MOLECULAR AND CELLULAR BIOLOGY, Sept. 2006, p. 6880–6889
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 26, No. 18
Polycomb Group and SCF Ubiquitin Ligases Are Found in a
Novel BCOR Complex That Is Recruited to BCL6 Targets†
Micah D. Gearhart,1Connie M. Corcoran,1Joseph A. Wamstad,2and Vivian J. Bardwell1,2*
Department of Genetics, Cell Biology and Development and Cancer Center1and Molecular, Cellular, Developmental Biology
and Genetics Graduate Program,2University of Minnesota, Minneapolis, Minnesota 55455
Received 11 April 2006/Returned for modification 30 May 2006/Accepted 26 June 2006
The corepressor BCOR potentiates transcriptional repression by the proto-oncoprotein BCL6 and sup-
presses the transcriptional activity of a common mixed-lineage leukemia fusion partner, AF9. Mutations in
human BCOR cause male lethal, X-linked oculofaciocardiodental syndrome. We identified a BCOR complex
containing Polycomb group (PcG) and Skp–Cullin–F-box subcomplexes. The PcG proteins include RING1,
RYBP, NSPC1, a Posterior Sex Combs homolog, and RNF2, an E3 ligase for the mono-ubiquitylation of H2A.
BCOR complex components and mono-ubiquitylated H2A localize to BCL6 targets, indicating that the BCOR
complex employs PcG proteins to expand the repertoire of enzymatic activities that can be recruited by BCL6.
This also suggests that BCL6 can target PcG proteins to DNA. In addition, the BCOR complex contains
components of a second ubiquitin E3 ligase, namely, SKP1 and FBXL10 (JHDM1B). We show that BCOR
coimmunoprecipitates isoforms of FBXL10 which contain a JmjC domain that recently has been determined
to have histone H3K36 demethylase activity. The recruitment of two distinct classes of E3 ubiquitin ligases and
a histone demethylase by BCOR suggests that BCOR uses a unique combination of epigenetic modifications
to direct gene silencing.
The BCL6 gene encodes a sequence-specific transcriptional
repressor (17, 23, 65) that is highly expressed in germinal
center B cells. Germinal centers are maturation sites within
lymphoid tissues where antigen-stimulated B cells proliferate,
hypermutate their immunoglobulin (Ig) genes, undergo Ig
class switch recombination, and give rise to progeny plasma
cells that produce antibodies with high affinity for antigen (63).
BCL6 plays a central role in this process, modulating the tran-
scription of genes involved in lymphocyte activation, cell cycle
arrest, apoptosis, and differentiation (5, 22, 49, 54, 59–61, 66,
75, 76). Deregulated expression of BCL6 in germinal center B
cells plays an oncogenic role in non-Hodgkin’s lymphomas (4,
16), presumably by inhibiting apoptosis and enhancing prolif-
BCL6 belongs to a subclass of zinc finger proteins with a
POZ/BTB domain at the N terminus and Cys2-His2zinc fingers
at the C terminus (3, 70, 87). BCL6 can interact with a variety
of corepressors via several domains, including the POZ do-
main, a central repression domain, and the zinc fingers (19, 24,
25, 29, 36, 45, 82). The central domain of BCL6 recruits the
corepressor MTA3 and its associated HDAC-containing chro-
matin remodeling complex (Mi-2/NuRD) (29). Importantly,
MTA3 knockdown in B cells derepresses BCL6 targets that are
upregulated upon differentiation into plasma cells (29). The
POZ domain of BCL6 interacts with NCOR, SMRT, and
BCOR in a mutually exclusive fashion (37). In BCL6-positive
lymphoma cells, peptides that bind to the POZ domain of
BCL6 and block interactions with NCOR, SMRT, and BCOR
cause apoptosis and cell cycle arrest. The peptides do not,
however, cause plasma cell differentiation (61). This suggests
that the functions of BCL6 may be segregated among different
corepressors, with NCOR, SMRT, and/or BCOR silencing
genes involved in apoptosis and cell cycle control and MTA3
silencing genes involved in plasma cell differentiation (29, 51, 61).
While the highly related NCOR and SMRT corepressors are
found in complexes containing HDAC3 and the JmjC domain
protein JMJ2DA (32, 48, 80, 86), the repression mechanisms
used by the unrelated corepressor BCOR are less well under-
stood (37). We previously identified BCOR in a yeast two-
hybrid screen, and aside from three ankyrin repeats it contains
no other recognizable domains. In transient-transfection lucif-
erase reporter assays, BCOR potentiates BCL6 repression,
and BCOR tethered to DNA can repress transcription inde-
pendently of BCL6. Certain isoforms of BCOR, generated by
use of an alternative splice acceptor site, can interact with AF9
and suppress its transcriptional activation. In humans, BCOR
plays multiple important roles in development, as evidenced
by the complex phenotypes seen in oculofaciocardiodental
(OFCD) syndrome females heterozygous for mutations in this
X-linked gene. Nevertheless, specific target genes regulated by
BCOR have not yet been identified.
To help elucidate the mechanisms by which BCOR represses
transcription, we purified the BCOR complex and performed
biochemical and functional analyses. We found that the BCOR
complex contains Polycomb group (PcG) proteins, including a
histone H2A ubiquitin E3 ligase and an SCF ubiquitin E3
ligase. BCOR is also able to associate with a JmjC domain
histone H3 K36 demethylase-containing protein. We find that
the BCOR complex and the mono-ubiquitylated form of his-
tone H2A localize to several BCL6 targets, including P53
(TP53) and Cyclin D2 (CCND2), in lymphoma cells. The en-
zymatic activities of the BCOR complex, through epigenetic
* Corresponding author. Mailing address: Department of Genetics,
Cell Biology and Development, 6-160 Jackson Hall, 321 Church St. SE,
Minneapolis, MN 55455. Phone: (612) 626-7028. Fax: (612) 626-7031.
† Supplemental material for this article may be found at http://mcb
modifications of chromatin, provide a mechanism for silencing
of BCL6 targets that is distinct from the HDAC and chromatin
remodeling activities of the SMRT, NCOR, and MTA3 com-
plexes (29, 32, 48, 80).
MATERIALS AND METHODS
Cloning, plasmids, and antibodies. BCOR, NSPC1, RING1, and RNF2 were
cloned from preexisting clones (37, 53) or the expressed sequence tag (EST)
clones referenced by accession numbers BI457391, BC002922, and BC012583,
respectively. FBXL10 was cloned by PCR amplification from ESTs BM473316
and BC008735 and cDNA from HEK293 cells. Isoform A of human BCOR
contains all possible coding exons. Isoform C of human BCOR is generated by
use of an alternate splice acceptor site and results in a protein that is shorter by
34 amino acids. BCOR(A) (1-1476) and BCOR(C) (1-1442) mimic a splice
acceptor mutation observed in OFCD patients that is presumed to result in an
in-frame stop codon six amino acids C-terminal to residues 1476 and 1442 in
isoforms A and C, respectively.
N-terminally Flag-tagged and C-terminally hemagglutinin (HA)-tagged
BCOR isoform A and N-terminally Flag-tagged human NSPC1 were cloned into
pZOME-1N (Cellzome) to create ProtA2-TEV-CBP-flag-BCOR(A)-HA- and
ProtA2-TEV-CBP-flag-NSPC1-encoding retroviruses. All open reading frames
or deletions were cloned into a modified version of pGEX-2T (GE HealthCare),
T7plink-NTAG, EFplink2 (provided by Richard Treisman, Cancer UK, London,
England), or EFplink-Flag (provided by Caroline Hill, Cancer UK, London,
England) for bacterial, in vitro, or mammalian expression. All nucleotides of
inserts were verified by sequencing. Rabbit polyclonal antibodies were raised
against BCOR, FBXL10, and NSPC1 using glutathione S-transferase (GST)
fusions of human BCOR(C) (1035-1230), human FBXL10 (726-817), and human
NSPC1 (128-189) and subsequently affinity purified.
