a novel n-cor complex contains components of the mammalian

A novel N-CoR complex contains components of the mammalian SWI/SNF complex and

the corepressor KAP-1.

Caroline Underhill1, Majdi S.Qutob3, Siu-Pok Yee 2,3, Joseph Torchia1,2*

Cancer Research Laboratories, London Regional Cancer Centre, Dept. of Pharmacology and Toxicology1 and Oncology2 and Biochemistry 3 The University of

Western Ontario, London, Ontario, Canada.

*Corresponding Author: Dr. Joseph Torchia Cancer Research Laboratories. Rm 4020 London Regional Cancer Centre 790 Commissioners Road East London, Ontario Canada, N6A 4L6 Fax: (519) 685-8646 Tel: (519) 685-8692 E-mail: [email protected]

Running Title: Purification of Corepressor Complexes.

Key Words: repression, nuclear hormone receptors, deacetylase, purification.

Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on September 29, 2000 as Manuscript M007864200 by guest on M

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Summary

Transcriptional silencing by many transcription factors is mediated by the Nuclear

Receptor Corepressor (N-CoR). The mechanism by which N-CoR represses basal

transcription involves the direct or indirect recruitment of histone deacetylases (HDACs).

We have isolated two multiprotein N-CoR complexes, designated N-CoR-1 and N-CoR-2,

which possess histone deacetylase activity that is mediated by distinct HDACs. Based on

western blotting using antibodies against known subunits, the only HDAC found in the N-

CoR-1 complex is HDAC3. In contrast, N-CoR-2 contains predominantly HDAC1 and

HDAC2, as well as several other subunits that are found in the Sin 3A/HDAC complex.

Using mass spectrometry and western blotting, we have identified several novel

components of the N-CoR-1 complex which includes the SWI/SNF-related proteins BRG1,

BAF 170, BAF 155, BAF 47/INI1, and the corepressor KAP-1 that is involved in silencing

at heterochromatin. Indirect immunofluorescence has revealed that both KAP-1 and N-

CoR co-localize throughout the nucleus. These results suggest that N-CoR is found in

distinct multiprotein complexes, which are involved in multiple pathways of transcriptional

repression.

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Purification of corepressor complexes

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Introduction

Cellular proliferation and differentiation is critically dependent on the ability of sequence

specific DNA-binding transcription factors to activate or repress the transcription of target genes

in a coordinated fashion. This is accomplished through transcription factor-mediated recruitment

of coactivators and corepressors which can regulate transcription through the modification of the

local chromatin environment and by specifically interacting with components of the core

transcriptional machinery (1,2). The nuclear hormone receptor superfamily provides a unique

model to study the mechanism of transcriptional activation and repression and the role of

reversible chromatin modification in the control of gene expression. In the absence of ligand,

nuclear hormone receptors such as the thyroid hormone (T3R) and retinoic acid receptors (RAR)

function as potent repressors by interacting with specific corepressor proteins (3,4). Ligand

binding induces a conformational change in these receptors that results in corepressor release and

the recruitment of coactivator proteins (2,5). The Nuclear Receptor Corepressor (N-CoR), and its

related family member Silencing Mediator for Retinoid and Thyroid hormone receptor (SMRT),

were initially identified by yeast two hybrid screening using unliganded T3R or RAR as bait,

respectively (3,4). Several lines of evidence suggest that both N-CoR and SMRT mediate the

repressive effects of nuclear hormone receptors. First, both N-CoR and SMRT contain two

nuclear receptor interaction domains (IDs) in the carboxy terminus. Molecular characterization

of the ID’s reveals the presence of a signature CoR box motif that is necessary and sufficient for

receptor interaction and ligand-induced release of N-CoR or SMRT (6-8). More recently, a

strong correlation between repression mediated by the T3R and recruitment of N-CoR or SMRT

have also been demonstrated in Xenopus oocytes (9,10). Second, microinjection of anti-N-CoR

antibodies into living cells blocks T3R- and RAR-mediated repression (11).


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N-CoR and SMRT contain three highly conserved repression domains designated RD1,

RD2 and RD3 (3,4,12-14). The lack of homology between these domains suggests that their

mechanism of action is mediated via distinct pathways. RD1 serves as a major interacting

surface for the corepressor Sin3A, which, in turn, can directly interact with the histone

deacetylase 1 and 2 (HDAC1 and 2), suggesting that repression by N-CoR and SMRT is linked

to the deacetylation of histones (11,14,15). Single cell microinjection studies using antibodies

against mSin3, or its associated histone deacetylases, inhibits repression by unliganded T3R and

RAR (11). Transcriptional repression by nuclear hormone receptors is also blocked by

deacetylase inhibitors such as Trichostatin A (TSA) (11). Taken together, these studies suggest

that the repressive effects of N-CoR and SMRT are mediated, in part, by a Sin3/HDAC complex.

In contrast, the RD3 of SMRT and N-CoR interacts with the class II histone deacetylases that are

structurally related to the yeast Hda1 protein (16,17). The interaction between SMRT and class II

HDACs occurs in vivo and in vitro and correlates with the repressor activity of RD3.

