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INTRODUCTION |
In eukaryotic cells, DNA in association with histones and other
nuclear proteins forms a DNA-protein complex or chromatin that exhibits
varying levels of compaction (1, 2). Chromatin is folded
hierarchically, with the basic level represented by a repeated
structural unit, the nucleosome, comprising 200 ± 40-bp1 DNA of which 146 bp
make about 1.7 superhelical turns around the histone octamer (3), and
the remainder (linker DNA) is not constrained by core histones. Linker
DNA is usually associated with the ninth (linker) histone, which brings
the core-proximal segments of linker DNA together (4-6). In
vitro, nucleosome arrays fold into ~30-nm-wide chromatin fibers
having a characteristic zigzag organization at low ionic strength with
separate nucleosomes connected by extended linkers (7-10). If the
extent of DNA charge shielding or neutralization is increased
(e.g. by increasing the salt concentration in the medium),
the zigzag chromatin fibers first become more compact and then
self-associate to form the next level of higher order folding, which
involves closer contacts between nucleosomes (11-15). Modulation of
chromatin folding constitutes a potentially important regulatory
mechanism that may influence the locus-specific accessibility of DNA
templates to the trans-acting factors mediating transcription (16-19),
replication (20), transposition (21), and perhaps other
template-dependent functions.
It has been known for many years that, in interphase nuclei, the
chromatin is not uniformly decondensed but contains a considerable amount of more compact material, called heterochromatin (22), which
generally increases during the terminal stages of eukaryotic cell
differentiation (23). Much cytological and genetic evidence suggests
that chromatin compaction (spreading of heterochromatin) is associated
with position-specific genetic inactivation (24-28). A surprisingly
high diversity of structurally distinct heterochromatin-associated proteins has been reported. For example, SIR3, SIR4, and RAP1 proteins
are involved in telomeric silencing in Saccharomyces cerevisiae (29, 30), Pdd1p is involved in chromatin condensation and elimination in Tetrahymena thermophila (31), and the
numerous Su(var) and Polycomb Group proteins of Drosophila
melanogaster (26, 32, 33) are implicated in chromosomal
locus-specific genetic repression mediated by chromatin
structure, as are the related "chromo" domain-containing vertebrate
proteins (34, 35).
Although several studies have indicated that stable alterations of
chromatin higher order folding distinguish the organization of
euchromatin and heterochromatin (13, 36, 37), the structural determinants and the molecular mechanism of heterochromatin formation and spreading are still obscure. Terminal cell differentiation provides
a convenient system for biochemical studies of the coordinated formation of heterochromatin and associated genetic inactivation. In particular, blood cell differentiation or hemopoiesis has been extensively studied both for its applicability to gene regulation and
for its intrinsic significance for oncology (38, 39). In the normal
developmental sequence, the number of expressed genes becomes
drastically reduced (40, 41), the nuclear chromatin undergoes a
considerable increase in condensation (see reviews in Ref. 42), and
nuclear matrix components, which are associated with transcriptional
activity, are progressively lost (43).
Based on the hypothesis that the extensive heterochromatin spreading in
blood cells is caused by one or more of the relatively few proteins
that are still expressed during terminal differentiation, we have
sought highly abundant nuclear proteins that specifically interact with
repressed chromatin and are confined to the late stages of cell
differentiation. Our study has yielded an abundant developmentally
regulated 42-kDa nuclear protein, MENT (myeloid and
erythroid nuclear termination
stage-specific protein). This protein is expressed in terminally
differentiated blood cells, is associated with repressed chromatin, and
is able to induce large scale condensation of nuclear chromatin and the
dissociation of inactivated chromatin from the nuclear matrix in
vitro (44, 45). MENT is present in all three main avian blood cell
types (erythrocytes, lymphocytes, and granulocytes) and is
especially abundant in granulocytes, where it becomes the predominant
nuclear nonhistone protein (~2 molecules/nucleosome) and is
concentrated in the compact peripheral heterochromatin (46). MENT-like
polypeptides have also been found in chromatin of mammalian
leukocytes.2
We have isolated the compact granulocyte chromatin in a soluble form
and applied cryoelectron microscopy, a powerful imaging technique
permitting the visualizing of the native organization of unfixed
biological material (47) to the study of heterochromatin conformation.
For the first time, we have documented a direct link between
heterochromatin and higher order folding of nucleosome arrays that is
attributed to the accumulation of a single nonhistone protein, MENT, in
chromatin fibers. We have cloned the MENT cDNA, deduced the protein
primary structure, and identified the protein structural motifs that
may account for the molecular interactions of MENT. The combined
results suggest a molecular basis for the formation of MENT-directed heterochromatin.
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MATERIALS AND METHODS |
Cells, Nuclei, and Chromatin--
Polymorphonuclear granulocytes
were isolated from peripheral blood of adult white leghorn chicken as
described (46). COS-7 cells (CV-1 simian cells transformed with
origin-defective SV-40 virus expressing wild-type T antigen, ATCC
number CRL-1651) were grown to 80% confluency in Dulbecco's modified
Eagle's medium (D-5796; Sigma) containing 10% fetal calf serum
(F-2442; Sigma) and 1 mM pyruvate. Cell cultures were
washed 2 times in PBS containing 0.14 M NaCl, 2.7 mM KCl, 10 mM Na2HPO4,
1.7 mM KH2PO4, pH 7.5, and the
cells were resuspended in PBS using a cell scraper. To isolate the
nuclei, cell suspensions of granulocytes or COS-7 cells in PBS were
centrifuged for 3 min at 1000 × g and resuspended in
reticulocyte standard buffer containing 10 mM NaCl, 3 mM MgCl2, 10 mM Tris-HCl, pH 7.5, plus 0.5% Nonidet P-40 (Nonidet P-40; Life Technologies, Inc.) and 1 mM phenylmethylsulfonyl fluoride. With granulocyte nuclear
preparations, the NaCl concentration varied between 0 and 0.3 M. The cell suspensions were homogenized by 20-30 strokes
of pestle A in a Dounce homogenizer over 30 min on ice. Nuclei were
centrifuged for 10 min at 7600 × g, and the nuclear
pellets were resuspended in reticulocyte standard buffer plus 1 mM phenylmethylsulfonyl fluoride. Isolated nuclei could be
stored for a week at +2 °C without a detectable DNA or protein degradation.
