg/ml streptomycin-penicillin. SW48 cells, stably

transfected with patch-methylated GFP, were cultured in IL-15 medium with 10% fetal

bovine serum and 100 µg/ml streptomycin-penicillin. Cells were lysed in mild lysis buffer

(150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and 10 mM Tris/HCL [pH 7.5]). Lysates

were separated by SDS-PAGE, and transferred onto PVDF membranes for Western

analysis, to track the induction of Fas and GFP expression. The induction of GFP

expression in living cells was followed by fluorescent microscopy, using a Olympus IX50

inverted microscope.

Individualized HDAC inhibition assays All five full-length recombinant human HDACs (rhHDACs) were expressed as His6-

tagged and GST-fusion proteins in insect High5 cells using a baculoviral expression

system, and were purified using Ni-NTA (QIAGEN). The deacetylase activity of rhHDAC

was assayed according to previous reports with minor optimization (Wegener et al,

2003; Riester et al, 2007). Briefly, HDAC 1, 3, 8, 10 and 11 were assayed with a

HDAC substrate (Ac-Lys-Tyr-Lys(ε-acetyl)-AMC). The total HDAC assay volume

was 25 µl and all assay components were diluted in Hepes buffer (25 mM Hepes, 137

mM NaCl, 2.7 mM KCl and 4.9 mM MgCl2, pH 8.0). The HDAC assay mixture contained

HDAC substrate (5-50 µM, 5 µl), HDAC protein (20-200 nM) and 10HDA or reference

inhibitor SAHA (1 µl) and the reaction was carried out in black 384-well plates

(OptiPlateTM-384F, PerkinElmer). The assays for HDAC 8, 10 and 11 were carried out at

room temperature for 3 h, and those for HDAC 1 and 3 were incubated for 24 h. The

reaction was quenched with the addition of 25 µl Trypsin (diluted to final

concentration 0.3125%). The plates were incubated for 30 min at room temperature to

allow the fluorescence signal to develop and then the fluorescence generated was monitored at

wavelengths 355 nm (excitation) and 460 nm (emission) using Envision (PerkinElmer).

Chemical synthesis of 10HDA and 9ODA

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(E)-10-hydroxy-2-decenoic acid (10HDA, 1, Figure S7A) was prepared starting from

commercially available 1,8-octanediol (3), which was monoacetylated with acetic

anhydride in dichloromethane and then oxidized with Dess-Martin periodinane to the

aldehyde (5). Doebner condensation of malonic acid with the latter, followed by mild

alkaline hydrolysis yielded 10HDA. On the other hand, (E)-9-oxo-2-decenoic acid

(9ODA, 2, Figure S7B) was obtained starting from the commercially available 1,5-

pentanediol (7), which was monobrominated with hydrobromic acid and toluene.

Knoevenagel condensation of the resulting bromide (8) with ethyl acetoacetate in the

presence of sodium and ethanol, followed by decarboxylation gave the ω-

hydroxyketone (9), which was then oxidized with Dess-Martin periodinane to the

corresponding ketoaldehyde (10). Doebner condensation of the latter with malonic acid

finally furnished 9ODA.

All chemicals were purchased from Sigma-Aldrich (Milan, Italy) or from Alfa

Aesar GmbH (Karlsruhe, Germany) and were of the highest purity. All solvents were

reagent grade and, when necessary, were purified and dried by standard methods. All

reactions requiring anhydrous conditions were conducted under a positive atmosphere

of nitrogen in oven-dried glassware. Standard syringe techniques were used for

anhydrous addition of liquids. Reactions were routinely monitored by TLC performed on

aluminum-backed silica gel plates (Merck DC, Alufolien Kieselgel 60 F254) with spots

visualized by UV light (λ = 254, 365 nm) or using a KMnO4 alkaline solution.

Concentration of solutions after reactions and extractions involved the use of a rotary

evaporator operating at a reduced pressure of ~10 Torr. Organic solutions were dried

over anhydrous sodium sulfate. Chromatographic separations were performed on silica

gel (Silica gel 60, 0.063-0.200 mm; Merck DC). Melting points were determined on a

Gallenkamp melting point apparatus in open capillary tubes and are uncorrected.