Recent studies have also implicated N-CoR and SMRT in transcriptional repression that

is mediated by several other families of transcription factors including Notch (18), the

homeodomain proteins RPX and Pit-1 (19), p53 (20) and the antagonist-bound estrogen and

progesterone receptors (21, 22). Abnormal recruitment of N-CoR and SMRT has also been

linked to pathological disorders such as acute promyelocytic leukemia (PML) that is associated

with rearrangements of the gene encoding the RARα receptor and the PML zinc finger protein.

This results in the formation of a PML-RAR fusion protein that blocks myeloid differentiation.

Treatment with retinoic acid causes the release of N-CoR/SMRT and abolishes the RAR-PML-

mediated differentiation block (23, 24).


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Biochemical purification of the mammalian Sin3 has identified a Sin3 complex which

consists of 8-10 proteins and includes HDAC1 and 2, RbAp46/48, Sap 18 and Sap 30 (25, 26).

However, neither N-CoR nor SMRT were identified as components of the mSin3/HDAC

complex, or a related complex known as NURD/Mi2/NRD, which contains both histone

deacetylase and nucleosome remodelling activity (27-29). Based on these studies, N-CoR may

not be a stable component of a deacetylase complex and may function as an adaptor protein for

sequence-specific transcriptional repressors upon activation of specific signalling pathways.

Alternatively, it has been suggested that N-CoR may be targeting a different subpopulation of

cellular mSin3 or HDACs (30).

To address this issue, we have undertaken a biochemical purification of endogenous N-

CoR from HeLa cell nuclear extracts by combining conventional chromatography with

immunoaffinity purification using an affinity purified anti-N-CoR antibody. We have isolated

two major N-CoR-containing complexes designated N-CoR-1 and N-CoR-2 which, on the basis

of gel filtration chromatography, are each approximately 2.0 MDa. Both complexes possess

intrinsic histone deacetylase activity based on their ability to deacetylate core histones. We

demonstrate that N-CoR-2 contains proteins that are found in the Sin3/HDAC and

NURD/Mi2/NRD complexes, which are not found in N-CoR-1 suggesting that N-CoR-1 is a

novel N-CoR-containing complex. Using mass spectrometry we have identified several

components of the N-CoR-1 complex that includes several subunits of the SWI/SNF-related

chromatin remodelling complexes as well as the corepressor protein KAP-1. Taken together,

these findings implicate N-CoR in multiple pathways of transcriptional repression that may

involve the recruitment of distinct corepressor complexes.


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Experimental Procedures

Western blotting and antibody production

Subcellular fractions of cells were prepared according to standard methods (31). Western

blotting was performed as described previously (32). Protein samples were separated by SDS-

PAGE, transferred to nitrocellulose and detected by Enhanced Chemiluminescence according to

the manufacturers recommendations (Amersham). The HDAC2 antibody and the Sin3A antibody

were purchased from Santa Cruz Biotechnology. The RbAp48 antibody was from Upstate

Biotechnology. The N-CoR polyclonal antisera was raised against a His-tagged recombinant N-

CoR (aa 2232-2453) protein and was purified by protein A Sepharose chromatography. To

further purify the anti-N-CoR antibody, the IgG fraction was passed through an affinity column

consisting of the His-tagged recombinant N-CoR protein (aa 2232-2453) crosslinked to

Sepharose 4B, and the specific antibody was eluted with 100 mM glycine (pH 2.8).

Purification of the N-CoR complexes

Forty litres of HeLa cells, grown to mid-log phase, were obtained from the National Cell Culture

Centre (Minneapolis, MN). Nuclear extracts were prepared according to standard methods (31).

To purify the nuclear N-CoR complexes, the nuclear extract was dialyzed against buffer A (20

mM Tris-HCl, pH 7.9, 0.5 mM EDTA, 0.5 mM EGTA, 10% glycerol, 0.5 mM DTT, 0.2 mM

PMSF and 5 µg/ml each of leupeptin, aprotinin and pepstatin A) containing 100 mM KCl and

was loaded onto an 80 ml P11 phosphocellulose column pre-equilibrated with the same buffer.

The flowthrough was collected and the column was washed sequentially in a stepwise fashion

using buffer A containing 0.3 M, 0.5 M and 1.0 M KCl. At each step, the column was washed

with two column volumes of buffer containing the corresponding salt concentration to allow UV


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absorbance to stabilize near baseline prior to collection of the next protein peak. The majority of

immunoreative N-CoR was found in the 0.3 and 0.5 M KCl fractions. The N-CoR-containing

fractions were pooled, dialyzed against buffer A containing 100 mM KCl and then passed

through a DEAE Sepharose column and eluted with an increasing KCl gradient. All fractions

were analyzed by western blotting using anti-N-CoR antibody, the N-CoR-containing fractions

were again pooled, concentrated by precipitating with 20-60% ammonium sulphate and then

applied to a Sephacryl S300 column pre-equilibrated with buffer A containing 100 mM KCl.