Micrococcal nuclease (MNase) digestion of all types of nuclei was
conducted as described (46) and terminated by adding EDTA to 10 mM. To obtain soluble chromatin, the nuclei were digested with MNase to obtain an average DNA fragment size between 400 and 6000 bp and centrifuged for 5 min at 10,000 × g. The
supernatant S1 was removed, and the pellet was resuspended in 1 ml of
TEN (Tris-EDTA-NaCl buffer containing 10 mM Tris-HCl, pH
7.5, 1 mM EDTA, 10 mM NaCl). Supernatant S2
containing more than 50% of the input nuclear chromatin was then
obtained after centrifugation for 5 min at 10,000 × g.
The nuclear pellets were washed once more with 1 ml of TEN buffer with
centrifugation to provide the S3 supernatant, and the nuclear remnant
fraction was obtained by dissolving the final pellet in 1 ml of 0.5% SDS.
Electrophoretic Techniques--
DNA electrophoresis in agarose,
polyacrylamide gel electrophoresis of proteins, detection of proteins
and nucleic acids, Western blotting, probing with anti-MENT antibodies,
and quantitative densitometry of electrophoregrams were conducted as
before (46). Protein to DNA ratios were estimated from parallel
measurements of DNA concentration by UV spectrophotometry
(A260 = 1 for 50 µg/ml DNA).
Ultracentrifugation--
For size fractionation of soluble
chromatin, 0.5 ml of S-150 sample containing 0.2 mg/ml DNA was loaded
on a 5-25% sucrose gradient (12 ml) containing TAEW (25 mM sodium acetate, 2 mM Na2-EDTA, 20 mM Tris acetate, pH 7.4) or HEN (HEPES-EDTA-NaCl buffer
containing 40 mM NaCl, 1 mM EDTA, 10 mM HEPES, pH 7.5). Ultracentrifugation was carried out in
an SW-41 rotor on a Beckman L8-50 ultracentrifuge for 3 h at
4 °C and 35,000 rpm. 1-ml fractions were collected and used for
protein and DNA electrophoreses. The chromatin pellet (fraction 1) was
resuspended from the bottom of the tube in 1 ml of 0.5% SDS.
Cryoelectron Microscopy--
Cryoelectron microscopy using
soluble chromatin samples (50-100 µg/ml in TEN buffer) was conducted
as described (5, 48). Specifically, 3-µl chromatin samples were
applied to holey carbon films, blotted with Whatman 52 filter paper,
and plunged into liquid ethane held just above its freezing point in
liquid nitrogen. Grids were transferred under liquid nitrogen to a
cryoholder (model 626; Gatan Inc., Pleasanton, CA), and observed at
170 °C in an electron microscope (CM10; Philips Electronic
Instruments Co., Mahwah, NJ) at a nominal magnification of × 45,000. Tilt pairs of micrographs (angular separation 30°) at
1.1-1.5-µm defocus, were recorded in low dose mode, on film S0-163
(Eastman Kodak Co., Rochester, NY) and developed in full-strength D-19
(Kodak) for 12 min.
Cloning and Sequencing of MENT cDNA--
MENT protein was
isolated from the nuclei of unfractionated chicken blood cells as
described (44). Five peptides derived from isolated MENT were
sequenced. For PCR-mediated cloning of MENT cDNA, we designed
redundant oligonucleotide primers as described (49). The
20-22-nucleotide-long primers were deduced from MENT regions with
minimally degenerate codons. Total RNA and poly(A)+
mRNA was isolated from 1 g of chicken bone marrow using Trizol reagent (Life Technologies, Inc.) and the Message Maker RNA isolation kit (Life Technologies), and the first strand cDNA synthesis was conducted using the Life Technologies Superscript preamplification system essentially as described in the vendor's manual. PCR
amplifications with degenerate primers were carried out using the
AmpliTherm polymerase and MasterAmp PCR optimization kit (Epicentre
Technologies, Madison, WI). Amplification cycles were carried out in
30-µl samples using a high melting wax for "hot start"
conditions. The first denaturation for 5 min at 94 °C was followed
with 35 cycles, each containing three 1-min steps at 94, 42, and
72 °C, and finally for 10 min at 72 °C. PCR products were cloned
into pCRII vector (Invitrogen) and sequenced using the Sequenase II
system (Amersham Pharmacia Biotech). One of the sequenced PCR products
amplified between the primers specific for peptides 1 (ATIGGIAA(C/T)TT(C/T)ACIGTIGA) and 3 (TGIAT(A/G)TT(C/T)TCIGC(C/T)TG(C/T)TC) also included the sequence
of peptide 2, indicating that this PCR product was derived from the
target MENT cDNA.
