Histone Chaperones ASF1 and NAP1 Differentially Modulate Removal of Active Histone Marks by LID-RPD3...

Molecular Cell

Article

Histone Chaperones ASF1 and NAP1 DifferentiallyModulate Removal of Active Histone Marksby LID-RPD3 Complexes during NOTCH SilencingYuri M. Moshkin,1 Tsung Wai Kan,1 Henry Goodfellow,3 Karel Bezstarosti,2 Robert K. Maeda,4 Maxim Pilyugin,4

Francois Karch,4 Sarah J. Bray,3 Jeroen A.A. Demmers,2 and C. Peter Verrijzer1,*1Department of Biochemistry, Center for Biomedical Genetics2Proteomics CenterErasmus University Medical Center, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands3Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK4Department of Zoology and National Research Center Frontiers in Genetics, University of Geneva, 30 quai E. Ansermet, 1211 Geneva-4,

Switzerland*Correspondence: [email protected]

DOI 10.1016/j.molcel.2009.07.020

SUMMARY

Histone chaperones are involved in a variety of chro-matin transactions. By a proteomics survey, we iden-tified the interaction networks of histone chaperonesASF1, CAF1, HIRA, and NAP1. Here, we analyzed thecooperation of H3/H4 chaperone ASF1 and H2A/H2Bchaperone NAP1 with two closely related silencingcomplexes: LAF and RLAF. NAP1 binds RPD3 andLID-associated factors (RLAF) comprising histonedeacetylase RPD3, histone H3K4 demethylase LID/KDM5, SIN3A, PF1, EMSY, and MRG15. ASF1 bindsLAF, a similar complex lacking RPD3. ASF1 andNAP1 link, respectively, LAF and RLAF to the DNA-binding Su(H)/Hairless complex, which targets theE(spl) NOTCH-regulated genes. ASF1 facilitatesgene-selective removal of the H3K4me3 mark byLAF but has no effect on H3 deacetylation. NAP1directs high nucleosome density near E(spl) controlelements and mediates both H3 deacetylation andH3K4me3 demethylation by RLAF. We concludethat histone chaperones ASF1 and NAP1 differen-tially modulate local chromatin structure duringgene-selective silencing.

INTRODUCTION

Regulated modulation of the chromatin structure is essential for

the transmission, maintenance, and expression of the eukaryotic

genome. The combined actions of ATP-dependent chromatin-

remodeling factors (remodelers), histone chaperones, and

histone-modifying enzymes drive chromatin dynamics. Histones

are subjected to a wide range of reversible posttranslational

modifications, including acetylation, phosphorylation, methyla-

tion, and ubiquitylation (Berger, 2007). Histone modifications,

in turn, can promote the recruitment of selective regulatory

factors and modulate chromatin accessibility. Chromatin remod-

elers control DNA accessibility by mediating nucleosome mobi-

782 Molecular Cell 35, 782–793, September 25, 2009 ª2009 Elsevie

lization either through sliding or by nucleosome (dis)assembly

(Mohrmann and Verrijzer, 2005).

Whereas originally considered mainly as mere chaperones, it

has become clear that histone chaperones play diverse roles

during chromatin transactions (De Koning et al., 2007; Eitoku

et al., 2008). Histone chaperones bind selective histones and

include the highly conserved H3/H4 chaperones ASF1, CAF1,

HIRA, and Spt6 and the H2A/H2B chaperones NAP1, Nucleo-

plasmin, and FACT (De Koning et al., 2007; Eitoku et al., 2008).

Although their biochemical activity, binding and release of

histones, appears rather mundane, in conjunction with other

factors, histone chaperones participate in a variety of chromatin

transactions and other cellular tasks. For example, yeast NAP1

participates in an extensive interaction network including a

diverse set of transcription initiation/elongation factors, chro-

matin remodelers, RNA-processing factors, cell-cycle regula-

tors, and other proteins (Del Rosario and Pemberton, 2008; Mo-

sammaparast et al., 2005; Walfridsson et al., 2007; Zlatanova

et al., 2007).

ASF1 is one of the major H3/H4 chaperones, and through asso-

ciation with other proteins, it contributes to diverse chromatin

transactions (De Koning et al., 2007; Eitoku et al., 2008). (1) In

conjunction with CAF1 and the MCM2-7 DNA helicase, ASF1

participates in replication-coupled chromatin assembly (Groth

et al., 2007; Tyler et al., 1999, 2001). (2) When associated with

HIRA, ASF1 participates in replication-independent chromatin

assembly and histone replacement (Green et al., 2005; Zhang

et al., 2007). (3) DNA-repair-associated chromatin assembly

requires the cooperation between ASF1 and the H3K56 acetyl-

transferase Rtt109 (Chen et al., 2008). (4) ASF1 functionally

cooperates with the Drosophila BRM chromatin remodeler

(Moshkin et al., 2002), and (5) interaction of ASF1 with transcrip-

tion activators stimulates histone eviction from promoter areas

and facilitates recruitment of chromatin-specific coactivator

complexes (Adkins et al., 2007). (6) ASF1 itself is one of the

targets of Tousled-like kinase (TLK), which controls cell-cycle

progression and chromatin dynamics (Carrera et al., 2003). (7)

Finally, ASF1 is involved in developmental gene expression

control by mediating transcriptional repression of NOTCH target

genes (Goodfellow et al., 2007). ASF1 is recruited to E(spl) genes

r Inc.

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Molecular Cell

Histone Chaperones in Histone Mark Removal

by the sequence-specific DNA-binding protein Su(H) and its

associated corepressor complex, harboring Hairless (H) and

SKIP.

NOTCH is the central component of a highly conserved devel-

opmental signaling pathway that is present in all metazoans.

NOTCH is a single-pass transmembrane protein that is activated

through ligand binding, resulting in the release of the NOTCH

intracellular domain (Nicd), which is targeted to the nucleus

to activate gene expression (Artavanis-Tsakonas et al., 1999;

Bray, 2006). The CSL (CBF1, Su(H), and Lag1) family of

sequence-specific DNA-binding proteins is the key targeting

factor of Nicd and coactivators and, in the absence of Nicd,

corepressors. The repression of NOTCH target genes involves

multiple chromatin-modifying activities including histone deace-

tylases, H3K9 methyltransferases, CtBP, NcoR/SMRT, and

Goucho (GRO) (Nagel et al., 2005; Perissi et al., 2008; Shi

et al., 2003). In the absence of the Nicd, loss of ASF1 leads to

derepression of the E(spl) genes, revealing its essential role in

silencing (Goodfellow et al., 2007).