The column was washed with buffer A at a flow rate of 0.4 ml/min. Each N-CoR complex eluted

as a single large molecular mass peak of approximately 2.0 MDa. The fractions corresponding to

each peak were analyzed for N-CoR by western blotting, pooled, and dialyzed against buffer A

containing 100 mM KCl without DTT. For affinity purification of the N-CoR complexes,

affinity purified N-CoR antibody was crosslinked to protein A Sepharose using

dimethylpalmilidate (DMP) according to standard procedures (33). The pooled fractions from the

gel filtration step were precleared using protein A Sepharose crosslinked to rabbit IgG. The

precleared sample was then loaded onto the affinity column at a flow rate of 0.4 ml/min and the

flowthrough was collected and reloaded onto the affinity column five times. The bound proteins

were washed with ten column volumes each of buffer A (without DTT) containing 0.1% NP40

and 0.3M KCl, 0.1% NP40 and 0.5M KCl, 0.5% NP40 and 0.7M KCl, and a final wash in buffer

A containing 100 mM KCl prior to elution with 100 mM glycine (pH 2.8). For mock purification

experiments, samples from the gel filtration step were loaded onto protein A sepharose alone, or

protein A sepharose crosslinked to an irrelevant antibody.


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Mass spectrometry

Proteins were separated by 7% SDS-PAGE and then stained with colloidal blue for one hour

followed by destaining in 25% MeOH for an additional 2 hrs. The protein bands were excised

and cut into 1 mm3 pieces. The gel pieces were washed twice in a 50% CH3CN solution for 5

min followed by two washes with a 250 µl solution consisting of 50% CH3CN/50 mM

NH4HCO3 for 30 min. The gel pieces were lyophilized, rehydrated in 10 mM NH4HCO3 (pH 8.5)

containing 0.1 µg/µl trypsin (Roche) and incubated overnight at 37EC. The tryptic fragments

were extracted by two 30 min washes with a solution containing 60% CH3CN/10% TFA. The

combined solutions were lyophilized using a speedvac, resuspended in 20 µl 0.5% TFA solution

and the peptide suspensions were purified using a ZipTip (Millipore) cartridge according to the

manufacturers instructions prior to analysis by MALDI-MS.

MALDI-MS analyses were carried out by using a Perseptive Biosystem Voyager-DE

STR mass spectrometer (Perseptive Biosystems Inc., Farmingham, MA), equipped with a pulsed

UV nitrogen laser (337 nm, 3-ns pulse) and a dual microchannel plate detector. The spectra were

acquired in reflectron-DE mode, acceleration voltage was set to 20 kV, grid voltage at 72% of

the acceleration voltage, guide wire voltage at 0.020%, delay time at 140 ns, low mass gate was

set at 250 Da, and the mass to charge ratio was calibrated internally with the dimer of α-cyano-4-

hydroxycinnamic acid ([2M+H]+ 380.09 Da) and trypsin autolysis peptide ([M+H]+ 2163.05 Da).

Two parts of α-cyano-4-hydroxycinnamic acid and one part of nitrocellulose were dissolved in

acetone-isopropanole (4:1) to final concentrations of 20 and 10 mg/ml, respectively. A 0.5 µl

volume of this solution was deposited on MALDI target and allowed to dry. 0.5 µl of 1% acetic

acid was placed on top of the matrix layer followed by addition of 1 µl of analyte solution. Mass


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spectra were recorded after evaporation of the solvent and processed using GRAMS software for

data collection and analysis.

Peptide sequence analysis was conducted by Post-Source Decay (PSD) technique. The

spectra were acquired at DE-reflectron mode. The accelerating voltage was set at 20 KV, grid

voltage at 75%, guide wire voltage at 0.024%, and delay time at 100ns. The timed ion selector

was pre-set to the protonated molecular weight of the analyte. The spectra were acquired in 10-

13 segments with mirror ratio 1.0 to 0.1342 and finally "stitched" together by the instrument

software. The tryptic peptides obtained were used to search for protein candidates in the

nonredundant protein sequence database with the program PROWL that is publicly available

(http://prowl.rockefeller.edu).

Immunofluorescence

HeLa cells were plated onto Fisherbrand microscope slides and maintained in DMEM

with 10% FBS at 37°C with 5% CO2 . Cells were allowed to adhere for approximately 18 hr

prior to fixation with 4% paraformaldehyde for 10 min. The cells were then permeabilized with

0.2% Triton X-100 in PBS and blocked for 30 min in 5% goat serum. After a brief wash, slides

were incubated with rabbit anti-N-CoR antibody (1:300 dilution) and anti-KAP-1 mouse

monoclonal IgG antibody supernatant (1:10 dilution) in 3% BSA at 4EC overnight. Cells were

then washed three times in PBS for 5 min, and then incubated for 4 hrs at room temperature with

anti-mouse IgG conjugated to rhodamine (1:500 dilution) and anti-rabbit IgG conjugated to

fluorescenin (1:500 dilution) (Santa Cruz). Images were captured using a Sensicam digital

camera on a Olympus Provis AX-70 microscope and analyzed using Image Pro Plus version 4.0.


