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J. Biol. Chem., Vol. 275, Issue 39, 30378-30386, September 29, 2000
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From the Department of Cell Biology and Anatomy and the
§ Program in Molecular Biology and Biotechnology,
University of North Carolina at Chapel Hill, North Carolina 27599
Received for publication, May 4, 2000, and in revised form, July 10, 2000
Nuclear autoantigenic sperm protein (NASP),
initially described as a highly autoimmunogenic testis and
sperm-specific protein, is a histone-binding protein that is a
homologue of the N1/N2 gene expressed in oocytes of Xenopus
laevis. Here, we report a somatic form of NASP (sNASP) present in
all mitotic cells examined, including mouse embryonic cells and several
mouse and human tissue culture cell lines. Affinity chromatography and
histone isolation demonstrate that NASP from myeloma cells is complexed
only with H1, linker histones. Somatic NASP is a shorter version of
testicular NASP (tNASP) with two deletions in the coding region arising
from alternative splicing and differs from tNASP in its 5' untranslated regions. We examined the relationship between NASP mRNA expression and the cell cycle and report that in cultures of synchronized mouse
3T3 cells and HeLa cells sNASP mRNA levels increase during S-phase
and decline in G2, concomitant with histone mRNA
levels. NASP protein levels remain stable in these cells but become
undetectable in confluent cultures of nondividing CV-1 cells and in
nonmitotic cells in various body tissues. Expression of sNASP mRNA
is regulated during the cell cycle and, consistent with a role as a
histone transport protein, NASP mRNA expression parallels histone
mRNA expression.
NASP,1 initially
described as a highly autoimmunogenic testis and sperm-specific
protein, is present in the nucleus of spermatozoa and spermatogenic
cells (1-3), hence the name nuclear autoantigenic sperm protein.
Previous studies (4) have demonstrated that human NASP contains three
functional histone binding sites: site I (amino acids 116-127),
site II, (amino acids 469-512), and site III (amino acids 211-244).
In vitro, recombinant NASP will bind both linker and core
histones (4).2 As a
histone-binding protein (4, 5), NASP appears to be a homologue of the
N1/N2 gene expressed in oocytes of Xenopus laevis (6-9)
because, in addition to the conserved coding regions, there is an
almost 60% identity between the 3' untranslated regions of rabbit NASP
and Xenopus N1/N2 mRNAs (2). In Xenopus
oocytes, the non-chromatin-bound core histones H3 and H4 are associated in a complex with N1/N2 (10, 11), providing a mechanism for the storage
of histones for DNA replication in the early embryo. Both mouse (12)
and sea urchin oocytes (13) also store histones in a
non-chromatin-bound form. Other mammalian histone-binding proteins, for
example the nucleosome assembly factor NAP-1 (14-17), the chromatin
assembly factor CAF-1 (18-21), and the transcription proteins
HIRA-HIRIP3 (22), not only bind histones but appear to be important for
the assembly of chromatin (19, 23-26). Consequently NASP may be
important not only for both the storage and transport of histones but
also for the assembly of chromatin.
Here, we report a somatic form of NASP (sNASP) present in almost all
tissues examined, including mouse embryonic cells and several mouse and
human tissue culture cell lines. Interestingly, an analysis of the
histones complexed with NASP in vivo revealed that in mouse
myeloma 66-2 cells only H1, linker histones, could be detected. Somatic
NASP is a shorter version of the testicular form of NASP (tNASP) with
two deletions in the coding region arising from alternative splicing
and differing from tNASP in its 5' untranslated regions. In both
embryonic and transformed cell lines in culture, we found that both
sNASP and tNASP are expressed. Unlike spermatogenesis in the testis, in
which DNA replication and histone synthesis are uncoupled, in somatic
cells histone synthesis and histone mRNA levels are closely coupled
to DNA replication (27-29). Having found that NASP is present in
somatic cells, we therefore also examined the relationship between NASP
mRNA expression and the cell cycle and report that sNASP mRNA
levels increase during S-phase and decline during G2,
concomitant with histone mRNA levels.
All reagents and chemicals used in this study were of molecular
biology grade. Alkaline phosphatase, peroxidase, and fluorescein isothiocyanate-labeled goat anti-rabbit (heavy and light chain) antisera were purchased from Organon Technica, Inc. (West Chester, PA).
DNA probes were labeled with either a North2South biotin-DNA labeling kit (Pierce) or [32P-rsqb]dCTP (ICN Biomedicals
Inc., Costa Mesa, CA) with Ready To Go DNA labeling beads (Amersham
Pharmacia Biotech). All oligonucleotides were synthesized at the
University of North Carolina-Chapel Hill (UNC-CH) Nucleic Acids Core
Facility. Restriction and modifying enzymes were purchased from New
England Biolabs, Inc. (Beverly, MA). Purifications of plasmid DNA and
PCR products were carried out using QIAprep Miniprep and QIAquick PCR
purification kits, respectively (Qiagen Inc., Valencia, CA).
