From the Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, North Carolina 27599-7090
Received for publication, September 16, 2002, and in revised form, December 4, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
NASP is an H1 histone-binding protein that is
cell cycle-regulated and occurs in two major forms: tNASP, found in
gametes, embryonic cells, and transformed cells; and sNASP, found in
all rapidly dividing somatic cells (Richardson, R. T.,
Batova, I. N., Widgren, E. E., Zheng, L. X., Whitfield,
M., Marzluff, W. F., and O'Rand, M. G. (2000) J. Biol. Chem. 275, 30378-30386). When full-length tNASP fused to
green fluorescent protein (GFP) is transiently transfected into HeLa
cells, it is efficiently transported into the nucleus within 2 h
after translation in the cytoplasm, whereas the NASP nuclear
localization signal (NLS) deletion mutant (NASP- Linker (H1) histones are involved in chromatin remodeling events
and frequently exchange between numerous linker-binding sites on
chromatin (2-4). Moreover, H1 histones have been shown to influence
G1/S phase progression (5) and in some cases inhibit transcription initiation (6). Consequently, understanding the role of
H1 histones is important for our understanding of both gene activation
and chromatin organization (7-9).
H1 histones not bound to DNA appear to be bound to the H1
histone-binding protein NASP, which has been characterized previously in vivo and in vitro (1, 10, 11) as an acidic
protein containing functional histone-binding sites, a leucine zipper,
ATP/GTP-binding sites, and a functional nuclear localization signal
(11, 12). NASP occurs in two major forms as follows: tNASP, found in
gametes, embryonic cells, and transformed cells; and sNASP, found in
all dividing somatic cells (1). Mouse sNASP (Mr
45,751) is identical to tNASP (Mr 83,934),
except that it lacks two internal regions of the protein (1, 10).
Similarly human tNASP and sNASP occur with identical deletions to those
found in the mouse (10, 13).
During the cell cycle, NASP mRNA expression in somatic
cells increases and decreases concurrently with histone mRNA
changes in expression (1). However, in rapidly dividing cells protein levels of NASP remain fairly constant and only decrease to undetectable levels in non-dividing cells (1). Reports that overexpression of H1
histones can influence progression through the cell cycle (5) prompted
us to investigate the effect on progression through the cell cycle by
overexpression of NASP and to examine the relationship of
NASP to H1 histones in HeLa cells. In this report we demonstrate that
1) the overexpression of tNASP does influence progression through the cycle, 2) tNASP is bound to H1 histones in HeLa cells, 3)
in vitro complexes of tNASP-H1 will transfer H1 to DNA, and 4) that NASP and H1 have virtually identical mobilities within the nucleus.
All chemicals and reagents used in this study were molecular
biology grade. Restriction enzymes were purchased from Roche Diagnostics. Purification of plasmid DNA and PCR products were carried
out using QIAprep Miniprep and QIAquick PCR purification kits
(Qiagen, Valencia, CA), and sequencing was performed at the University
of North Carolina, Chapel Hill, automated sequencing facility.
Affinity-purified goat anti-green fluorescent protein antiserum was
purchased from Rockland (Gilbertsville, Pa); rabbit anti-histone H1
(FL-219) polyclonal antiserum was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA); and rabbit anti-NASP antiserum was
prepared against either N-terminal (nucleotides 96-1099) or C-terminal
(nucleotides 1100-2414) recombinant proteins as described previously
(1).
Construction of Expression Vectors--
The entire coding
sequence of mouse tNASP (nucleotides 92-2405, GenBankTM
accession number AF034610) was amplified from mouse testis Quick-clone
cDNA (Clontech, Palo Alto, CA) using the Expand
High Fidelity PCR System (Roche Molecular Biochemicals) and cloned into
a KpnI/BamHI site in the pEGFP-N1 vector, which
contains the sequence for expressing green fluorescent protein
(GFP1;
Clontech, Palo Alto, CA). The nuclear localization
signal (NLS) deletion mutant (NASP-
For cell cycle studies, vectors lacking the GFP sequence were
constructed from pEGFP-N1 by excising the GFP by sequential digest by
BamHI and NotI. Digested ends were repaired by
Klenow enzyme and ligated by T4 DNA ligase. All constructs were
sequenced to verify the correct reading frame.
For in vitro H1-binding studies, full-length tNASP and
NASP- Cell Studies--
HeLa cells were maintained in Dulbecco's
modified Eagle's medium-H plus 10% calf serum. Cells were removed
from plastic dishes by trypsinization in EDTA. Synchronized
populations of HeLa cells were obtained by double thymidine blocking as
described previously (1).