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Purification of radiolabelled HeLa core histones

Core histones were purified from HeLa cells essentially as described with some

modifications (34). Briefly, 1 liter of HeLa cells were grown to a density of 0.8x106 cells/ml in

DMEM supplemented with 7% FBS. The cells were preincubated with 10 mM sodium butyrate

for 24 hrs before harvesting. The cells were then centrifuged and resuspended in 50 ml of PBS

supplemented with 100 µg/ml cyclohexamide, 10 mM sodium butyrate and 0.1 mCi/ml [3H]

acetic acid and incubated at 37EC for 2hrs with gentle stirring. The cells were then washed three

times with PBS (500 x g, 5 min) and then lysed in 10 ml of buffer B (20 mM Tris-HCl (pH 7.6) ,

2 mM MgCl2, 3mM CaCl2, 10 mM sodium butyrate, 0.2 mM PMSF and 1% NP40). Nuclei were

collected by centrifugation (2,000xg for 10 min), washed twice in 10 ml of buffer B and then

resuspended in buffer B without NP40 (buffer C). The nuclear proteins were extracted by adding

buffer C containing 0.42 M KCl, followed by centrifugation at 10,000xg for 30 min. The nuclear

pellet was resuspended in two volumes of buffer C and then extracted twice with 0.2 M H2S04

for 90 min in an icebath with gentle stirring then centrifuged (30,000xg for 20 min). The

supernatants were pooled, dialyzed extensively against 100 mM acetic acid and the histones were

precipitated with ten volumes of ice-cold acetone and resuspended in water to a concentration of

approximately 2 mg/ml.

Histone deacetylase activity

Histone deacetylase activity was monitored as described (35). Aliquots of the affinity purified

complexes were retained on 25µl of Protein A Sepharose affinity resin containing crosslinked

anti-N-CoR antibody. The beads were washed extensively with buffer A containing 500 mM

KCl and 0.5% NP40 and then resuspended in buffer D (20 mM Tris-HCl (pH 7.6) 100 mM KCl,


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0.1 mM EDTA, 0. 2 mM DTT and 0.2 mM PMSF) containing [3H]histones (20,000 cpm/rxn) to

a final volume of 200 µl. The reactions were allowed to proceed at 37EC for 90 min and stopped

by the addition of 0.05 ml of 0.1 M HCl/0.7 M acetic acid solution. Released [3H]acetate was

extracted with 0.6 ml ethyl acetate. After centrifugation, 0.3 ml of the upper organic phase was

used for liquid scintillation counting.

Results

Identification of N-CoR-containing complexes.

As a first step in determining whether endogenous N-CoR is a stable component of a corepressor

complex, we generated a polyclonal antibody against the carboxy terminus of N-CoR. The

affinity purified antibody recognized a single protein band of approximately 260 kDa that is

consistent with the size of N-CoR (Fig 2A). Nuclear extracts prepared from HeLa cells were

initially fractionated on a P11 phosphocellulose column using a step gradient with increasing salt

concentrations (Fig.1A and 1B). Western blot analysis of the eluates derived from this column

demonstrated that N-CoR was present in three fractions. A distinct N-CoR-containing fraction

was eluted with 0.3 M KCl buffer (N-CoR-1). N-CoR immunoreactivity was also found in

fractions eluted with 0.5 M (N-CoR-2) and a minor component was also detected in the 1.0 M

KCl (data not shown). To confirm that efficient protein separation was achieved with the P11

column, we analyzed the eluates for the transcriptional coactivator p/CIP (32), which is part of a

family of nuclear receptor coactivator proteins required for ligand dependent signalling. p/CIP

eluted from the phosphocellulose column with 0.1 M KCl. A similar elution profile was obtained

with other members of this family of coactivators (data not shown) suggesting that they are

found in complexes that are distinct from N-CoR.


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The relative abundance of Sin3A was also examined in the fractions eluted from the

phosphocellulose column. Although Sin3A was detected in all of the N-CoR-containing

fractions, the relative distribution was not consistent with the distribution of N-CoR. Based on

densitometric scanning, only 7% of the total amount of Sin3A was found in the 0.3 M KCl

fraction suggesting that the major N-CoR-containing fraction is not complexed to Sin3A but may

function in other contexts (16,17).

To characterize these complexes further, we subjected the P11-purified fractions to

purification by DEAE-Sepharose followed by gel filtration chromatography using a Sephacryl S-

300 column. Immunoblot analysis of the eluates derived from the Sephacryl S300 column

demonstrated that N-CoR-1 and N-CoR-2 eluted with the void volume corresponding to a

molecular mass of approximately 2.0 MDa (Fig. 1C). Since N-CoR is a 260 kDa protein, this

result strongly suggests that it is found as part of large multiprotein complexes. To purify these

complexes further, the affinity purified N-CoR antibody was crosslinked to protein A Sepharose

and the N-CoR-containing fractions isolated from the Sephacryl S300 column were pooled and

applied to the immunoaffinity column. After extensive washing with highly stringent buffer

containing 0.5% NP40 and 0.7M KCl, the retained proteins were eluted with 100 mM glycine

(pH2.8).

Western blot analysis of the immunopurified N-CoR-1 complex demonstrated that the

majority of N-CoR bound to the immunoaffinity column and that a single immunoreactive peak

was present in the eluate with no significant breakdown products detected (Fig. 2A). Silver

staining of an SDS-PAGE gel of the N-CoR-1 complex consistently identified approximately 20

proteins ranging in molecular weight from 35 to 350 kDa (Fig. 2B). The interaction of these

polypeptides appears to be specific since the majority of these are not retained when an


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analogous “mock” purification was performed using a control affinity column. Since the

mechanism of N-CoR action has been linked to the recruitment of a Sin3A/HDAC complex, we

initially tested the eluate by western blotting using antibodies against specific subunits found in

this complex (Fig. 2C). Although Sin3A, HDAC1 and 2 migrated through the Sephacryl S300

column as large molecular weight complexes and appeared to coelute with N-CoR (data not

shown), they were not retained by the anti-N-CoR immunoaffinity column. This suggests that

they are not intrinsic components of the N-CoR-1 complex and are most likely associated with

other multiprotein complexes. The only component that was detected by western blotting was

HDAC3, which is consistent with recent observations (9, 10, 36, 37). In addition, we were

unable to identify either RbAp48, which has been demonstrated to be a common subunit found in

several chromatin modifying complexes, or Sap 30 a component of the Sin3/HDAC complex.

