1

Supplemetary Materials

Materials and Methods

Stable knockdown of HDACs in murine embryonic fibroblasts

For establishing a transient knockdown of HDAC1, HDAC2 and HDAC3, immortalized

Hdac3 fl/- Mef cells were transfected with mouse pLKO.1-puro, or pLKO.1 encoding

shRNAs derived from the TRC1 library (http://www.broadinstitute.org/rnai/trc/lib):

pLKO.1-puro-shHdac1 (cloneIDs TRCN0000039399, TRCN0000039400,

TRCN0000039402, TRCN0000039403), pLKO.1-puro-shHdac2 (cloneIDs

TRCN0000039395, TRCN0000039396, TRCN0000039397, TRCN0000039398),

pLKO.1-puro-shHdac3 (cloneIDs TRCN0000039389, TRCN0000039390,

TRCN0000039391, TRCN0000039392). For transient transfection 3.5x104 Mef cells

were seeded in 48 well plates. 270ng plasmid DNA per well was transfected using

Lipofectamine® LTX and PlusTM reagent from InvitrogenTM, following the

manufacturer’s instructions. 24h post transfection, transfected cells were selected for 48h

by adding 1µg/ml puromycin.

Each shRNA-encoding vector was transfected in duplicate wells. One well was kept

untreated, while the second well was stimulated with IL-1α (10ng/ml) for 3h. After

harvesting the cells on ice in 1x PBS, the samples were prepared for RT-qPCR analysis

using the TaqMan® PreAmp Cells-to-CT™ Kit from Ambion®, following the

manufacture’s instructions. The expression of mCxcl2 (Mm00436450_m1) and mUbe2l3

(Mm00784559_s1) was determined by qPCR using the TaqMan® Fast universal PCR

master mix and 7500 Fast real time PCR system from Applied Biosystems. Relative

changes of mCxcl2 mRNA expression compared to the unstimulated pLKO.1 control

were normalized to the expression of mUbe2l3 and quantified using the 2-ΔΔCt method.

Chromatin immunopecipitation (ChIP)

One 175-cm2 flask of confluent KB cells (corresponding to 2.5 - 3.0 x107 cells), treated

as described in the figure legends, was used for each condition. Proteins bound to DNA

2

were cross-linked in vivo with 1% formaldehyde added directly to the medium. After 10

minutes incubation at room temperature, 0.1 M glycine was added for 5 minutes to stop

the cross-linking. Then, cells were collected by scraping and centrifugation at 1,610 x g

(5 minutes, 4°C), washed in cold PBS containing 1mM PMSF and centrifuged again at

1,610 x g (5 minutes, 4°C). Cells were lysed for 10 minutes on ice in 3ml ChIP lysis

buffer (1% SDS, 10mM EDTA, 50mM Tris pH 8.1, 1mM PMSF, Roche protease

inhibitor mix). The DNA was sheared by sonication (7 x 30s on / 30s off, 4 times;

Bioruptor, Diagenode) and lysates cleared by centrifugation at 16,100 x g at 4°C for 15

minutes. Supernatants were collected and stored in aliquots at –80°C for subsequent

ChIP. For determination of DNA concentration 20µl of sheared lysate was diluted with

100µl TE buffer including 10µg/ml RNAse A. After 30min at 37°C, 3.8µl proteinase K

(20mg/ml) and 1% SDS was added and incubated for at least 2h at 37°C followed by

overnight incubation at 65°C. Samples were resuspended in two volumes of buffer NTB

(Macherey & Nagel) and DNA was purified using Nucleo Spin columns (Macherey &

Nagel) according to the manufacturer’s instructions. DNA was eluted with 50µl 5mM

Tris pH 8.5 and concentration was determined by Nano Drop. For CHIP, the following

antibodies were used: anti-histone H3 (2µg, Abcam; ab1791), anti-acetyl-histone H3

(K9) (2µg, Millipore; 07-352), anti-NF-κB p65 (3µg, Santa Cruz; sc-372), anti-HDAC3

(4µg, Millipore; 17-10238), anti-phospho-Pol II (S5) (1.35µg, Abcam; ab5131), IgG

(2µg, Cell Signaling; 2729). Antibodies were added to precleared lysate volumes

equivalent to 25µg of chromatin. Then, 900µl of ChIP dilution buffer (0.01% SDS, 1.1%

