RESEARCH ARTICLE
2383
CHMP1 is a novel nuclear matrix protein affecting
chromatin structure and cell-cycle progression
Daniel R. Stauffer*, Tiffani L. Howard, Thihan Nyun and Stanley M. Hollenberg
Vollum Institute, L474, Oregon Health Sciences University, 3181 S. W. Sam Jackson Park Rd, Portland, OR 97201-3098, USA
*Author for correspondence (e-mail: [email protected])
Accepted 6 April 2001
Journal of Cell Science 114, 2383-2393 © The Company of Biologists Ltd
SUMMARY
The Polycomb-group (PcG) is a diverse set of proteins
required for maintenance of gene silencing during
development. In a screen for conserved partners of the PcG
protein Polycomblike (Pcl), we have identified a new
protein, human CHMP1 (CHromatin Modifying Protein;
CHarged Multivesicular body Protein), which is encoded
by an alternative open reading frame in the PRSM1 gene
and is conserved in both complex and simple eukaryotes.
CHMP1 contains a predicted bipartite nuclear localization
signal and distributes as distinct forms to the cytoplasm
and the nuclear matrix in all cell lines tested. We have
constructed a stable HEK293 cell line that inducibly
overexpresses CHMP1 under ecdysone control.
Overexpressed CHMP1 localizes to a punctate subnuclear
INTRODUCTION
Epigenetic modifications are used to record and stabilize
changes in the transcriptional activity of genes. One memory
system, first discovered in Drosophila, uses the antagonistic
action of repressors (Polycomb-group; PcG) and activators to
maintain earlier developmental decisions about the activity of
specific targets (Gould, 1997; Jacobs and van Lohuizen, 1999;
Pirrotta, 1999). The PcG comprises a large, structurally diverse
set of nuclear proteins, which functions both to select target
genes and regulate their transcription.
Both biochemical and genetic studies suggest that PcG
silencing is mediated through modification of chromatin
structure. For example, a purified PcG complex has been
demonstrated to inhibit nucleosome remodeling by SWI/SNF
proteins in vitro (Shao et al., 1999). Furthermore, one
component of this complex has been shown to directly interact
with the nucleosome core (Breiling et al., 1999). Changes in
histone covalent modification may also play a direct role in
silencing. Studies in Drosophila demonstrate a genetic
interaction between the PcG and dMi-2, which encodes a
component of a nucleosome remodeling/histone deacetylase
complex (Kehle et al., 1998; Wade et al., 1998; Zhang et al.,
1998). Finally, one member of the PcG, human EED, has been
demonstrated to interact physically with a histone deacetylase
(van der Vlag and Otte, 1999).
Genetic analysis and theoretical considerations suggest that
establishment or maintenance of PcG silencing requires the
activity of proteins during DNA replication and mitosis. The
Drosophila PcG gene cramped is expressed only during S-
pattern, encapsulating regions of nuclease-resistant,
condensed chromatin. These novel structures are also
frequently surrounded by increased histone H3
phosphorylation and acetylation. CHMP1 can recruit a
PcG protein, BMI1, to these regions of condensed
chromatin and can cooperate with co-expressed vertebrate
Pcl in a Xenopus embryo PcG assay; this is consistent with
a role in PcG function. In combination, these observations
suggest that CHMP1 plays a role in stable gene silencing
within the nucleus.
Key words: MeSH, Gene silencing, Nuclear matrix, Chromatin,
Histones, DNA replication, S phase
phase and interacts with the DNA replication gene mus209
(Yamamoto et al., 1997). At mitosis, the Drosophila PcG
protein CCF is associated with mitotic chromosomes and is
required for their proper condensation (Kodjabachian et al.,
1998). By contrast, at mitosis the majority of transcription
factors and most PcG proteins are no longer chromatin bound
(Buchenau et al., 1998). The temporal relationship between the
action of cramped, ccf and other PcG genes has not been
defined.
Most of the 15 identified Drosophila PcG proteins have been
demonstrated to have structural and functional counterparts in
vertebrates (Gould, 1997) and some PcG target genes may also
be conserved (Bel et al., 1998; Hanson et al., 1999; Satijn and
Otte, 1999). By contrast, only a few PcG proteins have
structural relatives in Caenorhabditis elegans (Holdeman et al.,
1998; Kelly and Fire, 1998; Korf et al., 1998), Arabadopis
(Preuss, 1999) or budding yeast (Nislow et al., 1997; Stankunas
et al., 1998). The absence of conserved PcG components in
these organisms suggests that specific aspects of this silencing
mechanism arose later in evolution. In addition, it may indicate
that highly conserved, multifunctional PcG components
common to all eukaryotes might be under-represented in
Drosophila genetic screens for new PcG members.
The PcG has been estimated to include 30-40 gene products
(Jurgens, 1985; Landecker et al., 1994), with about half of
these molecularly identified. These chromatin-associated
proteins have been shown to function together through cisacting regulatory elements (Chan et al., 1994; Chiang et al.,
1995; Christen and Bienz, 1994), but are found in several
biochemically distinct multiprotein complexes. The best
2384
JOURNAL OF CELL SCIENCE 114 (13)
characterized is a complex of eight polypeptides called PRC1 (Shao et al., 1999) and a second, distinct complex that
includes Enhancer-of-Zeste and Extra Sex Combs (Denisenko
et al., 1998; Jones et al., 1998; Sewalt et al., 1998; van
Lohuizen et al., 1998). Some PcG components have not been
included in either complex and may have distinct roles. For
example, Pcl shows genetic interactions with many PcG genes
and encodes a protein (Polycomblike) that colocalizes with
other PcG proteins on polytene chrososomes, but does not copurify with other known PcG proteins. Identification of
physical partners for Pcl may help to unify our understanding
of the action of PRC-1 and Enhancer-of-Zeste complexes and
might reveal distinct gene regulatory mechanisms.
We used the yeast two-hybrid system, with mammalian Pcl
as bait, to identify a novel potential component of the PcG,
called CHMP1 (pronounced chimp-1; CHromatin Modifying
Protein; CHarged Multivesicular body Protein). CHMP1 is
widely conserved across eukaryotes and plays a cytoplasmic
role in protein sorting to the multivesicular body (Howard et
al., 2001). Endogenous mammalian CHMP1 is also present
in the interphase nuclear matrix and mitotic chromosome
scaffold. When overexpressed, CHMP1 has potent affects on
nuclear structure and DNA replication. In addition, exogenous
CHMP1 targets a PcG protein to condensed chromatin and
functions like a PcG protein in a Xenopus misexpression assay.