Complex purification. Stable cell lines (BCOR) or pools (NSPC1 and GFP-
puro) of HeLa S3 or HEK293 cells were generated by infection with ProtA2-
TEV-CBP-flag-BCOR(A)-HA, ProtA2-TEV-CBP-flag-NSPC1, and green fluo-
rescent protein (GFP) retroviruses. Cells were cultured in Joklik’s medium
(Biosource) or minimal essential medium (Invitrogen) with 5% calf serum (Bio-
source) and 1 ?g/ml puromycin. Nuclear extracts (26) were supplemented with
0.1% NP-40, 0.1% Tween, 2.0 mM EGTA, and 0.5 mM EDTA and incubated
with M2-agarose (Sigma) overnight. Beads were washed in 50 mM Tris pH 8.0,
1.0 mM MgCl2, 1.0 mM imidazole, 0.1% NP-40, 20% glycerol, 2.0 mM dithio-
threitol (DTT), 2.0 mM phenylmethylsulfonyl fluoride (PMSF), 2.0 mM EGTA,
0.5 mM EDTA, protease inhibitor cocktail (Complete EDTA-free; Roche), and
either 350 mM (BE-350) or 700 mM KCl (BE-350). Complexes were eluted with
?30% yields using 2 mg/ml Flag peptide, substituting 2.0 mM CaCl2for EGTA
and EDTA in the 350 mM KCl buffer (BC-350) and recaptured with calmodulin-
Sepharose (GE HealthCare). Calmodulin beads were washed with BC-350 and
BC-700 and eluted with ?30% yields in BE-350 for an overall yield of ?10%. For
mass spectrometry analysis, eluants were precipitated with 0.02% deoxycholate
and 20% trichloroacetic acid overnight, resolved on a 10% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, and visualized by
silver staining (SilverQuest; Invitrogen). Excised bands were reduced, alkylated,
trypsinized, and extracted for analysis by matrix-assisted laser desorption ioniza-
tion–time of flight analysis (QSTAR XL; Applied Biosystems). Proteins were
identified using Mascot (58), and amenable peptides were confirmed by tandem
mass spectrometry fragmentation. Alternatively, eluants were resolved on a
Superose 6 gel filtration column in BE-350 at a yield of approximately 2% and
deoxycholate-trichloroacetic acid precipitated for SDS-PAGE analysis.
Immunoprecipitations and in vitro protein interactions. Immunoprecipita-
tions and GST pull-down assays were performed as described elsewhere (37)
with minor modifications. Approximately 60 million Ramos or HEK293 cells
were used for immunoprecipitations of the endogenous BCOR complex (see Fig.
3B, below) with 2 to 4 ?g of normal rabbit IgG (Santa Cruz Biotechnology) or
BCOR antibody. Rabbit TrueBlot horseradish peroxidase (eBiosciences) was
used to visualize NSPC1 in immunoprecipitation experiments (see Fig. 2A and
3B, below). Glutathione S-transferase fusion proteins were expressed in
BL21(DE3) Escherichia coli. Protein concentrations were normalized by Coo-
massie staining on SDS-PAGE gels. Proteins were translated in vitro with
[35S]methionine (Amersham) and the T7 TNT quick-coupled system (Promega).
Two ?l of radiolabeled proteins was preincubated with 110 ?l binding buffer (20
mM HEPES pH 7.9, 100 mM KCl, 2 mM EDTA, 0.1% NP-40, 10% glycerol,
0.5% nonfat dry milk, 5 mM DTT) plus 30 ?l of blocked glutathione agarose
(50% slurry) for 30 min at room temperature and centrifuged at 1,000 ? g for 3
min to pellet beads. Supernatant was transferred to a clean tube and centrifuged
at 21,000 ? g for 10 min. One hundred ?l of the high-speed supernatants was
transferred to a new tube. Fifteen microliters of GST-fusion beads (a 50% slurry
containing a total of approximately 3.0 ?g of full-length GST fusion protein) was
added, and the mixtures were incubated for 1 h at room temperature. The beads
were centrifuged at 1,000 ? g for 3 min, washed twice with binding buffer and
once with binding buffer minus nonfat dry milk, boiled in SDS loading buffer,
resolved by SDS-PAGE, and imaged by autoradiography.
siRNAs transfections. Nonspecific small interfering RNA (siRNA; Dharma-
con D-001210-01) or a pool of siRNAs targeting human FBXL10 (Dharmacon
M-014930) was transfected into HEK293 cells at a concentration of 100 nM using
Dharmafect 1 lipid reagent and harvested using TRIzol reagent (Invitrogen).
Chromatin immunoprecipitation. Log-phase Ramos cells were cross-linked
with 1% formaldehyde for 10 min at 37°C before quenching with 0.125 M glycine
for 5 min. Cells were washed twice in phosphate-buffered saline (PBS) containing
Complete protease inhibitors (Roche) and 1.0 mM PMSF. Cells were resus-
pended in cell lysis buffer containing 10 mM HEPES pH 7.9, 0.5% NP-40, 1.5
mM MgCl2, 10 mM KCl, 0.4 mM DTT, protease inhibitors (Complete; Roche),
and 1.0 mM PMSF for 10 min on ice. Cells were centrifuged for 5 min at 1,500 ? g
at 4°C and resuspended in nuclear lysis buffer containing 20 mM HEPES pH 7.9,
25% glycerol, 0.5% NP-40, 0.420 M NaCl, 1.5 mM MgCl2, 0.5 mM EDTA,
protease inhibitors (Complete; Roche), and 1.0 mM PMSF for 20 min on ice.
DNA was sheared using a sonic dismembrator (model 500; Fisher Scientific)
equipped with a double-stepped microtip at 30% power for 5 s on, 10 s off for a
total of 8 min of “on” time to generate fragments between 500 and 6,000 bases.
Lysates were centrifuged for 10 min at 21,000 ? g at 4°C. Supernatants were
diluted into an equal volume of dilution buffer containing 20 mM Tris-HCl pH
7.9, 1% Triton X-100, 2 mM EDTA, 50 mM NaCl, protease inhibitors (Com-
plete; Roche), and 1.0 mM PMSF and precleared for 1 h at 4°C with protein
A-Sepharose (GE HealthCare) that had been blocked with 0.5 mg/ml bovine
serum albumin and 200 ?g/ml sheared salmon sperm DNA. Diluted and cleared
extracts corresponding to 2 ? 106Ramos cells were incubated with each of the
following antibodies: no-antibody control, 2 ?g normal rabbit IgG (Santa Cruz
sc-2027), 2 ?g BCL6 (N3; sc-858; Santa Cruz), 2 ?g rabbit polyclonal antibodies
generated against human BCOR, 4 ?g SKP1 (610530; BD Biosciences), 4 ?g
mono-ubiquitylated H2A (Ub-H2A; E6C5; Upstate), 25 ?l RNF2 monoclonal
hybridoma (1), or 4 ?g RYBP (AB3637; Chemicon). Rabbit antibodies against
mouse IgG or IgM (Upstate) were added to samples 1 h before immune com-
plexes were recovered using blocked protein A-Sepharose. Beads were washed
with low salt (20 mM Tris-HCl pH 8.0, 1% Triton X-100, 2 mM EDTA, 150 mM
NaCl), high salt (20 mM Tris-HCl pH 8.0, 1% Triton X-100, 2 mM EDTA, 500
mM NaCl), and LiCl (10 mM Tris, pH 8.0, 1% NP-40, 1% deoxycholic acid, 0.25
M LiCl, 1 mM EDTA), and twice with TE (10 mM Tris-HCl pH 8.0, 1 mM
EDTA) before being eluted from beads with freshly prepared elution buffer (100
mM NaHCO3, 1% SDS). After cross-links were reversed in the presence of 200
mM NaCl at 65°C for 5 h, samples were treated with RNase and proteinase K
and DNA fragments were purified using a PCR Clean-Up column (QIAGEN).
Genomic DNA was quantitatively amplified from each sample in duplicate using
SYBR Green QPCR reagent (Stratagene) on an MX3000P thermocycler (Strata-
gene) and the following primers: BCL6, 5?-GCAGTGGTAAAGTCCGAAG
C-3? and 5?-AGCAACAGCAATAATCACCTG-3?; Cyclin D2, 5?-GGGGGAG
CCGGACCTAATC and 5?-CTCGCCCCTGCATCTGCTGAC-3?; P53, 5?-GG
AGAAAACGTTAGGGTGTG-3? and 5?-GCTTTTGCGTTTGCTCTCAG-3?;
P53 5? region, 5?-GTTTTCAGCCCTTTCCTTCC-3? and 5?-GTGCCCTTCCC
H2A ubiquitylation assays. Eluants from the tagged NSPC1 purification were
incubated with rabbit E1, Ubc5c, Flag-tagged ubiquitin, and oligonucleosome
substrate as described previously (78).
TS cell culture and immunofluorescence. Trophoblastic stem (TS) cells were
cultured and prepared for immunofluorescence as described previously (21).