Infrared (IR) spectra (KBr) were recorded on a Shimadzu FTIR-8000 instrument. 1H

NMR spectra were recorded at 300 MHz on a Bruker Avance 300 spectrometer;

chemical shifts are reported in δ (ppm) units relative to the internal reference

tetramethylsilane (Me4Si). Mass spectra were recorded on a Finnigan LCQ DECA

TermoQuest (San Jose, CA, USA) mass spectrometer using an electrospray ion source

(ESI-MS). Combustion analysis on target compounds were performed by our Analytical

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Laboratory at the University of Salerno. All compounds showed ≥98% purity. For all

compounds, analytical data were in excellent agreement with those previously reported

(Ishmuratov et al, 2008; Ishmuratov et al, 2002; Sisido et al, 1962). As a rule, samples

prepared for physical and biological studies were dried in high vacuum over P2O5 for 20

h at temperatures ranging from 25 to 110°C, depending on the sample melting point.

Preparation of 8-hydroxyoctyl acetate (4). To a stirred solution of 1,8-octanediol (240

mmol) in dichloromethane (175 mL), acetic anhydride was added portionwise (0.25

equiv every 2 days, 240 mmol total). The mixture was kept stirring at room temperature

for a total of 7 days. The solution was then washed with 5% aqueous Na2CO3 (3 × 100

mL) and brine (3 × 80 mL). The organic phase was dried over anhydrous sodium sulfate

and evaporated. The crude residue was purified by column chromatography on silica

gel (gradient, DCM/AcOEt, 9:1 to 8:2) to afford pure 4 as a pale yellow oil (Yield: 40%).

Preparation of 8-oxooctyl acetate (5). A solution of 4 (1.9 mmol) in anhydrous

dichloromethane (6 mL) was added to a stirred suspension of Dess-Martin periodinane

(2.0 mmol) in dichloromethane (10 mL). The resulting mixture was stirred at room

temperature for 3 hours. The slurry was then quenched with saturated NaHCO3 (30 mL)

and Na2S2O3 (6.00 g) and stirred for an additional 15 min. The mixture was filtered on

paper and, after separation, the aqueous phase was extracted with DCM (3 × 20 mL).

The combined organic phases were washed with saturated NaHCO3 (15 mL) and brine

(15 mL), then dried over anhydrous sodium sulfate and evaporated to give pure 5 as a

colorless oil (Yield: 100%).

Preparation of (E)-10-acetoxydec-2-enoic acid (6). To a round-bottom flask charged

with the aldehyde 5 (5.66 mmol) and cooled in an ice bath, pyridine (5.87 mmol) and

malonic acid (portionwise, 0.25 equiv every 2 days, 5.66 mmol total) were added. The

resulting mixture was stirred at room temperature for 7 days. Then, pyridine was

neutralized with HCl 3 N and the mixture was extracted with AcOEt (3 × 10 mL). The

combined organic phases were washed with brine (3 × 10 mL), then dried over

anhydrous sodium sulfate and evaporated. The crude residue was purified by flash

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column chromatography on silica gel (DCM/AcOEt, 8:2) to afford pure 6 as a pale

yellow oil (Yield: 59%).

Preparation of (E)-10-hydroxydec-2-enoic acid (1). To a solution of acid 6 (0.38

mmol) in methanol (2 mL), K2CO3 (0.42 mmol) was added, and the resulting mixture

was stirred at room temperature for 12 hours. Then the solvent was evaporated under

reduced pressure and the residue taken up with water (5 ml) and acidified to pH≈ 1 with

HCl 1 N. The aqueous phase was extracted with AcOEt (3 × 5 mL). The combined

organic phases were washed with brine (3 × 10 mL), then dried over anhydrous sodium

sulfate and evaporated to give pure 10HDA as a white solid in quantitative yield.

Preparation of 5-bromopentan-1-ol (8). To a stirred mixture of 1,5-pentanediol (57.60

mmol) in toluene (170 mL), hydrobromic acid 9 M (7.6 mL, 69.13 mmol) was added and

the resulting mixture was refluxed for 9 hours. After cooling to room temperature and

dilution with diethyl ether, the two phases were separated and the organic one was

washed with NaOH 1 N, brine and phosphate buffer (pH 7). The solution was dried over

anhydrous sodium sulfate and evaporated to give pure 8 as a colorless oil. (Yield: 81%).

Preparation of 8-hydroxyoctan-2-one (9). Ethyl acetoacetate (0.726 mL, 5.98 mmol)

was added to a stirred solution of Na (137.5 mg, 5.98 mmol) in anhydrous ethanol (3.15

mL) under a N2 atmosphere flask. The resulting mixture was refluxed for 1 h, then 5-

bromopentan-1-ol 8 (5.98 mmol) was added and heating maintained for an additional 3

hours. The solvent was then removed under reduced pressure and the crude residue

was taken up with NaOH 1 M (15 mL), and the mixture was stirred up to complete

dissolution. Then H2SO4 0.2 M (45 mL) was added, and the resulting mixture was stirred

at 50°C for 30 min. After cooling to RT it was extracted with AcOEt (3 × 25 mL). The

combined organic phases were washed with brine (3 × 15 mL), then dried over

anhydrous sodium sulfate and evaporated to give pure 9 as a white solid (Yield: 83%).