To restore the 5'-end of the cDNA, we employed the 5'-rapid
amplification of cDNA ends PCR technique (50) essentially as described (51). The 3'-end of the cDNA was restored using the 3'-rapid amplification of cDNA ends PCR method (50). The
PCR-amplified DNA fragments were inserted into pCRII vector
(Invitrogen) and sequenced on both strands, revealing a 1230-bp open
reading frame (ORF) encoding all five independently sequenced MENT
peptides. To verify the ORF sequence, PCR amplification was repeated
with two nonredundant primers flanking the MENT ORF, and the product was inserted in the pCRII vector and sequenced on both strands with the
Sequenase II system.
cDNA Expression and Cell Immunofluorescence--
MENT ORF
was amplified by reverse transcription-PCR with oligonucleotide primers
containing appropriate restrictase sites and inserted in pRc/CMV vector
(Invitrogen) to provide a MENT-expressing plasmid, pSG109. COS-7 cells
were transfected with pSG109 and SuperFect transfection media (Qiagen)
as described in the vendor's manual. Transfected cells were grown for
48 h. Fixing of cells attached to the cover glasses with
methanol/acetone and staining with anti-MENT antibodies and DNA dyes
(10 µg/ml Hoechst 33258) was performed essentially as described (45).
For double-staining with anti-MENT and anti-fibrillarin (52)
antibodies, cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature. Fluorescence and confocal microscopy were
conducted as described (45).
Protein Sequence Analysis and Three-dimensional Modeling--
A
search for DNA and protein homologies in the available data banks was
performed using the BLAST program (53). Sequence alignments were
conducted using the Wisconsin package version 9.1 (Genetics Computer
Group, Madison, WI). The protein pI was calculated as described (54).
To build the three-dimensional protein model, we employed the
ProModII software (55, 56). The model-building resources are
provided by the automated protein modeling server, Swiss Model (Glaxo
Wellcome Experimental Research, Geneva, Switzerland), which is
accessible through the Internet. The protein models were visualized
using the Swiss-PDB viewer program compatible with Windows
95TM. Modeling of electrostatic and surface properties of
the molecules was performed using the GRASP program (Graphical
Representation and Analysis of Surface Properties (57)) running on a
Silicon Graphics Indigo workstation with operating system IRIX 5.3.
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RESULTS |
Solubilization of Tightly Packed Granulocyte Chromatin--
Highly
condensed chromatin from chicken granulocyte nuclei has a nucleosome
repeat (192 ± 2 bp) and a core and linker histone content (46)
similar to that of many cells with much more active genomes (1). Unlike
most other types of chromatin, which are readily solubilized after
MNase digestion and removal of divalent cations, granulocyte nuclei
digested to DNA fragment sizes between 200 and 20,000 bp did not
release any soluble chromatin after repeated washing in low salt/high
pH media. Our previous work suggested that MENT, an extremely abundant
granulocyte chromatin protein (2.1 molecules/nucleosome) was the most
likely factor inhibiting chromatin solubility (46). To address the
impact of auxiliary proteins on chromatin conformation, it was
essential to isolate granulocyte chromatin from the nuclei while
maintaining its association with MENT. We therefore determined the
optimal salt concentration allowing chromatin solubilization with a
minimal loss of associated chromatin proteins by preparing a series of granulocyte chromatin fractions eluted from nuclease-digested nuclei
with varying concentrations of NaCl. An abrupt increase in chromatin
solubility occurred between 100 and 150 mM NaCl (Fig. 1, a and b). The
amount of MENT retained in chromatin also changed very considerably
between these two NaCl concentrations, as shown by Western blotting of
nuclear proteins probed with anti-MENT antibodies (Fig.
1c).

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Fig. 1.
Salt-dependent solubilization of
nuclease-digested granulocyte chromatin. a, percentage
of total nuclear DNA (A260) recovered as soluble
chromatin after MNase digestion of chicken granulocyte nuclei washed in
reticulocyte standard buffer media containing 0.5% Nonidet P-40 plus
the indicated NaCl concentrations. After MNase digestion, nuclei were
washed with TEN buffer and centrifuged to obtain soluble chromatin
(s), which corresponds to the combined fractions S1, S2, and
S3 and nuclear pellets (p) corresponding to the nuclear
remnant fractions (see "Materials and Methods"). b,
electrophoresis of DNA from the fractions of soluble and insoluble
chromatin shown in a. The left lane
contains molecular weight standards (Life Technologies). c,
anti-MENT binding to a Western blot of proteins from granulocyte nuclei
washed with reticulocyte standard buffer containing the NaCl
concentrations as indicated above.
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Granulocyte nuclei washed with 150 and 300 mM NaCl (samples
CG-150 and CG-300) retained, respectively, 25 and 1.4% of the original
MENT and were used for isolating soluble chicken granulocyte chromatin
after limited MNase digestion. Further characterization was carried out
on the soluble chromatin fraction S2 (see "Materials and Methods"),
which contained about 50-90% of total nuclear DNA. The MENT/DNA ratio
was the same in the TEN-washed nuclei as in the soluble chromatin
fractions. Depending on the extent of micrococcal nuclease digestion,
the average size of nucleosome chains (Nav) in
S2 preparations varied between 3 and 50 without affecting the protein
composition of the solubilized material (data not shown).
MENT Is Integrated into Soluble Polynucleosome Arrays--
Since
the isolation of granulocyte chromatin required a considerable
depletion of MENT, it was important to determine if the residual
protein was bound firmly enough to be considered as an integrated
architectural element of soluble polynucleosomes. We subjected soluble
granulocyte polynucleosomes (CG-150, Nav = 20) to ultracentrifugation in 5-25% sucrose gradients containing 50 mM monovalent ions and analyzed the gradient fractions by
DNA electrophoresis and Western blotting (Fig.
2, a and b). All
input MENT cosediments with chromatin (Fig. 2b), none being
found at the top of the gradient. Thus, the behavior of MENT is in
sharp contrast to the ubiquitous architectural nonhistone chromatin protein, HMG-1, most of which does not co-sediment with chromatin under
these conditions (58).

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Fig. 2.
