Regulation of histone acetylation and nucleosome assembly ... - Nature

© 2006 Nature Publishing Group http://www.nature.com/nsmb

ARTICLES

Regulation of histone acetylation and nucleosome assembly by transcription factor JDP2 Chunyuan Jin1,2,8, Kohsuke Kato3, Takahiko Chimura4, Takahito Yamasaki1, Koji Nakade1, Takehide Murata1, Hongjie Li1,2, Jianzhi Pan1, Mujun Zhao5, Kailai Sun2, Robert Chiu6, Takashi Ito7, Kyosuke Nagata3, Masami Horikoshi4 & Kazunari K Yokoyama1 Jun dimerization protein-2 (JDP2) is a component of the AP-1 transcription factor that represses transactivation mediated by the Jun family of proteins. Here, we examine the functional mechanisms of JDP2 and show that it can inhibit p300-mediated acetylation of core histones in vitro and in vivo. Inhibition of histone acetylation requires the N-terminal 35 residues and the DNA-binding region of JDP2. In addition, we demonstrate that JDP2 has histone-chaperone activity in vitro. These results suggest that the sequence-specific DNA-binding protein JDP2 may control transcription via direct regulation of the modification of histones and the assembly of chromatin.

Histone acetyltransferases (HATs) regulate transcription by covalently modifying histone proteins. Their activities are counteracted by histone deacetylases (HDACs) and regulated by certain cellular and viral proteins as well as by post-translational modification1,2. For example, the basic helix-loop-helix protein Twist and the adenoviral oncoprotein E1A inhibit the acetyltransferase activities of p300/CBP and PCAF3,4, and TAT, the transactivator protein of human immunodeficiency virus type 1, represses acetylation that involves Tip60 and TAFII250 (refs. 5,6). One potential mechanism for the inhibition of HATs is the direct binding of acetyltransferases to regulatory factors. Alternatively, phosphorylation of GCN5 by the Ku DNA-dependent protein kinase inhibits its HAT activity7, whereas phosphorylation of p300/CBP and ATF-2 stimulates their HAT activities8–10. Recently, a new type of human cellular complex, termed inhibitor of histone acetyltransferases (INHAT), has been shown to inhibit the HAT activities of p300/CBP and PCAF by binding histones, thereby preventing them from serving as substrates for acetyltransferases11. One type of INHAT complex, known as template-activating factor-1b (TAF-1b; also known as the myeloid leukemia–associated oncoprotein Set), has been shown initially to have nucleosome-assembly activity12,13. JDP2, a component of the transcription factor AP-1, has a basic leucine zipper (bZIP) motif in its C-terminal region. JDP2 can dimerize with itself or with c-Jun, JunB, JunD or ATF-2, thereby inhibiting transactivation by Jun, ATF-2 and C/EBPg14–16. These observations suggest that JDP2 may be a repressor module within AP-1. JDP2 is

involved in a variety of transcriptional responses associated with AP-1, such as UV-induced apoptosis17, cell differentiation18,19, tumorigenesis and antitumorigenesis20,21. JDP2 can also function as a repressor by recruiting HDAC3 to the promoter region of the c-jun gene18. Moreover, it can act as a coactivator of progesterone receptors22, a phenomenon that suggests that JDP2 might have multiple functions. We set out to examine in detail the way in which JDP2 functions. Here, we demonstrate that mouse JDP2 can inhibit HAT activity of p300 both in vitro and in vivo and that the region of JDP2 including the DNA-binding domain has an indispensable role in the repression of HAT activity. Moreover, we show that JDP2 also has histonechaperone activity in vitro and is involved in nucleosome assembly in vivo. Our results indicate that JDP2, a sequence-specific transcription factor, regulates gene expression via direct control of the modification of histones and the assembly of nucleosomes. RESULTS JDP2 inhibits HAT activity in vitro and in vivo We have reported previously that JDP2 represses transactivation by histone acetyltransferase p300 and by ATF-2 (ref. 18). We postulated that JDP2 might function as a transcriptional repressor by directly inhibiting these enzymatic activities. To investigate this possibility, we examined the effect of JDP2 on the p300-mediated acetylation of histones23 in filter-binding assays. We first characterized the biochemical parameters of the HAT activity of p300 in this assay to

1Gene Engineering Division, Dept. of Biological Systems, BioResource Center, RIKEN (The Institute of Physical & Chemical Research), Tsukuba Science City, Ibaraki 305-0074, Japan. 2Dept. of Medical Genetics, China Medical University, Shenyang 110001, China. 3Dept. of Infection Biology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba 305-8575, Japan. 4Institute of Molecular & Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. 5Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Science, Chinese Academy of Sciences, Shanghai 20031, China. 6Dental Research Institute, University of California at Los Angeles School of Medicine, Los Angeles, California 90095-1668, USA. 7Dept. of Biochemistry, Nagasaki University School of Medicine, 1-24-4 Sakamoto, Nagasaki 852-8523, Japan. 8Present address: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, US National Institutes of Health, Bethesda, Maryland 20892, USA. Correspondence should be addressed to K.K.Y. ([email protected]).

Received 14 October 2005; accepted 17 January 2006; published online 5 March 2006; doi:10.1038/nsmb1063

NATURE STRUCTURAL & MOLECULAR BIOLOGY

VOLUME 13

NUMBER 4

APRIL 2006

331

ARTICLES Figure 1b. We further show that JDP2 can inhibit acetylation of core histones by other HATs, including CBP, PCAF and GCN5 (Fig. 1e). To explore the HAT-inhibitory activity of JDP2 in vivo, we examined the relationship between JDP2 occupancy and the status of histone acetylation in the JDP2-binding region using chromatin immunoprecipitation (ChIP) (Fig. 1f). All ChIP data was normalized by nucleosome density, which was measured by the binding of antibodies specific for the C-terminal tail of H3. Consistent with our previous study18, recruitment of JDP2 to the differentiation regulatory element (DRE) of the c-jun gene decreased B30% 48 h after the start of retinoic acid treatment of F9 cells transfected with

