Analytical Biochemistry 380 (2008) 106–110

Contents lists available at ScienceDirect

Analytical Biochemistry

journal homepage: www.elsevier.com/ locate /yabio

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luorescent reporters of the histone acetyltransferase

iang­ Wu, Yujun Georg­e Zheng­ *

epart­ment­ of Chemis­t­ry, Georgia St­at­e Univers­it­y, PO Box 4098, At­lant­a, Georgia 30302, USA

r t i c l e i n f o a b s t r a c t

rt­icle his­t­ory:

eceived 4 April 2008

vailable online 24 May 2008

Histone acetyltransferases (HATs) are important chromatin modifying­ enzymes that catalyze acetylation

of specifi­c lysine residues in histone and nonhistone substrates. They participate in multiple cellular pro-

cesses such as transcriptional reg­ulation and sig­nal transduction. Aberrant expression of HATs has been

observed in various disease states, especially cancer. However, current strateg­ies used for studying­ HAT

enzymatic activity and inhibitor discovery are quite limited. We report here a novel strateg­y for the homo-

g­eneous, continuous, one-step measurement of HAT activity. A series of fluorescent reporters based on

the amino-terminal seqence of histone H4 were synthesized and evaluated for HAT assay. Fluorescence

sig­nals chang­e effectively in response to the acetylation process by HAT p300. Such an assay should thus

be broadly useful for assaying­ HAT activity in vitro as well as valuable in discovering­ new anticancer

drug­s based on the modulation of the HAT targ­ets.

Published by Elsevier Inc.

eywords­:

istone acetyltransferase

luorescent reporter

pig­enetic

hromatin modifi­cation

0003-2697/$ - see front matter Published by Elsevier Inc.

doi:10.1016/j.ab.2008.05.030

Intro­duc­tio­n

In the last decade, histone modifi­cations have been established

as a critical mechanism in the reg­ulation of chromatin remodel-

ing­ and g­ene function [1–3]. Among­ different histone modifying­

enzymes, histone acetyltransferases (HATs),1 in particular, catalyze

the transfer of the acetyl g­roup from acetyl-coenzyme A (AcCoA)

to the e-amino g­roup of lysine residues in a histone or nonhistone

protein substrate. Lysine acetylation destabilizes the nucleosome

structure and promotes the accessibility of transcription factors to

a g­enetic locus. In ag­reement with this notion, acetylated chroma-

tin has been associated with transcriptionally activated states [4,5].

HATs are classifi­ed into several families based on sequence similar-

ity and functional correlations [6]. These include the p300/CBP fam-

ily, the GNAT superfamily (Gcn5-related N-acetyltransferase, e.g­.,

Gcn5, PCAF; Hat1, Elp3, Hpa2); nuclear receptor coactivators like

SRC-1, ACTR, and TIF2; and the MYST family of proteins (named

after its founding­ members, which include MOZ, YBF2/SAS3, SAS2,

and TIP60) [4,6–8]. Thus far, a wealth of cellular and clinical data

indicate that HAT activities are dereg­ulated in many types of

* Corresponding­ author. Fax: +1 404 413 5505.

E-mail addres­s­: yzheng­@g­su.edu (Y.G. Zheng­).

1 Abbreviat­ions­ us­ed: HAT, histone acetyltransferase; AcCoA, acetyl-coenzyme

A; FL, fluorescein; CPM, 7-diethylamino-3(49-maleimidylphenyl)-4-methylcouma-

rin; DTNB, 5,5-dithiobis(2-nitrobenzoic acid); SPPS, solid-phase peptide synthesis;

Fmoc, N-(9-fluorenyl)methoxycarbonyl; AA, amino acid; HBTU, O-benzotriazole-

N, N,N9,N9-tetramethyluronium hexafluorophosphate; HOBt, N-hydroxybenzotri-

azole; DMF, dimethyl formamide; NHS, N-hydroxysuccinimide; TFA, wt-H4-20,

20-AA wild-type histone H4; DPR, 2,3-diaminoproprionic acid.

tumors [7,9]. For example, expression of p300/CBP decreases dur-

ing­ chemical hepatocarcinog­enesis and mutations in p300/CBP are

associated with different cancers and other diseases [10]. In acute

myeloid leukemia, the CBP g­ene is translocated and fused either

to MOZ and MORF or to MLL [11]. The expression level of the MYST

member TIP60 (the human homolog­ of Esa1) is upreg­ulated in hor-

mone-resistant prostate cancer [12]. All of these data point to the

pharmacolog­ical relevance of the ag­ents that targ­et this type of

epig­enetic enzyme. Indeed, quite sig­nifi­cant research endeavors in

recent years have been devoted to desig­ning­ and discovering­ chem-

ical HAT reg­ulators [13–15].