Thus, CHMP1 has a potential dual function, with distinct roles
in membrane trafficking and nuclear gene regulation.
MATERIALS AND METHODS
Plasmid constructions
pLexA-mPcl1 and pLexA-dPcl were constructed for two-hybrid
analysis by introducing the full-length open reading frames from
mPcl1 (Yoshitake et al., 1999) and Drosophila Pcl (Lonie et al., 1994)
into the BamHI site of pLex-A. The Drosophila Pcl cDNA was kindly
provided by R. Saint. For subcloning, the human CHMP1 open
reading frame was isolated by reverse-transcription from human
placental RNA followed by PCR with primers 5′-GGCAGAATTCACCATGGACGATACCCTGTTCCAGTTGA-3′, and 5′-GGCAGGATCCATCACGGGGCAGAGGCGGTGCACA-3′. The resulting
EcoRI/BamHI fragment was subcloned into a modified version of
pCS2+ (a gift of David Turner) to generate pCS-hCHMP1. pINDCHMP1 was constructed by excising CHMP1 from pCS-hCHMP1
with HindIII/XhoI and introducing it into the corresponding sites of
pIND (Invitrogen).
Two hybrid screen
Saccharomyces cerevisiae strain L40, transformed with pLex-mPcl1,
was used to screen an E9.5/10.5 mouse embryo cDNA fusion library
as described previously (Hollenberg et al., 1995). The bait plasmid
(containing ADE2) was eliminated from each primary positive by
isolating a red colony (Chen et al., 1996). Each library positive was
then tested for β-galactosidase activity after mating with derivatives
of yeast strain AMR70 that express LexA-mPcl1 or LexA-dPcl.
Fractionation of cells and tissues
Nuclear matrix was isolated as described previously (Belgrader et al.,
1991), with minor modifications. Cells were washed and collected in
cold PBS and pelleted by centrifugation at 1000 rpm for 5 minutes.
From this point on, all buffers contained a protease inhibitor cocktail
consisting of 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride
(AEBSF), 1 mM E-64 and 1 mg/ml each of bestatin, leupeptin and
aprotinin (Sigma Immunochemicals). The cell pellet was resuspended
in 1.5 ml lysis buffer (10 mM Tris-HCl, pH 7.4, 2.5 mM MgCl2) and
transferred to ice for 15 minutes. Cells were lysed with 25-60 strokes
of a dounce homogenizer and the lysate was gently layered over a 1.5
ml sucrose cushion (0.5 M sucrose, 10 mM Tris-HCl, pH 7.4, 2.5 mM
MgCl2). Nuclei were pelleted by centrufugation at 5000 g for 10
minutes at 4°C in a Sorvall HB4 rotor. The upper layer consisting of
cytoplasmic extract was collected and concentrated to 100 µl in a
centricon 10 column. The nuclear pellet was stripped of membranes
by resuspension in 200 µl of Triton extraction buffer (0.5% Triton X100, 10 mM Tris-HCl pH 7.4, 2.5 mM MgCl2), followed by
incubation on ice for 15 minutes. Membrane depleted nuclei were
collected by centrifugation at 5000 g for 10 minutes in a microfuge
at 4°C. Chromatin and soluble proteins were removed by resuspension
in 150 µl of DNase I digestion buffer (10 mM Tris-HCl, pH 7.4, 150
mM NaCl, 5 mM MgCl2) containing 50 U of DNase I and incubated
at 4°C for 1 hour. This was followed by addition of 150 µl of 2× High
Salt buffer (10 mM Tris-HCl pH 7.4, 1.45 M NaCl), and further
incubation for 15 minutes at 4°C. The sample was centrifuged at
10,000 g for 10 minutes at 4°C and the supernatant saved as the DNase
I High Salt fraction. The nuclear matrix pellet was resuspended in 150
µl of RIPA buffer (50 mM Tris-HCl pH 7.4, 5 mM DTT, 150 mM
NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS).
Stable line construction and cell culture
The IND-CHMP1 stable line was obtained by LipofectAMINE
(Gibco BRL) transfection of EcR-293 cells (human embryonic kidney,
stably expressing the hybrid ecdysone receptor, Invitrogen). HEK293
cells were cultured on plates or eight-well Nunc SuperCell culture
slides (Intermountian Scientific) in Dulbecco’s modified eagle
medium (DMEM) with 10% fetal calf serum, 100 units/ml penicillin
G, and 100 µg/ml streptomycin (Gibco BRL), 300 µg/ml Zeocin
(Invitrogen) and 800 µg/ml G418 (Calbiochem). Cells were allowed
to recover for 18 hours after plating before muristerone (10 µM)
induction.
Immunocytochemistry and antibodies
Cultured cells were fixed with 4% paraformaldehyde in PBS, washed
with PBS and allowed to dry on the slide. Slides were incubated 30
minutes in blocking solution (PBS with 10% normal sheep serum, 0.1
mg/ml bovine serum albumin, and 0.05% Triton X-100). Primary
antibodies in blocking solution and secondary antibodies in PBS with
0.05% Triton X-100 were added consecutively for 1 hour each at room
temperature.
Rabbit polyclonal anti-CHMP1 (1:300) was made by injection into
rabbits of GST-CHMP1 fusion protein mixed with Freund’s
incomplete adjuvant (Pocono Rabbit Farm & Laboratory Inc.). The
antibody was subsequently purified over a MBP-CHMP1 Affigel-10
column and eluted with low pH. Other antibodies include polyclonal
phospho-histone H3 (Cat# 06-570 1:200) and polyclonal antiacetylated histone H3 (Cat# 06-599, 1:100) from Upstate
Biotechnology, and monoclonal anti-BMI1 (a kind gift from Maarten
van Lohuizen, 1:300). Secondary antibodies are anti-rabbit Alexa
488 (Molecular probes, 1:300) and Cy3 (Jackson ImmunoReseach
Laboratories Inc.; 1:300) or anti-mouse Cy3 (Jackson
ImmunoReseach Laboratories Inc.; 1:300). Hoechst 33258 and
propidium iodide (Molecular Probes) were included at 1.0 µg/ml.