Mouse embryonic fibroblasts (MEFs) were irradiated and plated at 2 ? 106
cells/10-cm dish. TS cells were grown on MEFs in TS medium (RPMI 1640 with
20% fetal bovine serum, 1 mM sodium pyruvate, 10 ?M ?-mercaptoethanol, 2
mM L-glutamine) plus 25 ng/ml FGF4, and 1 ?g/ml heparin was added on the day
of use as described previously (72). Cells were cultured in a 5% CO2incubator
at 37°C and passaged 1:15 every 4 days. For immunofluorescence experiments,
TS cells were grown on glass coverslips for 2 days from a 1:10 passage. Cells were
rinsed three times with PBS and fixed with 2% paraformaldehyde in PBS for 15
min at 25°C. Cells were then rinsed three times with PBS and permeabilized with
0.4% Triton X-100. Cells were rinsed three times with PBS and blocked with 0.2%
fish skin gelatin in PBS for 30 min at 25°C. Cells were then incubated with
primary antibodies in 5% normal goat serum in PBS for 1 h at 25°C in a humid
chamber. Dilutions of antibodies were as follows: BCOR, 1:25; RNF2 (1), 1:2.
Post-antibody incubation, cells were washed three times for 3 min each with 0.2%
VOL. 26, 2006 TRANSCRIPTIONAL REPRESSION BY BCOR6881
fish skin gelatin in PBS. Cells were then incubated with appropriate secondary
antibodies (GAR-568 1-11036 and GAM-488 A-11029; Molecular Probes) for 30
min at 25°C in a humid chamber. Cells were then washed three times for 3 min
each with 0.2% fish skin gelatin in PBS, followed by two rinses with PBS. Cells
were finally mounted in PermaFluor antifade reagent (Immunon) and stored at 4°C.
Purification and identification of BCOR complex components.
To help reveal BCOR-dependent repression mechanisms, we
performed biochemical analyses of the BCOR complex. BCOR-
interacting proteins were identified from HeLa S3 and HEK293
cell lines stably expressing multiply epitope-tagged BCOR at an
approximately 1:1 ratio to the endogenous protein. Although
these cells do not express BCL6, they can easily be produced in
large quantities suitable for biochemical purification. Size-exclu-
sion chromatography of a two-step affinity-purified BCOR com-
plex indicates that HeLa S3-tagged BCOR is in an ?800-kDa
complex and coelutes with at least six protein bands (Fig. 1A).
Mass spectrometric analysis (see Table S1 in the supplemental
material) of copurifying bands after a two-step purification from
HEK293 cells identified HSP70 and the PcG proteins NSPC1
(PCGF1), RING1, and RNF2 (Fig. 1C), which are homologs of
proteins found in the PcG PRC1 complex (46). We also identified
SKP1 (SKP1A, isoform b), a component of Skp1–Cullin–F-box
(SCF) E3 ubiquitin ligases (14). Additional bands at 24 kDa, 32
kDa, and 97 kDa were specifically observed in the tagged BCOR
FIG. 1. BCOR and NSPC1 are obligate partners in an ?800-kDa complex. (A) Western blotting and silver stain analysis of size-exclusion
fractionation (Superose 6) of the BCOR complex purified from a tagged BCOR HeLa S3 cell line. Bands at 20 kDa, 25 kDa, 32 kDa, 35 kDa,
50 kDa, and 70 kDa and tagged BCOR elute as a tightly migrating ?800-kDa complex but at a 2% yield. (B) Silver stain analysis and protein
identifications from purifications of HEK293 cells stably expressing untagged GFP (Mock), TAP-Flag (FL)-tagged BCOR, or TAP-FL-
tagged NSPC1. Bands immediately below BCOR and at 40 kDa were identified as BCOR and tagged NSPC1 degradation products,
respectively. All identifications were confirmed by tandem mass spectrometry fragmentation except for the four observed peptides from
RYBP, which were not amenable to fragmentation. HSP70 has been observed in other PcG complexes (46). The band at 24 kDa (●) was
present in both purifications, but insufficient data prevented definitive identification. (C) Schematic diagram of proteins found in the BCOR
complex. The amino terminus of FBXL10 (shown in gray) containing the JmjC domain was not detected by mass spectrometry. RYBP has
fewer amino acids than NSPC1 but has been shown to run aberrantly at 32 kDa in previous studies (30). The following abbreviations were
used: A, ankyrin; C, CXXC; P, PHD; F, F-box; L, leucine-rich repeat; ZF, zinc finger. Two alternatively spliced isoforms of BCOR (A and
C) used in this study differ by 34 amino acids.
6882 GEARHART ET AL.MOL. CELL. BIOL.
FIG. 2. Mapping of protein-protein interactions within the BCOR complex. (A) The C terminus of BCOR is necessary and sufficient for
interaction with NSPC1. Left: A GST fusion of NSPC1 was used to pull down in vitro-translated radiolabeled BCOR(C) (1-1721),
BCOR(C) (1-1442), or BCOR(C) (1428-1721). Right: Transfected myc-BCOR(A) (1-1755) but not myc-BCOR(A) (1-1476) precipitates
endogenous NSPC1 from HEK293 cells. Additional bands in the NSPC1 Western blots (*) correspond to nonspecific interactions and
cross-reactivity with protein A-Sepharose. (B) Upper left: GST-NSPC1 interacts with in vitro-translated radiolabeled RING1 and RNF2.
Lower left: A weak interaction was observed between GST-RING1 and in vitro-translated RNF2. Upper right: GST-RYBP interacts with
in vitro-translated RING1 and RNF but not luciferase. Lower right: GST-FBXL10 (1052-1336) interacts with in vitro-translated SKP1.
(C) Cotransfected full-length myc-BCOR(A) (1-1755) but not myc-BCOR(A) (1-1476) coimmunoprecipitates with transfected Flag-FBXL10
(1-1336) and Flag-FBXL10 (481-1336) in HEK293 cells. Full-length BCOR is enriched in the presence of full-length and N-terminally
truncated FBXL10 (lanes 4 and 5, respectively) relative to background (lane 2). In contrast, C-terminally truncated BCOR is not enriched
relative to background (compare lane 6 to lane 3). We consistently observe that the abundance of full-length BCOR, but not C-terminally
truncated BCOR, is reduced upon cotransfection with FBXL10 (compare input lane 2 to lanes 4 and 5 and lane 3 to lane 6). However,
endogenous BCOR levels are only modestly increased upon siRNA knockdown of FBXL10 (data not shown; see Fig. 3A). (D) Model of
protein-protein interactions in the BCOR complex. FBXL10 is tentatively drawn (hashed lines) to have direct contacts with BCOR and
NSPC1, but direct interactions between either molecule have been difficult to dissect.
VOL. 26, 2006 TRANSCRIPTIONAL REPRESSION BY BCOR6883
purifications, but quantities were insufficient for identification.
HDACs and AF9 (MLLT3), previously shown to interact with
BCOR (37, 69), were not detected by silver staining or immuno-
blot analysis (not shown), perhaps reflecting a transient or chro-
NSPC1 is a homolog of the Drosophila melanogaster PRC1
component Posterior Sex Combs (Psc) protein (31, 55). Be-
cause this homolog of Psc had not been found in other PcG
complexes, we hypothesized that NSPC1 is a unique compo-
nent of the BCOR complex, and we subsequently performed a
reciprocal tagging experiment in HEK293 cells. Purification of
tagged NSPC1 isolated and identified all of the associated
bands observed in the tagged BCOR complex (Fig. 1B).
Higher yields in the tagged NSPC1 purification also permitted
the identification of the 32-kDa and 97-kDa bands as RING1-
YY1-binding protein (RYBP) and FBXL10 (Fig. 1C). The
24-kDa band remains unidentified. The similarity in constitu-
ents between the tagged BCOR and tagged NSPC1 purifica-
tions suggests that BCOR and NSPC1 are requisite partners in
HEK293 cells and that tagging either BCOR or NSPC1 results
in purification of the same complex. Aside from the chaperone
protein HSP70, all proteins identified in the BCOR complex
fall into two categories: PcG-associated proteins or SCF ubiq-
uitin ligase components.
In vitro and in vivo determinations of protein-protein inter-
actions within the BCOR complex. To determine how BCOR
and NSPC1 are associated in this complex, we performed GST
pull downs and coimmunoprecipitation experiments. We
found that in vitro-translated full-length BCOR, but not C-
terminally deleted BCOR (1-1442), interacts efficiently with
bacterially produced GST-NSPC1 (Fig. 2A, left panel). The C
terminus of BCOR (1428-1721) is sufficient to interact with
GST-NSPC1 (Fig. 2A, left panel). Endogenous NSPC1 is im-
munoprecipitated by transfected full-length myc-BCOR, but
not by C-terminally truncated myc-BCOR in HEK293 cells
(Fig. 2A, right panel). We conclude that the C terminus of
BCOR is both necessary and sufficient for interaction with
Like other mammalian Psc homologs (12, 35, 71), GST-
NSPC1 interacts with both in vitro-translated RING1 and
RNF2 (Fig. 2B, left upper panel). RING1 and RNF2, in turn,
interact with each other (Fig. 2B, left lower panel) and RYBP
(Fig. 2B, upper right panel) (30). Since we have not detected a
direct interaction between BCOR and RING1 or RNF2 (data
not shown), we conclude that NSPC1 bridges the interaction
from BCOR to RING1 and RNF2, which in turn interact with
RYBP (Fig. 2D). Together NSPC1, RING1, RNF2, and RYBP
form a PcG subcomplex within the BCOR complex.