Preparation of 7-oxooctanal (10). A solution of 9 (2.9 mmol) in anhydrous

dichloromethane (6 mL) was added to a stirred suspension of Dess-Martin periodinane

(3.1 mmol) in dichloromethane (10 mL). The resulting mixture was stirred at room

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temperature for 3 hours. The slurry was then quenched with saturated NaHCO3 (30 mL)

and Na2S2O3 (10.00 g) and stirred for an additional 15 min. The mixture was filtered on

paper and, after separation, the aqueous phase was extracted with DCM (3 × 20 mL).

The combined organic phases were washed with saturated NaHCO3 (15 mL) and brine

(15 mL), then dried over anhydrous sodium sulfate and evaporated to give pure 5 as a

yellow oil in quantitative yield.

Preparation of (E)-9-oxodec-2-enoic acid (2). To a round-bottom flask charged with

the aldehyde 10 (2.46 mmol) and cooled in an ice bath, pyridine (2.55 mmol) and

malonic acid (portionwise, 0.25 equiv every 2 days, 2.46 mmol total) were added. The

resulting mixture was stirred at room temperature for 7 days. Then, pyridine was

neutralized with HCl 3 N and the mixture was extracted with AcOEt (3 × 10 mL). The

combined organic phases were washed with brine (3 × 10 mL), then dried over

anhydrous sodium sulfate and evaporated. The crude residue was purified by flash

column chromatography on silica gel (petroleum ether/AcOEt, 1:1) to afford pure 9ODA

as a pale yellow oil (Yield: 53 %).

References Ishmuratov G, Yakovleva M, Tambovtsev K, Legostaeva Y, Kravchenko L, Ishmuratova N, Tolstikov G (2008). Two approaches to the synthesis of 9-oxo-and 10-hydroxy-2 &lt;i&gt;E&lt;/i&gt; -decenoic acids, important components of queen substance and royal jelly of honeybees &lt;i&gt;Apis mellifera&lt;/i&gt. Chemistry of Natural Compounds 44(1): 74-76 Ishmuratov GY, Kharisov RY, Botsman OV, Ishmuratova NM, Tolstikov GA (2002). Synthesis of 9-Oxo- and 10-Hydroxy-2E-decenoic Acids. Chemistry of Natural Compounds 38(1): 1-23 Riester D, Hildmann C, Grunewald S, Beckers T, Schwienhorst A (2007). Factors affecting the substrate specificity of histone deacetylases. Biochemical and Biophysical Research Communications 357(2): 439-445 Sisido K, Kawanisi M, Kondô K, Morimoto T, Saitô A, Hukue N (1962). Syntheses of 9-Keto- and 10-Hydroxy-trans-2-decenoic Acids and Related Compounds. The Journal of Organic Chemistry 27(11): 4073-4076. Wegener D, Wirsching F, Riester D, Schwienhorst A (2003). A Fluorogenic Histone Deacetylase Assay Well Suited for High-Throughput Activity Screening. Chemistry & Biology 10(1): 61–68

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Supplemental Figures Figure S1. The three different epigenetic reporter systems used in this study. A) K-ras

transfected NIH 3T3 cells harbor an epigenetically silenced Fas locus that can be

reactivated with treatment of 5-Aza, HDACi or PMTi (protein methyltransferase

inhibitors - like BIX). B) Generation of pig ear fibroblasts with epigenetically silenced

GFP expression. In this system, GFP expression can be restored with treatment of 5-

Aza, HDACi or PMTi. C) CMV-GFP patch methylated SW48 cells have an epigenetically

silenced GFP locus that can be reactivated with small molecule inhibitors of epigenetic

regulators.

Figure S2. Control panel for Figure 2. This bright field shows that an equal number of

cells are observed in each well.

Figure S3. 10HDA and royal jelly reactivate p21(WAF1) expression without reducing

DNA methylation levels. A) RT-qPCR was performed on RNA isolated from SW48 colon

cancer cells treated with 5-Aza (50 nM), 1% royal jelly and a combination of the two.