Indirect Immunofluorescence--
HeLa cells were grown on
chamber slides with polystyrene wells (Falcon® CultureSlide, BD
Biosciences), washed twice with cold (4 °C) PBS (phosphate-buffered
saline, pH 7.0), fixed with chilled ( Chemical Transfection--
Plasmid-DNA complexes were
transiently transfected into HeLa cells using Effectene Transfection
Reagent according to the manufacturer's instructions (Qiagen,
Valencia, CA). This method is based on a non-liposomal lipid
formulation and resulted in low cytotoxicity and high transfection
efficiency (~97%) as determined by FACS analysis. Cells were
transfected for 4-6 h with tNASP-GFP, NASP- Fluorescent-activated Cell Sorting (FACS) Analysis--
Control
cells and transfected cells 2, 4, 6, 8, and 24 h after release
from the double thymidine block were washed with PBS, trypsinized, and
fixed with 70% ethanol for Immunoaffinity Chromatography--
Affinity chromatography was
carried out with rabbit anti-NASP (C-terminal) antibodies (30 mg)
coupled to Reacti-Gel 6× beads (Pierce; coupling efficiency >80%).
HeLa cells were trypsinized, washed twice with PBS, and solubilized by
incubation in lysis buffer: 0.01 M sodium phosphate buffer,
pH 7.2, 0.15 M NaCl, containing 1% Triton X-100,
0.5% sodium deoxycholate, and protease inhibitor mixture (Sigma) for
30 min on ice. Following centrifugation (10 min, 6000 × g), the pellet was sonicated, added to the supernatant, and
centrifuged again (10 min, 6000 × g). The supernatant
was collected and incubated with antibody-coupled beads overnight at
4 °C. After washing with PBS, the bound proteins were eluted from
the beads with ImmunoPure® Elution Buffer (Pierce). The eluate pH was
neutralized, and the sample was concentrated by vacuum centrifugation.
Samples were separated by SDS-PAGE on 10-20% Tris-Cl Criterion
gradient minigels (Bio-Rad) under reducing conditions, blotted to
Immobilon-P (Millipore Inc., Bedford, MA), and probed for NASP and H1
histones. A C-18 reverse phase HPLC column (Waters, DeltaPak, 15 µM, 300 Å, 3.9 × 300 mm) equilibrated with
acetonitrile (ACN) and 0.1% trifluoroacetic acid was used for the
identification of histone H1 subtypes as described previously (1).
Briefly, proteins were eluted at 28 °C with a multistep ACN in 0.1%
trifluoroacetic acid gradient (0.7 ml/min). Gradients were developed
from 18.5 to 100% buffer B (90% ACN in 0.1% trifluoroacetic acid)
and 81.5 to 0% buffer A (10% ACN in 0.1% trifluoroacetic acid).
DNA Supercoiling Assay and H1-NASP Complexes--
To assess the
ability of H1-NASP complexes to transfer H1 to DNA, H1-NASP complexes
were prepared in vitro and analyzed by a DNA supercoiling
assay modified from the method of Kleinschmidt et al. (14).
Calf thymus H1 histones (Roche Molecular Biochemicals) were
biotinylated using an EZ-Link Sulfo-NHS-biotin kit (Pierce). Biotinylated H1 histones (1 µg) and tNASP (3 µg) were incubated (30 min at 37 °C in PBS containing 1 mM ATP) and
chromatographed in 1× SSC buffer, pH 7.4, on Micro Bio-Spin 30 chromatography columns (Bio-Rad) to remove unbound H1 histones, which
are retained by the column. Samples were evaluated by SDS-PAGE
(10-20% Tris-HCl Criterion gels, Bio-Rad) and Western blotting to
Immobilon-P membranes (Millipore, Bedford, MA). Biotinylated H1
histones were detected using alkaline phosphatase-conjugated avidin,
and tNASP was detected using anti-NASP antibodies (N-terminal) and an
alkaline phosphatase-conjugated secondary antibody (Cappel, West
Chester, PA).
Supercoiled SV40 DNA (Invitrogen) was relaxed by incubation with
topoisomerase I (Invitrogen). H1 histones, tNASP, or H1-NASP complexes
from the Micro Bio-Spin 30 chromatography columns (described above)
were incubated with the relaxed SV40 DNA for 30 min at 37 °C in TOPO
buffer (50 mM Tris-HCl, 50 mM KCl, 10 mM MgCl2, 0.1 mM EDTA, 0.5 mM dithiothreitol, 1.0 mM ATP, pH 7.5, with 30 µg/ml bovine serum albumin). The reactions were terminated by the
addition of a Sarkosyl-proteinase K (2% and 0.3 mg/ml final concentration, respectively) mixture, which was incubated for 15 min at
37 °C. The DNA was subsequently phenol-chloroform-extracted, ethanol-precipitated, and analyzed by 1% agarose gel electrophoresis in 4× TAE and ethidium bromide-stained for visualization.