1.4

b

1.2 1.0 0.8 0.6

18 7

Relative HAT activity

d

2 – – – +

3 – – + –

4 – + – –

1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

1 H2A + p300 – JDP2 –

6 + + – –

7 8 + + ++ – – + – –

9 10 11 + + + – – – ++ – – – + ++

2 + + –

1.2 1.0 0.8 0.6 0.4 0.2

0.0 1 H2B + p300 – JDP2 –

3 + + +

2 + + –

3 + + +


0.8 0.6 0.4 0.2 4 + – – – – +

5 – + – – – –

6 – + – – + – 7

1.0

6 Fold difference

1.2 0.8 0.6 0.4 0.2

8 – – + – – –

9 – – + – + –

10 – – + – – +

11 – – – + – –

5 4 3 2 1

0.0 1 RA –

7 – + – – – +

2 +

3 –

4 +

Empty vector JDP2 Anti-H3

0

1 RA –

12 – – – + + –

2 +

3 –

4 +

Empty vector JDP2 Anti-JDP2

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

2 + + –

1.0 0.8 0.6 0.4 0.2

0.0 1 p300 – JDP2 – Histones +

4 + – +

3 + + +

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

1 H4 + p300 – JDP2 –

2 + + –

2 + – +

3 + + –

3 + + +

1.0 0.8 0.6 0.4 0.2

0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 p300 + + + + + + + + + + + + + + + + + + JDP2 – – – + + – – + + – – – + + – – + +

13 – – – + – +

Nucleosomes –

–

CRE CRE mCRE mCRE DRE DRE mDRE mDRE bZIP–ATF-2 + + + + + + + + + + + + + + + + + + 5

6

4

5

Fold difference

3 + – – – + –

Fold difference


1.0

2 + – – – – –

3 + + –

1.2

1.2

1.2

1 – – – – – –

2 + – –

1 H3 + p300 – JDP2 –

g

1.4

p300 CBP PCAF GCN5 JDP2 GST

1 – – –

p300 JDP2 GST

1.6

0.0

Fold difference

5 + – – –


1 – – – –


0.2

p300 JDP2 GST BSA

f

H3 H2B H2A H4

0.4 0.0

e

c Size (kDa)



a



determine the optimal experimental conditions (Supplementary Data and Supplementary Fig. 1 online). Acetylation by p300 was inhibited specifically and dose-dependently by exogenous JDP2 containing a GST tag (Fig. 1a). These results were confirmed by gel-electrophoretic analysis (Fig. 1b). JDP2 itself was not acetylated by p300 (Fig. 1c). In addition, the GST tag does not contribute to the inhibition activity, as JDP2 fused to a histidine tag (His-JDP2) also inhibited p300 activity (Supplementary Fig. 2 online). We next tested whether the HAT-inhibitory activity of JDP2 is specific for particular core histones. JDP2 inhibited the acetylation of all core histones tested (Fig. 1d), consistent with the results in

3 2 1 0

RA

1 –

2 +

3 –

4 +

5 –

6 +

7 –

8 +

Empty vector JDP2 Empty vector JDP2 Anti-AcH3

4 3 2 1 0

RA

1 –

2 +

3 –

4 +

5 –

6 +

7 –

8 +

Empty vector JDP2 Empty vector JDP2

Anti-AcH4

Anti-AcK8H4

9 –

10 +

N91A

Anti-AcK16H4

Figure 1 Inhibition by JDP2 of histone acetylation. (a) Filter-binding assays were performed with 2 mg of core histones and 10 pmol (+) or 30 pmol (++) of indicated proteins. Lane 1, buffer; lanes 5–11, 4 pmol of p300. (b) Analysis by PAGE of HAT activity. (c) JDP2 was not acetylated by p300. (d) Filterbinding assays were performed without (–) or with (+) GST-JDP2. (e) Each histone acetyltransferase (3 pmol) was incubated without or with JDP2 before the addition of core histones. (f) ChIP assays were performed with indicated antibodies (Ac prefix denotes acetylated protein), 48 h after treatment (+) or not (–) with retinoic acid. (g) Filter-binding assays were performed with indicated reconstituted mononucleosomes (even-numbered lanes, 50 ng; odd-numbered lanes, 100 ng), in the absence or presence of GST-JDP2. Lanes 1 and 10, buffer. In panels a and c–f, each bar represents an average from two or three experiments, with s.d. shown.

332

VOLUME 13

NUMBER 4

APRIL 2006


data not shown). When JDP2 is first incubated with the bZIP domain of ATF2 (bZIP-ATF2) and then added to reconstituted nucleosomes, the heterodimer appreciably binds (Supplementary Fig. 3) and inhibits the acetylation of nucleosomes containing CRE or DRE by 90–95% (Fig. 1g). By contrast, we detected a reduction of B50% when reconstituted mononucleosomes contained mCRE or mDRE. This reduction in the extent of inhibition might be due, at least in part, to the fact that mCRE or mDRE still bind weakly to JDP2. Our data indicate that the binding of JDP2 as a heterodimer to nucleosomes is indispensable for its HAT-inhibition activity.

empty vector. During retinoic acid–induced cellular differentiation, histones H3 and H4 became hyperacetylated. In contrast, in both undifferentiated and differentiated F9 cells stably expressing JDP2, we observed an increase in the level of JDP2 at the DRE (Fig. 1f). The increase in the occupancy of JDP2 in JDP2-transfected F9 cells correlates with reduced levels of acetylated histones H3 and H4 induced by retinoic acid treatment (Fig. 1f). Exposure of F9 cells to retinoic acid led to acetylation of H4 Lys8 and Lys16 but not Lys5 and Lys12 (Fig. 1f and data not shown). Overexpression of JDP2 apparently repressed the retinoic acid–induced acetylation of H4 Lys8 and Lys16 (Fig. 1f). These data suggest that JDP2 can inhibit the acetylation of histone H3 and acetylation at specific residues of H4 in vivo.

JDP2 binds directly to core histones Examining the association of JDP2 with histones, we showed that in vitro–purified GST-JDP2 but not GST alone was efficiently immunoprecipitated by histone-specific antibodies (Fig. 2a). Moreover, GST-JDP2 in a crude lysate of Escherichia coli also specifically bound core histones (Fig. 2b). To identify histones that can bind JDP2, we performed immunoprecipitation followed by western blotting analysis using GST-JDP2 and purified individual core histones. GST-JDP2 interacted strongly with histones H3 and H4 and weakly with H2A and H2B (Fig. 2a). As the HAT-inhibition activity of JDP2 on core histones does not depend on ionic strength (data not shown), nonspecific electrostatic interactions seem unlikely to explain the binding of JDP2 to histones (Supplementary Data). We next performed immunoprecipitation and western blotting analysis with a nuclear extract from Cos1 cells transiently expressing JDP2 to determine whether JDP2 can interact with core histones in vivo. The results reveal the presence of all four core histones in immunoprecipitates of extracts prepared with antibodies specific for JDP2 (Fig. 2c).