MENT is firmly associated with soluble
granulocyte chromatin. a, agarose electrophoresis of
DNA from sucrose gradient fractions 1-13 obtained after
ultracentrifugation of long granulocyte chromatin
(Nav = 20) in HEN buffer (ethidium bromide
stain). b, Western blotting of the material isolated from
combined sucrose gradient fractions: 1 (lane 1), 2-4
(lane 2), 5-7 (lane 3), 8-10
(lane 4), and 11-13 (lane 5).
Detection with anti-MENT antibodies. c, SDS-polyacrylamide
gel electrophoresis of proteins from total granulocyte nuclei
(lane 1); soluble granulocyte chromatin, CG-150
(lane 2); and the oligonucleosome pellet obtained
after ultracentrifugation of CG-150, Nav = 3 (lane 3) (Coomassie R-250 stain).
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We observed a significant enrichment in MENT (0.84 molecules of
MENT/nucleosome) in the fastest sedimenting fraction containing long
(>20-mer) polynucleosomes. Small oligonucleosomes contained less MENT,
with practically none recovered from the mononucleosome fraction. As
discussed below, the distribution of MENT is consistent with the
greater compaction of long polynucleosomes (Fig.
3, a-f).

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Fig. 3.
Cryoelectron microscopy of unfixed, unstained
polynucleosomes. a-i, preparation of MENT-containing
granulocyte chromatin (CG-150, Nav = 20)
vitrified in TEN buffer. a-d, typical images of long
granulocyte polynucleosomes (>20-mers) comprising highly compact
chromatin fibers. The arrows (b and c)
point to the axial structures discussed in the text. e-i,
smaller and less compact oligonucleosomes (<20-mers) from the
same sample. j-l, cryoelectron microscopy
of soluble chromatin from COS-7 cells also vitrified in TEN buffer.
Note the much more open three-dimensional zigzag architecture.
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The low level of MENT in short oligonucleosomes appears to reflect
their origin in less condensed "euchromatin" rather than their
inability to bind the protein; when we ran a sucrose gradient of a
granulocyte chromatin sample from the same nuclei (CG-150) but obtained
after extensive cleavage by MNase (Nav = 3),
MENT was abundant in both the oligonucleosome and mononucleosome
fractions, and again no free protein was detected (data not shown).
Therefore, the most likely explanation for the preferential association
of MENT with long polynucleosomes is that this fraction originated from
heterochromatic nuclear domains relatively protected from nuclease
digestion (MENT inhibits MNase digestion and is unevenly distributed in
granulocyte nuclei, being preferentially associated with compact
heterochromatic areas (46)). As the heterochromatin becomes more
extensively digested, MENT then appears in the oligonucleosome and
mononucleosome fractions.
To determine whether any other proteins besides MENT are associated
with compact granulocyte chromatin, we analyzed the protein composition
of nuclear and soluble chromatin released after the 150 mM
NaCl treatment (Fig. 2c, lanes 1 and
2) and after sedimentation of oligonucleosomes
(Nav = 3) through a sucrose gradient
(lane 3). Densitometry of the Coomassie-stained
gel shows that soluble CG-150 chromatin (lane 2)
retains 26% of the MENT present in untreated granulocyte nuclei
(lane 1), which, from the previous estimation of
2.1 MENT molecules/nucleosome in granulocytes (46), indicates that
about 0.5 molecules of MENT/nucleosome are associated with total
soluble chromatin. After centrifugation, MENT remains the only
prominent band among the nonhistone proteins (Fig. 2c,
lane 3). All other abundant nonhistone proteins
such as actin and Mim-1 present in the crude nuclear preparation (46)
were lost during sedimentation, indicating that they have a low
affinity for chromatin. The core and linker histone content is typical
of somatic nuclei, with three major subfractions of histone H1 giving a
total of ~1 linker histone/nucleosome in all chromatin fractions
(Fig. 2c), demonstrating that MENT is added to, but does not
replace, other histones in granulocyte nuclei. In contrast,
heterochromatin in mature avian erythrocytes contains ~0.5 H1
molecules/nucleosome, the H1 being partially replaced by H5 during
development (1).
We thus conclude that MENT is the single protein in granulocyte
chromatin whose abundance and distribution in chromatin is consistent
with it being a principal factor in the extensive heterochromatization that occurs in these cells. In native granulocyte nuclei, this protein
is present at a concentration that renders the chromatin completely
insoluble; by removing 75% of the nuclear MENT, we were able to
isolate a chromatin fraction suitable for biochemical and structural analysis.
MENT-associated Granulocyte Polynucleosomes Retain a Compact Higher
Order Folding in Low Ionic Strength Media--
Cryoelectron microscopy
has recently emerged as a powerful technique allowing visualization of
the solution conformation of biological material suspended in the
buffer of choice. The three-dimensional configuration can be recovered,
and the common artifacts associated with fixation, staining, and
flattening inherent in standard transmission electron microscopy
techniques are avoided (47, 48). This technique has been used to obtain
a detailed characterization of the three-dimensional zigzag
conformation of polynucleosomes in low salt media (5).
For cryoelectron microscopy, we used the long chromatin
(Nav = 20) from granulocyte nuclei, which
retained about 0.5 molecules of MENT/nucleosome (Fig. 1c).
When vitrified in 20 mM monovalent ions, these granulocyte
polynucleosomes (Fig. 3, a-d) display a considerably more
compact structure than do other types of chromatin under similar
conditions (Fig. 3, j-l; see also Refs. 5 and 59),
appearing as compact fibers ranging in diameter from 30 to 50 nm.
Nucleosome disks are seen predominantly at the periphery of the compact
fibers and do not form close contacts with each other. When visible,
the entry/exit sites of the linker DNA segments are always oriented
toward the fiber interior. The arrangement of nucleosomes in the
compact chromatin fibers is nonuniform, with no evidence of symmetry or
a helical architecture, but is consistent with higher order folding
based on a three-dimensional zigzag that allows a high degree of
nonuniformity of the structure (9).