Triton X-100, 1.2mM EDTA, 167mM NaCl, 16.7mM Tris/HCl pH 8.1) were added and

the samples were rotated at 4°C overnight. Thereafter, 30µl of a protein A/G sepharose

mixture, pre-equilibrated in ChIP dilution buffer was added to the lysates and incubation

continued for 2h at 4°C. Beads were collected by centrifugation, washed once in 900µl

ChIP low salt buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris pH 8.1,

150mM NaCl), once in 900µl ChIP high salt buffer (0.1% SDS, 1% Triton X-100, 2mM

EDTA, 20mM Tris pH 8.1, 500mM NaCl), once in 900µl ChIP LiCl buffer (0.25M LiCl,

1% NP40, 1% desoxycholate, 1mM EDTA, 10mM Tris pH 8.1) and twice in 900µl ChIP

TE buffer (10mM Tris pH 8.1, 1mM EDTA) for 5 minutes at 4°C. Beads were finally

resuspended in 100µl TE buffer including RNAse A (10mg/ml). In parallel, 1/10 volume

3

(2.5µg) of the initial lysate (input samples) were diluted with 100µl TE buffer including

10µg/ml RNAse A. After 30min at 37°C, 3.8µl proteinase K (20mg/ml) and 1% SDS

were added and both, input and immunoprecipitates were incubated for at least 2h at

37°C followed by overnight incubation at 65°C. Samples were resuspended in two

volumes of buffer NTB (Macherey & Nagel) and DNA purified using Nucleo Spin

columns (Macherey & Nagel) according to the manufacturer’s instructions. DNA was

eluted with 50µl 5mM Tris pH 8.5 and stored at –20°C until further use.

Quantification of ChIP DNA by real-time PCR

PCR products derived from ChIP were quantitated by real time PCR using the Fast ABI

7500 instrument (Applied Biosystems). The following primers were used as described in

(37). Hhuman IL-8 promoter, sense 5’-aagaaaactttcgtcatactccg-3’, antisense 5’-

tggctttttatatcatcaccctac-3’; human IL-8 upstream (negative control; gene free region

940bp upstream of IL-8 transcriptional start site): sense 5’-atcatgggtcctcagaggtcagac-3’,

and antisense, 5’-ggtgggagggaggtgttatctaatg -3’. The reaction mixture contained 2µl of

ChIP or input DNA (diluted 1:10 to represent 1% of input DNA), 0.25µM of primers and

10µl of Fast Sybr Green Mastermix (2x) (Applied Biosystems) in a total volume of 20µl.

PCR cycles were as follows: 95°C (20s), 40x (95°C (3sec), 60°C (30sec)). Melting curve

analysis revealed a single PCR product.. Calculation of enrichment by

immunoprecipitation relative to the signals obtained for 1% input DNA was performed

according to the following equation: percent of (input)=2-(Ct sample-Ct input).

4

Supplementary Figures

Fig. S1. Apicidin inhibits IL-1-induced IL-8 expression in tumor cells.

KB cells (upper graph) or A549 cells (lower graph) were pretreated for 24 h with

increasing concentrations of the HDAC3 inhibitor apicidin, solvent control (DMSO, (1

%), or were left untreated. Thereafter cells were stimulated for 3 h with IL-1 as shown.

RT-qPCR was used to determine IL-8 mRNA expression, bars show the mean IL-8

expression ±SEM relative to the unstimulated control (KB cells n=2, A549 cells n=3).

Fig. S2. Tamoxifen has no impact on IL-1-inducible Cxcl2 expression.

Two immortalized Mef lines were treated for 72h with 10µM tamoxifen followed by 30

min of IL-1 treatment as shown or were left untreated as indicated. Total RNA was

isolated and Cxcl2 mRNA expression was quantified by RT-qPCR.

Fig. S3. shRNA-mediated suppression of HDAC3 in murine fibroblasts impairs IL-

1-induced Cxcl2 expression.

Hdac3 fl/- Mefs were transfected with empty pLKO.1 or with pLKO.1 containing the

indicated shRNA-expression cassettes directed against HDAC3. Cells were selected for

two days in puromycin (1µg/ml), stimulated for 3 h with IL-1 or were left untreated.

Thereafter mRNA expression of Cxcl2 was analyzed by RT-qPCR and normalized to the

expression of Ube2l3 as described in the Methods section. Shown are mean fold changes

+/- SEM relative to the vector control from duplicate determinations.