The molecular mechanism by which ASF1 achieves gene-

specific transcription repression and the potential roles of other

histone chaperones in developmental gene regulation remains

largely unknown. To address these issues, we first performed

a proteomics survey of the protein interaction networks of

ASF1, CAF1, HIRA, and NAP1 in Drosophila embryos. Our

analysis revealed that ASF1 and NAP1 interact with two related

but distinct corepressor complexes: LAF and RLAF. LAF,

comprising LID/KDM5 SIN3A, PF1, EMSY, and MRG15, associ-

ates with ASF1 (forming LAF-A). RLAF, comprising LAF plus

RPD3, interacts with NAP1 (forming RLAF-N). Through a combi-

nation of biochemistry and developmental genetics, we estab-

lished that LAF-A and RLAF-N are tethered to NOTCH target

genes by the Su(H)/H complex and mediate gene-selective

silencing. Both ASF1 and NAP1 are required for the targeted

removal of the positive H3K4me3 mark by facilitating LID/

KDM5 recruitment to chromatin. Furthermore, NAP1 mediates

nucleosome assembly at regulatory elements of NOTCH target

genes and histone deacetylation by RLAF. Our results uncover

extensive crosstalk between distinct histone chaperones and

histone-modifying enzymes in developmental gene regulation.

RESULTS

ASF1 Is a Hub in a Protein-Protein Interaction NetworkTo determine the ASF1 regulatory network, we made an inven-

tory of its associated proteins in developing Drosophila embryos

and S2 cells. We prepared nuclear extracts (NE) from 0–12 hr

embryos, encompassing up to 15 stages of embryonic develop-

ment, and from S2 cells. Nuclear extracts were incubated with

protein A Sepharose beads coated with either affinity-purified

a-ASF1 antibodies or a-GST antibodies as control. Following

extensive washes with a buffer containing 600 mM KCl and

0.1% NP-40, bound and unbound material were resolved by

SDS polyacrylamide electrophoreses (SDS-PAGE) and visual-

ized by Coomassie staining (Figures 1A and 1B and Tables S1

and S2 available online). Mass spectrometric analysis revealed

the association of ASF1 with known as well as additional factors.

Previously documented ASF1-binding proteins include the CAF1

Molec

complex (Tyler et al., 1999, 2001), HIRA and its associated factor

yemalpha/UBN1 (Banumathy et al., 2009), and the core histones.

TLK (Carrera et al., 2003) and MCM2 (Groth et al., 2007) were

only detected in the ASF1 purification from Drosophila embryo

nuclear extract (Figure 1A). The ASF1-associated factors identi-

fied in both experiments included SIN3A (Silverstein and Ekwall,

2005), histone H3K4me2/3 demethylase LID/KDM5 (Eissenberg

et al., 2007; Lee et al., 2007; Secombe et al., 2007), EMSY/

CG15356 (Hughes-Davies et al., 2003), PF1/CG3815 (Yochum

and Ayer, 2001), and MRG15 (Yochum and Ayer, 2002). To

obtain more evidence for stable association of ASF1-binding

proteins, we partially purified embryo nuclear extract by conven-

tional column chromatography prior to the ASF1 immunopurifi-

cation step. ASF1 containing peak fractions on the final S300

column were pooled, immunopurified, and analyzed by mass

spectrometry (Figure 1C and Table S3). SIN3A, PF1, EMSY,

MRG15, and the histone H3K4 demethylase LID were confirmed

as prominent ASF1-associated proteins, in addition to the well-

established CAF1 and HIRA.

To establish the in vivo significance of the interactions

between ASF1 and some of the ASF1-associated factors, we

used a genetic assay. Overexpression of ASF1 in the fly eye

leads to mildly disrupted eye development characterized by

reduced size and disorganized facets, providing us with a conve-

nient tool for genetic interaction analysis (Goodfellow et al.,

2007; Moshkin et al., 2002). To drive ASF1 overexpression in

the developing eye, we used an eyeless-Gal4, UAS-Asf1 fly

strain (ey-ASF1). The ey-ASF1 line was crossed with available

mutants, namely lid and Sin3A. In heterozygous combinations,

two different lid mutant alleles (lid10424 and lidk06901) and the

Sin3A08269 allele showed significant suppression of the ey-ASF1

rough-eye phenotype (Figure 1D). These genetic interactions

indicate that ASF1 cooperates with SIN3A and LID in vivo. These

results suggest that the network of ASF1-interacting factors is

substantially more extensive than previously anticipated.

Proteomic Characterization of ASF1-Interacting FactorsSephacryl S-300 size-exclusion chromatography revealed that

the ASF1-interacting proteins SIN3A, PF1, EMSY, and LID eluted

in fractions corresponding to apparent molecular masses

ranging from �440–900 kDa (Figure 2A). Thus, their migration

is consistent with their incorporation into large multisubunit

assemblages. The chromatin remodelers BRM and ISWI served

as a reference. Because ASF1 forms two mutually exclusive

complexes with CAF1 and HIRA, we wondered whether the

ASF1 interactors might associate with either of these two

complexes. To test this hypothesis, we immunopurified CAF1

and HIRA followed by mass spectrometric analysis (Figures 2B

and 2C and Tables S4 and S5). ASF1 and a number of previously

uncharacterized CAF1- or HIRA-interacting proteins were identi-

fied. Importantly, none of the additional ASF1-associated factors

were detected, suggesting that they bound neither CAF1 nor

HIRA. We note that the CAF1 and HIRA purifications share

a few proteins, suggesting potential crosstalk between these

two histone chaperones.