Fluorescence Recovery after Photobleaching (FRAP)--
HeLa
cells growing on coverslips (25-30% confluent) were transfected by
pEGFPN1-NASP as described above. Cells were observed 24 h after
transfection. Coverslips with cells were mounted in metal chambers and
observed with a ×40 objective in a Zeiss 410 confocal microscope using
the 488-nm laser line of an argon-krypton laser. In a FRAP experiment
one scan of the unbleached cell was acquired, followed by a single
bleach pulse of 5-10 s using a spot of rectangular shape and differing
in size depending upon the size of the bleached structure (usually 50 or 100% of the nuclear area). Images were collected from the same
section initially at 5- or 10-s intervals (1 min) followed by 30-s
intervals for 4 min. For imaging, the laser power was attenuated to
0.3% of the bleach intensity. FRAP recovery curves were obtained from background-subtracted images using GelExpert Software (Nucleotech Corp., San Carlos, CA). The relative fluorescence in the nuclear area
measured was determined as defined by Phair and Misteli (16). The
relative fluorescence is Irel = T0It/TtI0, where T0 is the total nuclear fluorescence
intensity before bleaching, and I0 is the
fluorescence intensity of the nuclear area to be bleached.
Tt and It are the fluorescence
intensities at time t. Approximately 1% of the fluorescence
was lost during imaging.
Localization of NASP--
Indirect immunofluorescent staining of
unsynchronized HeLa cells as well as synchronized cells 2, 4, 6, and
8 h after being released from the double thymidine block
demonstrated that immediately after mitosis and continuing throughout S
and G2 phases NASP is localized in both the nucleus and
cytoplasm (Fig. 1A). In the nucleus NASP is unevenly distributed and appears to be excluded from
nucleolar regions. In the cytoplasm NASP is most abundant in dense
aggregations in the perinuclear regions but often can be seen as rows
of aggregates reaching the peripheral cytoplasm (Fig. 1A).
Transfection of cells with tNASP-GFP resulted in strong nuclear
staining (Fig. 1B) 2 h after transfection (97% of the
cells were transfected after 24 h). Cytoplasmic staining remained
weaker than the nuclear staining throughout the 24 h of
observation. Transfection of cells with NASP- Association of H1 Histones with NASP--
In myeloma 66-2 cells H1
histones were found to co-purify bound to native NASP (1). Similarly in
HeLa cells, isolation of NASP by HPLC size chromatography or affinity
chromatography with anti-NASP antibodies resulted in the
co-purification of H1 histones (Fig. 2, C and D).
Histone analysis by reverse phase HPLC of H1 histones extracted from
H1-NASP complexes identified subtype H1d/e (H1.2) as the H1
histone (data not shown). Histone H1.2 is the most abundant subtype in
HeLa cells (15); however, other histone H1 subtypes may not have been
detected because of limited amounts of sample.
Effect of the Overexpression of NASP and NASP-
Table I presents the data collected from
6 independent experiments at 4 and 6 h after release from the
double thymidine block. At 4 and 6 h after release there were
significantly fewer HeLa cells, which were overexpressing tNASP, in the
G2 and S phases than there were non-overexpressing HeLa
cells (71.45 versus 50.8%; p = 0.01 at
4 h, and 79.85 versus 60%; p = 0.01 at
6 h; Fig. 4 and Table I). Similarly
there were significantly fewer overexpressing tNASP- DNA Supercoiling by H1-NASP Complexes--
To test the ability of
NASP to transfer H1 histones to DNA, calf thymus H1 histones were
biotinylated and bound to tNASP in vitro, and the H1-NASP
complexes were subsequently separated by chromatography on Bio-Rad
Micro Bio-Spin 30 (MBS30) columns. MBS30 columns separate H1-NASP
complexes from unbound histones. Fig. 5
demonstrates that biotinylated H1 histones are retained on the MBS30
column (lanes 1 and 2); however, when H1 is
complexed with NASP, both are eluted from the column (lanes
4 and 6). When NASP-
The separated H1-NASP complexes were incubated with relaxed SV40 DNA to
test the ability of H1 histones to supercoil the SV40 DNA (Fig.
6). Supercoiled and relaxed SV40 DNA are
shown in Fig. 6, lanes 1 and 2. The addition of
H1 histones to relaxed SV40 DNA causes supercoiling (lane
3), whereas the addition of tNASP does not (lane 4). As
shown in Fig. 6, lanes 6 and 7, the addition of
H1-NASP complexes, which have passed through the MBS30 column, to
relaxed SV40 DNA results in supercoiling the DNA. If H1 only is
chromatographed and the resulting eluate incubated with relaxed SV40
DNA, no supercoiling occurs because the H1 histones are retained by the
column (lane 5). If increasing amounts of tNASP are added at
the time of the addition of H1-NASP complexes to relaxed SV40 DNA, then
there is a reduction in the intensity of the supercoiled DNA bands
(Fig. 7). This indicates that tNASP
competes with DNA for H1 binding.