Taken together, these observations suggest that the N-CoR-1 complex contains novel

components.

The finding that HDAC3 is a component of the N-CoR-1 complex prompted us to

examine whether N-CoR-1 complex also possesses deacetylase activity. [3H]-labelled core

histones were purified from HeLa cells, preincubated with immunopurified N-CoR-1 and the

deacetylase activity was monitored by quantifying the release of radiolabelled acetyl CoA. We

observed that N-CoR-1 can deacetylate core histones in this assay, consistent with the presence

of HDAC3, and that the deacetylation activity is blocked when the complex was preincubated

with the deacetylase inhibitor TSA (Fig. 2D).

Mass spectrometric analysis of the N-CoR-1 complex.


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To identify novel components of the N-CoR-1 complex, SDS-PAGE analysis was performed and

several of the protein bands were excised and subjected to in-gel tryptic digestion. The peptides

were then isolated and analyzed by mass spectrometry (Table 1, Fig. 3). The majority of the

tryptic fragments derived from the 300 kDa protein band corresponded to N-CoR. We also

detected several tryptic fragments corresponding to the related family member SMRT although

these were relatively minor components Interestingly, several of the proteins which consistently

copurified with N-CoR have been found as intrinsic components of unrelated, yet biologically

important complexes. For example, SAP 130 and SF3a120 are subunits of protein complexes

essential for spliceosome assembly (38, 39). However, a function for SAP 130 and SF3a120 in

transcriptional regulation has not been previously uncovered.

Four of the proteins identified by mass spectrometry belong to the SWI/SNF family of

proteins and include the BRG1-Associated Factor (BAF) 170, BAF 155, BAF 47/INI1 and the

SWI/SNF Related CBP Activator Protein (SRCAP) (40). BAF 170 and BAF 155 are highly

similar homologues of the yeast SWI3 protein whereas BAF 47/INI1 represents the mammalian

homologue of the yeast SNF5 protein. All three proteins have been identified as subunits of

several SWI/SNF-related chromatin remodeling complexes including the human BRG1 and

hBRM complexes (41,42). We were unable to obtain definitive evidence for the BRG1

component of the SWI/SNF complex using mass spectrometry. However western blotting using

an anti-BRG1 antibody subsequently demonstrated that the p190 subunit is BRG1 suggesting

that the four core components of the SWI/SNF complex are found in the N-CoR-1 complex (Fig.

4).

To verify that these components co-localize with N-CoR, western blotting was performed

using polyclonal antibodies that recognize either BRG1, BAF 170, BAF 155 or BAF 47. We


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found that all of these proteins eluted as a high molecular weight complex from the Sephacryl

S300 column. Although the overlap with N-CoR was clearly significant, it was not identical

suggesting that the association of N-CoR with SWI/SNF is not 100%. (Fig. 4A). Importantly, all

of these same proteins are found in the immunoaffinity purified N-CoR-1 complex (Fig. 4B),

consistent with the mass spectrometry data.

KAP-1 and N-CoR colocalize in vivo.

The 110 kDa protein identified by mass spectrometry corresponds to Krab-associated

protein 1 (KAP-1) also known as TIF1β (43,44). KAP-1 is a 97 kDa transcriptional corepressor

that interacts with proteins containing the Kruppel associated box domain (KRAB), a common

motif found in many DNA binding transcriptional repressor proteins. Recent observations have

also demonstrated that KAP-1 interacts in vivo and in vitro with members of the heterochromatin

protein 1 (HP-1) family, which are important for regulating heterochromatin-mediated gene

silencing (45,46). HP-1 proteins have also been implicated in position effect variegation, a

euchromatic silencing mechanism exhibited by genes placed within or adjacent to

heterochromatin (47). Interestingly, it has been shown that TSA can interfere with KAP-1/TIF1β

mediated repression (48). This suggests that the mechanism of repression mediated by KAP-1

also involves histone deacetylation and is consistent with the presence of HDAC3 in the N-CoR-

1 complex.

To verify that KAP-1 co-localizes with N-CoR, western blotting was performed using

polyclonal antibodies which recognize either N-CoR or KAP-1. We found that a significant

component of KAP-1 also copurified with N-CoR as a high molecular weight complex from the

Sephacryl S300 column (Fig. 5A), and was also present in the immunoaffinity purified N-CoR-1


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complex (Fig. 5B). To define a potential in vivo role for KAP-1/N-CoR association, we

performed indirect immunoflourescence in asynchronously growing HeLa cells using a

monoclonal antibody raised to KAP-1( kindly provided by Dr. F. Rauscher III) together with

affinity purified anti-N-CoR antibody. Both KAP-1 and N-CoR staining were found throughout

the nucleus as an even speckled pattern (Fig. 5C). In some regions, both N-CoR and KAP-1 were

concentrated in micropunctate-like structures which may represent regions of pericentromeric

heterochromatin. When KAP-1 and N-CoR staining were directly compared, a significant

component of the signals overlap particularly in the granular regions presumed to be

pericentromeric heterochromatin. Taken together, these data indicate that a major fraction of

KAP-1 and N-CoR are found in the same complex in vivo and may function through a similar

mechanism.

