Supplemental Information for - Nature · Supplemental Information for . Acetylation site...

Supplemental Information for

Acetylation site specificities of lysine deacetylase inhibitors in human

cells

Christian Schölz, Brian T. Weinert, Sebastian A. Wagner, Petra Beli, Miyake Yasuyuki, Jun Qi,

Lars J. Jensen, Werner Streicher, Anna R. McCarthy, Nicholas J. Westwood, Sonia Lain, Jürgen

Cox, Patrick Matthias, Matthias Mann, James E. Bradner, Chunaram Choudhary

This file includes:

- Supplemental Figures 1-14

- Supplemental Table 1

- Supplemental Notes 1-2

Nature Biotechnology: doi:10.1038/nbt.3130

a

b

Supplemental Figure 1

NO2NH2

Br

SB(OH)2

NO2NHBoc

Br

NO2NHBoc

S

NaH, (Boc)2O

DMF, 0 °C60 %

NH2NHBoc

S

NH

O

HCTU, DIPEA,DMF, 23 °C

64%

NHNHBoc

S

O

NH

OOH

O

NHNH2

S

O

NH

O4M HCl,

dioxane/THF

23 °C95%

SI-1 SI-2 SI-3

SI-4 SI-5 JQ12

Pd(PPh3)4, Na2CO3DME/H2O, 120 °C

80 %

10% Pd/C, H2

EtOAc, 23 °C94%

Log[conc(M)]-10 -8 -6 -4

0

50

100

150HDAC1HDAC2HDAC3HDAC4HDAC5HDAC6HDAC7

HDAC8HDAC9

Nor

mal

ized

Act

ivity

c

HDAC1 HDAC2 HDAC3 HDAC4 HDAC5 HDAC6 HDAC7 HDAC8 HDAC99.20 77.2 1856 >4000 >4000 >4000 >4000 >4000 >4000

JQ12 IC50 (nM)

Supplemental Figure 1 | Synthesis and biochemical analysis of JQ12. a, Synthesis of JQ12. The amino group on commercially available 4-bromo-2-nitroaniline (SI-1) was �rst protected as tert-butyl carbamate. The resulted compound SI-2 underwent a palladium (II) catalyzed Suzuki coupling reaction with thiophen-2-ylboronic acid to establish the 4-thiophen-2-yl-phenyl core, and generate compound SI-3. The nitro group on SI-3 was then reduced to amino group under palladium(0) catalyzed hydrogenation to a�ord mono-protected diaminophenyl compound SI-4. The coupling reaction between the amino group on SI-4 with 4-acetamidobenzoic acid under standard amide coupling condition gave amide SI-5. Removal of the Boc protecting group on SI-5 furnished the �nal product JQ12. b, Inhibition of HDAC1-9 by JQ12. Puri�ed enzymes were incubated for 3 h with JQ12 and subsequently �uorogenic release of 7-amino-4-methylcoumarin from substrate upon deacetylase and trypsin enzymatic activity was determined. c, IC50 values of JQ12 for HDAC1-9 derived from b.