Fig. S4. TSA triggers histone H3 acetylation.

The indicated HEK293IL-1R control and HDAC3 knockdown cells were treated with

TSA (300ng/ml, 24 h) or IL-1 as shown. Nuclear extracts were analyzed by Western

blotting for TSA-mediated hyperacetylation of histones by use of antibodies against

acetylated histone H3 (Ac-(K9/14)-H3). Antibodies against beta actin were used as a

loading control, a representative experiment is shown.

5

Fig. S5. HDAC3-knockdown in human cells suppresses expression of several IL-1-

response genes.

The indicated HEK293IL-1R control and HDAC3 knockdown cells were stimulated for

various periods with IL-1 as indicated. RT-qPCR was used to quantify mRNA expression

of the indicated human genes. Changes of mRNA expression are displayed relative to the

untreated control. Error bars represent SEM from two independent experiments.

Fig. S6. Microarray-based identification of HDAC3-dependent IL-1-response genes

in murine cells.

Hdac3 fl/+ or Hdac3 fl/- Mefs were treated with tamoxifen- or IL-1 as described in the

legend for Fig. 2C. RNA was isolated and labeled cRNAs were hybridized to whole

murine genome Agilent microarrays. Fluorescence intensity values were used to calculate

ratios of gene expression. (A) Depicted are color-coded ratio values for 95 genes which

were regulated by IL-1 by at least 1.5-fold in the same direction in both independent

experiments. Green vertical bars indicate all genes with at least twofold regulation by IL-

1. Yellow bars indicate genes whose expression was reduced in the HDAC3 knockout by

a least twofold in two independent experiments. (B) Depicted are color-coded ratios

values for 490 genes which were regulated by at least twofold in response to tamoxifen.

In this group, only 7 genes (1.4%) were also responsive to IL-1 as indicated by red bars.

Fig. S7. Identification of p65 K4Q or p65 K5Q-suppressed or -induced genes.

The entire data set which was retrieved by sequential filtering of the expression values of

the microarray experiments as described in the legend of Fig. 9C and D is shown.

Depicted are color-coded ratio values for IL-1-regulated (green vertical bars) and p65

NF-κB-dependent genes (A) and for genes regulated by p65 K4Q or by K5Q mutants (B).

Numbers 1-24 indicate experimental conditions as described in the legend of Fig. 9C, D.

For further details see the Material & Methods section.

Fig. S8. DNA-binding of p65 is not required for HDAC3-mediated deacetylation.

HEK293T cells were transfected with expression plasmids encoding His-tagged p65 wild

type or a DNA-binding mutant (p65 E39I-His) along with YFP-CBP and increasing

6

concentrations of an expression vector for HDAC3-Flag (0.3µg and 1µg) as shown. One

fraction of cells was purified under denaturing conditions by Ni-NTA columns and

analyzed for p65 acetylation as displayed, another fraction was analyzed for adequate

protein expression.

Fig. S9. p65 NF-κB is deacetylated by HDAC1 or HDAC2 but not by HDAC5 and

HDAC8.

HEK293IL-1R cells were transiently transfected to express wild type His-tagged p65

along with untagged p300, HA-tagged HDAC3, FLAG-tagged HDAC2, 5, 8 or MYC-

tagged HDAC1. Cells were lyzed under denaturing conditions, proteins were precipitated

by TCA, redissolved and equal amounts were analyzed for general acetylation of p65

with a pan-acetyl-lysine specific antibody or with antibodies specific for acetylated p65

K310, K314 and K315 as shown. Expression of p65, HDACs and p300 was validated as

indicated and equal loading of lanes was confirmed by anti-β-actin antibodies. The

position of a molecular weight marker is indicated at the left.

Fig. S10. shRNA-mediated suppression of HDAC2, or HDAC3 in murine fibroblasts

impairs IL-1-induced Cxcl2 expression.

Hdac3 fl/- Mefs were transfected with empty pLKO.1 or with pLKO.1 containing the

indicated shRNA-expression cassettes directed against HDAC1 (A), or HDAC2 (B).

Cells were selected for two days in puromycin (1µg/ml), stimulated for 3 h with IL-1

(10ng/ml) or were left untreated. Thereafter mRNA expression of Cxcl2 was analyzed by

RT-qPCR and normalized to the expression of Ube2l3 as described in the Methods

section. Shown are mean fold changes +/- SEM relative to the vector control from

duplicate determinations.