+

+ +

+ +

H3

+ +

H4

+ +

+ +

Input

GST-JDP2 GST + Histones +

+

IP

+ + +

+

Input

+

JDP2

–

+

–

+

–

+ WB: anti-H3

GST-JDP2

42

c

IP: anti-histones

+

P2

H2B

JD

H2A

ti-

GST GST-JDP2

b

IP: anti-histones Histones

An

Input Histones

Ab

a

N

Inhibition of acetylation of reconstituted nucleosomes As JDP2 did not inhibit acetylation of nucleosomes by p300 (Supplementary Fig. 3 online), we tested whether the DNA-binding activity of JDP2 is involved in its HAT inhibition function by using reconstituted nucleosomes containing JDP2-binding sites. We reconstituted mononucleosomes with 147-base-pair (bp) DNA fragments that contained either the DRE or the cAMP-response element (CRE) by salt dialysis13. We also reconstituted mononucleosomes with DNA containing mutated DRE and CRE (mDRE and mCRE). In a previous study, we have shown that JDP2 alone binds directly to both CRE and DRE but not to mCRE and mDRE16,18. Electrophoretic mobility shift assays (EMSAs) confirmed the sequence-specific interaction between JDP2 alone and the 147-bp DRE-containing fragment (Supplementary Fig. 3). However, JDP2 alone only weakly binds mononucleosomes containing DRE or CRE, and it does not inhibit acetylation of the reconstituted nucleosomes by p300 (Supplementary Fig. 3 and

GST-JDP2

42

WB: anti-H4 32

32

GST

(kDa) Size

1

3

2

4

6 7 8 5 WB: anti-GST

9

10

11

(kDa) Size

12

GST

WB: anti-H2A


WB: anti-H2B WB: anti-JDP2

2

3

4

5

6

00

P2 JD ti-

t An

pu

G Ig

In

200

ATF-2 42 32 18

124 83 1

5

p3

Size (kDa)

83 JDP2

4

3

ti-

t pu

p300

32 18

2

An

G

In

Size (kDa)

Ig

Input

Input

Size (kDa)

1

e

IVT p300 GST-p300C

GST-p300M

Size (kDa)

GST-p300N

IVT JDP2

GST-JDP2

d GST-ATF-2


ARTICLES

42 32 1

p300 JDP2

200

1 2 3 WB: anti-p300

1 2 3 WB: anti-HA

2

Figure 2 Interaction of JDP2 with histones. (a) Binding of histones to JDP2 in vitro. GST-JDP2 was incubated with core histones or with each histone and immunoprecipitated (IP) with histone-specific antibodies, then western blotted (WB) with GST-specific antibodies. (b) Interaction between purified histones and GST-JDP2 in crude lysates of E. coli. (c) JDP2 associated with histones in vivo. Cell lysates from JDP2-overexpressing Cos-1 cells were subjected to IP without (NAb) or with antibodies against JDP2, then western blotted with indicated antibodies. (d) JDP2 did not interact with p300 in vitro. Left, GST pull-down results after in vitro–translated (IVT) 35S-labeled JDP2 was incubated with GST–ATF-2 or the N-terminal (p300N), middle (p300M) or C-terminal region (p300C) of p300. Right, GST pull-down results after IVT p300 was incubated with GST-JDP2. (e) JDP2 did not associate with p300 in vivo. Cell lysates prepared from HeLa cells expressing HA–ATF-2 or HA-JDP2 were immunoprecipitated with antibodies to p300 or JDP2 and bound proteins were detected with indicated antibodies.


VOLUME 13

NUMBER 4

APRIL 2006

333

To examine whether JDP2 can bind acetyltransferases directly, we performed binding assays using in vitro–translated 35S-labeled JDP2 and several GST-p300 constructs. By contrast to the interaction between 35S-labeled JDP2 and GST-ATF2, we detected no binding of JDP2 to several truncated GST-p300 variants (Fig. 2d). Similarly, we observed no interaction between GST-JDP2 and in vitro–translated p300 (Fig. 2d). To examine the interaction between JDP2 and p300 in vivo, we performed immunoprecipitation and western blotting analysis using nuclear extracts from HeLa cells that expressed hemagglutinin (HA)-tagged forms of ATF-2 and JDP2. The results showed the presence of ATF2 but not JDP2 in immunoprecipitations prepared with antibodies to p300 (Fig. 2e). Similarly, p300 was not detected in immunoprecipitations prepared with antibodies to JDP2 (Fig. 2e). The domains for histone binding and HAT inhibition We prepared several recombinant JDP2 deletion constructs (Fig. 3a) to map the histone-binding domain of JDP2 (Fig. 3b). Proteins truncated from residue 70 or 102 (constructs NT70 and NT102), resulting in deletion of the bZIP domain, as well as the substitution

102

ZIP

135

Histone-binding activity +

INHAT activity

163

WT 35CT 70CT NT135 NT102 NT70

++++ +++ +/– ++++ +++ +

+ +

bZIP FL34R

+ +++

– +

INHAT domain Histone-binding domain

102 70

b

Input

Histones

+ GST

+ FL34R

+ bZIP

+ NT70

+ NT102

+ NT135

+ 70CT

+ 35CT

+ GST

+ FL34R

+ bZIP

+ NT70

IP: anti-Histones

+ NT102

+ NT135

+ 70CT

Size (kDa) 42

+ 35CT

Input

+ 35–70

35 35

+ – +

IP: anti-Histones + m35–102

Basic

+ 35–102

70

+ 35–70

35

+ GST

1

+ m35–102

GST

mutant (FL34R) in which the third and the fourth leucine residues were changed to arginines, retained histone-binding activity. However, the construct lacking residues 1–70 (70CT) and the construct with the bZIP domain only (designated bZIP) did not bind histones at all. These results indicate that the histone-binding domain of JDP2 is located between residues 35 and 70. Further deletion analysis shows that the region between residues 35 and 70 is sufficient for the histonebinding activity of JDP2 (Fig. 3b, right gel). Using the same set of JDP2 deletion constructs, we mapped the region of JDP2 that contains the HAT-inhibition activity. We first assessed the purity of each protein by SDS-PAGE (Supplementary Fig. 4 online). The HAT assay is shown schematically in Figure 3c. Deletion of the N-terminal 35 residues (construct 35CT), truncation from residue 102 or 135 (constructs NT102 and NT135) or mutations in the bZIP domain (construct FL34R) did not affect inhibition of the p300 HAT activity (Fig. 3d). By contrast, the GST-JDP2 variants lacking the histone-binding domain (constructs 70CT and bZIP) were virtually inactive. Moreover, the JDP2 variant that contains the histone-binding domain but lacks the basic region of the bZIP