Supplemental Figure S2

-4 -2 0 2 4

-4-2

02

4

Apicidin n = 1090 R = 0.57

-4 -2 0 2 4-4

-20

24

Bufexamac (1 mM) n = 2483 R = 0.85

-4 -2 0 2 4

-4-2

02

4

Bufexamac (50 µM) n = 2659 R = 0.84

-4 -2 0 2 4

-4-2

02

4

CI.994 n = 1080 R = 0.69

-4 -2 0 2 4

-4-2

02

4

JQ12 n = 1185 R = 0.52

-4 -2 0 2 4

-4-2

02

4

LBH589 n = 2020 R = 0.77

-4 -2 0 2 4

-4-2

02

4

MGCD0103 n = 905 R = 0.57

-4 -2 0 2 4-4

-20

24

MS275 n = 1420 R = 0.68

-4 -2 0 2 4

-4-2

02

4

NaB n = 1107 R = 0.74

-4 -2 0 2 4

-4-2

02

4

Nicotinamide n = 2162 R = 0.79

-4 -2 0 2 4

-4-2

02

4

Pandacostat n = 1045 R = 0.75

-4 -2 0 2 4

-4-2

02

4

PCI24781 n = 1900 R = 0.9

-4 -2 0 2 4

-4-2

02

4

PCI34051 n = 1658 R = 0.44

-4 -2 0 2 4-4

-20

24

PXD101 n = 1127 R = 0.68

-4 -2 0 2 4

-4-2

02

4

SAHA n = 1135 R = 0.55

-4 -2 0 2 4

-4-2

02

4

Sirtinol n = 1040 R = 0.9

-4 -2 0 2 4

-4-2

02

4

Tenovin-6 n = 966 R = 0.92

-4 -2 0 2 4

-4-2

02

4

TSA n = 718 R = 0.65

-4 -2 0 2 4

-4-2

02

4

Tubacin n = 1445 R = 0.76

-4 -2 0 2 4-4

-20

24

VPA n = 1618 R = 0.57

Log2 (SILAC Ratio KDACI/Ctrl. Experiment 2)

Log 2 (S

ILA

C R

atio

KD

AC

I/Ctrl

. Exp

erim

ent 1

)

Supplemental Figure 2 | Correlation between acetylation changes quanti�ed in KDACI experiments. The plots show the correlation between acetylation site changes in two independent biological replicate experiments for each KDAC inhibitor.


5810

This study

Supplemental Figure S3

Weinert et al. (2013)

2298 588

Supplemental Figure 3 | Overlap of identi�ed acetylation sites with previously published acetylation sites from HeLa cells. The Venn diagram shows the overlap of identi�ed acetylation sites from this study with acetyla-tion sites identi�ed from a previous study in HeLa cells23.


Acetyllysine

SMC3

HDAC8

0 1.0 2.5 5.0 10 25ø

PCI34501 (μM)


51

191

191kDa

a bFo

ld in

crea

se in

ace

tyla

tion

(KD

AC

I/Con

trol)

Apicidi

n

Bufexa

mac

CI-994

JQ12

LBH58

9

MGCD0103

MS275

NaBNAM

Panda

costa

t

PCI2478

1

PCI3405

1

PXD101SAHA

Sirtino

l

Tenov

in6 TSA

Tubac

inVPA

0

2

4

6

8K106acK105ac/K106ac

Supplemental Figure 4 | HDAC8 inhibitor PCI34051 increases SMC3 acetylation. a, The bar plot shows SILAC ratios of acetylated SMC3 peptides from cells treated with di�erent KDACIs for 16 hours. Ratios represent median ± SD of all detected peptides representing either acetylated SMC3 at position K106 or double-acetylated SMC3 at K105 and K106. b, Treatment of HeLa cells with PCI34501 causes dose-dependent increase in SMC3 acetylation. SMC3 was immunoprecipitated and subsequently acety-lation of SMC3 was visualized by immunoblotting with a pan-anti-acetyllysine antibody. Immuno-blots shown are representative of two independent experiments. IP antibody: anti-SMC3; Immunoblot anti-body: anti-acetyllysine. ø: control, using anti-c-myc antibody for IP. WCL: whole cell lysate.

IP:SMC3

WCL



−4 −2 4

−4

−2

0

2

4

0 2

Log 2S

ILA

C R

atio

NA

M 7

2h/1

6h E

xp.2

Log2SILAC RatioNAM 72h/16h Exp.1

n=1063,R=0.79

0 20 40 60 80transcriptionRNA splicing

RNA metabolic processchromosome organization

chromatinspliceosomechromosome

nucleus

percent of proteinsassociated with GO term

Proteins with upregulated acetylation

p=2.1e-13

p=4.7e-12

p=1.1e-11

p=9.2e-10

p=4.3e-40

p=8.9e-11

p=1.4e-08

p=1.3e-06GO

CC

GO

BP

a b c

16h/

cont

rol0

20

40

60

80

100

AcK

site

s (%

)