Fig. S11: HDAC3 deletion does not affect TNF-induced genes.

(A) Wild type (Hdac3 fl/+) or knockout (Hdac3 fl/-) Mefs were treated and analyzed

exactly as described in the legend of Fig.2B except that cells were stimulated for 30 min

with TNF (20ng/ml) instead of IL-1. RT-qPCR was used to quantify Cxcl2 mRNA

expression. Shown are the mean –fold changes compared to the untreated Hdac3 fl/+

7

control line, error bars represent SEM from three different experiments performed in

duplicates. (B) Hdac3 fl/+ or Hdac3 fl/- Mefs were stimulated as described in (A) followed

by RNA isolation and analysis of gene expression on whole murine genome Agilent

microarrays. Fluorescence intensity values were used to calculate ratios of gene

expression. Depicted are color-coded ratio values for 76 genes which were regulated by

TNF by at least 1.5-fold in the same direction in both independent experiments. Purple

vertical bars indicate all genes with at least twofold regulation by TNF. Blue bars indicate

genes whose expression was reduced in the HDAC3 knockout by a least twofold in two

independent experiments. Green bars indicate the overlap with IL-1-induced genes as

shown in suppl. Fig.6B.

8

Supplementary Tables

Table S1. Overlap between HDAC3-dependent IL-1 response genes and p65-

dependent genes.

The data set shown in Fig. S6 was used to extract 40 genes which are IL-1-dependent and

whose expression is reduced by twofold in at least one of the two experiments. Using

data described in Fig.9 and suppl. Fig. 7 the dependency of these genes on p65 NF-κB or

its mutants is depicted.

Table S2. HDAC3 is a global regulator of the transcriptional IL-1 response in

human cells.

Shown are gene identifiers, fluorescence intensity values and relative ratios of gene

expression corresponding to the results depicted in Fig. 7B. Twofold regulated genes are

indicated by yellow or green colors, respectively.

Table S3. Microarray-based identification of HDAC3-dependent IL-1-response

genes in murine cells..

Shown are gene identifiers, fluorescence intensity values and relative ratios of gene

expression corresponding to the results depicted in Fig. S6A. Twofold regulated genes

are indicated by yellow or green colors, respectively.

IL-8

mR

NA

(-f

old

)

0

20

40

60

80

100A549

IL-8

mR

NA

(-f

old

)

0200400600800

100012001400 KB

+

.1

+++

+

110

+ +IL-1

apicidin (µg/ml, 24h) .1 1 10

DMSO

Figure S1

Figure S2

MEF MK2/5 wt mCxCL-2

Cx

cl2

mR

NA

(-f

old

)

0

10

20

30

40-IL-1

MEF NCor-DAD wt mCxCL2

Cx

cl2

mR

NA

(-f

old

)

0

1000

2000

3000-IL-1

- tamoxifen

Mef line 2Mef line 1

- tamoxifen

Cx

cl2

mR

NA

(-f

old

)

0

20

40

60

80- IL-1

pL

KO

.1

shH

DA

C3

(#1)

shH

DA

C3

(#3)

shH

DA

C3

(#4)

shH

DA

C3

(#2)

Figure S3

++++3h IL-1

++++TSA++++3h IL-1

++++TSA

Ac-(K9/14)-H3-actin

pS-Puro pS-HDAC3

IB: anti-(Ac)K9/14-H3IB: anti--actin

Figure S4

0

25

50

75

100

125

CX

CL

2

Figure S5

0

50

100

150

200

250

CX

CL

1

0

25

50

75

CX

CL

3

0

5

10

15

20

25N

FK

BIA

pS-HDAC3pS-Puro

mR

NA

(-f

old

)

- 0.5 1 3 8 16 24 IL-1(h) - 0.5 1 3 8 16 24 IL-1(h)