Two proteins in the BCOR complex, FBXL10 and SKP1,
have not previously been associated with PcG-mediated re-
pression but are presumed components of an SCF ubiquitin E3
ligase. FBXL10 is one of many F-box proteins containing the
SKP1-interacting F-box domain (39). In the FBXL subfamily,
leucine-rich repeats carboxy terminal to the F-box recognize
specific substrates, often dependent on posttranslational mod-
ification, and then mediate their ubiquitylation (14). As ex-
pected, in vitro-translated SKP1 interacts with a GST-FBXL10
(1052-1336) protein containing the F-box (Fig. 2B, lower right
panel). Although we thought the leucine-rich repeats might
facilitate additional contacts within the complex, we failed to
detect a strong interaction between GST-FBXL10 (1052-1336)
and any of the other components of the BCOR complex (data
not shown). However, transfected full-length Flag-FBXL10
can coimmunoprecipitate full-length myc-BCOR but not C-
terminally truncated myc-BCOR in HEK293 cells (Fig. 2C).
An N-terminally truncated Flag-FBXL10, roughly correspond-
ing to the 97-kDa protein we observed in the purifications, also
interacts with full-length myc-BCOR (Fig. 2C). Thus, the C
terminus of BCOR is necessary for interaction with NSPC1
and FBXL10 (Fig. 2A, C, and D). However, we do not know
whether FBXL10 interacts directly with BCOR or the PcG
subcomplex. Direct interactions with FBXL10 have been dif-
ficult to dissect in GST pull-down experiments, perhaps be-
cause the interaction of FBXL10 with another component(s)
of the BCOR complex requires a posttranslational modifica-
Full-length FBXL10 contains a JmjC domain, a CXXC zinc
finger, a plant homeodomain (PHD), the SKP1-binding F-box,
and leucine-rich repeats. However, the 97-kDa band in the
tagged-NSPC1 purification contained peptides from only the
C-terminal two-thirds of full-length FBXL10; thus, the protein
in this band lacks the N-terminal JmjC domain (Fig. 1C).
Analysis of EST databases indicates that the FBXL10 gene
encodes multiple isoforms due to the use of alternative pro-
moters and splice sites. To determine which isoforms of
FBXL10 are expressed in HEK293 cells, we produced an an-
tibody against a region of human FBXL10 immediately follow-
ing the PHD domain that is distinct from the highly related
FBXL11 (JHDM1A). This antibody recognizes one major
band at 97 kDa and five additional protein bands ranging from
88 to 172 kDa that are significantly depleted upon treatment with
siRNAs against FBXL10 (Fig. 3A). The 97-kDa band corre-
sponds in size to the band observed in the tagged NSPC1
To determine if the other isoforms of FBXL10 associate
with endogenous BCOR, we performed immunoprecipitations
in HEK293 cells. We found that BCOR antibodies coimmu-
noprecipitate all six isoforms of FBXL10 (Fig. 3B, lower
panel), including the 164-kDa and 172-kDa isoforms that,
based on EST data, must contain the JmjC domain. Since the
BCOR complex purification was carried out in HeLa S3 and
HEK293 cells, we tested whether a similar BCOR complex is
present in B cells. In Ramos cells, a Burkitt’s lymphoma B-cell
line, endogenous BCOR coimmunoprecipitates NSPC1 (Fig.
3B, upper panel) and all six isoforms of FBXL10 (Fig. 3B,
lower panel). This indicates that, like in HEK293 cells (Fig. 1B
and 3B, lower panel), both PcG and SCF components are
associated with BCOR in B cells. Although the 164-kDa and
172-kDa isoforms of FBXL10 are barely visible in the input,
they account for approximately one-fifth of the coimmunopre-
cipitated FBXL10 in Ramos cells. Thus, a significant fraction
of the BCOR complexes contain an isoform of FBXL10 har-
boring the JmjC H3 K36 demethylase domain (74).
Even though we have not mapped a direct interaction be-
tween the BCOR complex and FBXL10, four lines of evidence
indicate that the SCF and PcG proteins are in a single complex
with BCOR (Fig. 2D). First, bands corresponding in size to
SKP1 as well as PcG proteins comigrate with BCOR in the size
exclusion column (Fig. 1A). Due to the presence of SKP1,
which interacts directly with FBXL10 (Fig. 2B, lower right
6884GEARHART ET AL.MOL. CELL. BIOL.
panel), we presume that the isoforms of FBXL10 also comi-
grate with BCOR but are below the limit of detection of silver
staining in this analysis. Second, SKP1 and PcG proteins were
all identified in the tagged BCOR purification (Fig. 1B; see
also Table S1 in the supplemental material). Third, SKP1 and
FBXL10 were identified in the tagged NSPC1 purification
(Fig. 1B; see also Table S1). Fourth, FBXL10 and BCOR
coimmunoprecipitate, indicating that they can be found in the
same complex (Fig. 2C and 3B, lower panel). Together our
data indicate that BCOR associates in a single complex with
both PcG PRC1 proteins and SCF components that, depend-
ing on the incorporated FBXL10 isoform, can also contain a
JmjC histone demethylase domain.
The BCOR complex contains E3 ligase activity for histone
H2A. Of the BCOR complex PcG proteins, RNF2 is the only
one with a known enzymatic activity: an E3 ligase for the
histone protein H2A (12, 78). Ub-H2A is thought to be in-
volved in maintaining a repressed chromatin state (21, 28, 38,
78). Knockdown of the Drosophila homolog of RNF2 and
RING1, dRing, in S2 cells results in loss of histone H2A
ubiquitylation and upregulation of a PcG target gene (78). To
confirm this activity in the BCOR complex, purified complex
was incubated with E1, E2, Flag-tagged ubiquitin, and nucleo-
somal substrate. Consistent with the presence of RNF2, the
BCOR complex catalyzes the addition of Flag-tagged ubiquitin
onto H2A in a concentration-dependent manner (Fig. 4, upper
FIG. 3. Six FBXL10 isoforms associate with BCOR in HEK293 and Ramos B cells (A) FBXL10 knockdown and immunoblotting with
FBXL10-specific antibodies identifies six FBXL10 isoforms in HEK293 cells. Western assays of total cell extracts blotted with BCOR, actin
(Sigma), and FBXL10 antibodies are shown. FBXL10 knockdown results in only a modest increase in BCOR levels. (B) Antibodies to BCOR
coimmunoprecipitate all six isoforms of FBXL10 from HEK293 and Ramos nuclear extracts (lower panel). BCOR coimmunoprecipitation of
NSPC1 is also seen in Ramos cells (upper panel). The lower band in the NSPC1 Western blot (*) is due to cross-reactivity with protein
FIG. 4. Role of the BCOR complex in H2A mono-ubiquitylation.
The BCOR complex purified from the NSPC1-tagged cell line cata-
lyzes the addition of Flag-ubiquitin (flag-Ub) onto histone H2A in a
dose-dependent manner. RNF2 has been previously shown to ubiqui-
tylate H2A, generating the Flag-tagged product (78). The band at 26
kDa is the only band that increases in a BCOR complex-dependent
manner and is recognized by antibodies against the Flag epitope (up-
per panel) and Ub-H2A (lower panel). Additional Flag-ubiquitylated
proteins (*), which are observed even in the absence of the BCOR
complex above and below Flag–Ub-H2A, presumably reflect ubiquitin
ligase activities present in the oligonucleosome preparation.
VOL. 26, 2006 TRANSCRIPTIONAL REPRESSION BY BCOR6885
panel). Identity of the Flag–Ub-H2A band was confirmed by
Western blotting with antibodies specific for ubiquitylated Ub-
H2A (Fig. 4, lower panel). These results show that in addition
to the potential H3 K36 demethylase activity, the BCOR com-
plex has E3 ligase activity for histone H2A.