72h/

cont

rol

Supplemental Figure 5 | E�ect of long-term nicotinamide treatment on cellular acetylation. a, Correla-tion of NAM-regulated acetylome after 16 h (short-term) and 72 h (long-term) treatment. The scatter plot shows the correlation between acetylation sites quanti�ed in HeLa cells treated with NAM for 16 h and 72 h. Correlation was determined with Pearson correlation coe�cient. b, Subcellular distribution of proteins with NAM-upregulated acetylation sites after 16 h and 72 h treatment. The bar plot shows the fraction of NAM-up-regulated acetylated proteins annotated with the indicated Gene Ontology cellular compartment (GOCC) terms. red: nuclear; grey: cytoplasmic; blue: mitochondrial. c, Functional annotation of proteins containing NAM-upregulated acetylation sites after 72 h treatment with NAM. Signi�cantly enriched GO cellular com-partment (GOCC) and biological process (GOBP) terms are indicated.



LIG1

HMGB2

RANBP9

KAT5

POLD3

RAD23A

CLSPN

CASP3

PDCD5

DPY30

CASC5

SMARCC1

POU2F1 BRD1

ARID1B

SMARCA2

MN1

HNRNPC

SF3A1

FUS

SURF6

HMGA1

NPM1NR3C1

SUCLG2

MLL3

MLL2

H3F3B

SRSF3

EFTUD2

CENPV

CREBBP

HMGB1

CBX5

CBX3 TRIM33

HCFC1

NUP50

BAZ1B

EP300

TMPOHNRNPD

HNRNPF

ACIN1AKAP8L

PTBP1

ILF3

EP400

SETMYST1 MCRS1

ATF6DDX17

TRIM28RNF2

CCDC101

STAT1

MBIPENO1

ING4CHD9

YEATS2

WHSC2

SUB1

CTDP1

MYST2

C20orf20

BRD4

NCOA3

ETS1

NCOA2

COBRA1

SATB1TSPYL1

SP1

JUNBHMGB3

TAF3

AFF4

PSMC3

GTF3C3

WAC

PDLIM1

BRF1

EDF1

CRTC2

SP3

ZYX

PES1

DCAF13

HINT1

EEF1B2

EIF3H

RPL19

NOP58

RPS17

NHP2

TARS

S100A11

ALDH7A1

ALDH18A1

ST13

HSP90AA1

CAD

AHSA1

SUMO3

NUMA1

TPX2

CENPF

ORC6L

MAD2L1

MCM4

HIRA

KIF4A DYNC1LI1 SENP1

NSUN2

DUT

SSBP1

AK4

PPP1CB

NEDD8

CKS1B

PNPT1 NUDC

MRPL12

EBNA1BP2

RPS16

NCL

MKI67

DKC1

FBL

MYST3

MYST4

ING3

ANXA2

RPL38

TUFM

RAB8A RPL14BRD8

NOP14

SNCA

XRCC6

XRCC5

WRN

HYRC

NBNRAD50

RANBP2

BIN1

PARP1

EIF4HRSL24D1

IARSUBAP2L

NUP98

HCLS1

CDC37

UBE2M

FNBP4

SFRS18

SRSF2IP

DDX42

SRRM2

RBM17

PARN

ACTN4VIM

NONO

NCBP2U2AF2

SF3B1SNRPC

PRPF4B

RBM39

EZR

CFL1

PLEC

CALD1

KRT18

SFPQ

SPTAN1

ACTN1

FOSL2

PRPF4HNRNPA0

HNRNPA1CDK11A

SF1DDX5

CRTC1BCOR

NOL7

SSRP1CCNT1

SLC25A5

PDHX

COX4I1HIST1H1E

BAZ2A

RBBP7

SEC23IPTRIM25

LMNA

HNRNPU

Log2 SILAC Ratio (H/L)

1 5

Number of upregulated acetylation sites

1

2-3

4-5

6+

`Transcription´

`Acetylation´`Translation´

`Cell Cycle´

`DNADamage´

`ChromatinModification´

`Splicing´

`Cyto-skeleton´

PCBP1

HNRNPA2B1

SF3B2

DHX9

RBM10MATR3

PSIP1

SNW1

DHX38

KHSRP

DDX46

HNRNPM

ATF4

MAML1

GTF2H1TCERG1

TCEA1RUNX1

MEF2A

ETS2

INTS1

a

KPNB1

Light(R0, K0)