Cxcl2Cxcl1Ccl20Cxcl10Ccl7Ccl2Zfp36Irf1NfkbizTnfRnd1Tnfaip3Ch25hCxcl5Birc3Serpine1Il6Ptgs21810011O10RikMap3k8Ier3Mapk6B2mTlr2Gadd45bIer2Btg2Bcl10FosJunbKlf10Zc3h12a5830469G19RikAtf3Phlda1Rgs16Slc25a25Csrnp1UncxSdk2Edn1Gdf15Nav3Tnk1Olfr1222Tnip3Egr2NfkbiaIgf1Pde4bAmmecr1l2510017J16RikDusp1TslpNfkbidGdap56330407A03RikDusp8ItgavMagixRbbp8Pim1Vcam1Ccrl2JunSlc43a28030431J09RikIcam1Neurl3Dusp28430408G22RikElf3Olfr661Olfr391-psPax7Ntsr1Olfr971Ttyh1SrrAnks61700012D14RikCcl28GalcSatb1Wdr38Tcstv19530013L04RikMxd4Prickle2Olfr9752310035P21Rik2610001A08RikPitx2Hk3Msh2

A

Figure S6

1 2 3 4 5 6 7 8

log2 ratio

-5 0 5

29 IL-1-regulated genes suppressed by 2-fold in Hdac3 -/- cells in exp. 1

31 IL-1-regulated genes suppressed by 2-fold in Hdac3 -/- cells in exp. 2

35 IL-1-regulated genes by 2-fold in exp. 1 and exp. 2

tamoxifen + + + +

IL-1 + + + +

Hdac3 fl/-

Cre-ER + + + + + + + + +

exp. 1 1 1 1 2 2 2 2

B

Figure S6

tamoxifen + + + +

IL-1 + + + +

Hdac3 fl/-

Cre-ER + + + + + + + + +

exp. 1 2 1 2 1 2 1 2

1 2 3 4 5 6 7 8

log2 ratio

-5 0 5

IL-1-regulated genes:

Neurl3Il6NfkbiaCxcl1Elf3Ptgs2Icam1

Cxcl1BC017612Pkm2Aldoart2Slc5a3NrcamAngptl4Cdhr1Fam5cAnkrd37Lgi2AplnGm129Prss2Pcdh9Klhl1NpyPrl2c5Prl2c3Ly6aLy6fPodnl1Casp4VwfItgb2Mmp10Gsta3Ampd34930452B06RikHmgcrFdft1PmvkLy6iSlc6a12AI504432EregSox17Fgf21Tnfsf9Prdm8Il18rapPtprbLonrf3Sgsm1Mt2Hsd17b7Ccnd1FdpsPrl7a2Cyp51Lrrc27Mmp12Fgl2Hmgcs1Eno3CstaKif1aCcbe1Pbp2Jakmip2Prkg2LdlrKif21bPdlim2Crybb1Pcyt2Ctla2bMvkFabp4Cd53Ero1lDach2Klra12Tmem171Arap2VegfaCldn15Slc25a37Gm1966Dock5Stard42010005H15RikAdcy7Unc93aHas2Erdr11700016C15RikMarch11Ccdc88bPolr3gGaleSh3bgrl2Gramd1bSpnStom1600014C23RikElovl6H2-DMb1Dhcr7Htr2bMmabSyngr1FasnSerpinb6bSqleAcsl4Sc5dTrib3Prelid2PappaVcan6330407J23RikDusp4PtpreCd44Fgf2Fxyd5Gvin1H2-M3AacsBnip3Car5bPrl7a1Mtap2Adssl1Ubash3b1700027L20RikFgf7Adcyap1r1Fosl1Ptgs2AregPhexIdi1Vmn2r89Prss46Havcr2MslnGchfrMvdEpas1Klra22Klra7Klra15Klra202310002L13RikGzmeGsta1Gsta2Rnf183Scd1Acss2Angptl6Aldh1l1Arhgef16Unc13aLy6c2LssAcat2NsdhlPcsk9Ly6c1CtswCst6Lrp8Trim54Apobec1Cdca7lSlc2a6Egln3Tnni3Insig1Acat31700003F12RikAldocAk4Speer3Il6Car6Slc7a11Acsbg1Tm7sf2Slc16a3Mcpt84930583H14RikA630095E13RikNfkbiaIcam1Neurl3GemElf3Pag1Jazf1Col5a3Tgfa4930444P10RikMs4a2Ptplad2Rbp4Ccdc80Klf15Kcnj16Entpd2Cd1d2Mfsd7c5430407P10RikCpxm1EnpepCoblSh3tc2Tacc1Efr3bEfna2Il15LcorlSmagpAtl2Synpo2Wipf3Plcb1Ano1Lgr6Mettl7a2Rbm20Galntl1Ceacam1Rasl11bIl6st9030409G11RikHs3st3b1Lama2Mansc1Mboat2Lect2Mfap5Slc16a9VldlrCldn2Syt1Lama3Ccnjl4930523C07RikBmp8aTnfaip6Tmtc1Slc35f2Pid1Npr1Ramp1Camk4D8Ertd82eAplnrLmod3Col4a3Calml4Tacstd2Phyhip4833424O15RikBtbd3Pfkfb3Irf8Klhl31Samd5Lce3cDusp26Sp5TinagLtbp2Fam19a2Pkhd1Id4Rgs6Tnfrsf19Sfrp2AardItm2aSlnBean1NppaCd79bHist3h2baFam89a2310043M15RikCxcr6Slc16a7SynmTnnt3Wnt6Lsp1D3Bwg0562ePrr15Ccdc88cAbca1Pdzk1Esr1Hey1FibinRspo2Ppp4r4Abi3bpSpock2Mylk4933404M02Rik2310015B20Rik2900011O08RikZbtb8b2310057J16RikTgfbiD630039A03RikAgtLumLOC100503474Keg1Chrdl2Adam5Actr3bSncaipMcamArhgap24Cysltr1CftrKcnn2Popdc3OmdUpk3bSlc1a6Csf1rPgrHspa12aNcaldSt6galnac2Irx1NovFlrt3AnpepItga8ScelAdam12Aldh1a2D630045M09RikCol6a2Tspan8Adamts3Gm106Tmem86aRai2Padi2MmeGja1Adra1bAmdhd1Abcb11Gbp3FryIldr2BvesItgb6Il1r1FstCd55Gpr160Card10Kctd12bPde3aGnai1Chn2Ramp2Fgf5Ncf2Hey2Col4a4DcnFgf18IcoslNat8lClstn2Ggt6Pcsk6Cys1Tspan33Crb2BC023202Rassf10SdprDcdc2aPpfia2Kcnk4Cdo1Sox2Krtap1-5KdrKcng2NgfrTrdnTmem56Ceacam2Slc35f1Gap43C77370B3gnt8Cldn10Myh7BcamKrt7Trpc6Gabrb1Kcnf11700024P16RikFam70aCyp2s1Kcne4Gabra4AI118078C230098O21RikLrtm2Cpne4P4ha3Gna14Myh14Zfp185Dkk2Tspan13Thsd4Dkk1Dcaf12l1E330013P04RikTtc22Adamts16Ptpn5Mmp17Sept4Akr1c20Fam19a1Igfbp2Gc6330403K07RikIgf2Slc35d3Rcsd1Smtnl2Tekt1Prss12Nkain4C1qtnf3Lrp2Gm16485Elmod14930449I04RikSh2d4aGreb1Ptx3Col2a1Aldh1a3Hist3h2bb-psCxcl171700011H14RikNpy2rOxtrCorinWnt3Clvs2Cd200Moxd1KitC4bpGpr182G6pc2H19HpgdLrrc55MroGrem2Serpina1cHpgdsChst8Dio2Pdzd2Slc16a61700019N12RikLplPcp4l1Akr1c14Gjb22010300C02RikC130060K24RikDhrs9Dab1Bmp3Rasd1Serpina1d

Cxcl1Ptgs2Il6NfkbiaIcam1Neurl3Elf3

Figure S71 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 241 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 241 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