BCOR does not colocalize to the inactive X chromosome in
trophoblastic stem cells. PcG proteins and H2A mono-ubiq-
uitylation also are associated with the inactive X chromosome
(Xi) in mouse XX trophoblastic stem cells (21, 28). To test
whether the BCOR complex is involved in X inactivation, we
performed immunolocalization experiments in trophoblastic
stem cells. BCOR does not localize to the intensely staining Xi
labeled by RNF2 antibodies but shows a diffuse nuclear stain-
ing (Fig. 5A). This suggests that BCOR is unlikely to play a
role in the maintenance of X inactivation in trophoblastic stem
cells. The non-Xi diffuse nuclear staining of RNF2 likely re-
flects its association with BCOR as well as other PcG com-
plexes, such as E2F6.com-1 (56).
The BCOR complex is recruited to BCL6 targets in B cells.
Although BCOR can potentiate BCL6 repression in transient-
reporter assays (37), it is not known whether BCOR is re-
cruited to any of the BCL6 target genes. Therefore, we inves-
tigated the occupancy of the BCOR complex on endogenous
BCL6 target genes (57, 59, 61, 66, 79) in Ramos cells using
chromatin immunoprecipitation (ChIP) assays. We found that
BCL6, BCOR, SKP1, RYBP, and RNF2 specifically immuno-
precipitate a negative autoregulatory region in the first exon of
the BCL6 gene (Fig. 5B), indicating that the entire BCOR
complex, including PcG and SCF components, are present at
this locus. In addition, ChIP analyses showed that BCL6 and
BCOR are present at the BCL6-binding sites of Cyclin D2 and
P53 but not a region 14 kb 5? of P53 (Fig. 5C and D). The
FIG. 5. The BCOR complex occupies promoters of BCL6 target genes. (A) BCOR does not colocalize with the inactive X chromosome.
Immunofluorescence of XX trophoblastic stem cells using BCOR and RNF2 (1) antibodies is shown. RNF2 has been shown to localize to Xi (21,
28). The Xi is indicated with white arrows. (B) ChIP experiments show that BCL6, BCOR, BCOR complex components SKP1, RNF2, and RYBP,
and the monoubiquitylated form of H2A are associated with the autoregulatory region of the BCL6 gene. The anti-Ub-H2A antibody (Upstate)
is specific for the monoubiquitylated form of histone H2A (12, 77, 78). (C and D) BCL6, BCOR, and the monoubiquitylated form of H2A are
present at the promoters of Cyclin D2 and P53 but not a region 14 kb 5? of the P53 gene.
6886 GEARHART ET AL.MOL. CELL. BIOL.
higher efficiency of ChIP at the BCL6 locus relative to Cyclin
D2 and P53 may be due to cooperative binding of BCL6 to the
multiple binding sites in the first exon of this gene (57, 79), as
POZ domain-dependent cooperative binding by BCL6 has
been reported to three sites in the murine germ line ε pro-
moter (33). Importantly, our data suggest that BCL6, via
BCOR, can target PcG and SCF proteins sequence-specifically
Monoubiquitylated H2A is present together with BCOR at
BCL6 targets in B cells. Because we have not been able to
efficiently knock down BCOR or BCOR complex components
using RNA interference in cultured B cells, we do not know
whether the BCOR complex is essential for repression of
BCL6 targets. However, in ChIP experiments using monoclo-
nal antibodies that specifically recognize the ubiquitylated
form of H2A (12, 77, 78), we found that Ub-H2A is enriched
at BCL6 exon 1, Cyclin D2, and P53 relative to a region 14 kb
5? of the P53 gene (Fig. 5B to D). The presence of PcG
proteins in the BCOR complex, together with localization of
Ub-H2A to BCL6 targets in vivo, strongly suggests that the
BCOR complex is regulating these genes with its H2A mono-
We have identified a novel set of proteins that associate with
the corepressor BCOR in a single 800-kDa complex that is
recruited to a subset of BCL6 targets. The proteins in the
BCOR complex include the PcG and PcG-associated proteins
NSPC1, RING1, RNF2, and RYBP as well as components of
an SCF ubiquitin ligase, SKP1, and FBXL10. Our biochemical
and functional analyses indicate that BCOR recruits a unique
combination of enzymatic activities to chromatin targets: a
PcG E3 ubiquitin ligase for histone H2A, a demethylase for
histone H3 K36, and an SCF E3 ubiquitin ligase. Corepressor
recruitment by the POZ domain of BCL6 is required for the
proliferation and survival of BCL6-positive lymphoma cells
(61). However, the relative contributions of the deacetylase
and demethylation activities of the NCOR and SMRT com-
plexes (32, 48, 80, 81, 86) versus the histone ubiquitylation and
demethylation activities of the BCOR complex to repression of
individual BCL6 targets remain to be determined. Our ChIP
results indicate that histone H2A ubiquitylation activity of the
BCOR complex is present at the Cyclin D2 and P53 promoters,
suggesting that BCOR likely contributes to the repression of
these genes and perhaps to the enhanced proliferation and
survival of BCL6-positive lymphoma cells.
Our finding that the BCOR complex contains PcG proteins
which may contribute to BCL6-driven oncogenesis is consis-
tent with a growing number of observations indicating a role
for PcG proteins in stem cell identity, mammalian develop-
ment, cell cycle regulation, and cancer (11, 62). PcG proteins
are known to be part of a memory system that relies on epi-
genetic modification of chromatin to ensure faithful transmis-
sion of cell identities through cell division (10). In addition to
its potential roles in oncogenesis with BCL6 and MLL-AF9,
BCOR is clearly an essential corepressor in multiple develop-
mental pathways. OFCD patients, who have mutations in
BCOR, have severe craniofacial, digital, and cardiac defects.
Peripheral blood lymphocytes from female patients show
strongly biased inactivation of the X chromosome carrying the
mutant BCOR allele (53), indicating that BCOR is also re-
quired for normal hematopoiesis. The diverse phenotypes ob-
served in OFCD patients and the widespread expression of
BCOR suggest additional tissue-specific transcription factors
recruit the BCOR complex and its associated PcG and SCF
The recruitment of BCOR complex PcG proteins to target
genes by BCL6 in B cells suggests that BCL6 functions as a
PcG-targeting factor. Interestingly, BCL6 is related to the
Drosophila POZ zinc finger transcription factor GAGA, which
is thought to play a role in PcG and TrxG protein recruitment
to DNA (6, 50, 52). Typically, histone H3 K27 methylation,
mediated by the ESC-E(Z) methyltransferase complex, creates
a binding site for the chromo domain of the Pc protein in the
PRC1 complex (13). Although the BCOR complex, unlike
other PRC1-like complexes (46, 56, 78), does not copurify with
a chromo domain-containing protein, the complex does con-
tain protein domains with the potential to bind chromatin.
CXXC domains like the one found in FBXL10 can bind un-
methylated CpG sequences (9, 40, 68). PHDs can bind nucleo-
somes (8, 27), and in two recent examples this binding was
specific to H3 K4-trimethylated nucleosomes (67, 83). These
domains of FBXL10 could stabilize the association of the
BCL6-recruited complex with chromatin or may allow for
BCL6-independent recruitment of the BCOR complex. Simi-
larly, the BCOR-interacting protein AF9 (69) interacts with
the chromo domain protein MPc3 (Cbx8) (34), which may
facilitate chromatin contacts (7). The BCOR complex may
function analogously to the E2F6.com-1 complex recruited by
the transcription factor E2F6 (56). Like the BCOR complex, this
complex includes RING1, RNF2, and a Psc homolog (MBLR)
and also copurifies with the chromatin-binding domain protein
(HP1?), which may stabilize binding of E2F6.com-1 to chro-
matin trimethylated at K9 of histone H3 (56).
The interaction of BCOR with PcG proteins as well as an
F-box protein with a JmjC domain demethylase is particularly
striking. JmjC domain demethylase enzymes are able to re-
move methyl groups from lysines that have been mono-, di-, or
trimethylated (18, 20, 43, 73, 74, 81, 85). The specificity of
FBXL11 (JHDM1A) has been thoroughly characterized, and the
protein has been found to demethylate mono- and dimethyl
lysine 36 of histone H3 (74). FBXL10 (JHDM1B) has been
reported to also demethylate H3 K36 (74), but its specificity
remains to be fully established. The presence of methylated H3
K36 correlates with transcriptional elongation (44, 47, 84),
suppression of intragenic transcription (15, 41, 42), and the
defining of actively transcribed regions (2, 64). BCOR com-
plexes containing the demethylase activity of the full-length
FBXL10 may therefore additionally contribute to transcrip-
tional regulation by altering H3 K36 methylation at target
The F-box and the leucine-rich repeats at the C terminus of
FBXL10 are presumed components of an SCF E3 ubiquitin
ligase. Although we see a modest FBXL10-dependent effect on
endogenous BCOR abundance (Fig. 3A), the direct target,
nature (mono versus poly), and outcome (signaling versus deg-
radation) of the ubiquitylation activity of FBXL10 are un-
known. The presence of SKP1 at the BCL6 locus (Fig. 5B)
indicates that SCF ubiquitin ligase components are found on
VOL. 26, 2006 TRANSCRIPTIONAL REPRESSION BY BCOR 6887
chromatin with the BCOR complex. Future characterization of
the SCF E3 ligase activity of FBXL10 will provide important
insights into BCOR complex-dependent gene silencing.