“Control”

Medium(R6, K4)“KDACi”

Heavy(R10, K8)

“KDAC KO”

In-solution protein digest

SCX chromatography LC-MS/MS analysis

Acetyl-lysine IP

Cell lysisMix proteins 1:1:1

b c

SIRT1

Tubulin

MEF wt

KOkDa

97

51

Sirt6 (123bp)

Gapdh (72bp)

d

−5

0

5

−5 0 5 −5 0 5

Log 2S

ILA

C R

atio

Sirt

6 -/- /

WT

Exp

.2

Log2SILAC RatioSirt6 -/- / WT Exp.1

−5

0

5

Log 2S

ILA

C R

atio

Sirt

6 -/- /

WT

Log2SILAC RatioNAM / Ctrl.

n=729,R=0.72

n=1931,R=0.10

Sirt2 (143bp)

Gapdh (72bp)

Supplemental Figure 6 | Nicotinamide causes hyperacetylation of nuclear proteins. a, Functional interac-tion networks of proteins with NAM-upregulated acetylation sites (after 16 h of NAM treatment). Network analy-sis was performed using protein interaction information from the STRING database and networks were visualized using Cytoscape. The color code re�ects the highest SILAC ratio of hyperacetylated sites on each protein and the color of circle boundaries indicates the number of upregulated sites for each protein. Clusters of functionally related proteins are highlighted. b, Schematic representation of triple SILAC experiments. Wildtype (WT) MEFs labeled with “light” SILAC were treated with vehicle control, MEF WT cells labeled with “medium” SILAC were treated for 16 h with KDAC inhibitors, and knockout (KO) cells were labeled with “heavy” SILAC and were treated with vehicle control. Subsequently, proteins were extracted and proteolysed using Lys-C and trypsin. Acetylated peptides were enriched with anti-acetyllysine antibodies and samples were analyzed by mass spectrometry. c, Veri�cation of Sirt1, Sirt2 and Sirt6 deletion in KO cells. Deletion of Sirt1, Sirt2 and Sirt6 in KO cells was con�rmed via immuno-blotting (SIRT1) or QPCR (Sirt2 and Sirt6). d, Correlation of acetylation site changes identi�ed in Sirt6-/- cells. The scatter plots show the correlation between acetylation changes observed in biological replicate experiments in Sirt6-/- cells, and NAM-treated MEF cells and Sirt6-/- cells, respectively. Correlation was determined with Pearson correlation coe�cient.Nature Biotechnology: doi:10.1038/nbt.3130


−4 −2 4

−4

−2

0

2

4

0 2

Log 2

SIL

AC

Rat

ioN

icot

inam

ide/

cont

rol

Log2SILAC RatioEX-527/control

n=1574,R=0.68

Supplemental Figure 7 | NAM and EX-527 increase acetylation of shared targets. The scatter plot shows the correlation between acetylation sites quanti�ed in NAM- and EX 527-treated HeLa cells. Correlation was deter-mined with Pearson correlation coe�cient.



−4 −2 4

−4

−2

0

2

4

0 2

Log 2S

ILA

C R

atio

Sirt

inol

/ C

trl. E

xp.2

Log2SILAC RatioSirtinol / Ctrl.Exp.1

n=752,R=0.37

−4

−2

0

2

4

Log 2S

ILA

C R

atio

AG

K2

/ Ctrl

. Exp

.2

−4 −2 40 2Log2SILAC Ratio

AGK2 / Ctrl. Exp.1

n=774,R=0.46

−4

−2

0

2

4

Log 2S

ILA

C R

atio

Sirt

2 -/- /

wt E

xp.2

−4 −2 40 2Log2SILAC RatioSirt2 -/- / WT Exp.1

n=751,R=0.71

−4 −2 4

−4

−2

0

2

4

0 2

Log 2S

ILA

C R

atio

Sirt

2 -/- /

WT

Log2SILAC RatioNAM / Ctrl.