Mobility of NASP in the Nucleus--
Given our results that NASP
and H1 histones co-purify from HeLa cells and that NASP can transfer H1
histones to DNA in vitro, it is possible that H1 histones
not bound to DNA are bound to NASP. If NASP-H1 complexes were present
in the nucleus, then we would expect that the mobility properties of
NASP would be similar to those of H1. Consequently, we used FRAP to
determine the time necessary for NASP to move into a bleached area of
the nucleus. Bleaching ~50% of a nucleus containing tNASP-GFP (see
also Fig. 1B) resulted in a recovery of fluorescence to a
plateau level of 92.5 ± 4.6% in 220-225 s (n = 3 experiments). Fig. 8 shows a typical
experiment in which 50% of the nucleus was bleached (Fig.
8A), and the fluorescence recovery was 96.5 ± 1.2% in
225 s (Fig. 8B). This compares favorably to the
200-250 s necessary for recovery of H1-GFP fluorescence in the nucleus
(4). As fluorescence intensity increases in the bleached area, there is a concomitant decrease in intensity in the unbleached area (Fig. 8A), indicating that NASP is redistributed within the
nucleus. Because HeLa cells transfected with tNASP-GFP have very little tNASP-GFP in the cytoplasm (Fig. 1B), photobleaching the
entire nucleus resulted in no recovery of fluorescence in 300 s
(n = 3 experiments), indicating that essentially no
tNASP-GFP entered the nucleus from the cytoplasm in these
experiments.
In this study we have demonstrated that the H1 histone-binding
protein NASP is present in the nucleus and cytoplasm of HeLa cells and
can be directed to the nucleus by its NLS (Fig. 1). Even though
transfection of cells with tNASP-GFP resulted in almost complete
nuclear localization of the construct, endogenous NASP is clearly not
exclusively in the nucleus (Figs. 1 and 2). From Western blotting (Fig.
2) it would appear that there is less tNASP in the nucleus than in the
cytoplasm, whereas sNASP is more evenly distributed between the two
compartments. Because tNASP occurs in rapidly dividing cell populations
(gametes, embryonic cells, and transformed cells), its presence in the
cytoplasm may serve as a storage site for excess linker (H1) histones.
Alternatively, the majority of NASP in the cytoplasm could simply
reflect a lagging degradation mechanism in rapidly dividing cells
because in normal somatic cells there is little or no NASP in the
cytoplasm (1).
Overexpression of tNASP significantly delays the progression of cells
through the cell cycle, and surprisingly, overexpression of NASP- Additionally, this study has demonstrated in vitro that
H1-NASP complexes can cause the supercoiling of relaxed SV40 DNA by transferring H1 from NASP to DNA and importantly that excess tNASP can
compete with DNA for H1. Kleinschmidt et al. (14) first demonstrated the transfer of histone to DNA from the Xenopus
laevis form of NASP (N1/N2). Consequently, our results are
consistent with the hypothesis that an equilibrium exists between
NASP-H1 histone complexes and H1 histones bound to DNA. This
equilibrium influences the availability of H1 histones to DNA and
affects the progression of the cell through the G1/S phase
transition. It can be upset by overexpression of tNASP with its extra
histone-binding site and is consistent with the observation that H1
histone overexpression has been shown to slow G1/S phase
progression (5) and in some cases inhibit transcription initiation
(6).
Recent reports of the mobility of H1 histones in the nucleus (3, 4)
concluded that an intermediate (4) or modulating protein (3) might be
present to regulate the movement of H1. By using FRAP experiments on
HeLa cells, this study has demonstrated that the mobility of NASP in
the nucleus is essentially the same as that of H1 histones (Fig. 8).
Although it could be fortuitous that the recovery times are similar,
one interpretation of this result is that NASP and H1 have identical
mobility characteristics because they are in a complex together in the
nucleus. This interpretation is supported by the results from the
co-precipitation experiments (Fig. 2). The recovery times of NASP and
H1 (~225 s) differ markedly from other nuclear proteins. For example,
GFP-HMG-17 and GFP-SF2/ASF have recovery times on the order of 30 s, whereas histone H2B has no recovery over 90 s (16). The less
mobile (immobile) fraction determined in a FRAP experiment is a
reflection of the residence time of the molecule in a compartment or
complex and can be modified by various treatments such as acetylation
(4). Small immobile fractions (~10%, e.g. GFP-SF2/ASF
(16)) indicate that most of the molecules are replaced within the
measured time, while large immobile fractions indicate that very few of
the molecules are replaced. Histone H2B would appear to be replaced in
the nucleosome very infrequently (16), whereas H1 histones are replaced
every 200-250 s (4). The immobile fractions of NASP and H1 vary
(~3.5% NASP versus ~9-26% for H1), but their recovery
times (~225 s) are similar, which may indicate populations of H1 and
NASP that are not complexed with each other or are in different
phosphorylation or acetylation states. Phosphorylation of linker
histones is known to inhibit chromatin remodeling (2, 18), and NASP may
serve to mediate such interactions. Taken together, the results
presented in this study indicate that NASP is most likely the
H1-regulating protein in the nucleus and that a dynamic equilibrium
exists between NASP-H1 complexes and DNA.