+ 35–102

a

+ GST

Size (kDa) 42

JDP2

32

32 4

5

6

7

8

9

10

11

12 13 14 15

17

16

18

19

d p300, [3H]AcCoA Histones JDP2

30 °C

30 °C

15 min

45 min

Analysis

21

e

22

23

24

1.2 1.0 0.8 0.6 0.4 0.2 0.0

bZIP

FL34R

NT70

NT102

70CT

NT135

35CT

WT

1 2 3 4 5 6 7 8 9 10 p300 – + + + + + + + + + JDP2 – –

77

92

35–102: 35

102

m35–102: 35

102


c

20

WB: anti-GST

WB: anti-GST

1.2 1.0 0.8 0.6 0.4 0.2 0.0

1 p300 – JDP2 –

2 + –

3

4

5

35–102 +

3

m35–102 +

2

35–70 +

1



ARTICLES

Figure 3 Mapping of the histone-binding and HAT-inhibition domains of JDP2. (a) Schematic representation of wild-type and mutant forms of JDP2 and a summary of the results of analysis of the histone-binding and HAT-inhibition activities of JDP2. HAT-inhibition activity is designated as ++++ (80–100% of the wild type), +++ (60–80%), ++ (40–60%), + (20–40%) or +/ (0–20%); histone-binding activity as + or –. (b) Mapping of the histone-binding domain. Core histones were incubated with GST or various derivatives of GST-JDP2, then immunoprecipitation (IP) was performed with anti-histones. Bound proteins were detected with anti-GST. (c) Schematic representation of the design of the experiment for measuring HAT-inhibition activity. AcCoA, acetyl CoA. (d) Mapping of the HAT-inhibition activity of JDP2 using filter-binding assays. (e) The basic region of JDP2 is essential for the HAT-inhibition activity of JDP2. p300 was incubated separately with various mutant forms of JDP2, namely 35–70, 35–102 and m35–102, in the presence of core histones. In d and e, each bar represents an average from three experiments, with s.d. shown.

334

VOLUME 13

NUMBER 4

APRIL 2006



ARTICLES only in the presence of core histones. Notably, JDP2 is as active as CIA1 (Fig. 4a). Next, we analyzed the chromatin-assembly activity of JDP2 by EMSA using a 198-bp fragment of 5S DNA that contains a well-characterized nucleosome-positioning signal. JDP2 facilitated the formation of mononucleosomes on the DNA fragment (Fig. 4b) in a dose-dependent manner, although the efficiency was lower than that of TAF-1b. In support of nucleosome assembly, western blotting analysis revealed that mononucleosomes eluted from the gel contained histone H3 (Fig. 4b, lower gel). Together, our results indicate that JDP2 has histone-chaperone activity in vitro. To address whether the assembly or disassembly of chromatin occurs around the JDP2-binding site in vivo and whether JDP2 is involved in these processes, we compared the density of nucleosomes near the DRE region in F9 cells transfected with empty vector and in cells that overexpressed JDP2. We monitored the hybridization signals with a DRE probe on chromatins extensively digested by micrococcal nuclease. The hybridization signal of mononucleosomes obtained from JDP2-overexpressing F9 cells was higher than that obtained from control cells (Fig. 4c, lower gel, compare lanes 1 and 2 with lanes 4 and 5), suggesting that JDP2 may be involved in nucleosome assembly. To examine the organization of nucleosomes near the DRE region in detail, we performed MNase digestion followed by

motif (construct NT70) also did not inhibit histone acetylation by p300. These results indicate that the histone-binding domain alone is essential but not sufficient for the HAT-inhibition activity of JDP2. To test whether the basic region of JDP2 is necessary for its HATinhibition activity, we generated two recombinant mutant proteins: 35–102, which included both the histone-binding domain and the basic region, and m35–102, in which residues at positions 88, 90 and 92 within the basic region were mutated (R88G, R90E and K92E). Both 35–102 and m35–102 bound histones efficiently (Fig. 3b), but only 35–102 inhibited histone acetylation by p300 (Fig. 3e). These results indicate that the DNA-binding region is also required for the HAT-inhibition activity of JDP2. JDP2 has intrinsic nucleosome-assembly activity The finding that JDP2 interacts directly with core histones and inhibits the acetylation of histones, as does TAF-1b, raises the possibility that JDP2 may also have histone-chaperone activity. To investigate this possibility, we performed supercoiling assays in vitro12. JDP2 was incubated with core histones; relaxed circular DNA was then added to the reaction mixture. As controls, both yeast Cia1p and human CIA1 were shown to introduce supercoils in the presence of core histones (Fig. 4a)24,25. JDP2 also introduces supercoils into circular DNA, but

a

b Core histones (+)

Buffer GST yCia1p CIA1 Buffer GST JDP2

Core histones (–)

Core histones –

–

–

+

His-TAF-1β –

+

–

–

GST-JDP2 –

–

+

–

Buffer GST JDP2 Buffer GST JDP2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

1

2

3

4

5

6

7

8

+ –

+

+

+

+

–

–

–

–

9 10 11 12

Relaxed/nicked DNA Supercoiled DNA

Relaxed/nicked DNA Supercoiled DNA

Nucleosomes

tRNA (carrier) tRNA (carrier)

c

JDP2 MNase (U)

– 30 15

d

+ – 30

15

75

–

Free DNA JDP2 RA

+ –

– +

–

+

C

T

A

1

G

2

3

4

5

6

7

8

9

(197 bp) Histone H3

1 Size (bp) 1,000 800 600 400

2

3

4

5

6

7

8

9

91 bp

1

200 100

2

3

4

5

6

7

8

–RA/F9 cells –214

Primer 4 –188 –158

–198 –174

+43 Primer 3 +77 –109 –101

+1 –74–66

–144 –136

+82

+RA/F9 cells –109 –101

–214 –198 –174 M1 M2 1

2

3

4

5

6

–144 –136

+1

+82

–74 –66

7

Figure 4 The histone-chaperone activity of JDP2. (a) In the presence of core histones, GST-JDP2 specifically introduced supercoils into circular DNA. (b) Top, nucleosome assembly in the presence of JDP2 on a fragment of 5S DNA. Bottom, the portions of the gels corresponding to the mononucleosomes (upper gel) were excised, eluted and subjected to western blotting (WB) analysis with antibodies for histone H3. (c) MNase-digested DNA and genomic DNA from permeabilized empty vector–transfected F9 cells (–) and from JDP2-overexpressing F9 cells (+) was subjected to agarose gel electrophoresis and stained with ethidium bromide (upper gel). Southern blotting was then performed with a DRE probe (lower gel). M1, 100-bp ladder of standard markers; M2, HindIII-digested l DNA; lanes 3 and 7, BamHI-digested genomic DNA. (d) The mononucleosomal DNA was purified and allowed to anneal to radiolabeled primer 4. Extension and LMPCR allowed the detection of extended products. A sequencing gel provided size markers. The schematic illustration shows the positions of nucleosomes in the absence (upper) and presence (lower) of retinoic acid. Small circles, putative SP-1–binding sites; small hatched circles, putative CTF-binding sites; short arrows, retinoic acid and E1A response element (upstream two are DREs)27.