Next, we purified biotin-tagged LID (bioLID) from S2 cells

using NeutrAvidin-coated beads and immunopurified LID, PF1,

and EMSY from embryo nuclear extracts using the appropriate

ular Cell 35, 782–793, September 25, 2009 ª2009 Elsevier Inc. 783

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affinity-purified antibodies, followed by mass spectrometric

analysis (Figures 2D–2G and Tables S6–S9). Our results revealed

that RPD3, LID, SIN3A, PF1, EMSY, and MRG15 form a stoichio-

metric complex, which we refer to as RLAF. The following RLAF

subunits have been implicated in gene repression: (1) SIN3A,

a transcriptional corepressor that is targeted to a variety of

promoters (Silverstein and Ekwall, 2005), (2) PF1, a PHD-finger

protein, (3) MRG15, which has been implicated in gene repres-

sion together with PF1 and SIN3A (Yochum and Ayer, 2001,

2002), (4) EMSY, originally identified as a human BRCA2-inter-

acting protein that can mediate transcriptional repression

(Hughes-Davies et al., 2003), and (5) LID/KDM5, a JARID1-type

demethylase that can remove the active H3K4me2/3 mark

(Eissenberg et al., 2007; Lee et al., 2007; Secombe et al.,

2007). (6) Finally, RPD3 is a histone deacetylase (Pile et al.,

2002) with a significant mascot score in our RLAF purifications

but that is absent from our ASF1 purifications. Thus, it appears

A

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Histones

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s)

PF1; CAF1-105EMSY; yemα

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(IgG)

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SIN3A

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(S30

0 #14

-16)

*

LID; CAF1-180

CAF1-75

CAF1-55(Hsc70-4)

* (IgG) MRG15

WT ey-ASF1

ey-ASF1/lidk06801 ey-ASF1/Sin3A08269

Figure 1. ASF1 Protein Interaction Network

(A) ASF1-associated proteins immunopurified from

Drosophila embryo nuclear extracts (NE). Embryo NE

was incubated with Protein A Sepharose beads coated

with either control a-GST or affinity-purified a-ASF1 anti-

bodies. Proteins were resolved by SDS-PAGE and visual-

ized by Coomassie staining. Indicated proteins present in

excised bands were identified by nanoflow LC-MS/MS

mass spectrometry. Their respective mascot score, emPAI

score, % coverage, and number of unique peptides

identified are listed in Table S1. Background proteins are

indicated with an asterisk.

(B) ASF1 and associated proteins were immunopurified

from S2 cell NE as described above. For mascot score

and number of unique peptides identified, see Table S2.

Background proteins are indicated with an asterisk.

(C) ASF1 peak fractions from a final Sephacryl S-300 size-

exclusion chromatography were pooled, followed by

a-ASF1 immunopurification as described above. ASF1-

associated factors were identified by mass spectrometry

(Table S3).

(D) Lid and Sin3A interact genetically with asf1. Represen-

tative scanning electron micrographs of adult eyes from

flies with the indicated genotypes are shown. Flies carrying

the UAS-asf1 transgene recombined with the eyeless-

GAL4 driver on the second chromosome (ey-asf1) show

distinctive small- and rough-eye phenotypes. The ey-asf1

phenotype was suppressed when combined with lidk06801

or with Sin3A08269 mutant alleles, but not with rpd304556

(data not shown).

that binding of ASF1 or RPD3 to LID and its

associated factors (LAF) is mutually exclusive.

(R)LAF components engage in additional inter-

actions, linking individual subunits to gene

silencing, replication, chromatin remodeling,

and other pathways (see Tables S7–S9).

In summary, we made a proteomic survey of

factors that associate with the histone H3/H4

chaperones ASF1, CAF1, or HIRA. In addition

to known associations, our analysis uncovered

several additional binding partners. Our obser-

vations emphasize that, rather than acting as

generic factors, distinct histone chaperones are involved in

highly specific chromatin processes. Here, we focus on an

ASF1-interacting complex harboring the histone H3K4me2/3

demethylase LID/KDM5, which we named LAF. In addition, we

identified RLAF, comprising LAF plus the histone H3 deacetylase

RPD3. However, association of RPD3 or ASF1 appears to be

mutually exclusive: ASF1 binds LAF, but not RLAF. Finally,

neither HIRA nor CAF1 interacts with LAF, suggesting that its

association with ASF1 is selective.

The Histone H3/H4 Chaperone ASF1 Binds LAF,whereas the H2A/H2B Chaperone NAP1 Binds RLAFTo further characterize the interaction between LAF and ASF1,

we performed a series of stringent coimmunoprecipitations

(coIPs) from embryo NE. Western immunoblotting showed

that SIN3A, PF1, EMSY, MRG15, and LID are stably associated

with ASF1 (Figure 3A). CAF1 and HIRA were also readily

784 Molecular Cell 35, 782–793, September 25, 2009 ª2009 Elsevier Inc.

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D

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mod(mdg4)

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OSA

MOR

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SNR1

*

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BAP55

Actin5C

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A

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EMSY

PF1

LID

SIN3A

BRM

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3 6 9 12 15 18

VoidFerritin440 kDa

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21 24 27 30 33 36 39 41

Aldolase158 kDa

CAF1-75

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05

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PAV

HNF4; CG16728

CG5199/CUT8

NOP5

TUMRanGAP

CG4557

C

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HTS; CG16728

MSPSTACC

yemα

ASF1

TRiC/CCT

α-HIR

A

(embry

o NE)

*

PAV; CG2918

CIN; TUM

TrlRanGAP

Ab

Ab

Figure 2. Protein-Protein Interaction Networks of ASF1-Associated Factors

(A) Copurifying ASF1-associated factors display overlapping elution patterns during Sephacryl S-300 size-exclusion chromatography. The indicated fractions

were resolved by SDS-PAGE, followed by immunoblotting. Voided volume (void) and elution of the markers ferritin (440 kDa) and aldolase (158 kDa) are indicated.

Detection of the BRM subunit of the Drosophila SWI/SNF complexes and ISWI serve as additional references.

(B and C) Immunopurification of CAF1 and HIRA from embryo NE using affinity-purified antibodies as described in Figure 1A. See Tables S4 and S5 for mass

spectrometric details. Background proteins are indicated with an asterisk. ASF1-associated factors are labeled in red, whereas proteins shared by CAF1 and

HIRA complexes are labeled in blue.

(D) Identification of LID-associated factors from NEs prepared from S2 cells expressing bio-tagged LID using Neutravidin beads. For details, see Table S6.

Proteins present in ASF1 purifications are labeled in red.

(E–G) Immunopurification of LID, PF1, and EMSY from NEs using affinity-purified antibodies as described in Figure 1A. Mass spectrometry details are in Tables

S7–S9. Proteins present in ASF1 purifications are labeled in red.

Molecular Cell 35, 782–793, September 25, 2009 ª2009 Elsevier Inc. 785

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A CB

α-PF1

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CAF1-105CAF1-75

USP7

MRG15

NAP1

Figure 3. ASF1 Binding to LAF, CAF1, or HIRA Is Mutually Exclusive

(A) CoIPs from Drosophila embryo NE with affinity-purified a-ASF1 antibodies confirmed the binding of LID-associated factors SIN3A, PF1, EMSY, LID, and

MRG15 to ASF1. ASF1 does not interact with RPD3, NAP1, or USP7. Proteins bound to the beads were resolved by SDS-PAGE and analyzed by immunoblotting

using the indicated antibodies. Lane 1 represents 10% of the input.