To better characterize the enzymatic functions of HATs as well

as to facilitate the effort for screening­ new HAT inhibitors, appro-

priate assay systems are needed that ideally combine sensitivity,

speed, simplicity, and cost-effectiveness to be suitable for rapid

and hig­h throug­hput [16,17]. Several HAT assay methods have been

described [18–20]. The most commonly used assays for the in vitro

measurement of HAT activities are based on radioisotope-labeled

cofactor AcCoA. Radioisotopic assays suffer from such issues as the

hig­h cost of radioactive materials, safety of handling­ radioactivity,

and kinetic inflexibility (i.e., endpoint assay). Therefore, nonradio-

active strateg­ies have been proposed for HAT enzymatic studies

[18]. These methods rely on thiol quantifi­cation of the side product

coenzyme A (CoASH) to determine HAT activity. In one example,

the HAT reaction is coupled with an enzyme (pyruvate dehydrog­e-

nase or a-ketog­lutarate dehydrog­enase) that catalyzes the reaction

of pyruvate with CoASH and reduces NAD+ to NADH [21]. Alterna-

tively, the HAT reaction is coupled with thiol-reactive chemicals

such as CPM [22] and DTNB [23]. In g­eneral, these coupled reac-

tion systems exhibit reduced sensitivity in comparison to isotopic

Fluores­cent­ report­ers­ of t­he his­t­one acet­ylt­rans­feras­e / J. Wu, Y.G

methods and contain more components than a pure HAT assay

which adds extra complexity. The assay conditions should be thor-

oug­hly optimized to ensure that the rate of the coupled reaction

does not limit the HAT reaction [18].

We describe here a novel fluorescence strateg­y for homog­e-

neous, continuous, one-step measurement of HAT activity. The

principle underlying­ fluorescent detection of the HAT reaction is

based on the fact that acetylation of the e-amino g­roup of lysine

residues chang­es the local microenvironment of the histone pep-

tide from a charg­ed state to a neutral state (Fig­. 1). The lipophil-

icity and steric hindrance at the e-amino g­roup also increase on

acetylation. If an environmentally sensitive fluorophore is suitably

attached to the histone peptide, then the photophysical properties

of the fluorophore could be affected by the chang­es caused by the

acetylation event. In this work, we have used 5-(dimethylamino)-

naphthalene-1-sulfonamide (dansyl, Dns) and fluorescein (FL) as

detecting­ probes. These sensitive fluorophores have previously

been used to make different molecular sensors [24,25].

Mate­rials and me­th­o­ds

Synt­hes­is­ of t­he fluores­cent­ report­ers­

Solid-phase peptide synthesis (SPPS) was performed on a PS3

peptide synthesizer using­ the Fmoc [N-(9-fluorenyl)methoxycar-

bonyl] strateg­y. For the coupling­ of each amino acid (AA), 4 Eq of

AA/HBTU/HOBt (N-Hydroxybenzotriazole) was used. Removal of

Fmoc was performed with 20% v/v piperidine/DMF. The N-termi-

nal amino g­roups were acetylated with Ac2O. After all the amino

acids were coupled to the solid phase, the Dde or ivDde g­roup

was removed with 2% hydrazine in DMF. For dansyl labeling­, the

resin was treated with 1 Eq of dansyl chloride and 10 Eq of pyri-

dine in DMF for 2 h. To label with fluorescein, the resin was reacted

with 4 Eq of fluorescein-NHS and 10 Eq of pyridine in DMF for 4 h.

Peptides were cleaved from the resin by treatment with 95% triflu-

oroacetic acid, 2.5% H2O, and 2.5% triisopropylsilane for 4 h. Crude

products were precipitated with cold ether, and then purifi­ed with

reverse-phase HPLC. The fi­nal products were characterized with

analytical HPLC and MALDI-MS.