Library Screening and Sequencing
The mouse testis cDNA library was described previously (30),
and the mouse ovary cDNA library was a gift from Dr. Jurrien Dean
(National Institutes of Health, Bethesda, MD). Screening by colony
hybridization was performed with 32P-labeled probes using
Express-Hyb Solution (CLONTECH, Inc., Palo Alto,
CA) according to the recommended protocol. Both strands of positive
clones were sequenced by the UNC-CH automated sequencing facility on a
model 377 DNA sequencer using the ABI Prism rhodamine terminator cycle
sequencing kit (Applied Biosystems Inc., Foster City, CA). The sequence
for mouse tNASP (Fig. 1) was obtained by screening a mouse testis
cDNA library with a rabbit tNASP probe (2). The sequence for sNASP
(Fig. 1) was obtained by screening a mouse ovary cDNA library with
mouse tNASP cDNA. Sequence manipulations and analyses were
performed using DNAsis software (Hitachi Software Inc., San Francisco, CA).
Northern Blots
Northern blots (Fig. 2, a multiple tissue Northern blot; Fig. 3,
an RNA master blot (CLONTECH)) were probed with
32P-labeled full-length tNASP, generated by PCR primers
specific to the coding region of tNASP (nt 92-2414), and hybridized
using Express-Hyb Solution according to the
CLONTECH protocol. Blots were exposed for 16 h
on a phosphorImager screen, scanned using a Molecular Dynamics Storm
860 PhosphorImager (Amersham Pharmacia Biotech), and analyzed and
quantitated using either ImageQuant software (ImageQuant, Inc.,
Sunnyvale, CA) or GelExpert software (Nucleotech, Inc., San Mateo, CA).
Northern blots of HeLa cell and mouse 3T3 fibroblast RNA were prepared
from 1.2% agarose gels loaded with 5 µg/well total RNA and
electrophoresed as described for low percentage formaldehyde gels in
the Qiagen RNeasy Mini Kit handbook. After electrophoresis, the gels
were capillary-transferred to Hybond-N (Amersham Pharmacia Biotech).
PCR Analysis
Gene expression in a variety of tissues was studied using the
multiple tissue cDNA panel (CLONTECH).
CLONTECH cDNAs are normalized to eight
different housekeeping genes. cDNAs from thymus and ovary were
prepared in our laboratory, but these were not normalized to different
housekeeping genes. Thymus and ovary cDNAs were prepared as
follows. RNA was isolated from the tissue according to the manufacturer's instructions using the RNeasy Mini Kit for Total RNA
(Qiagen) and cDNA was prepared from an oligo(dT) primer using a
SuperScript preamplication system for first-strand cDNA
synthesis (Life Technologies, Inc.). PCRs were performed using Ready To Go PCR beads (Amersham Pharmacia Biotech). Cycle conditions were set up
as described previously (23) using the following primers: for
amplification of the full-length NASP coding region, the sense primer
was 5'-ATGGCCACAGATTCTACAGCC-3' and the antisense primer was
5'-TTAACATGCAGTGCTTTT-3'; for amplification of a fragment specific for
tNASP, the sense primer was 5'-AATGAGTGTGGGGAAGCC-3' and the antisense
primer was 5'-TTCACTCTCAGAGGTAGC-3'.
Antisera
Rabbits were immunized with purified recombinant proteins as
described (31). Expression constructs were produced using PCR, cloned
into pQE-30, transformed to bacterial host M15 (pREP-4) for expression,
and purified on Ni-NTA resin as described (31). N-terminal (nucleotides
96-1099) and C-terminal (nucleotides 1100-2414) constructs from the
sequence of mouse tNASP (GenBankTM accession number
AF034610) were used for recombinant protein production.
Affinity-purified antibodies were prepared as described (31) using
immobilized N-terminal recombinant protein.
Western Blots
Proteins were separated on 10-20% gradient minigels (Bio-Rad)
and transblotted to Immobilon-P (Waters Inc., Bedford, MA), either
stained for protein or blocked, and probed with anti-NASP antiserum as
described (32). In blots used to assess the expression of NASP in HeLa
and 3T3 cells, the blots were stained with amido black for protein to
ensure equal loading, computer-imaged with a desktop scanner,
destained, and immunostained with anti-NASP. Protein lysates of mouse
embryo, spleen, and testis were made by grinding the minced tissue in a
Dounce homogenizer with PBS and Protease Inhibitor Mixture Set 1 (Calbiochem) at the recommended dilution. The suspension was frozen,
thawed, and centrifuged at 12,000 × g. After
centrifugation, the supernatant was extracted (twice in
chloroform and methanol (3:1)) and the volatile solvents removed by
vacuum centrifugation (10 min). Protein concentrations were determined
using Micro BCA protein detection reagents (Pierce).
Immunohistochemistry
Mouse tissue sections and embryos were fixed in Bouin's
solution, paraffin-embedded, sectioned, and incubated with rabbit anti-recombinant N-terminal NASP (affinity-purified) as primary antiserum. This antiserum recognizes both somatic and testicular NASP
protein. Immunostaining was carried out as described (33).