(B) Binding of ASF1 to LAF, CAF1, or HIRA is mutually exclusive. CoIPs using antibodies directed against CAF1-105 subunit and HIRA revealed mutually exclusive

binding to ASF1. CAF1 and HIRA do not bind each other, and neither binds LAF or NAP1.

(C) LID, SIN3A, PF1, EMSY, and MRG15 form a tightly associated complex (LAF) that binds either ASF1 (LAF-A) or RPD3 (RLAF) and NAP1 (RLAF-N), but not HIRA

or CAF1. CoIPs were performed using antibodies directed against PF1, EMSY, and LID.

detected, whereas RPD3, NAP1, or the unrelated USP7 did not

coimmunoprecipitate with ASF1. These results confirm our

proteomics analysis and substantiate the notion that ASF1 forms

a separate complex with LAF, lacking RPD3. We refer to this

complex as LAF-A. In conclusion, ASF1 engages in three mutu-

ally exclusive associations involving HIRA, CAF1, and LAF. As

predicted by our proteomics analysis, LAF does not associate

with CAF1 or HIRA but binds ASF1 or RPD3 (Figures 3B and 3C).

Mass spectrometric analysis of LID- and EMSY-associated

factors revealed NAP1, albeit with low mascot score (Tables

S6, S7, and S9). Indeed, coIPs using antibodies against PF1,

EMSY, or LID, followed by western immunoblotting, readily

revealed the association of NAP1 (Figure 3C). Next, we immuno-

purified NAP1 from NE prepared from Drosophila embryos and

S2 cells, followed by mass spectrometry detection (Figures 4A

and 4B and Tables S10 and S11). NAP1 associates with multiple

factors, including RLAF, Cohesin, RFC, and THO complexes.

CoIPs confirmed that NAP1 interacts with RLAF, but not with

ASF1 (Figure 4C). NAP1 binding to RLAF is substoichiometric

(Figures 2D–2G), suggesting that a portion of RLAF associates

with NAP1, forming RLAF-N. In conclusion, LID/KDM5 is part

of at least three mutually exclusive complexes: LAF-A, RLAF,

or RLAF-N (Figure 4D).

LAF-A and RLAF-N Repress Transcription of NOTCHTarget GenesRecently, we described that ASF1 functions in the NOTCH-

signaling pathway by contributing to the repression of NOTCH

786 Molecular Cell 35, 782–793, September 25, 2009 ª2009 Elsevier

target genes (Goodfellow et al., 2007). Therefore, we set out to

test whether the LAF complexes might also be involved in this

process. First, we used a well-established genetic approach

by analyzing the effect of combining the heterozygous Notch

allele N55e11 in trans with either lidk06801 or Sin3A08269. We found

that mutations in lid or Sin3A suppressed the dominant

thickening of the wing vein, caused by N55e11 (Figure 5A, left

panels). In contrast, heterozygous lidk06801 or Sin3A08269

enhanced the truncations of the L5 wing vein in flies heterozy-

gous for the HP141 mutation in transcriptional corepressor

Hairless (Figure 5A, right panels). These observations suggest

that, like ASF1, LID and SIN3A contribute to repression of

NOTCH target genes in vivo.

ASF1 is recruited to the E(spl) NOTCH target genes by the

sequence-specific DNA-binding Su(H)/H corepressor complex

(Goodfellow et al., 2007). Purification of NAP1-interacting

factors revealed subunits of the Su(H)/H corepressor complex

SKIP and CtBP (Figures 4A and 4B). Moreover, CtBP was also

detected in LID and PF1 purifications (Figures 2E and 2F).

Therefore, we decided to test whether LAF complexes interact

with Su(H)/H corepressor. To this end, we performed a series

of coIPs with antibodies against Su(H). Like ASF1, LAF was

efficiently coimmunoprecipitated with Su(H) as well as RPD3

and NAP1 (Figure 5B). In contrast, HIRA or USP7 were not

bound by Su(H). Conversely, Su(H) was also present in PF1,

EMSY, and LID immunopurifications (Figure 5B). We conclude

that LAF-A, RLAF, and RLAF-N bind the DNA-binding recruiter

Su(H).

Inc.

Molecular Cell


To address the role of the LAF complexes in gene expression

control, we treated S2 cells with dsRNA directed against

individual LAF subunits ASF1, NAP1, and RPD3. This leads to

a strong reduction of the targeted protein levels, without

appreciable destabilization of the others (Figure S1). Next, we

extracted RNA and, to monitor gene expression, used reverse

transcription followed by real-time PCR (RT-qPCR) using gene-

selective primers. Strikingly, depletion of ASF1, NAP1, individual

LAF subunits, or RPD3 resulted in a clear derepression of the

E(spl)m4, m7, and m8 genes (Figure 5D). All of these NOTCH

targets are silenced in an Su(H)/H-dependent manner in S2 cells.

Expression of non-NOTCH targets, such as cdc1 or elf23,

remained unchanged. In contrast to loss of ASF1 or NAP1,

HIRA depletion had no effect on NOTCH target gene expression.

Collectively, these results established a physical and functional

connection between LAF complexes and Su(H)/H corepressor

complex in NOTCH target silencing.

NAP1

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LID

MRG15

Rpd3

A B

C D

RLAF

LAF-A

RLAF-N

α-NAP1 (

S2 cell

s)

Contro

l Ab

175

83

62

47.5

32.5

25

PF1EMSY

LIDSIN3A

MRG15

RPD3

NAP1

Bx42/SKIP

CtBP; THOC6

SMC1;SMC3SA; dSFMBT

RAD21

THO2

HPR1THOC5

THOC7PROD

HP1

175

83

62

47.5

32.5

25

α-NAP1

(embry

o NE)

Contro

l Ab

PF1EMSY

LIDSIN3A

MRG15

RPD3

NAP1

Bx42/SKIP

CtBP; THOC6

SMC1;SMC3SA; dSFMBT

RAD21

THO2

HPR1THOC5

THOC7PROD

HP1

Figure 4. NAP1 Interactome Determination Re-

vealed Association with RLAF

(A and B) Immunopurification of NAP1 and associated

factors from embryo and S2 cell NEs using affinity-purified

antibodies directed against NAP1 was performed as

described above. Details are in Tables S10 and S11.