Because deregulated expression of BCL6 plays an oncogenic
role in non-Hodgkin’s lymphomas and BCL6 can recruit
BCOR to promoters involved in apoptosis and cell cycle reg-
ulation, we suggest that BCOR and its associated PcG and SCF
proteins may also play a role in BCL6-associated lymphomas.
Once a definitive functional role for BCOR is established, the
enzymatic activities of the BCOR complex may be attractive
therapeutic targets for non-Hodgkin’s lymphoma.
We thank Haruhiko Koseki (Riken, Japan) for RNF2 antibody,
Khanh Huynh and Jeannie Lee (Harvard University) for trophoblastic
stem cells, Jose Polo and Ari Melnick (Albert Einstein College of
Medicine, New York) for sharing BCL6 target primer sequences, and
Carrie Ketel and Jeff Simon (University of Minnesota) for oligonu-
cleosome substrate. We thank colleagues for helpful comments.
This work was supported by a grant to V.J.B. from the NCI (RO1-
CA071540). M.D.G. was supported by training grants from the NCI
and the NIDCR.
1. Atsuta, T., S. Fujimura, H. Moriya, M. Vidal, T. Akasaka, and H. Koseki.
2001. Production of monoclonal antibodies against mammalian Ring1B pro-
teins. Hybridoma 20:43–46.
2. Bannister, A. J., R. Schneider, F. A. Myers, A. W. Thorne, C. Crane-Robinson,
and T. Kouzarides. 2005. Spatial distribution of di- and tri-methyl lysine 36 of
histone H3 at active genes. J. Biol. Chem. 280:17732–17736.
3. Bardwell, V. J., and R. Treisman. 1994. The POZ domain: a conserved
protein-protein interaction motif. Genes Dev. 8:1664–1677.
4. Baron, B. W., J. Anastasi, A. Montag, D. Z. Huo, R. M. Baron, T. Karrison,
M. J. Thirman, S. K. Subudhi, R. K. Chin, D. W. Felsher, Y. X. Fu, T. W.
McKeithan, and J. M. Baron. 2004. The human BCL6 transgene promotes
the development of lymphomas in the mouse. Proc. Natl. Acad. Sci. USA
5. Baron, B. W., J. Anastasi, M. J. Thirman, Y. Furukawa, S. Fears, D. C. Kim,
F. Simone, M. Birkenbach, A. Montag, A. Sadhu, N. Zeleznik-Le, and T. W.
McKeithan. 2002. The human programmed cell death-2 (PDCD2) gene is a
target of BCL6 repression: implications for a role of BCL6 in the down-
regulation of apoptosis. Proc. Natl. Acad. Sci. USA 99:2860–2865.
6. Bejarano, F., and A. Busturia. 2004. Function of the Trithorax-like gene
during Drosophila development. Dev. Biol. 268:327–341.
7. Bernstein, E., E. M. Duncan, O. Masui, J. Gil, E. Heard, and C. D. Allis.
2006. Mouse polycomb proteins bind differentially to methylated histone H3
and RNA and are enriched in facultative heterochromatin. Mol. Cell. Biol.
8. Bienz, M. 2006. The PHD finger, a nuclear protein-interaction domain.
Trends Biochem. Sci. 31:35–40.
9. Birke, M., S. Schreiner, M. P. Garcia-Cuellar, K. Mahr, F. Titgemeyer, and
R. K. Slany. 2002. The MT domain of the proto-oncoprotein MLL binds to
CpG-containing DNA and discriminates against methylation. Nucleic Acids
10. Brock, H. W., and C. L. Fisher. 2005. Maintenance of gene expression
patterns. Dev. Dyn. 232:633–655.
11. Buszczak, M., and A. C. Spradling. 2006. Searching chromatin for stem cell
identity. Cell 125:233–236.
12. Cao, R., Y. Tsukada, and Y. Zhang. 2005. Role of Bmi-1 and Ring1A in H2A
ubiquitylation and Hox gene silencing. Mol. Cell 20:845–854.
13. Cao, R., and Y. Zhang. 2004. The functions of E(Z)/EZH2-mediated meth-
ylation of lysine 27 in histone H3. Curr. Opin. Genet. Dev. 14:155–164.
14. Cardozo, T., and M. Pagano. 2004. The SCF ubiquitin ligase: insights into a
molecular machine. Nat. Rev. Mol. Cell Biol. 5:739–751.
15. Carrozza, M. J., B. Li, L. Florens, T. Suganuma, S. K. Swanson, K. K. Lee,
W. J. Shia, S. Anderson, J. Yates, M. P. Washburn, and J. L. Workman.
2005. Histone H3 methylation by Set2 directs deacetylation of coding regions
by Rpd3S to suppress spurious intragenic transcription. Cell 123:581–592.
16. Cattoretti, G., L. Pasqualucci, G. Ballon, W. Tam, S. V. Nandula, Q. Shen,
T. Mo, V. V. Murty, and R. Dalla-Favera. 2005. Deregulated BCL6 expres-
sion recapitulates the pathogenesis of human diffuse large B cell lymphomas
in mice. Cancer Cell 7:445–455.
17. Chang, C. C., B. H. Ye, R. S. K. Chaganti, and R. Dalla-Favera. 1996. BCL-6,
a POZ/zinc-finger protein, is a sequence-specific transcriptional repressor.
Proc. Natl. Acad. Sci. USA 93:6947–6952.
18. Chen, Z., J. Zang, J. Whetstine, X. Hong, F. Davrazou, T. G. Kutateladze, M.
Simpson, Q. Mao, C. H. Pan, S. Dai, J. Hagman, K. Hansen, Y. Shi, and G.
Zhang. 2006. Structural insights into histone demethylation by JMJD2 family
members. Cell 125:691–702.
19. Chevallier, N., C. M. Corcoran, C. Lennon, E. Hyjek, A. Chadburn, V. J.
Bardwell, J. D. Licht, and A. Melnick. 2004. ETO protein of t(8;21) AML is
a corepressor for Bcl-6 B-cell lymphoma oncoprotein. Blood 103:1454–1463.
20. Cloos, P. A. C., J. Christensen, K. Agger, A. Maiolica, J. Rappsilber, T.
Antal, K. H. Hansen, and K. Helin. 2006. The putative oncogene GASC1
demethylates tri- and dimethylated lysine 9 on histone H3. Nature 442:307–
21. de Napoles, M., J. E. Mermoud, R. Wakao, Y. A. Tang, M. Endoh, R.
Appanah, T. B. Nesterova, J. Silva, A. P. Otte, M. Vidal, H. Koseki, and N.
Brockdorff. 2004. Polycomb group proteins Ring1A/B link ubiquitylation of
histone H2A to heritable gene silencing and X inactivation. Dev. Cell 7:663–
22. Dent, A. L., A. L. Shaffer, X. Yu, D. Allman, and L. M. Staudt. 1997. Control
of inflammation, cytokine expression, and germinal center formation by
BCL-6. Science 276:589–592.
23. Deweindt, C., O. Albagli, F. Bernardin, P. Dhordain, S. Quief, D. Lantoine,
J. P. Kerckaert, and D. Leprince. 1995. The Laz3/Bcl6 oncogene encodes a
sequence-specific transcriptional inhibitor: a novel function for the Btb/Poz
domain as an autonomous repressing domain. Cell Growth Differ. 6:1495–
24. Dhordain, P., R. J. Lin, S. Quief, D. Lantoine, J. P. Kerckaert, R. M. Evans,
and O. Albagli. 1998. The LAZ3(BCL-6) oncoprotein recruits a SMRT/
mSIN3A histone deacetylase containing complex to mediate transcriptional
repression. Nucleic Acids Res. 26:4645–4651.
25. Dhordain, P., O. Albagli, R. J. Lin, S. Ansieau, S. Quief, A. Leutz, J. P.
Kerckaert, R. M. Evans, and D. Leprince. 1997. Corepressor SMRT binds
the BTB/POZ repressing domain of the LAZ3/BCL6 oncoprotein. Proc.