n=1195,R=0.15

−4 −2 4

−4

−2

0

2

4

0 2

Log 2S

ILA

C R

atio

Sirt

2 -/- /

WT

Log2SILAC RatioSirtinol / Ctrl.

n=1577,R=0.11

−4 −2 4

−4

−2

0

2

4

0 2

Log 2S

ILA

C R

atio

Sirt

2 -/- /

WT

Log2SILAC RatioAGK2 / Ctrl.

n=1733,R=0.35

Supplemental Figure 8 | Correlation of acetylation site changes regulated by SIRT2 and sirtu-in inhibitors. The scatter plots show the correlation between acetylation site SILAC ratios from di�erent biological experiments for Sirt2-/- vs. WT MEFs; MEFs treated with sirtinol, AGK2 or NAM vs. control treated MEFs; as well as correlation between inhibitor-regulated and SIRT2-regulated sites. Correlation was determined with Pearson correlation coe�cient.



a b

HADHAECHS1

CoA

EHHADH

EHHADH HADHA

ACAA1

HADHB

ACAA2

CPT1A

CPT1B

CPT1C

CPT2

ACSL1

ACSL5

ACSL3

ACSL6

ACSL4

ACADMACOX3ACOX1

ACADL ACADVL

L-Palmitoyl-carnitine

Hexadecanoate(Fatty acid)

Hexa-decanoyl-CoA

trans-Hexadec-2-enoyl-CoA

(S)-3-Hydroxy-hexadecanoyl-CoA

3-Oxo-hexa-decanoyl-CoA

Acetyl-CoATetra-decanoyl-CoA

HADH

c d

-4 -2 0 2 4

0.0

0.5

1.0

1.5

Log2(SILAC Ratio Tenovin-6/Ctrl.)

Den

sity

nonmitochondrial

mitochondrial

P = 5.1e-153

Oxaloacetate

CSMDH1

MDH2

FH

SDHA

DLST

SUCLG1 OGDH

ThPP

Lipoamide-EDihydro-lipoamide-E

DLD

Citrate cis-Aconitate Isocitrate

Oxalosuccinate

IDH1

IDH2

IDH1

IDH2

2-Oxoglutarate

Phosphoenol-pyruvate

Acetyl-CoAPyruvate

S-Acetyldihydro-lipoamide-E

TCA cycle

ACLY

ACO1

ACO2

ACO1

ACO2

IDH3A

IDH3B

IDH3G

(S)-Malate

Fumarate

SuccinateSuccinyl-CoA

SUCLG2

SDHB

SDHC SDHD

OGDHL

OGDH

OGDHL

S-Succinyl-dihydrolipoamide

3-Carboxy-1-hydroxypropyl-ThPP

PCK1

PCK2Lipoamide-EDihydro-

lipoamide-E

DLD

DLAT

ThPP

PDHA1

PDHA2

PDHB

PDHA1

PDHA2

PDHB

PDHXPDHX

SUCLA2

Fraction of proteins involved (%)

Fraction of proteins involved (%)

membrane

cytoplasmic part

mitochondrion

mitochondrial nucleoid

mitochondrial membrane

mitochondrial matrix

mitochondrial part

5.4e−04

6.9e−05

1.3e−13

5.2e−16

4.5e−18

1.7e−26

1.3e−41

All identifiedUpregulated

0 20 40 60 80

fatty acid beta−oxidation

nucleoside triphosphate biosynth. process

amine catabolic process

tricarboxylic acid cycle

oxidation−reduction process

respiratory electron transport chain

cellular ketone metabolic process

4.6e−06

2.5e−06

6.8e−07

1.3e−08

2.1e−10

1.8e−11

1.3e−11

0 10 20 30 40

All identifiedUpregulated

GO

BP

term

sG

OC

C te

rms

Supplemental Figure 9 | Tenovin-6 selectively increases acetylation of mitochondrial proteins. a, The plot shows the distribution of SILAC ratios of acetylation sites on mitochondrial and non-mitochondrial proteins in tenovin-6 treated HeLa cells. Acetylation of mitochondrial proteins was selectively increased in tenovin-6 treated cells. b, The bar charts show enrichment of Gene Ontology cellular compartment (CC) or GO biological process (BP) terms among proteins with tenovin-6 upregulated acetylation sites. c and d Schematic overview of the TCA cycle and fatty-acid elongation pathways, respectively. Proteins that showed increased acetylation (>2-fold) in tenovin-6 treated HeLa cells are shown in red.