Samples were visualized with a Zeiss Axioplan microscope or by
confocal laser scanning microscopy on a BioRad MRC 1000.
Bromodeoxyuridine (BrdU) labeling was performed according to
instructions provided with the product (Becton Dickenson). TUNEL
was performed according to instructions included in the kit
(Boehringer Mannheim). Mitotic shake-off and spreads were
performed as described (Chandler and Yunis, 1978). The stabilized
nuclear matrix was prepared as described previously with
modifications (Nickerson et al., 1997). IND-CHMP1 cells were grown
on chamber slides and induced for 24 hours with 10 µM ponasterone.
Cells were washed with cold PBS and soluble proteins were removed
CHMP1 functions in the nucleus
RESULTS
by extraction in CSK buffer (10 mM Pipes, pH 6.8, 300 mM sucrose,
100 mM NaCl, 3 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100,
protease inhibitors) for 5 minutes at room temperature. Cells were
then crosslinked by treatment with 4% paraformaldehyde in CSK for
5 minutes at room temperature and washed three times with CSK
buffer for 5 minutes at room temperature. After washing in CSK, cells
were digested with 0, 25, 125, or 500 U/ml of DNase I for 50 minutes
at room temperature in digestion buffer (10 mM Pipes, pH 6.8, 300
mM sucrose, 50 mM NaCl, 3 mM MgCl2 protease inhibitors). Cells
were then washed with 0.25 M ammonium sulfate extraction buffer
(10 mM Pipes, pH 6.8, 250 mM ammonium sulfate, 300 mM sucrose,
3 mM MgCl2, 1 mM EGTA, protease inhibitors). The ammonium
sulfate extraction buffer was aspirated and cells were washed three
times with cold PBS followed by standard immunocytochemistry.
Identification of a conserved Pcl partner
The yeast two-hybrid system was used to screen for partners
of the mammalian PcG protein mPcl1 (Yoshitake et al., 1999).
The full-length mPcl1 open reading frame was fused to LexA
and used to screen a mouse embryo cDNA fusion library
(Hollenberg et al., 1995). Each positive was also tested for twohybrid interaction with Drosophila Pcl (Lonie et al., 1994) to
define the subset of proteins which interact with the conserved
structural features of mPcl1 (Yoshitake et al., 1999). This
procedure isolated a single library clone with equivalent Pcl
and mPcl1 two-hybrid signals, which corresponded to PRSM1
(Scott et al., 1996), a gene frequently deleted in sporadic breast
carcinoma (Whitmore et al., 1998). Surprisingly, a reading
frame alternative to that reported to encode the PRSM1
metallopeptidase was fused in-frame to the library activation
domain coding sequence. This distinct reading frame is
contained entirely within PRSM1 (Fig. 1A) and encodes a
novel protein of 196 amino acids, called CHMP1.
CHMP1 is closely related to another human protein,
CHMP1.5, and has structural counterparts in many eukaryotes
including Drosophila, C. elegans, Dictyostelium, Arabidopsis
and S. cerevisiae (Fig. 1B,C; D.R.S. and S.M.H., unpublished).
A search for structural motifs identifies only a predicted
bipartite nuclear localization signal (Fig. 1C; aa 20-35), which
is also present in human CHMP1.5 and Drosophila,
Dictyostelium and Arabidopsis CHMP1, but absent in CHMP1
from C. elegans and budding yeast. A BLAST similarity search
(Altschul et al., 1997) for other human proteins related to
CHMP1 shows that it is most closely related to BC-2, a protein
upregulated in the nuclear matrix of breast adenocarcinoma
(Keesee et al., 1999), followed by more distant proteins
involved in vesicle trafficking in S. cerevisiae (Babst et al.,
1998; Howard et al., 2001).
Western analysis
Antibodies used were affinity-purified polyclonal anti-CHMP1
(1:1000), monoclonal anti-α-tubulin (Research Biochemicals
International, 1:10,000), and chicken polyclonal anti-matrin-4 (kind gift
of Ronald Berezney, 1:2000). Bound antibodies were detected by HRPconjugated secondary antibodies and enhanced chemiluminescence.
Flow cytometry
A single-cell suspension was prepared by trypsin digestion and
tituration. The cells were fixed in cold 50% ethanol with vortexing
and stored at 4°C. After washing, the cells were stained with
a modified, standard propidium iodide staining protocol
(Thornthwaighte et al., 1980). Samples were then analyzed on a
FACScan flow cytometer (Becton-Dickinson, San Jose, CA). A total
of 20,000 cells were analyzed for each cell line and growth condition.
Raw single parameter histogram data was fit with the Multicycle
program (Phoenix Flow Cytometry, Inc., San Diego, CA) to estimate
the G1, S and G2/M phases of the cell cycle.
Xenopus injection assay
Embryos were injected with capped, in vitro transcribed RNA and
were analyzed morphologically and through RNA in situ
hybridization, as described previously (Yoshitake et al., 1999).
Fig. 1. CHMP1 is a
conserved eukaryotic
protein. (A) Conceptual
translation products
CHMP1 and PRSM1 from
alternative reading frames
of the human PRSM1
mRNA. (B) Phylogenetic
tree for CHMP1 and
CHMP1.5 from human
(H.s.), and CHMP1 from
Drosophila melanogaster
(D.m.), C. elegans (C.e.)
and S. cerevisiae (S.c.).
ClustalX was used to align
proteins and calculate the
distance between proteins
for tree construction
(Thompson et al., 1997).
(C) All CHMP1 proteins
from the tree are aligned.
Amino acids that are
identical in at least three
proteins are highlighted.