Indirect Immunofluorescence
Cell cultures were grown on microscope slides with wells
(Lab-Tek II chamber slides, Nalgene-Nunc, Naperville, IL). Medium was
removed by aspiration and the cells washed twice with cold (4 °C)
PBS. Methanol ( Cell Culture
66-2 mouse myeloma cells (34) were maintained in Dulbecco's
modified Eagle's medium-H plus 10% horse serum at 37 °C and 5% CO2. HeLa cells, mouse 3T3 fibroblasts, and CV-1 cells
were maintained in Dulbecco's modified Eagle's medium-H plus
10% calf serum.
Cell Cycle Studies
Thymidine Block--
To obtain populations of cells in
G2-phase, HeLa cells were arrested by double-thymidine
block. Cells were blocked for 18 h with 2 mM
thymidine, released for 9 h by washing out the thymidine, and
blocked again with 2 mM thymidine for 17 h to arrest
all of the cells at the beginning of S-phase. The cells were released by washing out the thymidine and total RNA and lysates prepared from
samples taken at specific time points after release from thymidine.
Serum Starvation--
At 72 h after plating 3T3 cells,
complete medium was removed, cells were washed three times with medium
alone and incubated with Dulbecco's modified Eagle's medium
containing 0.5% calf serum for 48 h. At time 0 h, the low
serum medium was removed, and the cells were stimulated by addition of
medium containing 10% serum.
The cell cycle positions of the HeLa and 3T3 cells were determined by
analyzing the DNA content of propidium iodide-stained cells from each
time point at the UNC Flow Cytometry Facility. Total cell RNA was
prepared for the cell cycle studies using the Ultraspec RNA isolation
system (Biotecx Laboratories Inc., Houston, TX). Cell lysates were
prepared by washing the collected cells in PBS followed by lysis in 1%
SDS and boiling for 5 min. In all cell cycle studies, equal RNA loading
was verified by probing Northern blots with G3PDH, and the position of
the cells in the cell cycle was monitored using FACS analysis (shown
for Fig. 6).
Affinity Purification
Rabbit antibodies to recombinant mouse NASP were affixed to
Reactigel 6X (Pierce) and were used to affinity purify native NASP and
its associated histones from lysates. Myeloma 66-2 cell pellets were
thawed on ice and resuspended in phosphate-buffered saline (PBS) with
the addition of Protease Inhibitor Mixture I and Proteasome Inhibitor I
(Calbiochem). Cell debris was pelleted in a microcentrifuge at
maximum speed, and the supernatant was mixed with the NASP:Reactigel
beads and rocked overnight at 4 °C. A column was made from the beads
and was washed extensively with several volumes of PBS before elution
with Immunopure elution buffer (Pierce). Collected fractions were
immediately neutralized with 1 M Tris-Cl, pH 8.5, and
elution of protein was tracked at 280 nm. Pooled fractions were
dialyzed in 10,000 molecular weight cutoff Slidealyzer cassettes
(Pierce) versus several changes of 1/20× PBS and lyophilized.
Isolation of Histones
H1 proteins from mouse myeloma cells or affinity-purified NASP
were isolated as described by Brown and Sittman (35). Core histones
were isolated from the perchloric acid-insoluble pellet by extraction
with 0.2 M H2SO4, precipitated with
trichloroacetic acid or 8 volumes of cold acetone, and washed with
acetone, 0.01 M HCl. All procedures were done at 4 °C.
Mouse histone H1 cDNAs (kindly provided by Dr. D. Sittman,
University of Mississippi Medical Center) were expressed from a pET-11d
vector. The histone H1 constructs were transformed into the BL21 (DE3)
bacterial host cell line and the recombinant proteins purified using
the T7 tag affinity purification kit (Novagen, Madison, WI) according
to the manufacturer's instructions.
Reverse-phase HPLC
Lyophilized histones were dissolved in 0.1% trifluoroacetic
acid and applied to a C-18 reverse-phase column (Waters,
DeltaPak, 15 µM, 300 Å, 3.9 × 300 mm) equilibrated
with acetonitrile and 0.1% trifluoroacetic acid. Proteins were eluted
at 28 °C with a multistep acetonitrile in 0.1% trifluoroacetic acid
gradient (0.7 ml/min). Gradients were developed from 18.5 to 100%
buffer B (90% acetonitrile in 0.1% trifluoroacetic acid) and 81.5 to 0% buffer A (10% acetonitrile in 0.1% trifluoroacetic acid). Protein elution was monitored at 220 nm. Fractions were collected, dried by
vacuum centrifugation, and analyzed by SDS-polyacrylamide gel electrophoresis.
Histone Labeling
Biotin labeling of histones was performed as described (4). The
binding of labeled histones to streptavidin-agarose was used to test
the efficiency of biotinylation, and bound and unbound fractions were
analyzed by SDS-polyacrylamide gel electrophoresis.
Co-immunoprecipitation
For binding experiments, rNASP (2 µg) was mixed with
biotin-labeled histone H1 recombinants in a 500-µl volume of
0.5× Tris-buffered EDTA and incubated for 1 h at room temperature
with gentle shaking. Precipitation was carried out with either
streptavidin or affinity-purified anti-rNASP antibodies. The samples
were loaded on 10-20% SDS-polyacrylamide gels.