NLS-GFP) is retained
in the cytoplasm. In HeLa cells synchronized by a double thymidine
block and transiently transfected to overexpress full-length tNASP or
NASP-
NLS, progression through the G1/S border is
delayed. Cells transiently transfected to overexpress the
histone-binding site (HBS) deletion mutant (NASP-
HBS) or sNASP were
not delayed in progression through the G1/S border. By
using a DNA supercoiling assay, in vitro binding data
demonstrate that H1 histone-tNASP complexes can transfer H1 histones to
DNA, whereas NASP-
HBS cannot. Measurement of NASP mobility in the
nucleus by fluorescence recovery after photobleaching indicates that
NASP mobility is virtually identical to that reported for H1 histones.
These data suggest that NASP-H1 complexes exist in the nucleus and that
tNASP can influence cell cycle progression through the G1/S
border through mediation of DNA-H1 histone binding.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
NLS; nucleotides 92-2192), which
lacked the nuclear localization signal (nucleotides 2215-2268), was
PCR-amplified and cloned into the same pEGFP-N1 vector. The entire
coding region of mouse sNASP (nucleotides 254-1384,
GenBankTM accession number AF095722) (1) was also cloned
into the pEGFP-N1 vector. The histone-binding site deletion mutant
(NASP-
HBS; nucleotides 527-1384), which lacked all histone-binding
sites (nucleotides 349-523) (11), was PCR-amplified and cloned into pEGFP-N1.
HBS (amino acids 567-773) were cloned into His6
tag-pQE30 vectors. Recombinant proteins were expressed in BL21 (DE3)
pLysS-competent Escherichia coli cells and purified
on nickel-nitrilotriacetic acid-agarose columns (Qiagen, Valencia, CA).
20 °C) methanol (20 min),
washed twice with cold PBS, and incubated in rabbit anti-NASP
(N-terminal) antiserum or preimmune serum (1:500) in PBS. Cells were
incubated for 45 min, washed in PBS (3×, 5 min each), and incubated in
fluorescein-conjugated, affinity-purified goat anti-rabbit IgG Fc
fragment (1:1000 in PBS; Cappel, West Chester, PA) for 30 min. Washed
cells were viewed with a Zeiss fluorescence microscope.
NLS-GFP, or GFP only.
Transfection was confirmed by Western blotting. Lysates from HeLa cells
transfected with tNASP-GFP or NASP-
NLS-GFP were separated by
SDS-PAGE, Western-blotted (1), and stained with rabbit anti-NASP
antibodies and affinity-purified goat anti-green fluorescent
protein polyclonal antibodies (Rockland, Gilbertsville, PA).
2 h on ice. Cells were washed in PBS,
stained (30 min at 37 °C) with 50 µg/ml propidium iodide in PBS
(containing 200 µg/ml RNase A and 0.1% Triton X-100), and incubated
overnight at 4 °C before analysis at the University of North
Carolina, Chapel Hill, Flow Cytometry Facility. For each sample at
least 10,000 cells were counted. After gating out doublets and debris,
cell cycle distribution was analyzed using Summit version 3.1 software (Cytomation, Inc., Fort Collins, CO). For each time point, at
least three different samples were examined.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (30K):
[in a new window]
Fig. 1.
Localization of NASP in HeLa cells.
A, immunofluorescent localization of NASP in HeLa cells
probed with rabbit anti-recombinant NASP. NASP appears in both the
nucleus and cytoplasm. B, fluorescent localization of NASP
in HeLa cells transfected with tNASP-GFP. C, fluorescent
localization of NASP in HeLa cells transfected with the nuclear
localization signal deletion mutant (NASP- NLS-GFP). D,
control. Fluorescent localization of GFP in HeLa cells transfected with
GFP only. Original digital images were taken with a ×40
objective.
NLS-GFP resulted in
strong cytoplasmic staining, particularly in the perinuclear region,
with no detectable stain in the nucleus (Fig. 1C). Control
transfection with GFP only resulted in staining in both the nucleus and
cytoplasm (Fig. 1D). Immunoprecipitation and Western
blotting of transfected cells with anti-NASP and anti-GFP antiserum
confirmed the expression of the constructs in these cells (Fig.