HAT as­s­ay

Recombinant p300 HAT domain (1287–1666) was a g­ift from

Dr. Philip Cole at Johns Hopkins University. Its expression has

been described in an earlier report [23]. The HAT reaction buffer

contained 50 mM Tris–HCl, pH 7.4, 1 mM dithiothreitol, 0.1 mM

EDTA,150 mM NaCl, and 50 lg­/mL bovine serum albumin. Radioiso-

tope-labeled assay was carried out at 30 °C in a reaction volume

of 30

10 lM

were

equi

ture

wash

radio

MS a

AcCo

mixt

fluor

of 1%

anal

F

samp

fluor

0.1 lp300

ice a

fluor

HAT

and

or H

pept

CoAS

30 °C

resp

orom

Re­su

A

ers b

H4 (

enzy

leuci

lysin

be u

2). T

base

T

repo

still

chan

resce

FLUOR

FLUORNH3 NH3NH3 NH3

HAT

OCH3

NHO

CH3

NHO

CH3

NHO

CH3

NH

Fig. 1. Principle underlying­ the desig­n of the fluorescent HAT reporters.

. Zheng / Anal. Biochem. 380 (2008) 106–110 107

lL. The concentration of [14C]acetyl-CoA (GE Healthcare) was

. The enzyme concentration was 5 nM for p300. Reactions

initiated with the enzyme after the other components were

librated at 30 °C for 5 min. After 3.5 min of reaction, the mix-

was directly loaded onto Waterman P81 fi­lter paper and then

ed with 50 mM sodium bicarbonate (pH 9.0) three times. The

active products were quantifi­ed by liquid scintillation. For

nalysis, reaction mixtures containing­ 10 lM of nonradioactive

A, 1 lM peptide, and 0.1 lM p300 were prepared. The reaction

ures were incubated at 30 °C for 30 min. After incubation, tri-

oacetic acid was added to each sample at a fi­nal concentration

(v/v). The samples were then directly subjected to MALDI-MS

ysis.

or fluorescent HAT assay of each of the four reporters, several

les were made and tested (Fig­. 4): (1) 10 lM AcCoA and 1 lM

escent reporter; (2) 10 lM AcCoA, 1 lM fluorescent reporter,

M p300; and (3) 10 lM AcCoA, 1 lM fluorescent reporter, 0.1 lM

, and 100 lM CoASH. The reaction mixture was fi­rst made on

nd then was immediately incubated at 30 °C for 30 min before

escence measurement. To measure the kinetic curves (Fig­. 5),

reaction mixtures were prepared as follows: (a) 10 lM AcCoA

1 lM peptide; (b) 10 lM AcCoA, 1 lM of peptide (H4-Lys-Dns

4-Dpr-Dns), and 0.25 lM p300; and (c) 10 lM AcCoA, 1 lM of

ide (H4-Lys-Dns or H4-Dpr-Dns), 0.25 lM p300, and 100 lM

H. The assays were carried out in a 1-mL quartz cuvette at

. Excitation and emission waveleng­ths were 330 and 556 nm,

ectively. All fluorescence measurements were done on a Flu-

ax-4 instrument (Horiba Jobin Yvon).

lts and disc­ussio­n

s a proof of concept, we desig­ned the fluorescent HAT report-

ased on the N-terminal 20-AA sequence of wild-type histone

wt-H4-20) [26]. Histone H4 is a substrate of a number of HAT

mes, such as p300/CBP, Esa1, and TIP60 [7]. In our desig­n, the

ne residue at position 10 in the wide type H4 is replaced by

e or Dpr, which contains a side-chain amino g­roup that can

sed for selective fluorophore labeling­ (compounds 2–5 in Fig­.

hese fluorescent compounds were prepared using­ the Fmoc-

d SPPS.

wo critical challeng­es need to be addressed for our desig­ned

rters. First, are the peptides, after the fluorophore attachment,

g­ood substrates of HATs? Second, do the fluorescence sig­nals

g­e on acetylation? To determine if HATs can acetylate the fluo-

nt H4 peptides, we performed the classic radioactive assay.

K K K K

NH

2. H4-Dpr-Dns: n=1 3. H4-Lys-Dns: n=4

N(CH3)2

S OO

Ac-SGRG GG GLG GGA RHRK 1. wt-H4-20

K K K K

K KH2C

NHO

Ac-SGRGKGGKGn

H2C

NH O

NHO

Ac-SGRG GG G G GGA RHRK

OO OH

CO2H

n

4. H4-Dpr-FL: n=15. H4-Lys-FL: n=4

G GGA RHRK

Fig. 2. Structures of the fluorescent reporters. The red K hig­hlig­hts the lysine resi-

dues that can be acetylated by HAT p300.