Two novel features were seen in the long compact chromatin. First, some
fibers showed a backbone-like axial structure that runs perpendicular
to the DNA linker segments and appears to be located toward the center
of the fiber (Fig. 3, b and c,
arrows). The axial structures, which were also seen by
conventional transmission electron microscopy of fixed granulocyte
oligonucleosomes (not shown) but never observed in MENT-depleted,
decondensed chromatin, may result from a juxtaposition of linker DNA
segments induced by MENT. Since DNA gives much more contrast than
protein in cryoelectron microscopy (5), it is unlikely that the axial
structures represent MENT itself. Another feature of MENT-containing
condensed chromatin is its tendency to form, in addition to "30-nm"
fibers (Fig. 3, b and d), thicker and more
electron-dense structures (Fig. 3, a and c). In
some cases, these appear to originate from the folding of a chromatin
fiber back on itself (Fig. 3c).
The small granulocyte oligonucleosomes (<10-mers) observed in the same
preparations are considerably more unfolded (Fig. 3, e-i)
and are closer in appearance to the typical low salt conformation observed with other chromatin types (Fig. 3, j-l; see also
Ref. 5). The more relaxed conformation of the small oligonucleosomes is
consistent with the differential distribution of MENT, which has a
strong preference for longer chromatin fragments and is practically
absent from small particles (Fig. 2, a and
b).
Although preferential binding of MENT to longer polynucleosomes might
involve protein redistribution similar to that reported for linker
histones (11, 60), other data suggest that the observed images are not
due to this potential artifact. For example, when we reassociated MENT
with soluble polynucleosomes, we observed compact but irregular
structures without any signs of side-by-side fiber organization
(46).
As another control, we studied the solution structure of
"euchromatin" derived from actively proliferating COS-7 cells. In the same 20 mM ionic strength buffer, this chromatin
(Nav = 20) had a typical open zigzag
organization (Fig. 3, j-l) similar to that observed with
chicken erythrocyte chromatin at low salt (5). Even in a 40 mM ionic strength buffer, the compaction of COS-7 nucleosome fibers was lower than with MENT-containing granulocyte chromatin at 20 mM (not shown). Thus, under similar ionic
conditions, the two types of chromatin display striking differences in
the extent of compaction.
MENT cDNA, Primary Structure, and Similarities to Other
Proteins--
We used information derived from the partial amino acid
sequence of five peptides isolated from purified MENT to design
redundant oligonucleotide primers and to amplify a MENT cDNA clone
from chicken bone marrow poly(A)+ mRNA (see
"Materials and Methods"). The cDNA clone obtained contained a
2074-bp nucleotide sequence including a 1230-bp ORF, which encoded a
410-amino acid polypeptide. These sequence data have been submitted to
the GenBankTM data base under accession number AF053401.
The protein sequence deduced from the ORF contained the sequences of
all five independently isolated peptides (the underlined
portions of the MENT sequence in Fig.
4a). The first ATG codon in
the ORF was also found at the start of one of the peptides, showing the
first ATG to be a probable translation initiation codon. The first
nonsense codon of the 1230-bp ORF was also the limit of serpin protein
homology (see below), apparently marking the C terminus of the protein. The predicted molecular mass of the 410-amino acid protein (47,383 Da)
is slightly higher that the molecular mass deduced from
SDS-polyacrylamide gel electrophoresis of MENT (42 kDa), further
confirming that the full sequence of MENT has been revealed.

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Fig. 4.
Amino acid sequence of MENT and comparison
with other proteins. a, alignment of the amino acid
sequence of MENT deduced from its cDNA with those of the related
serpins: human bomapin (accession no. P48595), horse leukocyte elastase
inhibitor (hlei; accession no. P05619), and chicken
ovalbumin (oval; accession no. P01012). Unshaded
areas show the MENT sequence and identical amino acid
residues in other proteins. The triangles indicate the
reactive site P1 residues in the active inhibitors: bomapin
and horse leukocyte elastase inhibitor. The asterisks show
the position of the putative NLS in MENT. The peptide sequences
determined by direct protein sequencing are underlined. The
M-loop domain (positions 60-91) is marked by a solid
box, and the "hinge" motif (positions 357-364) is
marked by carets. b, comparison of the amino acid sequences
of MENT and different vertebrate lamin chromatin binding regions (66,
67). Vertical lines and colons show
identities and similarities between each of the lamin protein sequences
and MENT. c, comparison of several A·T-rich DNA-binding
proteins having sequence similarities to the A·T hook motif (69). A
comprehensive survey of A·T hook proteins can be found in Ref.
70.
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The MENT ORF had no exact matches to other proteins in the available
data bases but revealed a highly significant sequence homology to a
number of proteins belonging to the ovalbumin subfamily of serpin
proteins (Fig. 4a). The similarity with serpins included most of the MENT sequence. Although among serpins there are a number of
proteins that fulfill different noninhibitory functions (61, 62),
including the recently discovered nuclease activity of leukocyte
elastase inhibitor in apoptosis (63), as far as we know, none has
previously been shown to be a chromatin-associated nuclear protein.
MENT thus appears to be a novel chromatin-binding structural protein.
Like ovalbumin, MENT does not inhibit proteases (46), and its
"reactive center" peptide bond, T-T, does not resemble reactive centers of any of the protease-inhibiting serpins (the
triangle in Fig. 4a shows the standard position
of the reactive center in serpins; see, for example, Ref. 62).