2A). Western blots of nuclear and cytoplasmic fractions from non-transfected HeLa cells confirmed the
presence of both s and t forms of NASP in the nucleus and cytoplasm
(Fig. 2B).
View larger version (35K):
[in a new window]
Fig. 2.
Western blot analysis of NASP in HeLa cells.
A, overexpression of tNASP-GFP (lanes 1 and
3) and NASP- NLS-GFP (lanes 2 and 4)
in HeLa cells. Lanes 1 and 2 probed with rabbit
anti-recombinant NASP. Lanes 3 and 4 probed with
anti-GFP. B, lanes 1 and 2, HeLa cell
nuclear and cytoplasmic fractions probed with rabbit anti-recombinant
NASP antibody. Lanes 3 and 4, HeLa cell nuclear
and cytoplasmic fractions probed with rabbit preimmune serum.
Lanes 5 and 6, Amido Black stain. Lanes were
loaded with equal amounts of protein. cyt, cytoplasmic;
nucl, nuclear fraction. C, lane 1,
eluate from an anti-recombinant NASP antibody affinity column loaded
with a HeLa cell lysate, probed with rabbit anti-recombinant NASP
antibody. Control blots of recombinant NASP (lane 2) and
affinity column eluate (lane 3) probed with anti-NASP
antibody absorbed with recombinant NASP. D, lane
1, commercial H1 histones. Lane 2, eluate from an
anti-recombinant NASP antibody affinity column loaded with a HeLa cell
lysate, probed with rabbit anti-H1 histone antibody. Control blots of
commercial H1 histones (lane 3) and affinity column eluate
(lane 4) stained with goat anti-rabbit immunoglobulin
(secondary antibody only).
NLS on the Cell
Cycle--
In experiments overexpressing NASP and NASP deletion
mutants in synchronized HeLa cells, we used constructs without the GFP sequence. Chemical transfection of cells with the vectors expressing NASP and NASP deletion mutants was done 24 h before the cells were
released from the double thymidine block to ensure complete expression
of the construct. Fig. 3 shows a typical
experiment in which cell cycle progression is analyzed by FACS analysis
up to 8 h after release from the double thymidine block.
Overexpression of tNASP clearly affects the progression of cells
through the cell cycle; compare Fig. 3, A and B.
Similarly overexpression of NASP-
NLS affects the progression of
cells through the cell cycle; compare Fig. 3, A and
C. Fig. 3, D and E, shows that cells transfected with NASP-
HBS, the mutant lacking histone-binding sites,
or sNASP do not delay progression through the cell cycle.
View larger version (25K):
[in a new window]
Fig. 3.
Cell cycle changes from overexpression of
NASP in HeLa cells. FACS analysis demonstrating double
thymidine-blocked HeLa cells allowed to progress in synchrony through
the cell cycle. A, normal progression through the cell
cycle. B, overexpression of tNASP affects progression
through the cell cycle. C, overexpression of NASP- NLS
(lacking nuclear localization signal) affects progression through the
cell cycle. D, overexpression of NASP-
HBS (lacking
histone binding sites) does not delay progression through the cell
cycle. E, overexpression of sNASP (somatic NASP) does not
delay progression through the cell cycle.
NLS HeLa cells
than non-overexpressing cells in the G2 and S phases (71.45 versus 53.17%; p < 0.01 at 4 h, and
79.85 versus 59.27%; p = 0.01 at 6 h;
Fig. 4 and Table I). There was, however, no significant difference
between tNASP and NASP-
NLS (Fig. 4). Data from 2, 8, and 24 h
showed no significant differences between any of the cells tested.
There was no difference in the progression of cells through the cell
cycle between untreated cells and cells treated with delivery reagents
only (Table I). At 4 and 6 h, cells transfected with NASP-
HBS
(p = 0.37 (4 h) and p = 0.48 (6 h)) or
sNASP (p = 0.34 (4 h) and p = 0.34 (6 h)) did not significantly delay progression through the cell cycle (Fig. 3, D and E; Table I). These data indicate
that the overexpression of tNASP is different from the overexpression
of sNASP in its effect on cell cycle progression and that the
histone-binding sites are necessary for the delay in cell cycle
progression. However, the presence of a NLS is not required for this
effect.
Percent of cells in S+G2 phase
NLS; HBS-, NASP-
HBS; sN,
full-length sNASP; t test = Student's t
test; NS, not significant.
View larger version (52K):
[in a new window]
Fig. 4.
Comparison of cells in S + G2 at
4 and 6 h after overexpression of tNASP or
tNASP- NLS in HeLa cells. Error bars
represent ± S.D. Cells overexpressing either tNASP or
tNASP-
NLS are significantly different from the non-overexpressing
HeLa cells but not from each other, see Table I.
HBS is substituted for tNASP, it
elutes from the column (lane 5) but does not bind H1
histones and does not non-specifically carry H1 histones through the
column (lane 3).