Identification of the N-CoR/Sin3/HDAC complex

A similar purification scheme was used to affinity purify the N-CoR-2 complex.

However, SDS-PAGE analysis revealed a different protein profile when compared to the N-

CoR-1 complex (Fig. 6A). In this case, approximately 12-15 proteins consistently copurified with

N-CoR. Surprisingly, we observed that several of the proteins found in the Sin3/HDAC complex

are also found in the immunopurified N-CoR-2 complex including Sin3A, HDAC1 and HDAC2

and Sap30 (Fig. 6B). HDAC3 was detected in the N-CoR-2 complex although it does not appear

to be a stoicheometric component. We could not detect RbAp48 although it was clearly present

in the Sephacryl S300 fraction.

To assess whether N-CoR-2 also possesses histone deacetylase activity, [3H]-labelled

core histones purified from HeLa cells were preincubated with the immunopurified complexes


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and the deacetylase activity was monitored by quantifying the release of radiolabelled acetyl

groups. N-CoR-2 deacetylated core histones in this assay, consistent with the presence of

HDAC1 and HDAC2, and the deacetylase activity is blocked when the complex was

preincubated with the deacetylase inhibitor TSA (Fig. 6C). These data provide direct

biochemical evidence for the existence of an N-CoR/Sin3/HDAC complex, consistent with

earlier reports (11,14).

Discussion

Functional studies using microinjection of neutralizing antibodies into living cells have

provided strong evidence suggesting that the repressive effects of N-CoR and SMRT are

mediated, in part, through the recruitment of a Sin3/HDAC complex (11,14). However,

biochemical purification studies have been unable to demonstrate the existence of an endogenous

N-CoR/Sin3A/HDAC complex (17,25,26). Two hypotheses have been proposed to explain these

results. First, it is possible that the interactions between N-CoR or SMRT and the Sin3/HDAC

complex are transient, or require the recruitment of additional factors to become stable

components (49). Second, that multiple N-CoR complexes exist which contain distinct subunits

and become associated with different classes of transcription factors. Partial evidence for this

hypothesis has come from studies using confocal microscopy which demonstrated that the

distribution of N-CoR is heterogenous and that only a small fraction of endogenous N-CoR is co-

localized with HDAC1(30).

In the present study, we have used phosphocellulose chromatography to separate

endogenous N-CoR into two chromatographically distinct fractions designated N-CoR-1 and N-

CoR-2. We have purified N-CoR-1 and N-CoR-2 to apparent homogeneity and have used both


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western blotting and mass spectrometry to demonstrate that each complex contains distinct

proteins. For example, although N-CoR-1 and N-CoR-2 both possess HDAC activity, this

activity is mediated by different HDACs. HDAC3 represents the major class I deacetylase in the

N-CoR-1 complex, whereas HDAC1 and 2 are found only in the N-CoR-2 complex. The

presence of different catalytic subunits in each of the N-CoR complexes suggests that they may

have unique functional roles in the cell.

Our results are consistent with recent findings demonstrating that HDAC3 is part of a

complex, which also includes N-CoR or SMRT (9,10,36,37). It is interesting that the number of

proteins which we have identified in the N-CoR complexes is considerably greater than the

number of proteins identified in the SMRT core complex. This suggests that there are intrinsic

differences between SMRT and N-CoRs ability to interact with other intracellular proteins.

In addition to HDAC3, the N-CoR-1 complex also contains several proteins found in the

SWI/SNF complex (50). The SWI/SNF complex is the prototypical chromatin remodelling

complex which has been shown to disrupt chromatin structure and facilitate the binding of

transcriptional activators to nucleosomal sites (51-53). The glucocorticoid receptor (GR) can

target the SWI/SNF complex to chromatinized templates containing GR binding sites in yeast

and in mammalian cells resulting in disruption of local nucleosomal structure (54, 55). Multiple

mammalian SWI/SNF complexes have also been identified which have diverse subunit

compositions although BRG1, BAF 170, BAF 155 and BAF 47 represent the core catalytic

components (41,42). BRG1 is homologous to the yeast SWI2/SNF2 and possesses DNA-

dependent ATPase activity, a necessary function carried out by all chromatin remodelling

complexes identified to date. Reconstitution studies have shown that BRG1 alone can remodel

nucleosomes, although this remodeling activity is enhanced when all four core components are


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present (56). Thus, one possibility is that the core BAFs may be involved in targeting or

stabilizing nucleosomes in a particular conformation that is conducive to remodelling activity by

BRG1.