RLAF subunits are labeled in red.

(C) CoIPs from embryo NEs using affinity-purified a-NAP1

antibodies confirmed the binding of NAP1 to the following

RLAFs: RPD3, LID, SIN3A, PF1, EMSY, LID, and MRG15.

NAP1 does not interact with either ASF1 or BRM.

(D) Cartoon summarizing the selective association of ASF1

and NAP1 with LAF and RLAF. RLAF comprises six tightly

associated subunits: RPD3, LID, SIN3A, PF1/CG3815,

EMSY/CG15356, and MRG15. LAF is similar to RLAF but

lacks RPD3. Binding of RPD3 or ASF1 to LAF is mutually

exclusive. Thus, ASF1 only binds LAF, forming LAF-A.

RLAF is bound by NAP1, forming RLAF-N.

LAF-A and RLAF-N Differentially AffectChromatin Structure at RegulatoryElements of NOTCH Target GenesBecause LID is an H3K4me2/3 demethylase,

we tested whether LAF complexes might be

involved in the control of global H3K4 methylation

levels. We performed RNAi-mediated depletion

of individual LAF subunits, as well as ASF1,

NAP1, and RPD3. In addition, HIRA knockdown

was performed as a control (Figure S1). Examina-

tion of bulk histone H3K4 methylation revealed

that loss of LID, but not of other LAF subunits,

caused an increase in H3K4me3 levels (Fig-

ure S2). Other histone methyl marks that we

examined were not significantly affected. Con-

versely, loss of RPD3, but not of LAF, or histone

chaperones ASF1 and NAP1 caused increased

histone H3 acetylation (Figure S2). These obser-

vations suggest that LAF-A or RLAF-N do not

play major roles in bulk histone H3K4me3 deme-

thylation or H3 deacetylation. Rather, they might

act in a gene-selective manner.

Therefore, we tested whether the Su(H)/H complex might facil-

itate LID recruitment to E(spl) genes by chromatin immunoprecip-

itations (ChIPs) quantified by qPCR. Chromatin was extracted

from S2 cells expressing bioLID that were depleted for H, ASF1,

NAP1, individual LAF subunits, and RPD3. We found that loss

of H, ASF1, NAP1, or LAF subunits, except RPD3, significantly

impaired LID recruitment to the E(spl) enhancers and promoters

(Figures 6A and S3). Previously, we reported that ASF1 is

recruited to the E(spl) genes by the Su(H)/H corepressor complex

(Goodfellow et al., 2007). Therefore, we tested whether NAP1

might also be targeted by the Su(H)/H complex in S2 cells.

Indeed, loss of H, but not of ASF1 or LID, caused a clear reduction

in NAP1 binding to E(spl) regulatory elements (Figures 6B and S4).

Thus, the Su(H)/H complex plays a key role in tethering the histone

chaperones ASF1 and NAP1, as well as the LAF complexes.

To examine the impact of LAF-A and RLAF-N on local chro-

matin structure at the E(spl)m4, m7, and m8 enhancers and


Molecular Cell


A

HP141/+

Sin3

A08

269 /+

lidk0

6801

/++

/+

N55e11

/+

wild

typ

eB

α-PF1

α-EMSY

α-LID

Contro

l Ab

input (

NE)

Su(H)

C

RNAi knockdown

Fold

up

reg

ula

tio

n

2

4

6

8

0

ASF

1

SIN

3A

EMSYPF

1

LID

HIR

A

E(spl)m4 E(spl)m7 E(spl)m8

Mo

ck

cdc2 elf23

RPD

3

NA

P1

ASF

1

SIN

3A

EMSYPF

1

LID

HIR

A

Mo

ck

RPD

3

NA

P1

ASF

1

SIN

3A

EMSYPF

1

LID

HIR

A

Mo

ck

RPD

3

NA

P1

ASF

1

SIN

3A

EMSYPF

1

LID

HIR

A

Mo

ck

RPD

3

NA

P1

ASF

1

SIN

3A

EMSYPF

1

LID

HIR

A

Mo

ck

RPD

3

NA

P1

D

ASF1

SIN3A

PF1

EMSY

LID

α-Su(H

)

Contro

l Ab

input (

NE)

HIRA

USP7

RPD3

NAP1

Figure 5. LAF-A and RLAF-N Are Required for Repression of NOTCH Target Genes

(A) Lid and Sin3A interact genetically with the Notch pathway. Mutations in lid (lidk06801/+) and Sin3A (Sin3A08269/+) suppress wing vein thickening in flies hetero-

zygous for Notch mutant allele (N55e11/+). Arrows mark the distal part of L4 and L5 where the phenotypes are most evident. Mutations in lid (lidk06801/+) and Sin3A

(Sin3A08269/+) enhance truncations of the L5 wing vein in flies heterozygous for the Hairless mutant allele (HP141/+). (Arrow) The end of L5 in HP141/+. (Arrowheads)

Additional truncations in transheterozygous mutants.

(B) LAF, ASF1, NAP1, and RPD3 bind Su(H). Immunoprecipitations with either control (a-GST) or a-Su(H) antibodies, followed by washes with a buffer containing

200 mM KCl and 0.1% NP-40, were resolved by SDS-PAGE and analyzed by western blotting. Ten percent of the input material was loaded.

(C) Su(H) binds PF1, EMSY, and LID, as detected by coIPs performed as described above.

(D) RLAF-N and LAF-A are selectively required for silencing of NOTCH target genes. To knock down the indicated (R)LAF subunits, ASF1, NAP1, and HIRA as

a control, S2 cells were incubated with dsRNAs directed against the appropriate mRNAs. RNA was extracted and quantified by RT-qPCR, using primers selective

for E(spl)m4, E(spl)m7, E(spl)m8, and the cdc2 and elf23 controls. For normalization, we performed qPCR for 18S rRNA on progressively diluted samples. Normal-

ized mRNA levels were expressed relative to those in mock-treated cells. All results are based on three independent biological replicate experiments. Error bars

represent SE of mean.

promoters, we performed a series of ChIP-qPCR experiments

combined with RNAi-mediated protein depletion. Changes in

H3K4me3 and H3 acetylation levels were normalized against

those of histone H3, as determined by using modification-inde-

pendent antibodies. All ChIP data presented here are the results


of at least three fully independent biological replicates. Loss of

ASF1, NAP1, individual LAF subunits, or H caused increased

H3K4me3 levels at enhancers and promoters of the E(spl)m4,

m7, and m8 genes (Figures 6C and S5). In contrast, depletion

of RPD3 did not affect H3K4me3. However, when we examined

r Inc.