Sequences of Somatic and Testicular Mouse NASP--
In the course
of our studies on tNASP, it was of interest to ask whether or
not the fertilizing spermatozoon would carry NASP into the unfertilized
egg (5). However, contrary to expectations (2), NASP was detected with
an anti-NASP antibody in the germinal vesicle of growing oocytes within
the ovary (see Fig. 9E). Subsequent screening of an ovarian
cDNA library resulted in the isolation of a cDNA encoding
sNASP, which differed from tNASP, suggesting that the ovarian cDNA
was an alternatively spliced transcript. As a result of this
observation, we began an extensive search for the presence of NASP in
embryonic and somatic tissues. Fig. 1A shows the relationship
between sNASP and tNASP cDNAs. The sNASP is identical to tNASP
except for the deletion of the two separate regions, shown in Fig.
1A, reducing the size of sNASP to 421 amino acids (45,752 Da) from the 773 amino acids of tNASP (83,934 Da). The sequence
for mouse tNASP (Fig. 1B), obtained by screening a mouse
testis cDNA library, yielded a 2543-bp consensus sequence. The
sequence for sNASP (Fig. 1B), obtained from the mouse ovary cDNA library yielded a consensus sequence of 1486 bp, which differs from tNASP cDNA in the 5' UTR as well as in the deletion of two portions of the coding region. To confirm the sequence of sNASP in
other somatic tissues, the complete sNASP coding region from spleen
cDNA, generated by PCR, was sequenced and found to be identical to
ovary sNASP (data not shown). The lack of two tNASP regions in the
sNASP mRNA and the presence of differing sequences in the upstream
portion of the 5' UTR of sNASP are most likely explained by
differential pre-mRNA splicing events and different promoters. Fig.
1C shows the relevant 5' UTR sequences for the two NASP
forms, indicating how the 5' ends of the transcripts are alternatively spliced from the genomic sequence. The 5' end of the testis mRNA arises by the splicing of two small exons (T1a and
T1b, Fig. 1C) onto the 5' end of exon one
(E-1). The unique portion of the 5' UTR of sNASP,
(S1, Fig. 1C) is contiguous with the sequence
(E-1) common to both sNASP and tNASP. A 3' splice is evident
at the end of S1 (Fig. 1C). The splice junctions of the
exons have been identified by sequencing genomic DNA (not
shown).
The deduced mouse tNASP protein sequence is 81% identical to human
tNASP and retains the three histone binding sites (Fig. 1A) previously identified in human tNASP (4). Additionally, the sNASP-deduced protein sequence has the same structural features found in all tNASP sequences (36), including a nuclear localization signal, a leucine zipper, and an ATP/GTP binding site.
Northern Blot Analysis--
Probing a multiple tissue Northern
blot (CLONTECH) of mouse tissues with the tNASP
coding region cDNA revealed a strong signal in mouse testis at 2.7 kb (<T) and a weaker signal at 1.7 kb (<S) in mouse liver, kidney,
spleen, brain, and heart (Fig.
2A). Probing 66-2 mouse
myeloma cell RNA revealed a strong 1.7-kb (<S) signal and a weaker
2.7-kb (<T) signal (Fig. 2B). To assay a wider variety of
tissues for NASP expression, a mouse mRNA master blot was probed with the tNASP coding region. As shown in Fig.
3A, testis mRNA showed the
strongest signal, at least five times that found in 11-day-old embryos.
Densitometeric analysis of the blot without the testis signal (Fig.
3B) revealed that the other most predominant signals were
found in the thymus, spleen, ovary, and all embryonic stages, with peak
expression on day 11. Epididymal tissue expressed approximately 20% of
the 11-day-old embryo level, whereas all other tissues, including
mRNAs from mouse brain, eye, liver, lung, kidney, heart, skeletal
muscle, smooth muscle, pancreas, thyroid, submaxillary gland, prostate,
and uterus, had weak signals (<20%).
PCR Analysis of NASP Transcript Tissue Distribution--
PCRs were
performed with primers specific for the coding region common to both
mouse sNASP and tNASP, employing a multiple tissue cDNA panel as
the source of cDNA templates, including cDNAs from heart,
brain, spleen, lung, liver, skeletal muscle, kidney, testis, and 7-, 11-, 15-, and 17-day embryos (Fig. 4).
Agarose gel analysis of the PCR products showed the presence of a
2319-bp tNASP band only in the testis (<T, Fig. 4A).
However, all tissues, including the testis, produced a 1263-bp sNASP
band (<S, Fig. 4A). The PCRs of testis RNA were clearly
inefficient in this assay, as the Northern blots show very little sNASP
compared with tNASP in the testis (Fig. 2A), probably
because of the large size of the amplified testis fragments. Therefore,
PCRs were performed using the cDNA panel and primers specific for a
725-bp sequence specific to tNASP from nucleotides 334 to 1059. Agarose
gel analysis of these products showed 725-bp bands only from the testis
and embryos, with a strong signal on day 11 but with faint signals in
the other embryonic stages (<T, Fig. 4B). Embryonic stage
cDNAs did not give a 2319-bp band when amplified with common
tNASP and sNASP coding region primers because of the inefficient
amplification of the tNASP cDNA.