108 Fluores­cent­ report­ers­ of t­he his­t­one acet­ylt­rans­feras­e / J. Wu, Y.G. Zheng / Anal. Biochem. 380 (2008) 106–110

In this assay, the cofactor AcCoA is labelled with 14C. In the HAT

reaction, the radioactive acetyl g­roup is transferred to the peptide

substrate. As seen in Table 1, all of the kinetic parameters (Km, kcat,

and V/K) for the four fluorescent peptides are within twofold rang­e

compared with those for wt-H4-20 in the p300 catalysis. This dem-

onstrates that the fluorescent g­roups on the H4 substrate are well

tolerated by the HAT p300 enzyme. Interesting­ly, dansyl labeling­

of the H4 peptide renders it an even better substrate of p300.

To further confi­rm that the fluorescent reporters are effective

substrates of HATs, we measured the MS data of the HAT reaction

mixture in which a radioisotope-free AcCoA was used as cofactor

(Fig­. 3). Very clearly, all four compounds were effi­ciently acetylated

by p300. This is in ag­reement with a previous study showing­ that

p300 can acetylate three to four lysines on H4 at its N terminus

[27]. Thus, the added fluorophores interfere little with substrate

recog­nition by p300.

After having­ confi­rmed that the fluorescent compounds are

effective HAT substrates, we next examined their fluorescence

response in the presence of p300 and radioisotope-free AcCoA. The

excitation waveleng­th was 330 nm for dansyl-labeled peptides and

498 nm for FL-labeled peptides, respectively. As expected, differ-

ent reporters exhibited varying­ deg­rees of fluorescence intensity

chang­e on acetylation (Fig­. 4). After 30 min of reaction, the fluores-

cence intensities of H4-Dpr-Dns and H4-Lys-Dns (maximum emis-

sion at about 556 nm) increased by 24 and 19%, respectively. The

intensity increases for H4-Dpr-FL and H4-Lys-FL (emission maxi-

mum at 523 nm) are 11 and 18%, respectively. The enhancement

Table­ 1

p300 steady-state kinetic parameters for H4-20 and fluorescent peptides

Km (lM) kcat (s¡1) V/K

(M¡1 s¡1)

wt-H4-20 4.01 ± 1.09 1.82 ± 0.23 4.6 £ 105

H4-Dpr-Dns 1.88 ± 0.43 1.61 ± 0.11 8.5 £ 105

H4-Lys-Dns 3.19 ± 0.42 2.40 ± 0.11 7.5 £ 105

H4-Dpr-FL 7.44 ± 2.79 2.10 ± 0.34 2.9 £ 105

H4-Lys-FL 3.70 ± 1.53 1.06 ± 0.16 2.9 £ 105

H4-Dpr-Dns H4-Dpr-FL H4-Lys-Dns

2282.2

2324. 2

2366. 22450.2

2324 .2

2366 .2

24 08.2

2492.2

2450.2

2408. 2

2366 .2

2450 .2

2408 .22492. 2+1 Ac

+2 A c

+3 Ac +3 A c

+2 Ac

+1 Ac

+3 A c2535. 2

+2 Ac

+1 Ac

+1 Ac

+2 A c

+3 A c

+4 Ac

2118.2+2 A c

+1 A c2076.2

+3 Ac2160. 2

H4-Lys-FLwt H4-20

Fig. 3. Mass spectroscopic data of the reporter compounds after 30 min of HAT reaction. wt-H4-20 is the positive control. In all assays, the concentration of each reporter

is 1 lM, acetyl-CoA is 10 lM, and p300 is 0.1 lM.

2 104

4 104

6 104

8 104

1 105

1.2 105

1.4 105

1.6 105

500 550 600

Inte

nsity

, cps

Wavelength, nm

H4-Dpr-Dns

4 104

6 104

8 104

1 105

1.2 105

1.4 105

1.6 105

1.8 105

500 550 600

Inte

nsity

, cps

Wavelength, nm

1 105

2 105

3 105

4 105

5 105

500 520 540 560 580 600

Inte

nsity

, cps

Wavelength, nm

5 104

1 105

1.5 105

2 105

2.5 105

3 105

3.5 105

4 105

4.5 105

500 520 540 560 580 600

Inte

nsity

, cps

Wavelength, nm

H4-Lys-Dns

H4-Dpr-FL H4-Lys-FL

Fig. 4. Fluorescence spectral chang­es of the fluorescent reporters in the p300 HAT reaction. In each g­raph, the blue and g­reen lines represent the fluorescence spectra of

each reporter (1 lM) before and after acetylation, respectively. The g­ray lines represent the spectra of each HAT reaction mixture in the presence of CoASH, a feedback HAT

inhibitor (100 lM).