A
1
2385
0.05
B
196
CHMP1
H.s. CHMP1
H.s. CHMP1.5
UAG
AUG
AUG
UAG
AAAAAA
D.m. CHMP1
PRSM1
C.e. CHMP1
1
318
S.c. Chm1p
C
NLS
H.s. CHMP1
MD D T L
H.s. CHMP1.5
MEK HL
D.m. CHMP1
MEK HL
C. e. CHMP1 M G A G E S S M A L E K H L
S. c. Chm1p
MS RN S A A GL E N T L
FQL
FNL
FNL
FDL
FQL
K
K
K
K
K
FT A KQL
FAAKEL
FAVKEL
FAA KQL
FT SKQL
E K L A K K A E K D S K A E QA K V K K A
S RSA K K CDK E EKA EKA K I K KA
E RN S K K C E K E E K L E K A K A K K A
EK NA QRC E KD EK V E KD K L T A A
QK QA N K A S K E E K Q E T N K L K RA
RMA S RV D A V A S K V Q T A V T MK GV T K N MA QV T K A L D K A L S T MD L
RM S A RV D A V A A R V Q T A V T M G K V T K S MA GV V K S MD A T L K T MN L
R M S A R V D A V A S R V Q S A L T T R K V T G S M A G V V K A M D A A M K GM N L
K MA A R I D A V A A RV Q T A A T Q K RV T A S M S GV V K A M E S A MK S MN L
K L A S R V D S V A S R V Q T A V T M R Q V S A S M G Q V C K GM D K A L Q N M N L
L
I
I
I
L
L QK N V E CA RV Y A
Q K GN M E V A R I H A
Q K GN MD V A R I H A
K K GN K E V A QV H A
NE- NED I SR I YA
H.s. CHMP1
H.s. CHMP1.5
D.m. CHMP1
C. e. CHMP1
S. c. Chm1p
WL
FL
YL
Y I
L
H.s. CHMP1
H.s. CHMP1.5
D.m. CHMP1
C. e. CHMP1
S. c. Chm1p
S M S S A T T L T T P Q E QV D S L I MQ I A E E N G L E V L D Q L S Q L P E GA S A V G E S S V R S Q E - D Q L
T M S S T T T L T T P QN QV D M L L Q E MA D E A G L D L N M E L P Q G - Q T G F V GT S - V A S A E Q - D E L
T M S D T V T T S V P Q GD V D N L L QQV A D E A G L E L N M E L P S GV Q S Q S V GA S T A V S Q E Q - D E L
T MD GT T V L N A P K S QV D A L I A E A A D K A G I E L N Q E L P S N V P T A L P T GT QA V S E D K - D - L
M G V N S D A M L V D N D K V D E L M S K V A D E N GM E L K Q S A K L D N V P E I K A K E V N V D D E K E D K L
ENA
ENA
ENA
ENA
SNA
Q K V S S V MD R F E QQV QN L
E K I S A L MD K F E H Q F E T L
EK I S S L MEK F E SQF ED L
E K V QQ L MD R F E RD F E D L
QQ I T M I MD K F E QQ F E D L
SRRL
SQRL
T QRL
T ERL
AQRL
I
I
I
I
I
R K K N E GV N
RQK N QA V N
RQK N QA V N
RK K N EA V N
RK K N E RL Q
DV HT SVMED
D V Q T QQM E D
DV QS SVMEG
DV T T K T MEK
DT SVNV YED
AAL
A RL
A RL
AAL
RA L
RN
RD QV
RQA E
RNM
RG
2386
JOURNAL OF CELL SCIENCE 114 (13)
Fig. 2. Distinct forms of
mammalian CHMP1 protein
are found in cytoplasmic
and nuclear compartments.
(A) The subcellular
distribution of CHMP1 was
determined by western blot
analysis with purified antiCHMP1 antiserum after
18% SDS-PAGE.
(B) Human cells lines
derived from cervical
(HeLa), prostate (LNCaP),
breast (MCF7), colon (HT29), and chorionic (JAR)
carcinomas were compared
with primary human
umbilical vein endothelial
cells (HUV-EC-C) for
CHMP1 expression levels
and subcellular distribution. (C) Expression levels of the nuclear and cytoplasmic forms of CHMP1 are compared in a mouse cell line (C2C12),
mouse embryo fibroblasts and the indicated adult mouse tissues. Tissue extracts are unfractionated and represent total protein. Blots were
stripped and re-probed with antibody to alpha-tubulin (cytoplasm-specific) and matrin 4 (nuclear matrix-specific) to demonstrate the accuracy
of the fractionation. * indicates a nonspecific band. c, cytoplasm; ds, nuclear proteins released by DNaseI-digestion plus 800 mM NaClextraction; m, nuclear matrix solubilized by detergent; w, whole cell with complete detergent solubilization (see Materials and Methods).
A modified form of CHMP1 is nuclear matrixassociated
The two-hybrid interaction between CHMP1 and a PcG protein
suggested a nuclear localization and function for CHMP1. To
test this possibility, we analyzed the endogenous subcellular
distribution of CHMP1 in a variety of established cell lines
using an affinity-purified polyclonal antiserum to CHMP1.
Western blot analysis of a whole-cell extract from human
HEK293 cells (Fig. 2A, ‘w’) shows a doublet consisting of 32
kDa and 35 kDa species. These two forms are likely to
represent differential CHMP1 post-translational modification.
Each can be competed by pre-incubating the CHMP1 antibody
with an excess of CHMP1 fusion protein (data not shown) and
both are increased by overexpression from a human CHMP1
cDNA in HEK293 cells (Fig. 3B). Subcellular fractionation of
HEK293 cells into cytoplasmic (c), nuclear DNaseI/high salt
(ds) and matrix (m) fractions reveals that the smaller and larger
CHMP1 forms are restricted to the cytoplasm and nuclear
matrix, respectively (Fig. 2A). A cytoplasmic marker, αtubulin, is undetectable in noncytoplasmic fractions, whereas
nuclear matrix marker matrin-4 (Nakayasu and Berezney,
1991) is absent from the cytoplasmic fraction, indicating the
fidelity of our fractionation procedure. This partitioning of the
two CHMP1 forms into distinct subcellular compartments also
occurs in each of six other human cell lines tested (Fig. 2B).
Although the matrix form of CHMP1 never localizes to the
cytoplasm, some lines (HeLa, MCF7, HT-29 and JAR) show a
detectable level of the 32 kDa form of CHMP1 in the nuclear
matrix. This suggests that both CHMP1 forms are sometimes
matrix-associated.