HIF1-α

T T

y₁₇

V P E E

y₁₃

E

y₁₂

L

y₁₁

N

y₁₀

P

y₉

acK

y₈

I

y₇

L

y₆

A

y₅

L

y₄

Q

y₃

N

y₂

A

y₁

Q R

b₂ b₃ b₇ b₈ b₉ b₁₀ b₁₁ b₁₂ b₁₄²⁺

y₁

b₂

b₃

y₂y₃

y₄

y₅y₆

b₇b₁₄²⁺

y₇

b₈

y₈

b₉

y₉

b₁₀y₁₀

b₁₁

y₁₁

b₁₂

y₁₂

y₁₃ y₁₇

02

4Intensity [10e6]

200 400 600 800 1000 1200 1400 1600 1800 2000m/z

050

100

Rel

ativ

e A

bund

ance

a

b

n = 3430R= 0.76

Log 2 S

ILA

C R

atio

Buf

exam

ac (1

mM

) / C

trl.

Log2 SILAC RatioBufexamac (50µM) / Ctrl.

−5

−5

0

5

0 5

MAP1LC3B

/B2

PBXIP1

IGFBP7

F11R

SERPINI1

COL12A

1

COL4A2

PLOD2

FAM162A

RIOK3

P4HA2

PPL

GFPT2

STAU1

COL3A1

TK1ENO2

ALDOC

P4HA1

COL5A1

HK2

ABHD4

NDRG10

2

4

6

8

10

Bufexamac (1

Bufexamac (50µM)

Fold

upr

egul

atio

n (B

ufex

amac

/Con

trol) mM)

c

Supplemental Figure 10 | Quanti�cation of acetylation sites in cells treated with high-dose bufexamac. a, Experimental correlation of acetylomes quanti�ed in response to high- and low-dose bufexamac treated HeLa cells. The dot plot shows correlation of average SILAC ratios at two di�erent concentrations of bufexa-mac (50 µM and 1 mM). b, Annotated MS/MS scan of acetylated HIF1-α peptide TTVPEEELNPK(ac)ILALQNAQR. c, Proteome analysis of SILAC-labeled HeLa cells treated with 50 µM and 1 mM bufexamac. Proteins shown are upregulated at 1 mM bufexamac (>2-fold increase in at least one experiment and at least >70% increase in two additional replicate experiments). Proteins labeled in blue are known to be associated with hypoxia. Error bars show SD of quanti�ed ratios.Nature Biotechnology: doi:10.1038/nbt.3130


FeCl3 (µM)

Cel

l Dea

th (%

)

0 25 50 100 150 200 2500

10

20

30Bufexamac (250 µM)Dfx (200 µM)

Supplemental Figure 11 | Iron supplementation prevents bufexamac-induced cell death in HL60 cells. The bar chart shows percentage of cell death in HL60 cells treated with bufexamac or DFX. Iron supplementation reduced cell death to background level. Cell viability was determined by propidium iodide (PI) staining of cells and subsequent analysis of PI �uorescence. Error bars represent SD of three independent experiments.



Wavelength (nm)

OD

300 400 500 600 7000.0

0.1

0.2

0.3

0.4

0 230µM

Bufexamac (µM)

OD 4

90

0 50 100 150 200 2500.00

0.05

0.10

0.15

0.20

0.25

ba

Kd: ~130 µM

Supplemental Figure 12 | Bufexamac is an iron chelator. a, UV/VIS spectroscopy of bufexamac titrated into FeCl3 solution. b, The �gure shows the derived iron binding curve measured at 490 nm in a. Data analy-sis resulted a KD of 136 ± 11 µM. All measurements were performed using methanol as solvent due to very low solubility of bufexamac in aqueous solutions. Therefore, these KD values may di�er in aqueous solu-tions.