VOLUME 13

NUMBER 4

APRIL 2006

335

ARTICLES

– + +

– +

– – + +

– +

42

[32P]-CRE

0.8

GST–JDP2 GST

0.6 0.4

2 +

3 +

4 +

5 + N91A

1 p300 + – GST-JDP2

8 9 10

I47A


Q5A

3

F9 RA–

F9 RA+

ATF-2 42 32

Free DNA

Empty vector JDP2

1.0 0.8 I47A RA+

0.4 0.2 0.0 1 – –

2 + –

3 + WT

5 4 6 + + + I47A N91A Q5A

N91A RA+

Q5A RA+

1 2 3 4 5 6

30 25 20 15 10 5 0

Empty vector JDP2

I47A N91A Q5A Collagen 4α1 mRNA

WT RA+

0.6

RA JDP2

6 7 8 9 10

f

Lamin B1 mRNA

Relative CAT activity


e

1.2

– Size + – – – – (kDa) 83

1 2 3 4 5

d

IVT ATF-2

NS

0.0 2

– + – – – –

[32P]-DRE

JDP2–DNA complex

0.2

WT

32 (kDa) 1 Size

c

1.0

Input GST GST-WT GST-Q5A GST-I47A GST-N91A

– +

1.2

WT Q5A I47A N91A

– +

b

WT Q5A I47A N91A

– +

N91A

I47A

N91A

– +

WT

WT

Q5A

GST-JDP2 – GST + Histones +

IP: anti-histones


Input

Q5A I47A

a

0

48

72

RA (h)

30 25 20 15 10 5 0

I47A N91A Q5A

0

48

72

RA (h)

-730/+873c-jun-CAT

Figure 5 Role of histone-binding and HAT-inhibition activities of JDP2 in transcription and differentiation of F9 cells. (a) Binding of core histones to JDP2 and its derivatives. Core histones were incubated with GST-fused wild-type JDP2 (WT) or its derivatives. Then immunoprecipitation (IP) was performed with anti-histones, followed by western blotting (WB) with anti-GST. (b) Filter-binding assays were performed to determine the HAT-inhibition activity of derivatives of JDP2. (c) Left, EMSA showing binding of WT and derivatives (Q5A, I47A or N91A) to [32P]CRE and [32P]DRE. NS, nonspecific complex. Right, GST-pull down assay showing heterodimerization of JDP2 and its derivatives with ATF-2. (d) Effects of JDP2 mutants on transcription. F9 cell lines, stably transfected with –730/+873c-junCAT, were transfected with vectors that encoded WT or its derivatives plus pRSVLacZ. After 24 h of transfection, cells were incubated with 10 6 M retinoic acid for another 48 h and the CAT activity was then determined. (e) Effects of JDP2 and its derivatives on retinoic acid–induced changes in the morphology of F9 cells. (f) Effects of derivatives of JDP2 on the expression of markers of differentiation. In b, d and f, each bar represents an average from three experiments, with s.d. shown.

primer extension and ligation-mediated PCR (LMPCR). When a primer corresponding to nucleotides –188 to –158 (primer 4, Fig. 4d) was allowed to anneal with mononucleosomal DNA prepared from JDP2-overexpressing F9 cells, the final product was 91 bp long, indicating that the 3¢ margin of the nucleosomes was at position –67 and the 5¢ margin was at position –213. These nucleosomes should contain the DRE sequence, which is located between positions –190 and –170. The band intensities of the LMPCR products showed greater deposition of mononucleosomes around the DRE in JDP2overexpressing F9 cells than in control cells (Fig. 4d, compare lanes 1 and 3). Moreover, whereas treatment of control F9 cells with retinoic acid decreases the density of nucleosomes in the DRE region (Fig. 4d, lanes 3 and 4), overexpression of JDP2 results in an increase in this density (Fig. 4d, compare lanes 2 and 4). The results with another primer corresponding to nucleotides +71 to +43 (primer 3, Fig. 4d) were similar to those obtained with primer 4 (data not shown). ChIP assays with antibodies to the C-terminal region of histone H3 confirmed that nucleosome density near the DRE region was reduced by 35% upon retinoic acid treatment of control cells but not of JDP2transfected cells (Fig. 1f). These results suggest that JDP2 is involved in nucleosome assembly in vivo. Roles of histone-binding and HAT-inhibition activities On the basis of systematic mutational analysis26, we generated point mutations I47A and N91A, which are expected to affect histonebinding and HAT-inhibition activity, respectively. As a control, we generated the Q5A mutant. The mutant proteins are presumably stable, as they seem to be resistant to proteolysis as demonstrated by SDS-PAGE (Supplementary Fig. 4). Mutant I47A had no histonebinding activity or HAT-inhibition activity. N91A had histone-binding activity but no HAT-inhibition activity, whereas Q5A retained both

336

VOLUME 13

activities (Fig. 5a,b). All three mutant proteins had both DNA-binding and dimerization activity (Fig. 5c). We next examined the effects of these mutations on transcription from the c-jun promoter. F9 cells transformed with the –730/+873c-jun-CAT reporter construct were established previously27. We transiently transfected these transformed cells with a plasmid encoding either wild-type or mutant JDP2. We then measured CAT activity after the cells had been incubated with 10 6 M retinoic acid for 48 h. Expression of wild-type JDP2 or Q5A completely repressed the CAT activity; in contrast, expression of I47A or N91A did not suppress the CAT reporter activity (Fig. 5d). We generated F9 cells that stably express wild-type JDP2, I47A, N91A or Q5A to examine the effects of these proteins on retinoic acid– induced differentiation. The JDP2- or Q5A-expressing cells showed minimal evidence of differentiation (Fig. 5e). By contrast, I47A- or N91A-expressing cells showed changes typical of differentiation and resembled, for the most part, retinoic acid–treated wild-type F9 cells. To confirm these results, we used real-time PCR to compare the respective levels of expression of genes that are markers of differentiation (Fig. 5f). The levels of retinoic acid–induced expression of genes for collagen type 4a1 and laminin B1, two markers of endoderm differentiation, were approximately six-fold lower in JDP2- and in Q5A-expressing cells than in control cells after a 72-h treatment with retinoic acid. However, I47A- and N91A-expressing cells did not show a similar delay in gene expression. These results indicate that both the histone-binding and the HAT-inhibition activity of JDP2 are involved in the retinoic acid–induced differentiation of F9 cells. DISCUSSION The HAT-inhibition activity of JDP2 The mechanism of HAT inhibition by JDP2 seems to differ from that by which the originally identified INHAT complex, or E1A, functions.