The presence of BRG1 strongly suggests that the N-CoR-1 complex possesses chromatin

remodeling activity. This hypothesis is consistent with recent studies demonstrating that

chromatin remodeling is required for transcriptional repression as well as activation. Genome-

wide expression analysis using DNA microarrays have shown that mRNA levels for many genes

are elevated in yeast mutants carrying mutations for SWI/SNF (57,58). In mammalian cells, Rb

forms a complex with hSWI/SNF to inhibit transcription of cyclin E and A, resulting in growth

arrest (59,60). Finally, several labs have identified multiprotein chromatin remodeling complexes

which are believed to be directly involved in transcriptional repression. One such complex,

known as NuRD or Mi2, possesses both histone deacetylase and nucleosome remodeling activity

suggesting that chromatin modifying enzymes can be coupled to regulate transcription in vivo

(27-29). Thus, chromatin remodelling may be necessary step to facilitate the binding of N-CoR

by specific factors associated with nucleosomal DNA. Interestingly, the gene encoding BAF

47/INI 1 has been found mutated or deleted in various rhabdoid sarcoma tumour cell lines and in

primary rhabdomyosarcoma (61). This suggests that the repressive effects mediated by the N-

CoR-1 complex represents a critical mechanism for the regulation of specific genes important for

muscle cell proliferation

We have also established that KAP-1 is an intrinsic component of the N-CoR-1 complex.

KAP-1 functions as a bona fide corepressor involved in mediating the repression of a large

family of Kruppel-like zinc finger proteins which contain the KRAB repression domain (KRAB-

ZFP’s) (41,42). KAP-1 binds to multiple KRAB-containing zinc finger proteins in vivo and in


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vitro and mutations that abolish repression also abolish interaction with the KRAB domain. A

mechanistic link between KAP-1 and histone modification has also been established with the

demonstration that TSA can interfere with KAP-1-mediated repression (46). It has recently been

demonstrated that KAP-1 associates with members of the heterochromatin protein 1 (HP1)

family in vivo and in vitro (43,45). HP1 proteins are conserved nonhistone chromosomal proteins

associated primarily with pericentric heterochromatin where it is believed they function as

regulators of hetrochromatin silencing. The finding that KAP-1 and N-CoR colocalize in vitro

and in vivo suggests that their mechanism of action is similar. Importantly, N-CoR, through

KAP-1-directed recruitment of HP-1 proteins, may be involved in the assembly and/or

maintenance of heterochromatin at specific sites within the genome suggesting an entirely novel

function for N-CoR.

Based on western blotting using selected antibodies, N-CoR-2 contains several proteins

found in the Sin3A/HDAC complex. These results are in contrast to studies which have failed to

demonstrate the presence of such a complex (9, 10, 36). There may be several possible

explanations for these differences. First, purification strategies, which rely on the use of specific

antibodies, could disrupt specific protein-protein interactions. Second, the presence of a pre-

existing complex may be cell cycle dependent, or its composition can be regulated through post-

translational modifications. Consequently, variations in growth conditions might determine the

relative abundance of a specific multisubunit complex. Finally, variability in the composition of

the initial starting material may be important for determining the existence of specific

complexes.

In conclusion, we have provided biochemical evidence for the existence of multiple

endogenous N-CoR complexes. The finding that these complexes possess distinct factors


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suggests that they may perform gene-specific functions by utilizing multiple mechanisms of

transcriptional regulation. The continued analysis of the megadalton N-CoR complexes is critical

to deciphering common and unique mechanisms of gene repression that are necessary for the

homeostatic control of differentiated cell function.


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Acknowledgements

We are grateful to Dr. D. Ayer for supplying the Sap 30 antibody, to Dr. W.D. Wang for the

BAF 170, BAF 155 and BAF 47 antibodies, Dr. S. Schreiber for the HDAC-1 antibody and

special thanks to Dr. Rauscher III for the anti-KAP-1 monoclonal and polyclonal antibodies.


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Table Legends

Table 1. Proteins in the N-CoR-1 complex unambigously identified by mass spectrometry.

Figure Legends

Figure 1. Purification of N-CoR-containing complexes. (A) Schematic representation of the

various chromatographic steps used to purify the N-CoR complexes. (B) Western Blot analysis

of the fractions eluted from the P11 phosphocellulose column. The salt concentrations used to

purify the individual fractions are indicated at the top. For each sample, 20 µg of protein were

separated by SDS-PAGE, transferred to nitrocellulose and then probed with the specific

antibodies indicated on the left. (C) Gel filtration chromatography of the N-CoR complexes

using a Sephacryl S300 column. The molecular weight standards are indicated at the top of the

panel; 660 kDa, thyroglobulin; 440 kDa, ferritin.

Figure 2. N-CoR-1 is a multiprotein complex. (A) Western blot of the immunopurified N-CoR-1

complex. N-CoR-1 eluted from the Sephacryl S300 fraction was immunopurified using an anti-

N-CoR affinity column or an irrelevant antibody column as a control. The purified proteins were

separated by SDS-PAGE and immunoblotted using affinity purified anti-N-CoR antibody. FT;

flowthrough fraction. (B) Silver stained SDS-PAGE gel of the affinity purified N-CoR-

1complex. The Sephacryl S300 fractions containing N-CoR were pooled and purified by affinity

chromatography using an anti-N-CoR affinity column as indicated in “Materials and Methods”.