Natl. Acad. Sci. USA 94:10762–10767.
26. Dignam, J. D., P. L. Martin, B. S. Shastry, and R. G. Roeder. 1983. Eukary-
otic gene transcription with purified components. Methods Enzymol. 101:
27. Eberharter, A., I. Vetter, R. Ferreira, and P. B. Becker. 2004. ACF1 improves
the effectiveness of nucleosome mobilization by ISWI through PHD-histone
contacts. EMBO J. 23:4029–4039.
28. Fang, J., T. Chen, B. Chadwick, E. Li, and Y. Zhang. 2004. Ring1b-mediated
H2A ubiquitination associates with inactive X chromosomes and is involved
in initiation of X inactivation. J. Biol. Chem. 279:52812–52815.
29. Fujita, N., D. L. Jaye, C. Geigerman, A. Akyildiz, M. R. Mooney, J. M. Boss,
and P. A. Wade. 2004. MTA3 and the Mi-2/NuRD complex regulate cell fate
during B lymphocyte differentiation. Cell 119:75–86.
30. Garcia, E., C. Marcos-Gutierrez, L. M. del Mar, J. C. Moreno, and M. Vidal.
1999. RYBP, a new repressor protein that interacts with components of the
mammalian Polycomb complex, and with the transcription factor YY1.
EMBO J. 18:3404–3418.
31. Gong, Y., X. Wang, J. Liu, L. Shi, B. Yin, X. Peng, B. Qiang, and J. Yuan.
2005. NSPc1, a mainly nuclear localized protein of novel PcG family mem-
bers, has a transcription repression activity related to its PKC phosphoryla-
tion site at S183. FEBS Lett. 579:115–121.
32. Guenther, M. G., W. S. Lane, W. Fischle, E. Verdin, M. A. Lazar, and R.
Shiekhattar. 2000. A core SMRT corepressor complex containing HDAC3
and TBL1, a WD40-repeat protein linked to deafness. Genes Dev. 14:1048–
33. Harris, M. B., J. Mostecki, and P. B. Rothman. 2005. Repression of an
interleukin-4-responsive promoter requires cooperative BCL-6 function.
J. Biol. Chem. 280:13114–13121.
34. Hemenway, C. S., A. C. de Erkenez, and G. C. D. Gould. 2001. The polycomb
protein MPc3 interacts with AF9, an MLL fusion partner in t(9;11)(p22;q23)
acute leukemias. Oncogene 20:3798–3805.
35. Hemenway, C. S., B. W. Halligan, and L. S. Levy. 1998. The Bmi-1 onco-
protein interacts with dinG and MPh2: the role of RING finger domains.
36. Huynh, K. D., and V. J. Bardwell. 1998. The BCL-6 POZ domain and other
POZ domains interact with the co-repressors N-CoR and SMRT. Oncogene
37. Huynh, K. D., W. Fischle, E. Verdin, and V. J. Bardwell. 2000. BCOR, a
novel corepressor involved in BCL-6 repression. Genes Dev. 14:1810–1823.
38. Jason, L. J. M., R. M. Finn, G. Lindsey, and J. Ausio. 2005. Histone H2A
ubiquitination does not preclude histone H1 binding, but it facilitates its
association with the nucleosome. J. Biol. Chem. 280:4975–4982.
39. Jin, J., T. Cardozo, R. C. Lovering, S. J. Elledge, M. Pagano, and J. W.
Harper. 2004. Systematic analysis and nomenclature of mammalian F-box
proteins. Genes Dev. 18:2573–2580.
40. Jorgensen, H. F., I. Ben-Porath, and A. P. Bird. 2004. Mbd1 is recruited to
both methylated and nonmethylated CpGs via distinct DNA binding do-
mains. Mol. Cell. Biol. 24:3387–3395.
41. Joshi, A. A., and K. Struhl. 2005. Eaf3 chromodomain interaction with
methylated H3-K36 links histone deacetylation to Pol II elongation. Mol.
6888 GEARHART ET AL.MOL. CELL. BIOL.
42. Keogh, M. C., S. K. Kurdistani, S. A. Morris, S. H. Ahn, V. Podolny, S. R. Download full-text
Collins, M. Schuldiner, K. Y. Chin, T. Punna, N. J. Thompson, C. Boone, A.
Emili, J. S. Weissman, T. R. Hughes, B. D. Strahl, M. Grunstein, J. F.
Greenblatt, S. Buratowski, and N. J. Krogan. 2005. Cotranscriptional Set2
methylation of histone H3 lysine 36 recruits a repressive Rpd3 complex. Cell
43. Klose, R. J., K. Yamane, Y. Bae, D. Zhang, H. Erdjument-Bromage, P.
Tempst, J. Wong, and Y. Zhang. 2006. The transcriptional repressor
JHDM3A demethylates trimethyl histone H3 lysine 9 and lysine 36. Nature
44. Krogan, N. J., M. Kim, A. Tong, A. Golshani, G. Cagney, V. Canadien, D. P.
Richards, B. K. Beattie, A. Emili, C. Boone, A. Shilatifard, S. Buratowski,
and J. Greenblatt. 2003. Methylation of histone H3 by Set2 in Saccharomyces
cerevisiae is linked to transcriptional elongation by RNA polymerase II. Mol.
Cell. Biol. 23:4207–4218.
45. Lemercier, C., M. P. Brocard, F. Puvion-Dutilleul, H. Y. Kao, O. Albagli, and
S. Khochbin. 2002. Class II histone deacetylases are directly recruited by
BCL6 transcriptional repressor. J. Biol. Chem. 277:22045–22052.
46. Levine, S. S., A. Weiss, H. Erdjument-Bromage, Z. H. Shao, P. Tempst, and
R. E. Kingston. 2002. The core of the polycomb repressive complex is
compositionally and functionally conserved in flies and humans. Mol. Cell.
47. Li, B., L. Howe, S. Anderson, J. R. Yates, and J. L. Workman. 2003. The Set2
histone methyltransferase functions through the phosphorylated carboxyl-
terminal domain of RNA polymerase II. J. Biol. Chem. 278:8897–8903.
48. Li, J. W., J. Wang, J. X. Wang, Z. Nawaz, J. M. Liu, J. Qin, and J. M. Wong.
2000. Both corepressor proteins SMRT and N-CoR exist in large protein
complexes containing HDAC3. EMBO J. 19:4342–4350.
49. Li, Z., X. Wang, R. Y.-L. Yu, B. B. Ding, J. J. Yu, X. M. Dai, A. Naganuma,
E. R. Stanley, and B. H. Ye. 2005. BCL-6 negatively regulates expression of
the NF-?B1 p105/p50 subunit. J. Immunol. 174:205–214.
50. Mahmoudi, T., L. M. P. Zuijderduijn, A. Mohd-Sarip, and C. P. Verrijzer.
2003. GAGA facilitates binding of Pleiohomeotic to a chromatinized Poly-
comb response element. Nucleic Acids Res. 31:4147–4156.
51. Melnick, A. 2005. Reprogramming specific gene expression pathways in
B-cell lymphomas. Cell Cycle 4:239–241.
52. Mulholland, N. M., I. F. G. King, and R. E. Kingston. 2003. Regulation of
Polycomb group complexes by the sequence-specific DNA binding proteins
Zeste and GAGA. Genes Dev. 17:2741–2746.
53. Ng, D., N. Thakker, C. M. Corcoran, D. Donnai, R. Perveen, A. Schneider,
D. W. Hadley, C. Tifft, L. Zhang, A. O. Wilkie, J. J. van der Smagt, R. J.
Gorlin, S. M. Burgess, V. J. Bardwell, G. C. Black, and L. G. Biesecker. 2004.
Oculofaciocardiodental and Lenz microphthalmia syndromes result from
distinct classes of mutations in BCOR. Nat. Genet 36:411–416.
54. Niu, H. F., G. Cattoretti, and R. Dalla-Favera. 2003. BCL6 controls the
expression of the B7-1/CD80 costimulatory receptor in germinal center B
cells. J. Exp. Med. 198:211–221.
55. Nunes, M., I. Blanc, J. Maes, M. Fellous, B. Robert, and K. McElreavey.
2001. NSPc1, a novel mammalian Polycomb gene, is expressed in neural
crest-derived structures of the peripheral nervous system. Mech. Dev. 102:
56. Ogawa, H., K. Ishiguro, S. Gaubatz, D. M. Livingston, and Y. Nakatani.
2002. A complex with chromatin modifiers that occupies E2F-and Myc-
responsive genes in G0cells. Science 296:1132–1136.