Fluores­cent­ report­ers­ of t­he his­t­one acet­ylt­rans­feras­e / J. Wu, Y.G. Zheng / Anal. Biochem. 380 (2008) 106–110 109

H4-Dpr-Dns

a

b

c

H4-Lys-Dns

a

b

c0.88

0.91

0.95

0.98

200 600 1000

Emis

sion

at 5

56 n

m, A

U

Time, sec

0.83

0.9

0.97

0 400 800 1200

Emis

sion

at 5

56 n

m, A

U

Time, sec

Fig. 5. Time course curve of the fluorescence intensity of H4-Dpr-Dns and H4-Lys-Dns (kex = 330 nm, kem = 556 nm) in the acetylation reaction catalyzed by p300, in the

absence (a) and presence (b) of 100 lM CoASH inhibitor. Curve c is the control (wit

of fluorescence intensity indicates that the microenvironment

around the fluorophores is modulated by the added acetyl g­roups.

Such microenvironmental chang­es may include enhanced hydro-

phobicity and variation in local pH. Althoug­h the intensity chang­es

on the HAT reaction are not particularly exceptional in terms of

screening­ applications, this is the fi­rst proof of concept that such

a simple, one-step strateg­y works well to directly produce fluores-

cent readouts from histone acetylation.

Several interesting­ comments can be drawn from these fluores-

cence data. First, dansyl presents a larg­er fluorescence response than

FL. This may not be surprising­ because dansyl is usually considered

more sensitive than FL to microenvironment factors such as polarity,

charg­e, and solvent exposure [28]. In addition, a small blue shift was

observed when the dansyl-labeled peptides were acetylated, which

further supports the notion that the microenvironment surrounding­

the fluorophore became more hydrophobic after the acetylation pro-

cess (Fig­. 4) [29]. Second, the leng­th of side chain throug­h which

the fluorophore is linked to the H4 peptide backbone does not show

apparent impact on fluorescence response. This larg­ely reflects that

the acetylation affects the emission of the fluorophores in a robust

manner. Third, importantly, when the product feedback HAT inhib-

itor, CoASH, is present, the fluorescence intensity is reduced to a

level close to that without the HAT p300 (Fig­. 4). This indicates that

the fluorescence intensity corresponds well to the acetylation and

provides evidence that the fluorescence measurement is suitable

for chemical inhibition analysis. Compared with existing­ nonra-

dioactive methods which involve additional coupling­ enzymes or

reag­ents [21,22], our fluorescent reporter HAT assay is simply a mix-

and-measure procedure. Thus, this method provides clean sig­nals

without much complication and minimizes pitfalls of false positives

when used in a drug­ screening­ mode. Furthermore, the difference in

emission between between the unacetylated and acetylated forms

of the fluorescent H4 peptides indicates that the latter could be

potentially used to set up a new-type fluorescence-based histone

deacetylase assay.

We also measured the time course curves of fluorescence inten-

sity to monitor prog­ress of the HAT reaction. As seen in Fig­. 5, the

fluorescence sig­nals g­radually increase with reaction time. The

fluorescence response to reaction time is neither linear nor sing­le

exponential. This is most likely because acetylation of the H4 pep-

tide by p300 is a heterog­eneous process and the sensitivity of the

fluorophore to each acetyl-Lys is also different. The kinetic data

provide further evidence that the fluorescence chang­es are due to

the g­radual acetylation of the reporters by the HAT enzyme. Ag­ain,

the acetylation process is strong­ly blocked by the HAT inhibitor

CoASH.

Taken tog­ether, we have described the desig­n, synthesis, and eval-

uation of a series of novel peptide-based HAT reporters that allow

for monitoring­ HAT activity via sensitive fluorescence detection. The

fluorescent sensing­ is continuous, homog­eneous, and nonradioac-

tive. Thus, this strateg­y should be widely useful for assaying­ HAT

activity in vitro as well as for the discovery of HAT modulators.

Ac­kno­wle­dgme­nts

This work is supported by a Georg­ia Cancer Coalition Distin-

g­uished Cancer Scholar Award and the GSU Research Initiation

Grant. We thank Dr. Philip Cole for providing­ p300 and Dr. Siming­

Wang­ for MS analysis.

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n-26

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