Subcellular compartmentalization of the two CHMP1
species also occurs in a murine system. An established mouse
cell line (C2C12 myoblasts) and primary mouse embryo
fibroblasts (MEF) show accurate fractionation of the two
CHMP1 subcellular forms (Fig. 2C). Whole-tissue extracts
from mouse indicate that both CHMP1 forms are widely
expressed, with increased levels of the 35 kDa form in adult
heart, kidney and liver (Fig. 2D). Overall, these analyses
demonstrate that CHMP1 is expressed in diverse mammalian
cell types as distinct cytoplasmic and nuclear forms.
The occurrence of two CHMP1 forms suggested that
CHMP1 might be modified in a cell-cycle-dependent fashion.
To test this possibility, HEK293 cells were synchronized and
fractionated at distinct phases of the cell cycle. Western
analysis of CHMP1 demonstrated that the ratio of cytoplasmic
to nuclear CHMP1 is largely invariant throughout interphase
(D.R.S., T.N. and S.M.H., unpublished). Within mitotic cells
the larger, 35 kDa form of CHMP1 is found in the chromosome
scaffold fraction. In contrast to this western blot analysis, Mphase is the only point during the cell cycle when nuclear
CHMP1 becomes visible by immunocytochemistry. During
telophase, CHMP1 is distributed in a punctate arrangement on
the condensed chromosomes, but cannot be seen in the nucleus
immunocytochemically at earlier stages of mitosis or in
interphase nuclei (D.R.S., T.L.H., T.N. and S.M.H.,
unpublished). Cytoplasmic CHMP1, however, is readily visible
by immunocytochemistry and exhibits a perinuclear
localization (Fig. 3C, 0 hours) (Howard et al., 2001). In
combination, western analysis and immunocytochemistry
demonstrate that nuclear CHMP1 is present within the nuclear
matrix or chromosome scaffold at all stages of the cell cycle,
but has an epitope that is usually masked from antibody
recognition in situ.
Exogenous CHMP1 is modified and nuclear matrixassociated
Overexpression in mammalian cells was used to study the
functional potential of CHMP1. All attempts to generate a
stable line that constitutively overproduces CHMP1 were
unsuccessful, regardless of the species origin of CHMP1
(D.R.S. and S.M.H., unpublished). Therefore, we relied upon
an ecdysone-inducible expression system (No et al., 1996) to
CHMP1 functions in the nucleus
2387
Fig. 3. Exogenous CHMP1 is tightly regulated and
enters the nucleus. All experiments use IND-CHMP1
cells and exogenous CHMP1 induction with 10 µM
muristerone. (A) Total RNA was harvested at the
indicated times of induction and analyzed by northern
blot for CHMP1, with CHO-B as an internal control.
Endogenous CHMP1 RNA does not comigrate with
exogenous and is not detected with this shorter
exposure. (B) Cells were harvested at 0 hours and 24
hours of induction, fractionated and analyzed as in Fig.
2. CHMP1 species that comigrate with endogenous
(closed arrows) or are novel (open arrow) are
indicated. (C) Cells were induced for the indicated
times, fixed and double labeled with anti-CHMP1 (red)
and Hoechst 33258 (blue). Note the cytoplasmic fibers
at 3 hours and 9 hours. (D) Cells were induced for 24
hours, including treatment with colcemid (200 ng/ml)
for the final 10 hours to enrich for mitotic cells.
Metaphase spreads were prepared (Chandler and Yunis, 1978) and stained for CHMP1 and DNA. The inset shows an enlarged region from the
spread. c, cytoplasm; m, nuclear matrix solubilized by detergent; w, whole cell with complete detergent solubilization.
generate a derivative of HEK293 cells, IND-CHMP1, which
conditionally overexpresses CHMP1. Northern blot analysis
demonstrates a tightly regulated, rapidly inducible CHMP1
(Fig. 3A). Exogenous CHMP1 RNA is virtually undetectable
prior to induction, whereas a strong signal appears at 1.5 hours
and is close to steady state by 3 hours (Fig. 3A, compare 0,
1.5, 3 and 6 hours).
Biochemical fractionation of IND-CHMP1 cells
demonstrates that the ratio of cytoplasmic to nuclear CHMP1
is not dramatically altered during induction (Fig. 3B). The
intensity of the cytoplasmic and nuclear matrix forms of
CHMP1 are both comparably increased (compare whole cell,
‘w’, for 0 and 24 hours). Overexpression does alter the ratio of
CHMP1 forms within the nuclear matrix fraction (m). In
addition to upregulation of the 35 kDa CHMP1 matrix form
(top arrow), both the 32 kDa form (bottom arrow) and a novel
intermediate form (open arrow) become prominent in the
nuclear matrix after a 24 hour induction, which suggests
saturation or disruption of normal modification mechanisms.
IND-CHMP1 was analyzed by immunocytochemistry at
various times of induction to define further the subcellular
distribution of exogenous CHMP1. At early times of induction
(Fig. 3C, 3-9 hours), CHMP1 produces a transient cytoplasmic
fiber pattern (3 hours) and mixed cytoplasmic fiber nuclear
punctate pattern (9 hours). At 24 hours post induction, nearly
all cells exhibit a very intense nuclear punctate CHMP1 pattern
and less intense cytoplasmic diffuse signal. The extreme
intensity of the punctate nuclear CHMP1 signal at 24 hours
post induction made simultaneous visualization of both the
nuclear and less intense cytoplasmic diffuse patterns
impossible without greatly over exposing the images (Fig. 3C,
24 hours). In contrast to the transient production of CHMP1
cytoplasmic fibers, the nuclear pattern of CHMP1 persists for
at least three days. This nuclear pattern is not reversible by
muristerone removal, even though exogenous CHMP1 mRNA
becomes undetectable within 6 hours, indicating that nuclear
CHMP1 is extremely stable in these cells (T.N. and S.M.H.,
unpublished). The suggestion that CHMP1 is closely
associated with chromatin in the nucleus is supported by
analysis of mitotic IND-CHMP1 cells. After induction,
metaphase spreads show that CHMP1 is localized to the
axial component of highly condensed chromosomes, the
chromosome scaffold (Fig. 3D) (Earnshaw and Laemmli,
1983). Therefore, immunocytochemical analysis of exogenous
CHMP1 in both interphase and mitotic cells strongly supports
the nuclear localization of endogenous CHMP1, detectable
only by western with our antibody during interphase.