Western Blot Analysis of Native NASP--
Western blots of lysates
(equal protein loading) from mouse testis, spleen, embryos, mouse 66-2 myeloma cell line, and mouse 3T3 cells were probed with anti-NASP
antibodies. A strong tNASP band of 138 kDa was present in the
testis, in myeloma cells, and in 3T3 cells, with a somewhat weaker band
in embryos (Fig. 5). An sNASP band of 62 kDa was present in all of the lysates (Fig. 5). These results support
the data presented in Figs. 2-4, which show that tNASP is present in
3T3 cells, myeloma cells, and embryos but not in the spleen (Figs.
2-4). Only sNASP is present in spleen.
The addition of protease inhibitors to the lysates did not prevent the
appearance of immunoreactive NASP breakdown products, particularly in
the myeloma extract. No significant bands were seen when blots were
probed with preimmune serum (Fig. 5). Recombinant 6-His-tagged
tNASP also migrated at 138 kDa (data not shown). The anomalous
migration of NASP and other similar nuclear proteins after
SDS-polyacrylamide gel electrophoresis has been observed and discussed
previously (1).
NASP mRNA Regulation during the Cell Cycle--
The level of
NASP mRNA was assayed in an asynchronous, exponentially growing
population of HeLa cells and in double thymidine-blocked cells allowed
to progress in synchrony through the cell cycle. In double
thymidine-locked cells at the G1/S border
(t = 0, Fig. 6A), there was little, if any,
sNASP and relatively little tNASP mRNA present. However, as cells
were released from the G1/S block and entered S-phase,
increased levels of NASP mRNA were detected (t = 2, 4, and 6 h, Fig. 6, A and C). Asynchronously
dividing cells expressed an intermediate level of NASP mRNA
(AS, Fig. 6A). As expected, histone H4 mRNA
levels were low at the G1/S border in blocked cells and
increased significantly as cells progressed through S-phase (Fig.
6A), particularly relative to the G3PDH controls.
In a second experiment, time points were taken from G1/S
through mitosis. After 6-6.5 h, NASP mRNA levels began to decrease and were almost undetectable by 10 h, late G2/M-phase
(Fig. 7, A and B).
Similarly, histone H4 mRNA levels began to decrease and were
relatively low by 10 h (Fig. 7). In Western blots of lysates from
parallel cultures of synchronous HeLa cells, NASP levels remained
constant throughout the cell cycle. After release from the thymidine
block, protein levels of sNASP and tNASP were not significantly
different from those at t = 0 (S>,
T>, Fig. 6B), consistent with NASP being a
relatively stable protein.
NASP and histone H4 mRNA levels were also assayed in 3T3 cells. In
cells starved for serum for 48 h (t = 0), very low
levels of NASP mRNA were detected (Fig.
8). After serum replenishment, some cells
entered S-phase at about 12 h, and the majority entered by 16 h, when there were significantly increased levels of both NASP and
histone H4 mRNA by 16 h (Fig. 8). Western blots of 3T3 cells
growing in culture demonstrated that NASP levels remained constant
throughout the cell cycle (Fig. 8B) in the same manner as
observed in HeLa cells (Fig. 6B). However, CV-1 cells that had been confluent for 8 days and were not dividing showed little or no
NASP present, indicating the eventual loss of NASP protein in
nondividing cells (Fig. 10E, see below). To ensure the
balanced loading of RNA in each well of each gel shown in Figs. 6-8,
all blots were also probed for the housekeeping gene
G3PDH. Similar G3PDH bands at all times
sampled indicated an equal loading of RNA at each time period.
Localization of NASP--
Fig. 9
demonstrates the localization of NASP in embryonic and somatic tissues.
In the 11.5-day mouse embryo (Fig. 9, A-D) most cells in
the embryo are positive for NASP. Nuclear staining was particularly
evident throughout areas of dense cell proliferation as seen in the
nasal process (NP) and brain (Fig. 9A) and in the cervical somites (S) and dorsal (D) root ganglia
(Fig. 9C). Control sections stained with preimmune serum
showed peroxidase-positive blood cells but no specific staining for
NASP (Fig. 9, B and D).
In the ovary, sNASP is present in the germinal vesicle ((GV)
nucleus) of primary oocytes in both primary and secondary follicles (Fig. 9E). Occasional staining was seen in oocytes within
primordial follicles. During the rapid proliferation of granulosa cells
in the growing follicle (F), NASP is evident in those cells
adjacent to the zona pellucida (corona radiata) and those lining the
developing antra (Fig. 9F).
In the spleen, sNASP is present in clustered groups of cells within
germinal centers of the white pulp (W, Fig. 9H).
The nuclear localization of NASP is clearly evident in these
proliferating lymphocytes (Fig. 9G). NASP is also seen in
cells scattered throughout the splenic cords of the red pulp
(R, Fig. 9H). Control sections showed no staining
(Fig. 9I).