ONH

OOH

HDAC6

TAT

acetylated microtubule

non-acetylated microtubule

Bufexamac

high-doselow-dose

Fe

ONH

OOHFe complexing iron

Proteasomaldegradation

HIF1-αHIF1-α

VHL

HIF1-αARNT

HIF1-αARNT

transcription of HIF1-α target genes

VEGF

a

b

c

d

e


AcAcAc

Cytoplasm

Nucleus

Supplemental Figure 13 | Proposed model describing the dose-dependent e�ects of bufexamac in cells. a, At lower doses bufexamac inhibits HDAC6, thereby increases acetylation of target proteins such as tubulin. b, At higher doses bufexamac inhibits HDAC6 activity, but additionally results in iron depletion by chelating cellular iron. c, Depletion of cellular iron by bufexamac mimics hypoxia, which leads to the stabili-zation of HIF1-α. d and e Stabilized HIF1-α accumulates in the nucleus where it binds to hypoxia response elements (HRE) and transcriptionally upregulates target genes involved in cellular responses to hypoxia, such as vascular endothelial growth factor A (Vegfa).


Class I

Class IIa

Class IIb

Class IV

Class III

SIRT1SIRT2SIRT3SIRT4SIRT5SIRT6SIRT7

HDAC1HDAC2HDAC3HDAC8

HDAC6HDAC10

HDAC11

HDAC4

HDAC5

HDAC7

HDAC9

MS275

NaB

Tubacin

Tenovin-6

Nicotinamide

Sirtinol

TSASAHAPXD101LBH589PandacostatPCI24781

Bufexamac

Apicidin

MG

CD

0103

CI994

PCI34051

JQ12

VPA


a bS

irtin

olN

icot

inam

ide

Teno

vin6

PC

I340

51P

CI2

4781

Buf

exam

acTu

baci

nN

aBut

yrat

eTS

AS

AH

ALB

H58

9P

XD

101

JQ12

Pand

acos

tat

MS

275

CI.9

94Va

lpro

ate

MG

CD

0103

Api

cidi

n

SirtinolNicotinamideTenovin6PCI34051PCI24781BufexamacTubacinNaButyrateTSASAHALBH589PXD101JQ12PandacostatMS275CI.994ValproateMGCD0103Apicidin

-1 10

Pearson

Supplemental Figure 14 | Comparison of in-vitro and in-vivo speci�city pro�les of KDACIs. a, Site-based speci�city analysis of KDACIs . SILAC ratios of all identi�ed sites from each Inhibitor were used to calculate pair-wise Pearson correlation coe�cients for all KDACIs. b, The �gure shows the agreement of KDACI-regulated acetylomes with their predicted acetylation pro�les. The type color indicates the level of agreement: Green: high agreement; yellow: partial agree-ment; red: little or no agreement (See also Supplementary note 2).


Supplemental Table 1 (List of KDAC Inhibitors)

Inhibitor CAS No. Chemical Name Alternative Name Conc. used in this study (µM) Solvent

AGK2 304896-28-4 2-Cyano-3-[5-(2,5-dichlorophenyl)-2-furanyl]-N-5-quinolinyl-2-propenamide 10 DMSO

Apicidin 183506-66-3 Cyclo[(2S)-2-amino-8-oxodecanoyl-1-methoxy-L-tryptophyl-L-isoleucyl-(2R)-2-piperidinexcarbonyl] 5 DMSO

Bufexamac 2438-72-4 2-(p -Butoxyphenyl)acetohydroxamic acid 0.1-1000 DMSOCI.994 112522-64-2 4-(acetylamino)-N-(2-aminophenyl)-benzamide Tacedinaline 50 DMSOJQ12 5 DMSO

LBH589 404950-80-7 (E)-N-hydroxy-3-(4-((2-(2-methyl-1H-indol-3-yl)ethylamino)methyl)phenyl)acrylamide Panobinostat 2 DMSO

MGCD0103 726169-73-9 N-(2-aminophenyl)-4-((4-(pyridin-3-yl)pyrimidin-2-ylamino)methyl)benzamide Mocetinostat 20 DMSO