NUMBER 4

APRIL 2006



ARTICLES E1A inhibits HAT activity by binding directly to the HAT domain of histone acetyltransferase3,4, whereas TAF-1b inhibits histone acetylation by binding directly to histones and preventing them from serving as substrates for acetylases11. The histone-binding domain of TAF-1b, which is rich in acidic residues, is sufficient by itself for INHAT activity11. However, the histone-binding region of JDP2, which is not rich in acidic residues, is essential but not sufficient for the HATinhibitory activity of JDP2. The basic region within the bZIP motif, which can bind DNA, is also required for the HAT-inhibition activity of JDP2. The observations that the N91A mutant has DNA-binding activity but no HAT-inhibition activity and that the basic region is essential for the HAT-inhibition activity of JDP2 in a reaction system without DNA suggest that the DNA-binding domain may have a function that is independent of DNA binding. In addition, the observation that JDP2 is able to inhibit acetylation by p300 in the presence of excess amounts of peptides derived from each histone (Supplementary Fig. 2) indicates that JDP2 might somehow interfere directly with the enzymatic activity of p300. The N91A mutant, which has histone-binding activity but no HATinhibition activity (Fig. 5a,b), does not inhibit retinoic acid–induced acetylation of H4 Lys16, suggesting that in vivo inhibition of retinoic acid–induced acetylation by JDP2 is due to its HAT-inhibition activity (Fig. 1f). JDP2 inhibits the p300-mediated acetylation of all core histones in vitro and the retinoic acid–induced acetylation of H3 and of H4 at Lys8 and Lys16 in vivo. These data suggest that JDP2 might inhibit histone acetylation at specific lysines of H4 by targeting specific enzymes in the c-jun promoter region. A previous study has shown that the enzymes that acetylate H4 Lys5 and Lys12 are different from those that acetylate H4 Lys8 and Lys16 (ref. 28). Acetylation of H4 Lys16 is catalyzed by specific HATs, such as MOF and its homologs29–31. Recent studies have shown that the acetylation of H4 Lys16 is crucial for chromatin condensation30 and activation of genes on the X chromosome of male Drosophila melanogaster30 and also that the loss of acetylation of H4 Lys16 is a common hallmark of human cancers32. It is possible that JDP2 might be involved in all these phenomena. JDP2 inhibits the acetylation of nucleosomes by p300 only when reconstituted nucleosomes contain recognition sequences for JDP2 (Fig. 1g). This observation suggests that the DNA-binding activity of JDP2 is also required for the HAT-inhibition activity of JDP2. Moreover, we have shown previously that homodimers of in vitro– translated JDP2 have relatively weak DNA-binding activity, as compared with that of the JDP2–ATF2 heterodimer when the DNA contains DRE16. Similar results were obtained with reconstituted templates. JDP2–ATF2 heterodimers bound much more strongly to reconstituted nucleosomes than did JDP2 alone (Supplementary Fig. 3). This difference could explain why bZIP-ATF2 was indispensable for effective repression of the acetylation of reconstituted nucleosomes by p300. JDP2 is a histone chaperone This is the first report, to our knowledge, of the characterization of a sequence-specific DNA-binding protein as a nucleosome-assembly factor. Our findings should facilitate efforts to understand some aspects of nucleosome assembly and the remodeling of chromatin. Histone H3 seems to be recruited to target sites in a DNA synthesis– dependent manner via the interaction of CAF-1 with proliferating cell nuclear antigen (PCNA)33,34, but it is unknown how histone variant H3.3 is incorporated into its target regions in the DNA synthesis– independent pathway. The observation that histone H3.3 is found in several other subcomplexes in addition to the HIRA-containing subcomplex suggests that H3.3 might be recruited to different sites


VOLUME 13

by different pathways35. Identification of a gene-specific DNA-binding protein, namely JDP2, as a nucleosome-assembly factor suggests that H3.3 could be directly deposited at specific locations by site-specific DNA-binding proteins that also have histone-chaperone activity. It will be of interest to examine the binding preference of JDP2 for histone H3 as compared to H3.3. Multiple interactions of JDP2 with DNA and histones We demonstrate here that the histone-binding activity (and presumably the nucleosome-assembly activity) and the HAT-inhibition activity of JDP2 seem to be involved in the regulation of transcription and cell differentiation. We have reported recently that repression of retinoic acid–mediated transcription by JDP2 depends on HDAC3 (ref. 18). We envision a model wherein the HAT-inhibitory activity, the histone-chaperone activity and the ability of JDP2 to recruit HDAC3 could all contribute to the silencing of the transcription complex via the modification of histone(s), the assembly of chromatin or both. Such a model could explain why loss of histone-binding activity or of HAT-inhibition activity alone did not entirely eliminate the inhibitory effects of JDP2 (Fig. 5d). This possibility is also supported by the fact that treatment of the cells with trichostatin A, an HDAC inhibitor, did not restore the transcription-inhibitory activity of JDP2 completely (data not shown). JDP2, a single transcription factor that has multiple activities, could be a good model system for clarifying the characteristics of such activities in the context of chromatin. METHODS Plasmids, proteins, reagents, preparation of cells and reconstituted mononucleosomes, and CAT, GST pull-down and electrophoretic mobility shift assays. See Supplementary Methods online. Assays of HAT activity. Filter-binding assays were performed as described elsewhere36 with minor modifications. Samples were incubated at 30 1C for 10–60 min in 25 ml of assay buffer, which contained 50 mM Tris-HCl (pH 8.0), 10% (w/v) glycerol, 0.1 mM EDTA, 1 mM DTT, 6 pmol [3H]acetyl-CoA (4.3 mCi mmol–1; Amersham Life Science) and core histones as indicated. In some cases, purified reconstituted nucleosomes, JDP2 and bZIP–ATF-2 were incubated first at 30 1C for 15 min. Then p300 (or CBP, PCAF or GCN5) prepared from E. coli or Sf9 cells and [3H]acetyl-CoA were added and incubation was continued for 45 min at 30 1C. Each reaction mixture was then spotted onto P-81 phosphocellulose filter paper (Upstate Biotechnology Co.). Filters were washed five times for 5 min each with 50 ml of 50 mM Na2HPO4 buffer and once in 50 ml of acetone for 5 min at room temperature. One pmol of p300, 5 pmol of JDP2 and 500 ng of core histones were used in each reaction except where specifically indicated. The radioactivity of air-dried filters was measured in a liquid scintillation counter. Samples were also analyzed by 18% (w/v) SDS-PAGE after reactions had been performed as described above, with the exception that we used 100 pmol of [14C]acetyl-CoA (55 mCi mmol–1; Amersham Life Science) instead of [3H]acetyl-CoA. Chromatin immunoprecipitation (ChIP) assays. ChIP assays were performed according to the protocol from Upstate Biotechnology Co.; details are provided in Supplementary Methods. Immunoprecipitation and western blotting analysis. Immunoprecipitation and western blotting were performed as described in ref. 18. GST-JDP2 (2 mg) was incubated with 8 mg of core histones or with 2 mg of each histone. Plasmid-supercoiling and nucleosome-assembly assays. We used 2 pmol of core histones from HeLa cells and 4 pmol or 8 pmol of GST or GST-JDP2 for assays of supercoiling in vitro, which were performed as described in ref. 24. Nucleosome-assembly reactions were performed essentially as described elsewhere13. We prepared a 197-bp fragment of 5S DNA from pB100-Uless/Strider DNA (a gift of J. Svejstrup, Cancer Research UK) by digestion with EcoRI, then