In 3T3 cells growing in culture, NASP was primarily localized in the
nucleus (Fig. 10, A-D). In
these cultures, which were approximately 50% confluent, every cell
contained NASP (compare Fig. 10, A and B,
C and D). In contrast, cultures of CV-1 cells that had been confluent for 8 days had very few mitotic cells (compare
Fig. 10, E and F) and showed little or no NASP
present in either the cytoplasm or nucleus (Fig. 10E) of
most cells. This finding is consistent with the loss of NASP mRNA
as cells exit S-phase and slow degradation of the NASP protein in the
arrested cells (Fig. 7). However, the occasional CV-1 cell that
entered mitosis in these cultures showed strong staining for NASP
(arrow, Fig. 10E), indicating a strong
correlation between the presence of NASP and the growth state of the
cells.
Histone Interaction with NASP--
To characterize the functional
role of NASP as a histone-binding protein in somatic cells, the
histones bound to native NASP in myeloma 66-2 cells were characterized.
Native NASP was isolated from the soluble fraction of mouse myeloma
cells by affinity-purified anti-recombinant NASP antibody column
chromatography (data not shown). Histones were extracted from the
isolated native NASP complex and the histone types present in the
affinity-purified NASP complex identified by reverse-phase HPLC. Based
on our standard elution profiles of purified mouse myeloma cell core
and linker histones, only the linker histones (variants H1a, H1b,
H1c, and H1d/e) were found to co-purify with native
NASP (Fig. 11A). No core
histones were present in the sulfuric acid extracts. To confirm the
binding of H1 subtypes, they were expressed in Escherichia coli as recombinants, purified, labeled with biotin, and used to
reconstitute the complex with recombinant NASP. Fig. 11B
shows that NASP was detected in the streptavidin-precipitated fraction when biotin-labeled recombinant H1c and recombinant NASP were incubated
together. Conversely, by immunoprecipitation of the complexes with
anti-NASP antibodies, the histone H1c protein was associated with the
immunoprecipitated fraction (Fig. 11C).
In this study, we have demonstrated that the histone-binding
protein NASP is present in dividing somatic cells and coupled to the
cell cycle. Importantly, it appears that in myeloma cells NASP is
complexed only with H1 linker histones and that the complex can be
reconstructed in vitro with recombinant NASP and histones, implying that NASP functions to transport and/or store H1 histones in
this cell line. sNASP is a shortened version of the testicular form of
NASP, lacking two separate regions of the tNASP protein sequence (Fig.
1). This is a result of alternative splicing of the NASP pre-mRNA,
skipping exons 4 and 6 (data not shown). Significantly, sNASP retains
its characteristic features, namely two of the three histone binding
sites, a nuclear localization signal, a leucine zipper, and an ATP/GTP
binding site (36). We have further demonstrated the presence of NASP
mRNA and protein in a variety of mouse somatic tissues, including
mouse embryonic cells (Figs. 2-5, 9, and 10). Moreover, NASP mRNA
accumulation is cell cycle-dependent, increasing during
S-phase, concomitant with increased histone mRNA accumulation, and
declining in G2, even in continually cycling cells (Figs. 6-8). Consequently, since we have previously demonstrated that NASP functions as a histone-binding protein with a nuclear localization signal (4), our present data suggest that sNASP plays an important role
during the cell division cycle, providing for the more rapid and
efficient transport of histones into the nucleus. Additionally, NASP
might also provide for non-chromatin-bound storage of linker histones.
Additional support for the presence of somatic NASP was found from
searches of GenBankTM (37), where EST sequences have been
reported from various developmental stages, skin, melanoma,
neuroepithelium, and mammary gland. Somatic NASP-like sequences also
appear in the human EST data base, and in GenBankTM there
is a partial sequence for an "autosomal cDNA variant of human
testis nuclear autoantigenic sperm protein from primary human
peripheral blood mononuclear cells" (GenBankTM accession
number AF035191), which has the same overall structure as mouse
sNASP. tNASP EST sequences have been reported from the testis, embryo,
and transformed cells.
The coupling of NASP mRNA accumulation to the cell cycle (Figs.
6-8) and the nuclear localization of NASP in dividing 3T3 cells (Fig.
10) in large lymphocytes (lymphoblasts) in the spleen (Fig. 9G) and in cells in growing embryonic tissues (Fig. 9,
A and C) imply that NASP has a role in the
cell's normal progression through its division cycle. This is also
evident from our observation that nonmitotic CV-1 cells and nonmitotic
cells in various tissues throughout the body do not have detectable
quantities of NASP. Even though NASP protein levels remained constant
in cultured cells when they were arrested for only 1 day, they
ultimately did lose their NASP (Fig. 10, E and
F). Hence, NASP remains present in cycling cells for some
period of time after cells leave the cycle, but eventually (8 days in
the case of CV-1 cells) NASP is no longer detectable.