57. Pasqualucci, L., A. Migliazza, K. Basso, J. Houldsworth, R. S. K. Chaganti,
and R. Dalla-Favera. 2003. Mutations of the BCL6 proto-oncogene disrupt
its negative autoregulation in diffuse large B-cell lymphoma. Blood 101:
58. Perkins, D. N., D. J. Pappin, D. M. Creasy, and J. S. Cottrell. 1999. Prob-
ability-based protein identification by searching sequence databases using
mass spectrometry data. Electrophoresis 20:3551–3567.
59. Phan, R. T., and R. Dalla-Favera. 2004. The BCL6 proto-oncogene sup-
presses p53 expression in germinal-centre B cells. Nature 432:635–639.
60. Phan, R. T., M. Saito, K. Basso, H. Niu, and R. Dalla-Favera. 2005. BCL6
interacts with the transcription factor Miz-1 to suppress the cyclin-dependent
kinase inhibitor p21 and cell cycle arrest in germinal center B cells. Nat.
61. Polo, J. M., T. Dell’Oso, S. M. Ranuncolo, L. Cerchietti, D. Beck, G. F. Da
Silva, G. G. Prive, J. D. Licht, and A. Melnick. 2004. Specific peptide
interference reveals BCL6 transcriptional and oncogenic mechanisms in
B-cell lymphoma cells. Nat. Med. 10:1329–1335.
62. Raaphorst, F. M. 2005. Deregulated expression of Polycomb-group onco-
genes in human malignant lymphomas and epithelial tumors. Hum. Mol.
63. Rajewsky, K. 1996. Clonal selection and learning in the antibody system.
64. Rao, B., Y. Shibata, B. D. Strahl, and J. D. Lieb. 2005. Dimethylation of
histone H3 at lysine 36 demarcates regulatory and nonregulatory chromatin
genome-wide. Mol. Cell. Biol. 25:9447–9459.
65. Seyfert, V. L., D. Allman, Y. S. He, and L. M. Staudt. 1996. Transcriptional
repression by the proto-oncogene BCL-6. Oncogene 12:2331–2342.
66. Shaffer, A. L., X. Yu, Y. He, J. Boldrick, E. P. Chan, and L. M. Staudt. 2000.
BCL-6 represses genes that function in lymphocyte differentiation, inflam-
mation, and cell cycle control. Immunity 13:199–212.
67. Shi, X., T. Hong, K. L. Walter, M. Ewalt, E. Michishita, T. Hung, D. Carney,
P. Pena, F. Lan, M. R. Kaadige, N. Lacoste, C. Cayrou, F. Davrazou, A.
Saha, B. R. Cairns, D. E. Ayer, T. G. Kutateladze, Y. Shi, J. Cote, K. F. Chua,
and O. Gozani. 2006. ING2 PHD domain links histone H3 lysine 4 methyl-
ation to active gene repression. Nature 442:96–99.
68. Shin Voo, K., D. L. Carlone, B. M. Jacobsen, A. Flodin, and D. G. Skalnik.
2000. Cloning of a mammalian transcriptional activator that binds unmeth-
ylated CpG motifs and shares a CXXC domain with DNA methyltransferase,
human Trithorax, and methyl-CpG binding domain protein 1. Mol. Cell.
69. Srinivasan, R. S., A. C. de Erkenez, and C. S. Hemenway. 2003. The mixed
lineage leukemia fusion partner AF9 binds specific isoforms of the BCL-6
corepressor. Oncogene 22:3395–3406.
70. Stogios, P. J., G. S. Downs, J. J. S. Jauhal, S. K. Nandra, and G. G. Prive.
2005. Sequence and structural analysis of BTB domain proteins. Genome
71. Suzuki, M., Y. Mizutani-Koseki, Y. Fujimura, H. Miyagishima, T. Kaneko,
Y. Takada, T. Akasaka, H. Tanzawa, Y. Takihara, M. Nakano, H. Masumoto,
M. Vidal, K. Isono, and H. Koseki. 2002. Involvement of the polycomb-group
gene Ring1B in the specification of the anterior-posterior axis in mice.
72. Tanaka, S., T. Kunath, A. K. Hadjantonakis, A. Nagy, and J. Rossant. 1998.
Promotion of trophoblast stem cell proliferation by FGF4. Science 282:2072–
73. Trewick, S. C., P. J. McLaughlin, and R. C. Allshire. 2005. Methylation: lost
in hydroxylation? EMBO Rep. 6:315–320.
74. Tsukada, Y., J. Fang, H. Erdjument-Bromage, M. E. Warren, C. H. Borchers, P.
Tempst, and Y. Zhang. 2006. Histone demethylation by a family of JmjC
domain-containing proteins. Nature 439:811–816.
75. Tunyaplin, C., A. L. Shaffer, C. D. Angelin-Duclos, X. Yu, L. M. Staudt, and
K. L. Calame. 2004. Direct repression of prdm1 by Bcl-6 inhibits plasmacytic
differentiation. J. Immunol. 173:1158–1165.
76. Vasanwala, F. H., S. Kusam, L. M. Toney, and A. L. Dent. 2002. Repression
of AP-1 function: a mechanism for the regulation of Blimp-1 expression and
B lymphocyte differentiation by the B cell lymphoma-6 protooncogene.
J. Immunology 169:1922–1929.
77. Vassilev, A. P., H. H. Rasmussen, E. I. Christensen, S. Nielsen, and J. E.
Celis. 1995. The levels of ubiquitinated histone H2A are Highly up-regulated
in transformed human cells: partial colocalization of Uh2A clusters and
Pcna/cyclin foci in a fraction of cells in S-phase. J. Cell Sci. 108:1205–1215.
78. Wang, H., L. Wang, H. Erdjument-Bromage, M. Vidal, P. Tempst, R. S.
Jones, and Y. Zhang. 2004. Role of histone H2A ubiquitination in Polycomb
silencing. Nature 431:873–878.
79. Wang, X., Z. Li, A. Naganuma, and B. H. Ye. 2002. Negative autoregulation
of BCL-6 is bypassed by genetic alterations in diffuse large B cell lymphomas.
Proc. Natl. Acad. Sci. USA 99:15018–15023.
80. Wen, Y. D., V. Perissi, L. M. Staszewski, W. M. Yang, A. Krones, C. K. Glass,
M. G. Rosenfeld, and E. Seto. 2000. The histone deacetylase-3 complex
contains nuclear receptor corepressors. Proc. Natl. Acad. Sci. USA 97:7202–
81. Whetstine, J. R., A. Nottke, F. Lan, M. Huarte, S. Smolikov, Z. Chen, E.
Spooner, E. Li, G. Zhang, M. Colaiacovo, and Y. Shi. 2006. Reversal of
histone lysine trimethylation by the JMJD2 family of histone demethylases.
82. Wong, C. W., and M. L. Privalsky. 1998. Components of the SMRT core-
pressor complex exhibit distinctive interactions with the POZ domain onco-
proteins PLZF, PLZF-RAR alpha, and BCL-6. J. Biol. Chem. 273:27695–
83. Wysocka, J., T. Swigut, H. Xiao, T. A. Milne, S. Y. Kwon, J. Landry, M.
Kauer, A. J. Tackett, B. T. Chait, P. Badenhorst, C. Wu, and C. D. Allis.
2006. A PHD finger of NURF couples histone H3 lysine 4 trimethylation
with chromatin remodelling. Nature 442:86–90.
84. Xiao, T. J., H. Hall, K. O. Kizer, Y. Shibata, M. C. Hall, C. H. Borchers, and
B. D. Strahl. 2003. Phosphorylation of RNA polymerase II CTD regulates
H3 methylation in yeast. Genes Dev. 17:654–663.
85. Yamane, K., C. Toumazou, Y. Tsukada, H. Erdjument-Bromage, P. Tempst,
J. Wong, and Y. Zhang. 2006. JHDM2A, a JmjC-containing H3K9 demeth-
ylase, facilitates transcription activation by androgen receptor. Cell 125:483–
86. Zhang, D. Z., H. G. Yoon, and J. M. Wong. 2005. JMJD2A is a novel
N-CoR-interacting protein and is involved in repression of the human tran-
scription factor achaete scute-like homologue 2 (ASCL2/Hash2). Mol. Cell.
87. Zollman, S., D. Godt, G. G. Prive, J. L. Couderc, and F. A. Laski. 1994. The
Btb domain, found primarily in zinc-finger proteins, defines an evolutionarily
conserved family that includes several developmentally-regulated genes in
Drosophila. Proc. Natl. Acad. Sci. USA 91:10717–10721.
VOL. 26, 2006TRANSCRIPTIONAL REPRESSION BY BCOR 6889