Remarkably, MENT exhibits a perfect sequence conservation within the
so-called "hinge region" consensus, GTEAAAT (amino acids 357-364
marked with carets in Fig. 4a) essential for the
large scale conformational transitions (S
R) typical of serpin
proteins (61). The hinge region is conserved in both inhibitory and
noninhibitory serpins retaining the S
R capability (64) but is
dramatically modified in serpins that lack both the inhibitory and the
S
R functions (65).
Two prominent structural features distinguish MENT from other serpins.
First, the MENT protein sequence is distinguished by a high content of
positively charged amino acids and a high pI value (9.4) compared with
a pI of 5-6.5 typical of serpins. As shown below, most of the basic
MENT amino acids reside on the solvent-exposed surface of the protein
and are probably involved in DNA binding. Second, the protein sequence
between amino acids 61 and 91 (designated here as the M-loop) bears no
resemblance to serpins (boxed portion in Fig.
4a). Amino acids 80-84 (asterisks in Fig.
4a) constitute a putative nuclear localization signal (NLS)
typical of many nuclear proteins and identical to the NLS of
Xenopus lamin II (66). The presence of an NLS is consistent with the nuclear localization of MENT (45, 46). The M-loop sequence has
a significant similarity with a number of nuclear lamina proteins in
the region including the NLS (Fig. 4b), which is also
involved in chromatin binding mediated by core histones (67). Nuclear
lamins constitute the bulk of insoluble nuclear structures (nuclear
envelope and nuclear skeleton), which bind selected regions of
chromatin often associated with transcriptionally active genes (68).
The nuclear envelope is in close contact with peripheral
heterochromatin, and the lamins have been shown to be involved in
heterochromatin formation in yeast (29, 30).
A short sequence in the M-loop upstream of the NLS bears no similarity
with lamins but is related to the A·T hook motif (Fig. 4c), which mediates binding to A·T-rich DNA by HMG(I/Y)
and is present in a number of eukaryotic chromosomal proteins (69, 70).
A·T-rich sequences are abundant in nuclear matrix binding DNA regions
(71) and are also prominent in satellite DNA, the principal component
of constitutive heterochromatin (72). The combination of the A·T hook
motif with the nuclear lamin-like chromatin binding region in the
M-loop domain may allow MENT to compete for nucleosomes with lamins and
other proteins of the nuclear skeleton, thus providing a possible
explanation for the previously reported activity of MENT toward
dissociation of chromatin-nuclear matrix bonds in maturing erythrocytes
(44).
The tertiary structures of a number of the serpin family members have
been solved by x-ray crystallography (61, 62, 73) and show a high
degree of structural conservatism. For example, the backbone of horse
leukocyte elastase (74) can be superimposed almost precisely with that
of
1-proteinase inhibitor and
1-anti-chymotrypsin, although it has only about 30%
sequence identity with the former. Thus, the polypeptide backbone
folding of MENT can be predicted with high confidence based on its
similarity to ovalbumin (35% identity), horse leukocyte elastase (48%
identity), and other serpins. We employed ProModII software (55, 56) to
build a three-dimensional model of MENT based on the three-dimensional structure of ovalbumin solved by x-ray crystallography in its native
uncleaved conformation with 1.95-Å resolution (75).
The inferred three-dimensional model of MENT showing the main secondary
structural elements is presented in Fig.
5A. The protein is roughly
40 × 55 × 70 Å in size. The three-dimensional structures of the two domains marked by the dashed boxes are
uncertain. One is the sequence between amino acids 352 and 379, which
corresponds to the reactive center domain (R-loop) and which undergoes
abrupt conformational transitions in serpins (61, 62). The other is the
nonserpin M-loop domain between amino acids 61 and 91, which is modeled
here as a protruding highly basic bipartite loop that could be involved
in interactions with DNA phosphates in a manner similar to the
DNA-interacting "wings" of histone H5 and transcription factor
hepatocyte nuclear factor 3 (76). It should be noted that the
propensity of this region to form either
-helix or
-strand is
approximately twice as low as to form a random coil as determined by
the approach of Garnier et al. (77). Therefore, the M-domain
has little potential to form a stable secondary structure and,
conceivably, may extend as much as 50 Å.

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Fig. 5.
Molecular modeling of MENT three-dimensional
structure. The protein backbone folding is based on the
three-dimensional crystal structure of ovalbumin (Protein Data Bank
entry DB 1OVA). A, three-dimensional rendering showing the
elements of the predicted secondary structure of MENT as flat
( -sheet) and helical ribbons ( -helices). The reactive center loop
domain (R-loop) and the MENT-specific loop domain (M-loop) are
indicated by dashed boxes. B, surface
electrostatic potential mapping by GRASP showing the regions that have
positive electrostatic potential in blue, negative
electrostatic potential in red, and neutral regions in
white. The molecular surface is viewed from the same side as
A on the left and from the opposite side on the
right. Positions of the M-loop (M) and the R-loop
(R) domains are indicated.
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Most of the positively charged amino acids are exposed on the surface
of MENT and may interact with DNA phosphates through ionic bonds. A
molecular surface potential model of MENT prepared using the GRASP
program (57) shows a strong clustering of positive and negative charges
on the protein surface. MENT appears to be a bipolar protein in which
the basic amino acid clusters tend to localize closer to the M-loop
domain, while the acidic and neutral clusters occupy the other side of
the molecule close to the R-loop (Fig. 5B). The highly basic
amino acid clusters are located on a convex protein surface, suggesting
that this domain may be inserted between negatively charged molecules,
perhaps between the nucleosome linkers, which are closely apposed at
the entry/exit site of linker histone-containing nucleosomes (5). The
negatively charged area in the vicinity of the R-loop may bind to basic
regions of other nucleosomal proteins, such as histones or even MENT
itself. It will be interesting to determine whether the R-loop of MENT,
which is not charged (Fig. 5b) and thus does not form ionic
bonds, is involved in the type of conformational transitions associated
with this structural domain in the serpins.