Molecular Cell


histone H3ac levels, we obtained strikingly different results.

Depletion of NAP1, RPD3, or H caused a strong increase in

H3ac levels at enhancers and promoters of the E(spl) genes

(Figures 6D and S6). SIN3A depletion had only a minor effect

on H3ac at the enhancers of E(spl) genes. However, neither

ASF1 nor other LAF subunits affected histone H3ac. In agree-

ment with its importance as part of the Su(H)/H recruiting

complex, loss of H caused both increased H3K4me3 and H3ac

levels.

We also examined the effect of LAF-A and RLAF-N on histone

density at the E(spl) regulatory elements. Strikingly, depletion of

NAP1, but not ASF1, caused a strong loss of histone H2B and H3

at the E(spl) promoters and enhancers (Figures 6E, 6F, S7, and

S8). This result suggests that NAP1 mediates a higher nucleo-

some density at the silenced E(spl) genes. Loss of NAP1 did

not affect general histone density (data not shown). Loss of H

also resulted in a drop of histone levels at these loci, again

emphasizing its importance for recruitment of the silencing

machinery. Other (R)LAF subunits did not appreciably affect

local histone density.

Collectively, these results revealed an intricate interplay

between sequence-specific DNA-binding factors, selective

histone chaperones, and histone-modifying enzymes in targeted

control of histone density and the removal of positive histone

marks during developmental gene silencing (Figure 7). We estab-

lished that the Su(H)/Hairless corepressor complex tethers

LAF-A and RLAF-N chromatin-modifying activities to NOTCH

target genes. ASF1, NAP1, and all LAF subunits assisted in LID

recruitment to the E(spl) genes and removal of the positive

H3K4me3 mark, whereas RPD3 played no role. Conversely,

only NAP1 and SIN3A were critical for H3 deacetylation by

RPD3. Thus, NAP1 cooperates with RLAF, stimulating both

H3K4 demethylation by LID and H3 deacetylation by RPD3.

Moreover, NAP1, but not ASF1, directs a higher histone density

at the repressed E(spl) regulatory elements. Thus, ASF1 and

NAP1 differentially modulate chromatin to mediate gene-selec-

tive silencing.

DISCUSSION

Our results emphasize that, rather than generic, redundant

factors, histone chaperones play highly specialized roles in

gene-specific regulation. We dissected the molecular mecha-

nism underpinning coordinate silencing of NOTCH target genes

by the histone H3/H4 chaperone ASF1 and the H2A/H2B chap-

erone NAP1 (Figure 7). ASF1 interacts with LAF, comprising

SIN3A, PF1, EMSY, MRG15, and the histone H3K4me2/3 deme-

thylase LID/KDM5, forming LAF-A. A closely related complex,

RLAF that includes the deacetylase RPD3, does not bind

ASF1. Instead, RLAF associates with NAP1, forming RLAF-N.

The chaperones ASF1 and NAP1 link, respectively, LAF and

RLAF to the Su(H)/H DNA-binding complex, tethering them to

the E(spl) genes. Both ASF1 and NAP1 bind the SKIP subunit

of the Su(H)/H complex (Figures 4A and 4B; Goodfellow

et al., 2007). Thus, at least in part, ASF1 and NAP1 facilitate

H3K4me3 demethylation activity at the E(spl) genes through

LID recruitment. Other LAFs might provide additional links to

the Su(H)/H complex by contacting GRO and CtBP, which them-

Molec

selves associate with the Su(H)/H complex (Nagel et al., 2005).

For example, mammalian PF1, MRG15, and SIN3A have been

reported to bind GRO (Yochum and Ayer, 2001). Here, we

identified CtBP in LID, PF1, and NAP1 immunopurifications,

providing an additional contact between the Su(H)/H complex

and (R)LAF.

ASF1 does not bind RLAF and has no effect on histone H3

deacetylation by RPD3. In contrast, NAP1 does associate with

RLAF and stimulates both H3K4 demethylation by LID and H3

deacetylation by RPD3. SIN3A had a mild effect, but the other

LAF subunits played no apparent role in deacetylation. Finally,

NAP1 depletion caused a dramatic loss of histones at the E(spl)

regulatory elements, whereas ASF1 depletion had no effect on

local histone density.

ASF1 has been proposed to function in chromatin assembly

by acting as a donor that hands off the H3/H4 tetramer to either

CAF1 or HIRA (De Koning et al., 2007). Because LAF-A does not

associate with either CAF1 or HIRA, this might explain that ASF1

does not modulate nucleosome density at the E(spl) genes. In

conclusion, the H3/H4 chaperone ASF1 mediates silencing of

NOTCH target genes by (1) providing a connection between

LAF and the Su(H)/H tether and (2) facilitating H3K4 demethyla-

tion by LID. The H2A/H2B chaperone NAP1 participates in E(spl)

silencing by (1) linking RLAF to Su(H)/H, (2) facilitating H3K4

demethylation by LID, (3) facilitating H3 deacetylation by RPD3,

and (4) directing high nucleosome density at repressed loci. The

functioning of the H2A/H2B chaperone NAP1 in demethylation

and deacetylation of histone H3 provides an example of trans-

histone regulation.

LID and its interacting factors appear to work in a context-

dependent manner. For example, LID facilitates activation of

dMYC target genes in a manner independent of its demethylase

activity (Secombe et al., 2007). Suggestively, we observed

a genetic interaction between ASF1 and dMYC, indicating

a potential role for LAF-A (data not shown). Recently, it has

been suggested that selective RLAF subunits could interact

with a homolog of GATA zinc-finger domain-containing protein

1 to facilitate expression of targets by inhibition of RPD3 activity

(Lee et al., 2009). In mammalian cells, LID homolog RBP2 and

MRG15 have been implicated in transcription elongation by

restricting H3K4me3 levels within transcribed regions (Haya-

kawa et al., 2007). Our identification of SIN3A as a LAF and

RLAF subunit provides a molecular explanation for the recent

observation that SIN3A is involved in genome-wide removal of

both H3K4 methyl and acetyl marks (van Oevelen et al., 2008).

Collectively, these findings suggest that LID and RPD3 enzy-

matic activities can be modulated through association with

specific partners. Our proteomics analysis of the LID, PF1, and

EMSY interaction networks further emphasizes the diverse

involvement of LAFs in regulation of chromatin dynamics.