CHMP1 arrests cells in S-phase
Exogenous CHMP1 produces strong effects on cell-cycle
progression. After treating IND-CHMP1 cells with
muristerone for 24 hours, the mitotic index drops tenfold (Fig.
4A). In addition, the fraction of cells replicating their DNA and
thus able to incorporate BrdU, decreases fourfold (Fig. 4A).
These effects are not a result of increased apoptosis, as
measured by TUNEL staining, and therefore must reflect
alterations in the cell cycle. To define the cell-cycle effect, the
percentage of cells in each phase was determined using flow
cytometry of propidium-iodide-stained nuclei. Fig. 4B shows
the distribution of cells in G1, S and G2/M. DNA content is
unaffected by muristerone in the parental activator line and is
very similar to that observed in IND-CHMP1 prior to
induction. By contrast, CHMP1 induction produces a
significant increase in S-phase cells by 24 hours (data not
shown), with about 80% of the cells showing an S-phase DNA
content at 48 hours (Fig. 4B). Not only are many more cells in
S-phase, but a greater fraction are in late S-phase relative to
early S-phase, as indicated by the steep upward slope of the Sphase peak. The poor correspondence between the fraction of
cells with S-phase DNA content (80%) and those actively
replicating DNA (10% BrdU positive) (compare Fig. 4A,B),
suggests that cells have arrested or are strongly inhibited in
their transit through S-phase, with a stronger effect during late
S-phase.
CHMP1 alters chromatin structure
The punctate pattern produced by CHMP1 in the nucleus is
accurately mirrored by both Hoechst 33258 and propidiumiodide fluorescence (Fig. 5A). As these two dyes bind to DNA
through distinct mechanisms (Harshman and Dervan, 1985;
Pjura et al., 1987; Wilson et al., 1985), the similar effect on
both strongly suggests that CHMP1 is locally increasing
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Fig. 4. Exogenous CHMP1 blocks cell-cycle progression in late S-phase. IND-CHMP1 cells were induced with 10 µM muristerone for the
indicated times. (A) Cells were scored for exogenous CHMP1 expression (triangles) and histone H3 phosphorylation (Ser10) with specific
antibodies (see Methods). The phospho-H3 (P-H3) pattern was scored as punctate nuclear (open squares), or uniform nuclear and therefore
mitotic (closed squares). DNA replication was measured by BrdU incorporation (asterisk) and apoptosis by TUNEL labeling (open circles).
(B) The IND-CHMP1 line and its parental activator line (Invitrogen) were induced with muristerone for the indicated times, fixed, stained with
propidium iodide and analyzed by flow sorting. The percentage of cells in each cycle phase is indicated in the corner of each panel.
nuclear DNA concentration through chromatin condensation.
Surprisingly, laser scanning confocal microscopic analysis
reveals that CHMP1 and the DNA dyes are not perfectly
colocalized. Instead, CHMP1 predominantly localizes to the
exterior regions of these new subnuclear structures, with
condensed chromatin inside (Fig. 5A). This lack of internal
antibody staining is not a consequence of general antibody
inaccessibility: a PcG antibody against BMI1 penetrates the
structure readily (Fig. 7).
Because modified chromatin structure is expected to
coincide with changes in histone modification (Kuo and Allis,
1998), the subnuclear distribution of phosphorylated (Ser10; PH3) and acetylated (Lys9,14; Ac-H3) histone H3 levels were
measured with specific antibodies (Hendzel et al., 1997). Prior
to induction, P-H3 occurs at high levels only in mitotic cells
and Ac-H3 shows a speckle pattern uniformly distributed
throughout the nucleus (Fig. 4A; Fig. 5B, 0 hours). CHMP1
induction produces localized increases in P-H3 and Ac-H3
levels (Fig. 5B, 24 hours), with these modified histones
accumulating in a relatively stable or very transient fashion,
respectively, in a subset of nuclei. Phospho-H3-positive regions
have no correspondence to centromeric heterochromatin as
demonstrated by double staining with a CENP-B antiserum
(D.R.S., T.L.H. and S.M.H., unpublished). Therefore, this does
not represent classical mitotic chromosome condensation that
initiates near centromeres (Hendzel et al., 1997; Van Hooser et
al., 1998). Neither CHMP1, P-H3, nor Ac-H3 localizes
predominantly within regions of de novo condensed chromatin.
Instead, each surrounds the Hoechst-dense DNA in a shell-like
structure in what are likely to be coincident patterns (Fig.
5A,B; Fig. 7). Therefore, overexpressed CHMP1 defines the
exterior of nuclear bodies, which show modified histones at the
periphery and condensed chromatin within the interior.
CHMP1 bodies are nuclease resistant
The nuclear CHMP1 structures have been further defined by
measuring the nuclease resistance of the components relative
to bulk chromatin. We have relied on a previous study which
demonstrated that chromatin in fixed nuclei is readily
accessible to nuclease digestion (Nickerson et al., 1997).
CHMP1-expressing cells were freed of membrane, fixed,
digested with increasing concentrations of DNase I for 1 hour
and analyzed by fluorescent microscopy for localized DNA
content relative to CHMP1 or P-H3. As shown in Fig. 6, DNase
I selectively degrades chromatin that is external to CHMP1
structures (compare Hoechst at 50 U vs. 0 U), supporting
the conclusion that increased dye binding corresponds
to condensed, nuclease-resistant chromatin. A higher
concentration of DNase I can efficiently remove Hoechststained, condensed chromatin (Fig. 6, 200 U) (Nickerson et al.,
1997), but has no impact on CHMP1 and P-H3 staining
intensity. This DNA-independent nuclear matrix localization of
CHMP1 is consistent with the cell fractionation analysis (Fig.
2A, ds). By contrast, the nuclease resistance of the P-H3 signal
is completely unexpected and significantly greater than on
mitotic chromosomes (D.R.S. and S.M.H., unpublished).