Ectopic Expression of MENT cDNA--
Actively proliferating
cells of either avian or mammalian origin appear to be devoid of MENT.
To explore the in vivo properties of MENT expression, we
transfected a mammalian cell line, COS-7, with a MENT-expressing vector
based on the cDNA sequence and containing its ORF placed downstream
of a cytomegalovirus promoter. After transfection and incubation of the
cells for 48 h, cytoplasmic and nuclear proteins were isolated and
analyzed by Western blotting with anti-MENT antibodies. Nuclei (but not
cytoplasm) of cells transfected with MENT cDNA produced a strong
42-kDa band (Fig. 6, lane
4) while the nontransfected cells, cells transfected with control vector DNA (lane 2), and cDNA without
an active promoter (lane 3) were MENT-negative.
This result shows that the cDNA encodes a unique protein with
molecular weight, antigenicity, and nuclear localization fully
consistent with the biochemical data and the sequence analysis of
chicken MENT.

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Fig. 6.
MENT synthesis from cDNA transfected in
COS-7 cells. Western blot of SDS-polyacrylamide gel
electrophoresis of proteins isolated from chicken granulocyte chromatin
(lane 1) and from the nuclei of COS-7 cells
transfected with a control vector plasmid (lane
2) and plasmids containing MENT ORF without an active
promoter (lane 3) and under the cytomegalovirus
promoter (lane 4) (detection with anti-MENT
antibodies).
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Many of the known heterochromatin-associated proteins are distributed
nonrandomly within cell nuclei, often being associated with foci of
condensed chromatin (28, 29). As is typical with the transient
expression procedure, about 10% of total cells transfected with
MENT-expressing vector developed various levels of MENT-specific signal
(detected with anti-MENT antibodies but not with preimmune serum (45)).
In all cells with a low and a moderate signal intensity, MENT was
confined entirely within the nucleus. In nontransfected cells as well
as in cells transfected with a control vector, no signal was detected.
Nuclei expressing relatively low amounts of MENT were similar in shape
and size to typical COS-7 nuclei (Fig. 7,
a-d). The strongest signal was associated with subnuclear
organelles that also bind antibodies against fibrillarin, a nucleolar
protein (e.g. see Ref. 52), showing that nucleoli are the
preferential sites for initial MENT accumulation (Fig. 7d).
Inside the nucleoli, Hoechst staining is relatively weak, indicating
that MENT recognizes specifically the nucleolar material rather than
merely following the local DNA concentration. It is noteworthy in this
respect that the yeast SIR3 and SIR4 proteins involved in yeast
"heterochromatin" are also found in the nucleolus, an association
that has a powerful influence on the cell life span (78). A nucleolar
localization of MENT has not been observed previously, since the
terminally differentiated cells in which it has been studied (nucleated
erythrocytes and granulocytes) lack nucleoli.

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Fig. 7.
Immunolocalization of MENT ectopically
expressed in COS-7 cells. Selected cells with relatively low
(a-d), and high (e-h) levels of MENT
immunostaining are shown. a, c, e, and
g, cells stained with rabbit anti-MENT antibodies and
fluorescein 5-isothiocyanate-conjugated goat anti-rabbit antibodies.
d, cells stained with mouse anti-fibrillarin antibodies and
donkey Texas Red-conjugated anti-mouse antibodies. b,
f, and h, cells stained with Hoechst 33258.
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In nonnucleolar regions, MENT is distributed nonrandomly, forming foci
of variable shape, many of which occur close to nucleoli. Staining by
the DNA-specific dye Hoechst 33258 shows that the signals from Hoechst
and from anti-MENT antibodies usually (but not always) coincide (Fig.
7, a, b, e, and f). Hoechst
preferentially stains A·T-rich heterochromatin (79), and its
co-localization with MENT foci may reflect a local heterochromatin
recruitment by the protein.
Within the most extensively MENT-expressing cells (which also show
MENT-positive staining in the cytoplasm), the nuclei are strongly
fluorescent and consistently smaller in size than control cells. Also,
more of the nuclear volume is occupied by MENT and Hoechst-positive
foci (Fig. 7, e-h). Taken together with our previous results showing that an addition of 1-2 molecules of MENT/nucleosome can induce a significant chromatin condensation and nuclear shrinkage in vitro (46), this suggests that the reduction in nuclear
size and the condensed nature of chromatin in these cells is the direct result of the high level of MENT and is likely to reflect its inherent
chromatin-condensing properties.
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DISCUSSION |
Chromatin condensation in terminally differentiated cells is a
widespread developmentally regulated phenomenon whose mechanism is
poorly understood. Here we report the first study of isolated condensed
chromatin where the highly compact state is linked to the accumulation
of a single nonhistone protein, MENT. The evidence that MENT is both
necessary and sufficient to effect chromatin condensation is
compelling: its expression is strictly limited to the terminal stage of
cell differentiation (44, 45); it is concentrated in peripheral
heterochromatin (45, 46); it brings about chromatin condensation when
either ectopically expressed in vivo (Fig. 7) or
reconstituted with isolated nuclei in vitro (46); and it is
the major nonhistone chromatin protein that is stably bound to compact
polynucleosomes (Fig. 2). This rather simple experimental system allows
a detailed study of the structure and formation of heterochromatin and
also provides important general insights into the mechanisms of
chromatin compaction and heterochromatin spreading.