In conclusion, our results emphasize the close interconnectiv-

ity between distinct chromatin transactions and reveal coopera-

tion between histone chaperones and targeted histone modifica-

tions during developmental gene control. Our proteomic survey

of ASF1, CAF1, HIRA, and NAP1 provides a starting point for

the functional analysis of the regulatory networks in which these

chaperones participate. As illustrated by our analysis of LAF-A

and RLAF-N, specific protein-protein associations and gene


Molecular Cell


Are

lati

ve L

ID C

hIP

(RN

Ai/

Mo

ck)

00.20.40.60.81.01.21.4

Enhancer Promoter

ASF

1

SIN

3A

EMSYPF

1

LID

HIR

A

Mo

ck

RPD

3

NA

P1 H

ASF

1

SIN

3A

EMSYPF

1

LID

HIR

A

Mo

ck

RPD

3

NA

P1 H

RNAi:

B

C

E F

Enhancer Promoter

0

0.4

0.8

1.2

1.6

H2B

ASF

1

SIN

3A

EMSYPF

1

LID

HIR

A

Mo

ck

RPD

3

NA

P1 H

RNAi:

ASF

1

SIN

3A

EMSYPF

1

LID

HIR

A

Mo

ck

RPD

3

NA

P1 H

D

Enhancer Promoter

0

0.4

0.8

1.2

1.6

H3

ASF

1

SIN

3A

EMSYPF

1

LID

HIR

A

Mo

ck

RPD

3

NA

P1 H

RNAi:

ASF

1

SIN

3A

EMSYPF

1

LID

HIR

A

Mo

ck

RPD

3

NA

P1 H

H3K

4me3

/H3

Enhancer Promoter

2

4

6

0

8

ASF

1

SIN

3A

EMSYPF

1

LID

HIR

A

Mo

ck

RPD

3

NA

P1 H

RNAi:

ASF

1

SIN

3A

EMSYPF

1

LID

HIR

A

Mo

ck

RPD

3

NA

P1 H

246

0

8H

3ac/

H3

Enhancer Promoter

ASF

1

SIN

3A

EMSYPF

1

LID

HIR

A

Mo

ck

RPD

3

NA

P1 H

RNAi:

ASF

1

SIN

3A

EMSYPF

1

LID

HIR

ARP

D3

NA

P1 H

1012

rela

tive

NA

P1 C

hIP

(RN

Ai/

Mo

ck)

00.20.40.60.81.01.21.4

ASF

1LI

D H

Mo

ckN

AP1RNAi:

Enhancer Promoter

ASF

1LI

D H

Mo

ckN

AP1

Figure 6. LAF-A and RLAF-N Differentially Modulate Removal of Active Histone Marks at NOTCH Target Genes

(A) Su(H)/H complex, ASF1, NAP1, and LAF subunits, but not RPD3, mediate LID recruitment to E(spl) genes. LID binding to E(spl) enhancers and promoters was

analyzed by ChIP-qPCR. Crosslinked chromatin was prepared from bio-LID-expressing S2 cells, which were either mock treated or incubated with dsRNA

directed against the indicated mRNAs. bio-LID was precipitated using NeutrAvidin beads. The abundance of E(spl)m4, E(spl)m7, and E(spl)m8 enhancer and

promoter DNA in the precipitates of treated cells was expressed relative to mock. Here, we depict the results for the E(spl)m4 gene. Similar results were obtained

for E(spl)m7 and E(spl)m8 (see Figure S3). All ChIP data in this manuscript are the result of at least three independent experiments. Error bars represent SE of

mean.

(B) The Su(H)/H complex mediates NAP1 recruitment to E(spl) genes. ChIPs were performed with a-NAP1 antibodies on either mock-treated S2 cells or cells

depleted for NAP1, ASF1, LID, or H. Results for the E(spl)m4 gene are shown, and similar results were obtained for E(spl)m7 and E(spl)m8 (see Figure S4). Error

bars represent SE of mean.

(C) H, ASF1, NAP1, and LAF subunits, but not RPD3, are required for H3K4me3 demethylation at NOTCH target gene enhancers and promoters. Crosslinked

chromatin was isolated from S2 cells that were either mock treated or incubated with dsRNA directed against the indicated mRNAs. H3K4me3 enrichment levels

at the enhancers and promoters of E(spl) genes were determined by ChIP-qPCR and normalized to histone H3. H3K4me3/H3 ratio in mock-treated cells was set

at 1. Results for the E(spl)m4 gene are shown. For E(spl)m7 and E(spl)m8, see Figure S5. Error bars represent SE of mean.

(D) RPD3, H, and NAP1, but not LAF-A, are required for histone H3 deacetylation at enhancers and promoters of E(spl) genes. ChIP-qPCR experiments were

performed as described above. The H3Ac/H3 ratio is strongly increased upon RNAi knockdown of H, NAP1, and RPD3 at both enhancers and promoters of

E(spl) genes. SIN3A only modestly affected H3ac at enhancers, but not at promoters. Neither other LAF subunits nor ASF1 affected H3ac at these sites. Results

for the E(spl)m4 gene are shown. For E(spl)m7 and E(spl)m8, see Figure S6. Error bars represent SE of mean.

790 Molecular Cell 35, 782–793, September 25, 2009 ª2009 Elsevier Inc.

Molecular Cell


targeting provide an intricate network of combinatorial gene

expression control.

EXPERIMENTAL PROCEDURES

Protein Purification, Mass Spectrometry, and In Vitro Interaction

Assays

Embryo NEs were prepared from 0–12 hr old Drosophila embryos or S2 cells,

and immunopurifications were performed as described (Chalkley and Verrijzer,

2004). In brief, extracts were incubated with affinity-purified antibodies

coupled by dimethylpimelimidate crosslinking to protein A Sepharose beads

(GE Healthcare 17-0963-03). We also used S2 cells transfected with BirA biotin

ligase and bio-tagged LID. Biotinylated LID was purified on NeutrAvidin coated

beads (Pierce 29202). Immuno- and biotin-purified complexes were washed

extensively with HEMG buffer (25 mM HEPES-KOH [pH 7.6], 0.1 mM EDTA,

12.5 mM MgCl2, 10% glycerol, and cocktail of protease inhibitors) containing

600 mM KCl and 0.1% NP-40 (HEMG/600). Anti-GST antibodies were used

for mock purifications. Proteins were eluted from the beads with 100 mM

NaCitrate buffer (pH 2.5), resolved by SDS-PAGE, and visualized by Coomas-

sie staining. Mass spectrometry was performed as described using an LTQ-

Orbitrap hybrid mass spectrometer (ThermoFischer) (Wilm et al., 1996). Data

analysis was performed using the Mascot search algorithm (version 2.1;

MatrixScience) searching against the FlyBase database (http://flybase.bio.

indiana.edu; version FB2007_03, released November 1, 2007). NE fraction-

ation by (NH4)2SO4 precipitation, POROS Heparin, and Sephacryl S-300

size-exclusion chromatography were performed as described (Mohrmann

et al., 2004).