View larger version (59K):
[in a new window]
Fig. 5.
Western blot showing binding of
H1 histones to tNASP but not NASP- HBS. Lanes 1-4,
biotinylated H1 histones stained with alkaline phosphatase
(AP)-conjugated avidin. Lanes 5 and 6,
NASP stained with rabbit anti-recombinant NASP and an alkaline
phosphatase-conjugated secondary antibody. Lane 1, control.
Biotinylated H1 (1 µg) not chromatographed on a Micro
Bio-Spin 30 column. Lane 2, negative. Biotinylated H1 (1 µg) chromatographed on and retained by a Micro Bio-Spin 30 column.
Lane 3, negative. Biotinylated H1 (1 µg) + NASP-
HBS (3 µg) chromatographed on a Micro Bio-Spin 30 column with the
biotinylated H1 retained. Lane 4, biotinylated H1 (1 µg) + tNASP (3 µg) chromatographed on a Micro Bio-Spin 30 column with the
biotinylated H1 carried by the tNASP. Lane 5, biotinylated
H1 (1 µg) + NASP-
HBS (3 µg) chromatographed on a Micro Bio-Spin
30 column with biotinylated H1 not bound by the NASP-
HBS, anti-NASP
probed. Lane 6, biotinylated H1 (1 µg) + tNASP (3 µg)
chromatographed on a Micro Bio-Spin 30 column with the biotinylated H1
carried by the tNASP, anti-NASP probed.
View larger version (57K):
[in a new window]
Fig. 6.
Transfer of H1 histones to DNA from tNASP
using a DNA-supercoiling assay. Lane 1, supercoiled SV40
DNA. Lane 2, relaxed SV40 DNA, supercoiled SV40 DNA relaxed
with topoisomerase I. Lane 3, relaxed SV40 DNA with
increased supercoiling due to addition of H1 (1 µg). Lane
4, relaxed SV40 DNA with no supercoiling induced by added tNASP (1 µg). Lanes 5-7, samples chromatographed on a Micro
Bio-Spin 30 column (see Fig. 5). Lane 5, H1 (1 µg),
control H1 was retained by the column and consequently no supercoiling
was detected. Lane 6, H1 (1 µg) + tNASP (2 µg).
Lane 7, H1 (2 µg) + tNASP (2 µg). In the presence of
tNASP, the H1-tNASP complex forms, passes through the column, and
subsequently increases DNA supercoiling. Lane 8, supercoiled
DNA standard sizes: 16,210, 14,174, 12,138, 10,102, 8,066, 7,045, 6,030, 5,012, 3,990, 2,972, and 2,067 bp.
View larger version (47K):
[in a new window]
Fig. 7.
Competition of DNA and tNASP for H1.
Lane 1, relaxed SV40 DNA, supercoiled SV40 DNA relaxed with
topoisomerase I. Lane 2, H1 (1 µg) + tNASP (2 µg),
supercoiling of relaxed SV40 DNA due to transfer of H1 from NASP.
Lane 3, H1 (1 µg) + tNASP (2 µg) + additional tNASP (2 µg); in the presence of additional (2 µg) tNASP, competition
between tNASP and DNA for H1-reduced DNA supercoiling. Lane
4, H1 (1 µg) + tNASP (2 µg) + additional tNASP (4 µg); in
the presence of additional (4 µg) tNASP, competition between tNASP
and DNA for H1 reduced DNA supercoiling further. Lane 5,
control supercoiled SV40 DNA. Lane 6, blank. Lane
7, supercoiled DNA standard sizes: 16,210, 14,174, 12,138, 10,102, 8,066, 7,045, 6,030, 5,012, 3,990, 2,972, and 2,067 bp.
View larger version (32K):
[in a new window]
Fig. 8.
FRAP of tNASP in HeLa cell nucleus.
A, bleach and recovery of fluorescent GFP-tNASP in a HeLa
cell nucleus. The HeLa cell was transfected with GFP-tNASP. The
rectangle in the bleach panel indicates the size
of the bleached area. B, relative recovery of fluorescence
intensity in the nucleus shown in A; 96.5% recovery in
225 s.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
NLS
also significantly delays progression through the cell cycle (Fig. 3
and Table I). However, overexpression of sNASP does not appear to have
a significant effect. The most obvious difference between tNASP
(Mr 83,934) and sNASP (Mr
45,751) is the presence of an additional histone-binding site (1, 11). The identical C-terminal sequences of tNASP and sNASP contain a leucine
zipper flanked by coiled coil regions downstream and possible
DNA-binding sites upstream; these structural features are often
indicative of protein dimers that bind DNA (17). If tNASP dimers form
in the cytoplasm and bind H1 histones, then the overexpression of tNASP
may result in insufficient linker histones for DNA replication. The
only other notable difference between tNASP and sNASP is the presence
of two additional ATP/GTP-binding sites in tNASP. These sites may be
important for the functional attributes of tNASP and its role in
chromatin remodeling.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. J. Gordon and B. Muller-Borer for help and expertise with the FRAP experiments.