MS275 209783-80-2 pyridin-3-ylmethyl 4-((2-aminophenyl)carbamoyl)benzylcarbamate Entinostat 5 DMSO

NaButyrate 156-54-7 Butyric acid sodium salt 5000 H2O

Nicotinamide 98-92-0 Pyridine-3-carboxylic acid amide Niacinamide, Vitamin B3, Vitamin PP 20000 H2O

Pandacostat(E)-3-[4-[[[(E)-(2,3-dihydroxy-4-oxocyclohexa-2, 5-dien-1-

ylidene)methyl]amino]carbamoyl]phenyl]-N-hydroxyprop-2-enamide

50 DMSO

PCI24781 783355-60-23-[(dimethylamino)methyl]-N-[2-[4-

(hydroxycarbamoyl)phenoxy]ethyl]-1-benzofuran-2-carboxamide

Abexinostat 1 DMSO

PCI34051 950762-95-5 N-hydroxy-1-[(4-methoxyphenyl)methyl]indole-6-carboxamide 10 DMSO

PXD101 414864-00-9 (E)-N-hydroxy-3-[3-(phenylsulfamoyl)phenyl]prop-2-enamide Belinostat 5 DMSOSAHA 149647-78-9 N'-hydroxy-N-phenyloctanediamide Vorinostat, Zolinza 2 DMSO

Sirtinol 410536-97-9 2-[[(Z)-(2-oxonaphthalen-1-ylidene)methyl]amino]-N-(1-phenylethyl)benzamide 100 DMSO

Tenovin6 1011557-82-64-tert-butyl-N-[[4-[5-

(dimethylamino)pentanoylamino]phenyl]carbamothioyl] benzamide

10 DMSO

TSA 58880-19-6 (2E,4E,6R)-7-[4-(dimethylamino)phenyl]-N-hydroxy-4,6-dimethyl-7-oxohepta-2,4-dienamide Trichostatin A 2 DMSO

Tubacin 537049-40-4 N-[4-[(2R,4R,6S)-4-[(4,5-diphenyl-1,3-oxazol-2-

yl)sulfanylmethyl]-6-[4-(hydroxymethyl)phenyl]-1,3-dioxan-2-yl]phenyl]-N'-hydroxyoctanediamide

10 DMSO

Valproate 99-66-1 2-propylpentanoic acid

Depakine, Depakene, Ergenyl, Dipropylacetic acid, Mylproin,

Convulex, Valproic Acid, Myproic Acid

5000 H2O

Supplemental Table 1 | List of KDAC Inhibitors used in this study. The table lists names of KDACIs and their concentration used in this study for global acetyl-lation analysis in HeLa cells. Used solvent and chemical name are also indicated.


Supplemental notes

Supplemental note 1

Synthesis of JQ12. The amino group on commercially available 4-bromo-2-nitroaniline (SI-1) was

first protected as tert-butyl carbamate. The resulted compound SI-2 underwent a palladium (II)

catalyzed Suzuki coupling reaction with thiophen-2-ylboronic acid to establish the 4-thiophen-2-yl-

phenyl core, and generate compound SI-3. The nitro group on SI-3 was then reduced to amino

group under palladium (0) catalyzed hydrogenation to afford mono-protected diaminophenyl

compound SI-4. The coupling reaction between the amino group on SI-4 with 4-acetamidobenzoic

acid under standard amide coupling condition gave amide SI-5. Removal of the Boc protecting

group on SI-5 furnished the final product JQ12. (Supplemental Figure 1A)

Supplemental note 2

Due to the lack of known bonafide targets for each of different KDACs it is not possible to compare

in-vivo specificities of KDACIs for acetylation sites with their in-vitro specificities for individual

deacetylases. We used the number of affected acetylation sites, the subcellular distribution of

KDACI-regulated proteins, known KDAC-specific acetylation sites, data from genetic knockout

cells, as well as comparative acetylation profiles for different KDACIs to obtain an indirect

comparison of in-vivo specificity profiles of KDACIs with their predicted cellular specificities

(based on the number and subcellular localization of target deacetylases).


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