Antibodies and Immunological Procedures

For antigen production, we cloned PF1 (encoding aa 437–600), EMSY (aa 173–

385), LID (aa 426–678 and 1226–1414), CAF1-105 (aa 431–583), and full-length

NAP1 and CAF1-55 cDNAs into pGEX4T-1. GST fusion protein expression,

purification, and subsequent immunization were performed as described

(Chalkley and Verrijzer, 2004). The remaining antibodies have been described:

a-ASF1 (Moshkin et al., 2002); a-SIN3A and a-RPD3 (Pile et al., 2002); a-HIRA

(Loppin et al., 2005); a-BRM, a-USP7, and a-ISWI (Mohrmann et al., 2004); and

a-Su(H) (sc-15813, Santa Cruz Biotechnology). CoIPs and immunoblotting

were performed using standard procedures (Mohrmann et al., 2004).

Drosophila Genetics

Fly strains for lid10424, lidk06801, Sin3A08269, rpd304556, and N55e11 were

obtained from the Bloomington stock center (http://flystocks.bio.indiana.

edu/). ey::Gal4, UAS::asf1/CyO stock and HP141 loss-of-function allele were

described previously (Goodfellow et al., 2007; Moshkin et al., 2002). Further

information on gene structure and chromosomal location of genes used is

present at FlyBase (http://www.flybase.org/). All crosses were performed at

25�C and repeated at least three times.

RNAi, RT-qPCR Expression Analysis, and ChIP-qPCR

RNAi and RT-qPCR experiments were performed as described (Moshkin et al.,

2007). In brief, Drosophila S2 cells were cultured in Schneider’s media

(Invitrogen 21720-024) and treated with double-stranded RNA (dsRNA) for

4 days. Double-stranded RNA for LAF-A subunits, RPD3, and HIRA were

synthesized using an Ambion Megascript T7 kit according to the manufac-

turer’s protocol. RNA samples from three fully independent experiments

were prepared and analyzed by RT-qPCR as described (Moshkin et al.,

2007). ChIP-qPCR assays were performed as described (Moshkin et al.,

2007; van der Knaap et al., 2005). In brief, crosslinked chromatin was prepared

from S2 cells and sheared by sonication to an average length of 0.5 kbp. Chro-

matin was then incubated with a-H3K4me3 (ab1791, Abcam), a-H3ac (17-615,

Upstate), a-H2B, or a-H3 (ab8580, Abcam) antibodies and extensively

washed. After IPs, recovered DNA was analyzed by qPCR with SYBR

green I, using the MyiQ single-color real-time PCR detection system (Bio-

Rad). In the case of LID, chromatin was prepared from S2 cells transfected

with BirA biotin ligase and bio-tagged LID. Chromatin fragments associated

with biotinylated LID were precipitated with Neutravidin beads (Pierce). Oligo-

nucleotide sequences used for RT-qPCR, qPCR, and dsRNA synthesis will be

provided upon request.

SUPPLEMENTAL DATA

Supplemental Data include seven figures and 11 tables and can be found with

this article online at http://www.cell.com/molecular-cell/supplemental/S1097-

2765(09)00516-4.

ACKNOWLEDGMENTS

We thank R. Eisenman and J. Secombe for discussions, sharing unpublished

results, and reagents; D. Wassarman for antibodies directed against SIN3A

and RPD3; and B. Loppin for anti-HIRA. This work was supported by grants

from the European Community EuTRACC, LSHG-CT-2007-037445 (to

C.P.V.); an EMBO Long Term Fellowship (Y.M.M.); and an MRC program grant

(to S.J.B.).

H

EMSY

me

PF1 SIN3A

LIDASF1

Su(H)GRO

SKIP

Rpd3

Ac

MRG15

EMSY

PF1 SIN3A

LID

MRG15

NAP1

CtBPme

H3K4

H3K4 H3

Figure 7. Model for Differential Chromatin Silencing at NOTCH

Target Genes

The Su(H)/H complex is the central targeting factor of NOTCH-regulated

genes. In the absence of Nicd, Su(H)/H binds several corepressors, including

CtBP and GRO. The Su(H)/H corepressor complex directs ASF1, NAP1, and

the LAF complexes to the E(spl) genes. H3/H4 chaperone ASF1 mediates

silencing of NOTCH target genes by providing a connection between LAF

and Su(H)/H and by facilitating H3K4 demethylation by LID. The H2A/H2B

chaperone NAP1 participates in E(spl) silencing by targeting RLAF to Su(H)/

H-bound genes and stimulating both H3K4 demethylation by LID and H3 de-

acetylation by RPD3. Finally, NAP1, but not ASF1, controls nucleosome

density at the repressed loci. Thus, ASF1 and NAP1 differentially modulate

chromatin to mediate gene-selective silencing. See the main text for details.

(E and F) H and NAP1 directly affect nucleosome density at E(spl) enhancers and promoters. ChIP-qPCR experiments were performed as above using antibodies

against histone H2B (E) or histone H3 (F). Levels of H2B and H3 are reduced upon RNAi-mediated knockdown of NAP1 or H at both enhancers and promoters of

E(spl) genes, but loss of ASF1 or (R)LAF subunits did not affect histone density at these sites. We depict results for the E(spl)m4 gene. Similar results were

obtained for E(spl)m7 and E(spl)m8 (see Figures S7 and S8). Error bars represent SE of mean.


http://flybase.bio.indiana.edu

http://flybase.bio.indiana.edu

http://flystocks.bio.indiana.edu/

http://flystocks.bio.indiana.edu/

http://www.flybase.org/

http://www.cell.com/molecular-cell/supplemental/S1097-2765(09)00516-4

http://www.cell.com/molecular-cell/supplemental/S1097-2765(09)00516-4

Molecular Cell


Received: December 19, 2008

Revised: April 16, 2009

Accepted: July 27, 2009

Published: September 24, 2009

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Histone Chaperones ASF1 and NAP1 Differentially Modulate Removal of Active Histone Marks by LID-RPD3...

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