NUMBER 4

APRIL 2006

337

ARTICLES


incubated 200 ng of this fragment at 37 1C for 60 min without or with 200 ng of core histones, which had been preincubated at 30 1C for 30 min without or with increasing amounts of His–TAF-1b (500 ng or 1.0 mg) or GST-JDP2 (300 ng, 600 ng or 1.2 mg). Samples were then subjected to electrophoresis on a nondenaturing 5% (w/v) polyacrylamide gel. Portions of the gel corresponding to mononucleosomes were excised, eluted and subjected to western blotting analysis with antibodies raised against histone H3 (Abcam). MNase analysis of chromatin structure. MNase analysis of chromatin structure was performed as described elsewhere37 with slight modifications. F9 cells (1 106), transfected with either pcDNA4/HisMaxC with no insert or pcDNA4/HisMaxC encoding wild-type JDP2, were fixed with 1.1% (v/v) formaldehyde for 30 min at room temperature. F9 cells were then permeabilized38 and digested extensively with MNase (30, 15, 7.5 or 0 U; Worthington Biochemical Co.). The MNase-digested DNA and genomic DNA were extracted and digested with BamHI, then analyzed by Southern blotting with 32P-labeled DRE oligonucleotide as the probe. For LMPCR, mononucleosomal DNA was purified from MNase-digested cells with a gel-extraction kit (Qiagen) and subjected to primer extension. The primer-extension mixture included 0.5 ml (1 mg ml–1) of gene-specific primers (primer 1, 5¢-GTCCGCGGACGACCAGG TTAGCCGAGT-3¢; and primer 6, 5¢-GAGCATTACCTCATCCCGTGAGCCT TCGCG-3¢), 0.5 ml (2,000 U ml–1) of Vent polymerase (NEB), 10 ml (1 mM) of dNTPs, 3 ml (25 mM) of MgCl2, 5 ml of 10 Therm buffer (NEB), 30.5 ml of H2O and 1 mg of mononucleosomal DNA. The DNA was denatured at 95 1C for 10 min, annealed at 55 1C for 30 min and extended at 72 1C for 30 min. The adaptor used for LMPCR included the sequence 5¢-GCGGTGACCCGGGA GATCTGAATTC-3¢ (LMPCR-1; top strand) and 5¢-P-GAATTCAGATCT-3¢ (LMPCR-2) and was ligated to the extension product for 16 h at 16 1C. Then PCR was performed with gene-specific primers (primer 2, 5¢-GGTTGGCCA AGTCCGTCCGTCTGTCTGTCT-3¢; primer 3, 5¢-TGGCCAAGTCCGTCCGTC TGTCTGTCTGTC-3¢; primer 5, 5¢-CCTCATCCCGTGAGCCTTCGCGGGCC CAGA-3¢; and primer 4, 5¢-CGTGAGCCTTCGCGGGCCCAGAGAAGAATC3¢) and LMPCR-1 (see above). A 6% (w/v) polyacrylamide sequencing gel, loaded with the extended products, was also loaded with the products of DNAsequencing reactions, which served as size markers. Real-time PCR. Real-time PCR was performed as described in ref. 18. Note: Supplementary information is available on the Nature Structural & Molecular Biology website. ACKNOWLEDGMENTS The authors thank V. Calhoun, K. Itakura, G. Gachelin, H. Ugai, Y. Shinozuka, M. Kimura, J. Svejstrup, K. Ura, J.L. Workman, K. Ikeda and G. Felsenfeld for reagents and/or many helpful discussions, suggestions and critical reading of the manuscript. This work was supported by grants from the RIKEN Bioresource Project and by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to K.K.Y.). COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. Published online at http://www.nature.com/nsmb/ Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions/ 1. Strahl, B.D. & Allis, C.D. The language of covalent histone modifications. Nature 403, 41–45 (2000). 2. Turner, B.M. Cellular memory and the histone code. Cell 111, 285–291 (2002). 3. Chakravarti, D. et al. A viral mechanism for inhibition of p300 and PCAF acetyltransferase activity. Cell 96, 393–403 (1999). 4. Hamamori, Y. et al. Regulation of histone acetyltransferases p300 and PCAF by the bHLH protein Twist and adenoviral oncoprotein E1A. Cell 96, 405–413 (1999). 5. Weissman, J.D. et al. HIV-1 tat binds TAFII250 and represses TAFII250-dependent transcription of major histocompatibility class I genes. Proc. Natl. Acad. Sci. USA 95, 11601–11606 (1998). 6. Creaven, M. et al. Control of the histone-acetyltransferase activity of Tip60 by the HIV-1 transactivator protein, Tat. Biochemistry 38, 8826–8830 (1999). 7. Barlev, N.A. et al. Repression of GCN5 histone acetyltransferase activity via bromodomain-mediated binding and phosphorylation by the Ku-DNA-dependent protein kinase complex. Mol. Cell. Biol. 18, 1349–1358 (1998).