Bound proteins were eluted from the affinity column using 100 mM glycine (pH 2.8) and

separated by SDS-PAGE. Approximate molecular weights of the isolated polypeptides are

indicated on the right. Molecular weight standards are indicated on the left. Control, aliquot from


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the mock affinity purification; N-CoR-1, aliquot from the fraction eluted from the affinity

column. (C) Western blot analysis of the affinity purified N-CoR-1 complex using specific

antibodies. Purified N-CoR-1 was separated by SDS-PAGE, transferred to nitrocellulose and

then probed with the specific antibodies indicated on the left. Control, aliquot from the mock

affinity purification; N-CoR, aliquot from the fraction eluted from the affinity column. (D)

Deacetylase activity of the immunopurified N-CoR-1 complex. Immunopurified N-CoR-1 was

preincubated either alone or with 100 nM Trichostatin A (TSA) and [3H]-core histones. The

reaction was allowed to proceed for 90 min prior to extraction and quantitation of the released

[3H]-acetyl CoA. The results are a representative experiment from at least two independent

purifications.

Figure 3. A representative MS spectrum that identifies the 170 kDa band found in the N-CoR-1

complex as BAF 170. The 170 kDa band was excised using a clean scalpel blade and digested

with trypsin overnight. The peptides were extracted and purified using a ZipTip column prior to

analysis by MALDI-TOF MS as described in “Materials and Methods”.

Figure 4. Components of the SWI/SNF complex copurify with N-CoR-1. (A) Gel filtration

chromatography of the N-CoR-1 associated proteins. The DEAE Sepharose fractions containing

N-CoR-1 were pooled, concentrated and passed through an S300 gel filtration column. Fractions

were collected and aliquots were separated by SDS-PAGE, transferred to nitrocellulose and then

probed with the specific antibodies indicated on the left. A fraction of the proteins identified as

components of the SWI/SNF complex co-elute with N-CoR and migrate as a large molecular

weight complex of approximately 2 MDa. (B) Western blot analysis of the affinity purified N-


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CoR-1 associated proteins. The pooled Sephacryl S300 fractions containing N-CoR were pooled

and purified by affinity chromatography using an anti-N-CoR affinity column as indicated in

“Materials and Methods”. Bound proteins were eluted from the affinity column using 100 mM

glycine (pH 2.8) and were separated by SDS-PAGE, transferred to nitrocellulose and then

probed with the specific antibodies indicated on the left. Input, containing an aliquot of the

pooled Sephacryl S300 fractions; Control, aliquot from the mock affinity purification; N-CoR-1,

aliquot from the fraction eluted from the anti−N-CoR antibody affinity column.

Figure 5. Colocalization of N-CoR and KAP-1. (A) KAP-1 and N-CoR copurify by gel filtration

chromatography. The DEAE Sepharose fractions containing N-CoR-1 were pooled, concentrated

and passed through an S300 gel filtration column. Fractions were collected and aliquots were

separated by SDS-PAGE, transferred to nitrocellulose and then probed with either N-CoR or

KAP-1 antibody as indicated on the left. A major fraction of KAP-1 and N-CoR coelute as a

large molecular weight complex of approximately 2 MDa. (B) KAP-1 is found in the affinity

purified N-CoR-1 complex. The pooled Sephacryl S300 fractions containing N-CoR were pooled

and purified by affinity chromatography using an anti-N-CoR affinity column as indicated in

“Materials and Methods”. Bound proteins were eluted from the affinity column using 100 mM

glycine (pH 2.8), separated by SDS-PAGE, transferred to nitrocellulose and probed with the

specific antibody indicated on the left. Control, aliquot from the mock affinity purification; N-

CoR-1, aliquot from the fraction eluted from the anti−N-CoR antibody affinity column. (C) A

component of N-CoR and KAP-1 colocalize in vivo. Indirect immunofluorescence of HeLa cells

stained with anti-N-CoR and anti-KAP-1 antibodies. Differential interference contrast images

were derived from a single population of HeLa cells grown asynchronously (panel 1) and stained


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with anti-N-CoR polyclonal antibody (panel 2), or anti-KAP-1 monoclonal antibody (panel 3). In

most cells a significant overlap was observed between KAP-1 and N-CoR staining (panel 4).

Figure 6. N-CoR-2 contains distinct subunits. The P11 500 mM KCl fraction (N-CoR-2) was

subjected to further purification by DEAE Sepharose, gel filtration and affinity chromatography

as described in “Materials and Methods”. (A) Silver stained SDS-PAGE gel of the N-CoR-2

complex. Approximate molecular weights of the isolated polypeptides are indicated on the right.

Molecular weight standards are indicated on the left. (B) Western blot analysis of the affinity

purified N-CoR-2 complex using specific antibodies. The specific antibodies used are indicated

on the left. Control, aliquot from the mock affinity purification; N-CoR-2, aliquot from the

fraction eluted from the affinity column. (C) Deacetylase activity of the immunopurified N-CoR-

2 complex. Immunopurified N-CoR-1 was preincubated either alone or with 100 nM Trichostatin

A (TSA) and in vivo [3H] labelled core histones. The reaction was allowed to proceed for 90 min

prior to extraction and quantitation of the released [3H]-acetylCoA. The results are a

representative experiment from at least two independent purifications.


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Caroline Underhill, Majdi S. Qutob, Siu-Pok Yee and Joseph Torchiaand the corepressor KAP-1

A novel N-CoR complex contains components of the mammalian SWI/SNF complex

published online September 29, 2000J. Biol. Chem.

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http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

a novel n-cor complex contains components of the mammalian

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