CHMP1 localizes PcG proteins to condensed
chromatin
CHMP1 was isolated as a Pcl partner and was therefore
CHMP1 functions in the nucleus
2389
Fig. 6. CHMP1 subnuclear domains are nuclease-resistant. INDCHMP1 cells were induced for 24 hours with muristerone, fixed and
digested with the indicated amount of DNaseI for 1 hour at room
temperature as described (Nickerson et al., 1997). Nuclei were
double-labeled for CHMP1 or phosphorylated (Ser10) histone H3
(P-H3), and DNA (Hoechst 33258) and visualized by fluorescence
microscopy.
Fig. 5. Exogenous CHMP1 locally condenses chromatin and alters
histone modification. IND-CHMP1 cells were induced with
muristerone (24 hours mur) or left uninduced (0 hours mur).
(A) Cells were fixed, then triple-labeled with anti-CHMP1, Hoechst
33258 and propidium iodide (PI) or (B) double-labeled for DNA and
antibody to modified forms of histone H3 (Upstate Biologicals).
Note the strong staining only in the mitotic cell at 0 hours. Ac-H3,
acetylated (Lys9,14); P-H3, phosphorylated (Ser10).
hypothesized to participate in PcG function. In some cell types,
such as the human osteosarcoma cell line U2OS, PcG proteins
are colocalized at punctate subnuclear structures (Alkema et
al., 1997). To investigate the relationship between these PcG
structures and CHMP1 nuclear bodies, U2OS cells were
transfected with CHMP1, then subsequently fixed and triplelabeled for CHMP1, the PcG protein BMI1 and DNA (Fig. 7).
Approximately 90% of the transfected cells that overexpress
CHMP1 no longer exhibit a punctate nuclear BMI1 pattern
(D.R.S. and S.M.H., unpublished). Given that BMI1 has been
demonstrated to lose its punctate distribution in late S-phase
(Voncken et al., 1999), this result is consistent with the action
of CHMP1 on cell-cycle progression in our inducible line (Fig.
4B). The remaining CHMP1-transfected cells show the
localized regions of chromatin condensation observed in our
inducible line (Fig. 3; Fig. 5; Fig. 6). In addition, they exhibit
a punctate BMI1 distribution that colocalizes with condensed
chromatin and is encapsulated by CHMP1 (Fig. 7). Transfected
CHMP1 also recruits human Polycomb2 to condensed
chromatin (D.R.S. and S.M.H., unpublished), suggesting that
many components of this PcG multiprotein complex (Alkema
et al., 1997) respond similarly to CHMP1 overexpression.
CHMP1 misexpressed in Xenopus embryos
produces a PcG phenotype
A Xenopus embryo assay was used to compare the functional
capabilities of CHMP1 and other PcG proteins. We showed
previously that dorsal injection of RNA encoding distinct PcG
proteins into four-cell Xenopus embryos produces similar
effects: repression of En-2 and Rx2A, and disruption of normal
anterior neural development (Yoshitake et al., 1999). This
assay was used to compare the ability of CHMP1 and mPcl1
RNA to produce this characteristic PcG phenotype. As shown
in Fig. 8A, unilaterally injected CHMP1 RNA can repress En2 or shift its site of expression posteriorly, frequently producing
more pronounced changes than mPcl1. The ability of mPcl1
and CHMP1 to cooperate in this assay was quantified using
suboptimal concentrations of each RNA. Fig. 8B demonstrates
a greater than additive effect on En-2 repression with coinjected CHMP1 and mPcl1, and an additive effect on shifting
the En-2 site of expression. Also consistent with PcG function,
CHMP1 strongly represses Rx2A and alters anterior neural
development in a manner indistinguishable from other PcG
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RNAs (T.L.H. and S.M.H., unpublished). Therefore, CHMP1
cooperates with mPcl1 and produces the entire spectrum of
phenotypes observed with four other PcG RNAs (Yoshitake et
al., 1999).
Fig. 7. CHMP1 subnuclear domains contain the PcG protein BMI1.
Human U2OS cells were transfected with pIND-CHMP1 and the
fusion trans-activator expression vector pVgRXR (Invitrogen) and
induced with muristerone. After 48 hours cells were fixed and triplestained for CHMP1 (green), BMI1 (red) and DNA (blue). Images
were collected by laser scanning confocal microscopy. Note that the
BMI1 signal is completely internal to CHMP1 (merge) and overlaps
with condensed chromatin in the transfected cell.
Fig. 8. CHMP1 produces a PcG phenotype upon misexpression in
Xenopus. (A) Four-cell-stage embryos were unilaterally injected
(arrowhead) with the indicated synthetic, capped RNA plus a
marker RNA encoding β-galactosidase and processed to measure
the side of injection (in situ β-galactosidase assay) and En-2
expression pattern (Yoshitake et al., 1999). (B) Embryos were
injected with 0.1 ng of each indicated RNA. At stage 20 embryos
were fixed and analyzed for En-2 expression by RNA in situ
hybridization. The percentage of embryos with the indicated effects
is the average of three independent experiments. Standard error is
indicated. Control embryos show less than 3% repression and 0.5%
shift.
DISCUSSION
On the basis of the assumption that the structural conservation
of Pcl from different phyla results from a conserved protein
interaction, we have used the yeast two-hybrid system to
identify CHMP1. This novel protein is found in a much broader
spectrum of eukaryotes than Pcl, and is thus likely to carry out
fundamental activities that are independent of Pcl. Our
analyses in this and the accompanying study (Howard et al.,
2001) are consistent with this notion and indicate that CHMP1
mediates distinct processes in both the cytoplasm and nucleus.
CHMP1 subcellular localization
Cell and tissue fractionation studies demonstrate that CHMP1
is not only ubiquitously expressed, but is always found in both
cytoplasmic and nuclear matrix fractions. The accompanying
study (Howard et al., 2001) demonstrates a cytoplasmic role
for CHMP1 in membrane trafficking, indicating that
cytoplasmic CHMP1 is not simply dormant protein awaiting
its nuclear calling. Consistent with distinct functions for
CHMP1 within the cell, we have observed a size difference
between the cytoplasmic and nuclear forms, which is
apparently the result of covalent modification. It is presently
unclear how CHMP1 is partitioned into cytoplasmic and
nuclear compartments. Although the putative modification may
regulate nuclear entry, our observation of significant levels of
the lower molecular weight, less-modified form of CHMP1 in
the nucleus in some cell types argues against this simple
distribution mechanism.