![]() |
FOOTNOTES |
---|
* This work was supported by NICHD, National Institutes of Health, through Cooperative Agreement U54HD35041 as part of the Specialized Cooperative Centers Program in Reproductive Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Integrated Toxicology Program, Nicholas School of
the Environment, A333 LSRC, Science Dr., Duke University, Durham, NC 27708.
§ To whom correspondence should be addressed: Dept. of Cell and Developmental Biology, CB 7090, University of North Carolina, Chapel Hill, NC 27599-7090. Tel.: 919-966-5698; Fax: 919-966-1856; E-mail: morand@unc.edu.
Published, JBC Papers in Press, December 30, 2002, DOI 10.1074/jbc.M210352200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GFP, green fluorescent protein; NLS, nuclear localization signal; HBS, histone-binding site; PBS, phosphate-buffered saline; FACS, fluorescent-activated cell sorting; HPLC, high pressure liquid chromatography; ACN, acetonitrile; FRAP, fluorescence recovery after photobleaching.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Richardson, R. T.,
Batova, I. N.,
Widgren, E. E.,
Zheng, L. X.,
Whitfield, M.,
Marzluff, W. F.,
and O'Rand, M. G.
(2000)
J. Biol. Chem.
275,
30378-30386 |
2. | Horn, P. J., Carruthers, L. M., Logie, C., Hill, D. A., Solomon, M. J., Wade, P. A., Imbalzano, A. N., Hansen, J. C., and Peterson, C. L. (2002) Nat. Struct. Biol. 9, 263-267[CrossRef][Medline] [Order article via Infotrieve] |
3. | Lever, M. A., Th'ng, J. P. H., Sun, X. J., and Hendzel, M. J. (2000) Nature 408, 873-876[CrossRef][Medline] [Order article via Infotrieve] |
4. | Misteli, T., Gunjan, A., Hock, R., Bustin, M., and Brown, D. T. (2000) Nature 408, 877-881[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Brown, D. T.,
Alexander, B. T.,
and Sittman, D. B.
(1996)
Nucleic Acids Res.
24,
486-493 |
6. |
Cheung, E.,
Zarifyan, A. S.,
and Kraus, W. L.
(2002)
Mol. Cell. Biol.
22,
2463-2471 |
7. |
Karetsou, Z.,
Sandaltzopoulos, R.,
Frangou-Lazaridis, M.,
Lai, C.-Y.,
Tsolas, O.,
Becker, P. B.,
and Papamarcaki, T.
(1998)
Nucleic Acids Res.
26,
3111-3118 |
8. |
Nagpal, S.,
Ghosn, C.,
DiSepio, D.,
Molina, Y.,
Sutter, M.,
Klein, E. S.,
and Chandraratna, R. A. S.
(1999)
J. Biol. Chem.
274,
22563-22568 |
9. |
Gunjan, A.,
Sittman, D. B.,
and Brown, D. T.
(2001)
J. Biol. Chem.
276,
3635-3640 |
10. | Richardson, R. T., Bencic, D. C., and O'Rand, M. G. (2001) Gene (Amst.) 274, 67-75[CrossRef][Medline] [Order article via Infotrieve] |
11. | O'Rand, M. G., Batova, I., and Richardson, R. T. (2000) in The Testis, From Stem Cell to Sperm Function (Goldberg, E., ed) , pp. 43-150, Springer-Verlag Inc., New York |
12. | Batova, I., and O'Rand, M. G. (1996) Biol. Reprod. 54, 1238-1244[Abstract] |
13. | O'Rand, M. G., Richardson, R. T., Zimmerman, L. J., and Widgren, E. E. (1992) Dev. Biol. 154, 37-44[Medline] [Order article via Infotrieve] |
14. |
Kleinschmidt, J. A.,
Fortkamp, E.,
Krohne, G.,
Zentgraf, H.,
and Franke, W. W.
(1985)
J. Biol. Chem.
260,
1166-1176 |
15. | Kratzmeier, M., Albig, W., Meergans, T., and Doenecke, D. (1999) Biochem. J. 337, 319-327[CrossRef][Medline] [Order article via Infotrieve] |
16. | Phair, R., and Misteli, T. (2000) Nature 404, 604-609[CrossRef][Medline] [Order article via Infotrieve] |
17. | Vinson, C. R., Sigler, P. B., and McKnight, S. L. (1989) Science 246, 911-916[Medline] [Order article via Infotrieve] |
18. |
Peterson, C. L.
(2002)
EMBO Rep.
3,
319-322 |