Although the DNA replication-coupled synthesis of histones has been
known for many years, early workers recognized that histone gene
expression could occur without DNA replication (38). Synthesis of mouse
oocyte histone H4 occurs independently of DNA replication during
oogenesis (12), and synthesis of testis-specific histones continues
during spermatogenesis, long after DNA synthesis for meiosis has
finished (4, 39, 40). A number of histone H1 proteins are also
synthesized independently of DNA replication (11), and H1 mRNAs,
particularly H1c and H1e mRNA, are present in nondividing cells
(41). The decoupling of DNA and histone synthesis in certain specific
cases such as spermatogenesis and oogenesis would seem to support the
need for non-chromatin histone storage proteins. The accumulation of
NASP is uncoupled from DNA replication during oogenesis (Fig. 9) and
spermatogenesis (5), tissues in which histone mRNA and protein
synthesis are also uncoupled from DNA replication. In somatic cells,
NASP mRNA appears to follow the histone pattern in that it is
coupled to DNA replication. Histone mRNA is regulated
transcriptionally and post-transcriptionally (41-43), and NASP
mRNA may also be similarly regulated. Indeed, it is quite possible
that common regulatory elements exist to ensure the concomitant
appearance and disappearance of both histone and NASP mRNAs as well
as the persistence of both NASP and histones when they are needed in
nondividing cells.
We thank Gail Grossman and the
Immunohistochemistry Core Facility of the Laboratories for Reproductive
Biology for their help and expertise.
*
This research 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF034610 (tNASP), AF095722 (sNASP).
¶
To whom correspondence should be addressed: Dept. of Cell
Biology and Anatomy, CB #7090, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090. Tel.: 919-966-5698; Fax:
919-966-1856; E-mail: morand@unc.edu.
Published, JBC Papers in Press, July 12, 2000, DOI 10.1074/jbc.M003781200
2
I. N. Batova and M. G. O'Rand,
unpublished observations.
The abbreviations used are:
NASP, nuclear
autoantigenic sperm protein;
sNASP, somatic form of NASP;
tNASP, testicular NASP;
PCR, polymerase chain reaction;
bp, base pair(s);
kb, kilobase;
PBS, phosphate-buffered saline;
G3PDH, glyceraldehyde-3-phosphate dehydrogenase;
FACS, fluorescence-activated
cell sorter;
HPLC, high pressure liquid chromatography;
UTR, untranslated region.
Characterization of the Histone H1-binding Protein, NASP, as
a Cell Cycle-regulated Somatic Protein*
,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C) was added to fix the cells (10 min, 20 °C). The cells were washed twice with cold PBS, blocked in 2%
normal goat serum (20 min), and washed with cold PBS (twice, 5 min
each). Rabbit anti-NASP or preimmune serum was added to each well
(1:500 in PBS and 0.1% bovine serum albumin), the cells were incubated
(45 min) and washed in PBS (3 times, 5 min each), and fluorescein
isothiocyanate-labeled goat anti-rabbit antiserum was added (1:1000 in
PBS and 0.1% bovine serum albumin) for a 30-min incubation followed by
three washes in PBS. The cells were also stained with propidium
iodide (0.01 mg/ml, 10 min) and washed (3 times in PBS) before the
plastic wells were removed.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (68K):
[in a new window]
Fig. 1.
Comparison of somatic and testicular
NASP. A, somatic and testicular NASP. The open
reading frames are identical except for the deletion of two separate
regions, reducing the size of tNASP from 773 to 421 amino acids in
sNASP. H1, H2, and H3, histone binding domains.
NLS, nuclear localization signal. B, the cDNA
sequence for mouse tNASP is 2543 bp with a start codon at nucleotides
92-94 and a TAA stop codon at nucleotides 2411-2413, encoding a
protein of 773 amino acids (Mr 83,934). The cDNA
sequence for somatic NASP is generated by substituting the dashed
underlined 5'UTR sequence (somatic 5'UTR) for the
single underlined portion of the sequence (testis 5'
UTR) and by deleting the two regions of the sequence that are
boxed. The sNASP cDNA contains the same start and stop
codons as tNASP and encodes a 421-amino acid protein (Mr 45,751). The histone binding domains are
single underlined, and the nuclear translocation signal is
double-underlined. Nucleotide numbers refer to
the tNASP sequence. The GenBankTM accession numbers are
AF034610 (tNASP) and AF095722 (sNASP). C, genomic
structure of NASP encoding the alternately spliced 5' UTRs of somatic
and testis NASP. The genomic sequence is diagrammed as the thin
centerline with exons shown as wider bands. In tNASP, the 5' UTR
includes T1a and T1b spliced onto the 5' end of
exon one (E-1). In sNASP, the 5' UTR includes E-1
and an additional 49 bp, S1, not present in tNASP. A TATA
box for the somatic form is indicated ~30 bp upstream of the
transcription initiation site.
View larger version (43K):
[in a new window]
Fig. 2.
Northern blot analysis of NASP expression The
blots were probed with a 32P-labeled cDNA representing
full-length tNASP. <T, tNASP, <S, sNASP.
A, 2 µg of poly(A)+ RNA from heart
(He), brain (Br), spleen (Sp), lung
(Lu), liver (Li), skeletal muscle
(Sk), kidney (Ki), and testis (Te).
B, 2 µg of poly(A)+ RNA from 66-2 mouse
myeloma cells.
View larger version (33K):
[in a new window]
Fig. 3.