Within the nucleus, both CHMP1 species are tightly
associated with the nuclear matrix. During mitosis, when the
nuclear matrix disassembles, CHMP1 remains associated with
the M-phase chromosome scaffold. Little is known about the
extent to which components of the nuclear matrix are recruited
for mitotic scaffold formation, although the biochemical
similarity of these structures suggests some shared proteins.
Further work will be required to determine if CHMP1 is
complexed with a common set of proteins in both the
interphase nucleus and mitotic scaffold. Given the proposed
role of CHMP1 in stabilizing gene activity during cell division,
it is tempting to speculate a function for CHMP1 in structuring
the condensed chromosome to facilitate gene memory through
M-phase. A similar role has been suggested for Drosophila
CCF, a protein that cooperates with other PcG proteins and is
essential for correct mitotic chromosome condensation
(Kodjabachian et al., 1998).
Chromatin modification through CHMP1 action
The suggestion that CHMP1 might play a role in mitotic
chromosome condensation is supported by its ability to
produce regions of visibly condensed chromatin in the
interphase nucleus. CHMP1 accumulates in specific
subnuclear regions and promotes increased DNA dye
fluorescence that is nuclease resistant. Surprisingly, CHMP1
does not perfectly colocalize with this structurally altered
CHMP1 functions in the nucleus
chromatin, but forms a shell around it. As this shell frequently
shows histone H3 phosphorylation and acetylation, it is likely
to represent a region of gene transcriptional activity (Cheung
et al., 2000; Clayton et al., 2000). In addition, it might be a
region of chromatin restructuring (Sauve et al., 1999). Perhaps
this region acts as a transition zone for genes that are either
being incorporated into the condensed chromatin structure or
are being transcriptionally activated. This shell would thus act
as a boundary that includes the demarcation between active and
inactive chromatin domains. The existence of CHMP1 within
this shell suggests a possible role for CHMP1 in sequestering
or sorting DNA elements. A functional interaction between
PcG proteins and boundary elements has been observed in
Drosophila and mammalian cells (Gerasimova and Corces,
1998).
One intriguing aspect of the CHMP1 structure is its strong
resistance to nuclease digestion. The interior of the structure is
more resistant to nuclease digestion, whereas the CHMP1positive shell is extremely resistant. This property is shared
with the phosphorylated histone H3 signal that surrounds
condensed chromatin. This observation suggests that histones
can become bound to the nuclear matrix at sites of chromatin
restructuring, perhaps as substrates for matrix-bound
remodeling complexes. Our analysis of histone H4 does not
show acetylation on the CHMP1 shell; instead, histone H4
within the CHMP1 structure exhibits weak hypoacetylation
(T.L.H. and S.M.H., unpublished). An understanding of this
apparent difference between histone H3 and H4 acetylation
awaits a more complete description of the changes in
nucleosome structure in specific contexts.
These profound changes in chromatin structure are
accompanied by strong effects on cell-cycle progression.
CHMP1 induction results in a large increase in the percentage
of cells with a late S-phase DNA content. This effect may stem
directly from the highly condensed and inaccessible chromatin
that CHMP1 overexpression produces. Many have noted a
strong correlation between replication timing and gene activity
(Sadoni et al., 1999), thus very late replication of chromatin
within CHMP1 bodies would be consistent with gene
silencing. Alternatively, CHMP1 overexpression may affect the
expression of genes required for DNA replication, or trigger an
S-phase checkpoint.
CHMP1 is a candidate PcG protein
Our observations suggest CHMP1 may function in PcG
silencing. Overexpressed CHMP1 encapsulates proteins in the
Polycomb complex and localizes them to visibly condensed
chromatin. In addition, CHMP1 behaves like other PcG
proteins in a Xenopus embryo injection assay. Finally, CHMP1
shows a two-hybrid interaction with Pcl proteins from
Drosophila and mouse. We have been unable to coimmunoprecipitate CHMP1 and mouse Pcl when
overexpressed, but this may be a consequence of inefficient
complex formation at elevated protein levels. In addition, this
interaction is likely to be transient, based on the close spatial
relationship between BMI1 and CHMP1 (Fig. 7), but absence
of colocalization. Because CHMP1 is more evolutionarily
conserved than most PcG proteins, it may function as a
component of an ancient, more fundamental gene regulatory
mechanism. This would be analogous to the use of the
NURD/Mi-2 complex in Drosophila PcG function (Kehle et
2391
al., 1998). A physical interaction between CHMP1 and the
PcG may promote a structural modification mediated by
multiprotein complex associated with CHMP1.
Our analysis suggests that CHMP1 is creating condensed,
nuclease-inaccessible chromatin domains. Others have not
seen evidence of nuclease resistance for PcG target genes,
although the assays used were quite distinct from ours
(Schlossherr et al., 1994). For example, our results with
CHMP1 overexpression may amplify smaller differences in
accessibility by increasing the volume of structurally modified
sequences. The presence of PcG proteins on condensed
chromatin is supported by immunocytochemical studies with
BMI1 (Voncken et al., 1999). Future analyses of PcG silencing
will promote our understanding of the relationship between
gene activity, chromatin structure and subnuclear localization.
Relationships between multivesicular body sorting
and gene silencing
A combination of our results (Howard et al., 2001) and the
work of others (Babst et al., 2000; Watanabe et al., 1998; Xie
et al., 1998) indicates that some proteins may play a dual role
in sorting membrane and protein to an endosomal derivative,
the multivesicular body, and in regulating nuclear gene
expression. Defining the subset of sorting proteins involved in
this dual activity, their mechanism of subcellular partitioning
and any mechanistic similarities between multivesicular body
formation and transcriptional regulation produced as a result
of dual protein activity are areas of future interest.
We thank the following generous contributors for reagents: R.
Berezney, R. Saint, E. M. Tan, and M. van Lohuizen. We greatly
appreciate the help of Tony Bakke with the flow cytometric analysis,
and thank Jan Christian for help with the Xenopus injections and their
interpretation. We also thank Matt Thayer for critical comments on
the manuscript and for his continued interest and encouragement. This
work was supported by a grant from the National Institutes of Health
(S. M. H. GM 52458-05).
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