Our cryoelectron microscopy observations allow a direct comparison
between heterochromatin-derived granulocyte polynucleosomes and
"euchromatin" from actively proliferating cells (Fig. 3). Two
principal differences stand out: the heterochromatin sample shows a
much closer packing of nucleosomes within the 30-nm fibers and also
contains fibers that vary in diameter up to 50 nm. In previous work,
changes in the compaction of polynucleosomes containing linker histones
were shown to be effected by changes in the degree of electrostatic
neutralization of DNA negative charges by counterions (14, 81). The
increase in counterions is known to affect the entry-exit angle of the
two linker DNA segments of a nucleosome consistent with a reduction in
the mutual repulsion of the two linker DNA segments causing a
longitudinal compaction (5, 18, 59). There is also an increased rate of
self-association (14), apparently through lateral contacts between the
fibers (14). In the present study, the different compaction levels of
the heterochromatin and euchromatin samples can also be traced to
differences in longitudal and lateral compaction, but for granulocyte
oligonucleosomes, high compaction levels do not require high salt but
rather the presence of MENT, a basic protein in which the positively
charged amino acids form potential DNA-interacting clusters at the
surface of the molecule (Fig. 5B).
The presence in the heterochromatin-derived polynucleosomes of larger
structures of varying diameter is indicative of inter- and intrafiber
interactions, including "fold-back" regions and side-to-side
self-associations, phenomena similar to those seen in tomographic
reconstructions of whole starfish sperm nuclei (80). Self-association
of chromatin fibers is also part of the continuum of salt-induced
chromatin compaction (14, 15, 81) and likely to predominate in compact
chromatin in vivo (82). Experiments in which we removed
about 75% of MENT in order to release soluble chromatin from
granulocyte nuclei, suggest that a more loosely bound fraction of
MENT is responsible for the interfiber associations. We propose that
both the lack of solubility of granulocyte chromatin with a full
complement of MENT and the increased fiber diameter of MENT-enriched
chromatin are due to the extensive interfiber interactions that occur
in vivo within electron-dense masses of heterochromatin.
The MENT serpin homology suggests that in addition to DNA charge
neutralization, the interfiber interactions may be dramatically strengthened by protein-protein interactions between the
nucleosome-associated molecules of MENT. Indeed, in some serpins,
especially those with an impaired inhibitory function, the S
R
transitions, instead of being intramolecular, involve spontaneous
polymerization through an interaction of the R-loop domain of one
protein with the A-sheet of another (83-85). In MENT, these domains
are oriented away from the putative DNA-binding basic surfaces (Fig.
5B). Thus, the association of MENT with DNA would not
interfere with potential protein-protein interactions. The conservation
of the "hinge region" consensus at the basis of the R-loop is a
very strong indication that the S
R capability may be present in
MENT. Indeed, besides being conserved among the inhibitory
serpins, this consensus is also present among noninhibitory serpins
retaining the S
R capability, such as hormone-binding globulins
(64), but is absent from ovalbumin and angiotensinogen, serpins
that lack both the inhibitory and the S
R functions (65).
When isolated from nuclei, MENT behaves as a monomeric protein during
gel filtration chromatography (data not shown). Thus, the suggested
multimerization should be promoted by association of the protein with
DNA and/or chromatin. Such an event has been recently observed in the
ternary complex of MAT
2 repressor with MCM1 transcriptional factor
and DNA (86), where a "chameleon" transition between
-helical
and
-strand conformations of a short amino acid motif in MAT
2 has
been revealed by x-ray crystallography. These authors pointed out that
serpins are another group of proteins capable of "chameleon"
transitions. It should be noted that although the change in secondary
structure is an essential element of the serpin S
R transition, the
latter also involves a larger scale rearrangement of the tertiary
structure that may be important in chromatin remodeling.
Another property of MENT that may be indicative of its in
vivo action is its ability to interfere in vitro with
the partition of specific genes to the insoluble nuclear matrix
fraction. Exogenously added MENT caused the c-myc
"housekeeping" gene to move from the nuclear matrix fraction to the
soluble fraction, but only after its expression had been shut down in
the course of erythropoiesis, while the transcriptionally active H5 and
-globin genes showed no response to MENT (44). The presence in MENT
of a nonserpin domain (M-loop) combining the features known to be
involved in binding A·T-rich DNA with localization in intranuclear
foci stained with an A·T-rich DNA-specific dye (Fig. 7) is consistent
with a direct interaction of MENT with such DNA. In particular, MENT may recognize specific DNA sequences such as the c-Myc
scaffold-associated region, which contains a prominent A·T-rich DNA
sequence (87). These elements may have a much stronger affinity for
DNA-binding proteins in the context of a nucleosome array than as naked
DNA as has been proposed for repeated A·T-rich sequences (88), thus accounting for the apparent absence of sequence-specific interactions of MENT with DNA in vitro.2
In a recent paper (71) it has been shown that an artificial protein
containing repeated A·T hook motifs, MATH20, can act as a general
chromatin activator by interfering with position effect variegation in
Drosophila. Based on their data, the authors suggested that
the binding of hypothetical chromatin compacting proteins to
scaffold-associated regions or certain A·T-rich satellites could
"lead to either chromatin folding, chromosome condensation (looping),
or formation of heterochromatin" (71). Their model, which involves a
component with a striking similarity to the major properties of MENT,
implies that the regulation of heterochromatin formation by competition
between A·T-binding activators and chromatin-condensing proteins may
be a general phenomenon not restricted to terminal differentiation.
The remodeling of chromatin architecture associated with
heterochromatin formation appears to be accomplished by a variety of
proteins that act at different levels of chromatin organization. MENT,
the first example that links heterochromatin with the higher order
folding of chromatin fibers, is confined to terminally differentiated blood cells. Further work may reveal new proteins, related or unrelated
to MENT, fulfilling similar functions in other cells and tissues. Their
identification and study, using either biochemical and ultrastructural
techniques similar to those that have proven successful here or genetic
and in vivo approaches now facilitated by the cloning of the
MENT cDNA, should provide a more general picture of the molecular
interactions regulating development- and position-specific chromatin condensation.