A

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

B

Figure S7

p65 K4Q-repressed genesp65 K4Q-induced genes

IL-1-induced genes

p65 K5Q-induced genesp65 K5Q-repressed genes

IB: anti-(Ac)K310-p65

IB: anti-FLAG

IB: anti-p65

IB: anti-p65

IB: anti-tubulin

input

Ni-NTA pulldown

Acetyl-K310 p65

p65-His

FLAG-HDAC3

p65-His

tubulin

++

+++++++YFP-CBP+

+

+

+

+

+p65-His + +

p65 E39I-His + +

FLAG-HDAC3 +

Figure S8

40 ‐actinIB: anti‐‐actin

p65-His + + + + + + +p300 + + + + + +

HDAC1-MYC +HDAC2-FLAG +

HDAC3-HA +HDAC5-FLAG +HDAC8-FLAG +

70

70 

70 

70 

70 

55

70

HDAC1‐Myc

IB: anti‐(Ac)K310‐p65

IB: anti‐(Ac)‐lysine

IB: anti‐p65

IB: anti‐HDAC3

IB: anti‐MYC

IB: anti‐FLAG

170

IB: anti‐p300

p300

unspecific

IB: anti‐(Ac)K314‐p65

IB: anti‐(Ac)K315‐p65

kDTCA lysates

Figure S9

70

95

130

55

HDAC2‐Flag

HDAC5‐Flag

HDAC8‐Flag

HDAC3‐HA

p65

pan acetyl‐lysine p65

acetyl‐K315 p65

acetyl‐K314 p65

acetyl‐K310 p65

Cx

cl2

mR

NA

(-f

old

)

0200400600800

100012001400

- IL-1

Cx

cl2

mR

NA

(-f

old

)

0

500

1000

1500

2000

2500- IL-1

pL

KO

.1

shH

DA

C1

(#1)

shH

DA

C1

(#3)

shH

DA

C1

(#4)

shH

DA

C1

(#2)

pL

KO

.1

shH

DA

C2

(#1)

shH

DA

C2

(#3)

shH

DA

C2

(#4)

shH

DA

C2

(#2)

Figure S10

A

B

Cx

cl2

mR

NA

(-f

old

)

0

200

400

600

800

+

+

+++

+

++

+

++Hdac3 fl/+Cre-ER +

tamoxifen +TNF + +

Hdac3 fl/- Cre-ER + + +

Figure S11

A

tamoxifen + + + +

TNF + + + +

Hdac3 fl/-

Cre-ER + + + + + + + + +

exp. 1 1 1 1 2 2 2 2

1 2 3 4 5 6 7 8

Figure S11

29 TNFα-regulated genes 2-fold in exp. 1 and exp. 2

10 TNFα-regulated genes suppressed by 2-fold in Hdac3 -/- cells in exp. 1

12 TNFα-regulated genes suppressed by 2-fold in Hdac3 -/- cells in exp. 2

60 IL-1-regulated genes 1.5-fold in exp. 1 and exp. 2

B log2 ratio

-5 0 5

Table S1

EntrezGeneID GenbankAccession GeneSymbol

expression reduced in HDAC3 -/-

expression suppressed in p65 -/-

IL-1-induced expression suppressed by p65 K4Q

IL-1-induced expression suppressed by K5Q

basal expression suppressed by K4Q

basal expression suppressed by K5Q

1 320897 AK033113 8030431J09Rik x2 213393 NM_145980 8430408G22Rik x x3 225339 AK030009 Ammecr1l x4 12010 AK086184 B2m x5 11796 BC011338 Birc3 x x6 20296 NM_011333 Ccl2 x x7 20297 NM_016960 Ccl20 x8 20306 NM_013654 Ccl7 x x9 54199 NM_017466 Ccrl2 x x x x

10 12642 NM_009890 Ch25h x x11 14825 NM_008176 Cxcl1 x12 15945 NM_021274 Cxcl10 x x13 20310 NM_009140 Cxcl2 x x x x14 20311 NM_009141 Cxcl5 x x15 13537 NM_010090 Dusp2 x16 18218 NM_008748 Dusp8 x17 13654 NM_010118 Egr2 x x x18 13710 NM_007921 Elf3 x19 14281 NM_010234 Fos x20 15894 NM_010493 Icam1 x x x21 16000 AK050118 Igf1 x22 16362 NM_008390 Irf1 x x x x23 16410 AK171739 Itgav x24 16476 NM_010591 Jun x25 50772 AK011505 Mapk6 x26 214854 NM_153408 Neurl3 x x27 18035 NM_010907 Nfkbia x x28 80859 NM_030612 Nfkbiz x x x29 18578 AK171700 Pde4b x x x30 18712 NM_008842 Pim1 x x x31 225182 AK087007 Rbbp8 x32 223881 NM_172612 Rnd1 x x x x33 18787 AK040662 Serpine1 x x x34 215113 AK090207 Slc43a2 x x35 24088 NM_011905 Tlr2 x x x x36 21926 NM_013693 Tnf x x x37 21929 NM_009397 Tnfaip3 x x x x x x38 53603 NM_021367 Tslp x x x39 22329 NM_011693 Vcam1 x x x x x x40 22695 NM_011756 Zfp36 x x

40 19 15 14 2 2