338

VOLUME 13

8. Kitabayashi, I. et al. Phosphorylation of the adenovirus-associated p300 kDa protein in response to retinoic acid and E1A during the differentiation of F9 cells. EMBO J. 14, 3496–3509 (1995). 9. Ait-Si-Ali, S. et al. Histone acetyltransferase activity of CBP is controlled by cycledependent kinases and oncoprotein E1A. Nature 396, 184–186 (1998). 10. Kawasaki, H. et al. ATF-2 has intrinsic histone acetyltransferase activity which is modulated by phosphorylation. Nature 405, 195–200 (2000). 11. Seo, S.B. et al. Regulation of histone acetylation and transcription by INHAT, a human cellular complex containing the Set oncoprotein. Cell 104, 119–130 (2001). 12. Kawase, H. et al. NAP-1 is a functional homologue of TAF-1 that is required for replication and transcription of the adenovirus genome in a chromatin-like structure. Genes Cells 1, 1045–1056 (1996). 13. Okuwaki, M. & Nagata, K. Template-activating factor-I remodels the chromatin structure and stimulates transcription from the chromatin template. J. Biol. Chem. 273, 34511–34518 (1998). 14. Aronheim, A., Zandi, E., Hennemann, H., Elledge, S.J. & Karin, M. Isolation of an AP-1 repressor by a novel method for detecting protein-protein interactions. Mol. Cell Biol. 17, 3094–3102 (1997). 15. Broder, Y., Katz, S. & Aronheim, A. The Ras recruitment system, a novel approach to the study of protein-protein interactions. Curr. Biol. 8, 1121–1124 (1998). 16. Jin, C. et al. Identification of mouse Jun dimerization protein 2 as a novel repressor of ATF-2. FEBS Lett. 489, 34–41 (2001). 17. Piu, F., Aronheim, A., Katz, S. & Karin, M. AP-1 repressor protein JDP-2: Inhibition of UV-mediated apoptosis through p53 down-regulation. Mol. Cell Biol. 21, 3012–3024 (2001). 18. Jin, C. et al. JDP2, a repressor of AP-1, recruits a histone deacetylase 3 complex to inhibit the retinoic acid-induced differentiation of F9 cells. Mol. Cell Biol. 22, 4815– 4826 (2002). 19. Ostrovsky, O., Bengal, E. & Aronheim, A. Induction of terminal differentiation by the c-Jun dimerization protein, JDP2, in C2 myoblasts and rhabdomyosarcoma cells. J. Biol. Chem. 277, 40043–40054 (2002). 20. Hwang,, H.C. et al. Identification of oncogenes collaborating with p27Kip1 loss by insertional mutagenesis and high-throughput insertion site analysis. Proc. Natl. Acad. Sci. USA 99, 11293–11298 (2002). 21. Heinrich, R., Livne, E., Ben-Izhak, O. & Aronheim, A. The c-Jun dimerization protein 2 inhibits cell transformation and acts as a tumor suppressor gene. J. Biol. Chem. 279, 5708–5715 (2004). 22. Wardell, S.E., Boonyaratanakornkit, V., Adelman, J.S., Aronheim, A. & Edwards, D.P. Jun dimerization protein 2 functions as a progesterone receptor N-terminal domain coactivator. Mol. Cell Biol. 22, 5451–5466 (2002). 23. Ogryzko, V.V., Schiltz, R.L., Russanova, V., Howard, R.H. & Nakatani, Y. The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 87, 953–959 (1996). 24. Munakata, T., Adachi, N., Yokoyama, N., Kuzuhara, T. & Horikoshi, M. A human homologue of yeast anti-silencing factor has histone-chaperone activity. Genes Cells 5, 221–233 (2000). 25. Umehara, T., Chimura, T., Ichikawa, N. & Horikoshi, M. Polyanionic stretch-deleted histone chaperone cia1/Asf1p is functional both in vivo and in vitro. Genes Cells 7, 59–73 (2002). 26. Wells, J.A. Systematic mutation analyses of protein-protein interfaces. Methods Enzymol. 202, 390–411 (1991). 27. Kitabayashi, I. et al. Transcriptional regulation of the c-jun gene by retinoic acid and E1A during differentiation of F9 cells. EMBO J. 11, 167–175 (1992). 28. Makowski, A.M., Dutnall, R.N. & Annunziato, A.T. Effects of acetylation of histone H4 at lysines 8 and 16 on activity of the Hat1 histone acetyltransferase. J. Biol. Chem. 276, 43499–43502 (2001). 29. Carrozza, M.J., Utley, R.T., Workman, J.L. & Cote, J. The diverse functions of histone acetyltransferase complexes. Trends Genet. 19, 321–329 (2003). 30. Dou, Y. et al. Physical association and coordinate function of the H3 K4 methyltransferase MLL1 and the H4 K16 acetyltranferase MOF. Cell 121, 873–885 (2005). 31. Taipale, M. et al. hMOF histone acetyltransferase is required for histone H4 lysine 16 acetylation in mammalian cells. Mol. Cell Biol. 25, 6798–6810 (2005). 32. Fraga, M.F. et al. Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer. Nat. Genet. 37, 391–400 (2005). 33. Shibahara, K. & Stillman, B. Replication-dependent marking of DNA by PCNA facilitates CAF-1-coupled inheritance of chromatin. Cell 96, 575–585 (1999). 34. Moggs, J.G. et al. CAF-1-PCNA-mediated chromatin-assembly pathways triggered by sensing DNA damage. Mol. Cell Biol. 20, 1206–1218 (2000). 35. Tagami, H., Ray-Gallet, D., Almouzni, G. & Nakatani, Y. Histone H3.1 and H3.3 complexes mediate nucleosome-assembly pathways dependent or independent of DNA synthesis. Cell 116, 51–61 (2004). 36. Brownell, J.E. & Allis, C.D. An activity gel assay detects a single, catalytically active histone acetyltransferase subunit in Tetrahymena macronuclei. Proc. Natl. Acad. Sci. USA 92, 6364–6368 (1995). 37. Lomvardas, S. & Thanos, D. Modification of gene expression programs by altering core promoter chromatin architecture. Cell 110, 261–271 (2002). 38. Pfeifer, G.P. & Riggs, A.D. Chromatin differences between active and inactive X chromosomes revealed by genomic footprinting of permeabilized cells using DNase I and ligation-mediated PCR. Genes Dev. 5, 1102–1113 (1991).

NUMBER 4

APRIL 2006