The primers for p21 were obtained from ABI (Hs01121168_m1). B) RT-qPCR was

performed on RNA isolated from SW48 colon cancer cells treated with 10HDA (1 mM)

and SAHA (20 µM), on their own or with 5-Aza (50 nM). C) SW48 colon cancer cells

were treated with royal jelly (0.4% & 4%) alone or in combination with 5-Aza (50 nM).

On its own, RJ does not reduce the level of DNA methylation at LINE-1 repeat

elements, as determined by pyrosequencing. The experiment in panels A, B & C were

performed twice, with three replicates each. D) SW48 colon cancer cells, with a

hypermethylated CMV promoter driving the expression of GFP, were treated with

10HDA (10 mM) alone or in combination with 5-Aza (50 nM). GFP-positive cell

percentages were measured using a BD FACSCalibur flow cytometer. 10,000 cells

were analyzed for each condition.

Figure S4. 10HDA does not inhibit the MAPK or PI3K pathways. A) The signal

transduction pathway down-stream of K-ras is depicted. In green, some of the shRNA

hits from the Gazin et al. study are highlighted. The knockdown of these green-boxed

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proteins results in the re-expression of Fas. B) It is of course possible that 10HDA is not

a direct regulator of epigenetic factors, but instead interferes with the ERK or PI3K

signaling pathways. After pretreatment of quiescent NIH 3T3 cells with 10HDA (10 mM),

U0126 (20 µM) and LY294002 (20 µM) for 1 h, the cells were stimulated with 10% FBS

for 30 min. Western analysis was performed on total cell lysates from these cells using

phospho-specific ERK and Akt antibodies. 10HDA treatment does not impact these

pathways.

Figure S5. A) 10HDA from different sources (commercially available from Matreya

LLC® [c] and synthesized by us [s]) shows similar effects on pig ear fibroblasts. The

queen bee pheromone (E)-9-oxodec-2-enoic acid (9-ODA), also synthesized by

Gianluca Sbardella [s], has the ability to restore GFP-expression when used under the

same conditions as 10HDA (5 and 1 mM). B) Quantitation via Western blot analysis

shows a dose-dependent effect of these compounds (5 and 1 mM). Both commercially

available and synthesized 10HDA show comparable effects. Actin serves as a loading

control. C) Structural similarity between 10-HDA and 9-ODA. D) Ability of SB and 9-

ODA to inhibit HDAC activity in-vitro. The data was generated using a HDAC

colorimetric activity assay kit. Error bars represent standard deviation calculated from

three independent experiments.

Figure S6. Screening for the ability of individual recombinant HDACs to be inhibited by

10HDA. Recombinant human HDAC1, 3, 8, 10 & 11 were expressed in insect cells. Ac-

Lys-Tyr-Lys(ε-acetyl)-AMC was used as a substrate in the presence of varying

concentrations of SAHA and 10HDA. The IC50s are given in the table. The

experiment was performed in triplicate to obtain error bars.

Figure S7. A) Synthesis scheme for 10HDA. Reagents and conditions: a) acetic

anhydride, DCM, rt; b) Dess-Martin periodinane, DCM, rt; c) malonic acid, pyridine, rt; d)

K2CO3, MeOH, rt, then HCl 1 M. B) Synthesis scheme for 9ODA. Reagents and

conditions: a) HBr, toluene, reflux; b) ethyl acetoacetate, Na, EtOH, reflux; c) NaOH 1

M, rt, then H2SO4 0.2 M, 50 °C; d) Dess-Martin periodinane, DCM, rt; e) malonic acid,

pyridine, rt.

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Figure S1

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Figure S2

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Figure S3

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Figure S4

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Figure S5

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Figure S6

The IC50 of SAHA and 10HDA on a panel of HDACs

HDAC1 HDAC3 HDAC8 HDAC10 HDAC11 SAHA (nM) 172 229 99 170 232

10HDA (mM) 6.4 6.5 6.6 8.2 5.8

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Figure S7 A

B

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Author Contributions: Mark T. Bedford – Conceived the experiments, organized the collaborations, and wrote the manuscript. Astrid Spannhoff – Performed the experiments depicted in Figures 1 & 3, S5D.

Yong Kee Kim – Performed the experiments depicted in Figures 2A & B, S2, S4B, S5A & B. Noel J-M. Raynal, Vazganush Gharibyan and Jean-Pierre J. Issa – Performed the experiments depicted in Figures 2C & S3. Ming-Bo Su, Yue-Yang Zhou and Jia Li – Performed the experiments depicted in Figures S6. Sabrina Castellano and Gianluca Sbardella – Synthesized 10HDA and 9ODA.