Dot blot analysis of NASP expression.
A, RNA master blot (CLONTECH)
representing 18 different mouse tissues and 4 stages of embryonic
development normalized to 8 different housekeeping genes probed with a
32P-labeled cDNA representing full-length tNASP. The
tissues included are brain (Br), eye (Ey), liver
(Li), lung (Lu), kidney (Ki), heart
(He), skeletal muscle (Sk), smooth muscle
(Sm), pancreas (Pa), thyroid (Tr),
thymus (Th), submaxillary gland (Sb), spleen
(Sp), testis (Te), ovary (Ov),
prostate (Pr), epididymis (Ep), uterus
(Ut), and embryonic stages 7, 11, 15, and 17 (E7, E11,
E15, E17). B, relative image intensities were
quantified using Gel Expert software. The intensities are normalized to
the day 11 embryo signal. The testis signal (not shown on graph) was
more than five times greater than the E11 signal.
View larger version (74K):
[in a new window]
Fig. 4.
Mouse multiple tissue cDNA panel.
Testis and somatic NASP were detected by PCR. cDNAs included are
from the heart (He), brain (Br), spleen
(Sp), lung (Lu), liver (Li), skeletal
muscle (Sk), kidney (Ki), and testis
(Te), as well as embryonic (E) stages from day 7, 11, 15, and 17. Additional cDNAs were prepared using thymus
(Th) and ovary (Ov), but these were not
normalized to the expression of housekeeping genes. In both A
panels, primers specific to the common coding regions of
testicular (2319 bp) and somatic (1363 bp) NASP were used. In both
B panels, primers specific to a 725-bp sequence of tNASP
were used. Standards were: S1, 
X174, HaeIII
digest; and S2, 
gt11, HindIII digest. <T,
tNASP, <S, sNASP.
View larger version (23K):
[in a new window]
Fig. 5.
Western blot analysis of NASP protein
expression. Lysates were prepared from mouse testis
(Te), spleen (Sp), day 11 embryos
(Em), 66-2 mouse myeloma cells, and mouse NIH-3T3 cells
(3T3). 20 µg of each lysate were run on a 10-20%
gradient SDS-polyacrylamide gel electrophoresis, blotted, and probed
with either anti-recombinant NASP or preimmune serum. The
arrowheads indicate sNASP at 62 kDa and tNASP at 138 kDa.
View larger version (39K):
[in a new window]
Fig. 6.
Cell cycle regulation of NASP expression in
HeLa cells. A, Northern blots of RNA isolated from an
asynchronous, exponentially growing population of HeLa cells and from
double thymidine-blocked cells allowed to progress in synchrony through
the cell cycle, probed with full-length tNASP. AS,
asynchronously dividing HeLa cells. t = 0, G1/S border; t = 2, 4, and 6 h as the
cells progressed through S-phase. The right panel shows the
FACS analysis demonstrating the progression of the cells through the
cell cycle for the time points assayed. <T, tNASP,
<S, sNASP. B, Western blot analysis of NASP
present in parallel cultures of synchronous HeLa cell lysates up to
10 h following release from the thymidine block. Imm,
anti-NASP; Pre, preimmune serum. T>, indicates
tNASP at 138 kDa, and S> indicates sNASP at 62 kDa.
C, bar graph representing the relative intensities of the
tNASP (<T) and sNASP (<S) bands shown in
A.
View larger version (65K):
[in a new window]
Fig. 7.
Cell cycle regulation of NASP mRNA in
HeLa cells. A, Northern blots of RNA isolated from
double thymidine-blocked cells allowed to progress in synchrony through
the cell cycle, probed with full-length tNASP. As in Fig.
6A, t = 0, G1/S border. NASP
mRNA was assayed over a 10-h period. S, S-phase,
G2, G2-phase, M, mitosis,
<T, tNASP, <S, sNASP. B, bar graph
representing the relative intensities of the tNASP (<T) and
sNASP (<S) bands shown in A.
View larger version (72K):
[in a new window]
Fig. 8.
Cell cycle regulation of NASP expression in
mouse 3T3 fibroblasts. A, the level of NASP mRNA
was assayed by probing Northern blots with full-length tNASP from
asynchronously growing mouse 3T3 fibroblasts (AS) from a
population of cells that were synchronized by serum starvation. In
serum-starved cells (t = 0), very low levels of NASP
expression were detected. After serum replacement, cells entered
S-phase and showed a peak of NASP mRNA by t = 16 h. <T, tNASP, <S, sNASP.
B, Western blot analysis of NASP expression in
parallel cultures of synchronized 3T3 cells. <T indicates
tNASP at 138 kDa, and <S indicates sNASP at 62 kDa.
View larger version (95K):
[in a new window]
Fig. 9.
Immunohistochemical localization of
NASP. Mouse tissues were probed with affinity purified rabbit
anti-recombinant NASP antibodies (A, C, E, F, G, and
H) or preimmune rabbit IgG (B, D, and
I). Sections were counter-stained with toluidine blue.
A-D, 11.5 day mouse embryo. A and C,
nuclear staining is evident throughout the embryo, particularly in
areas of dense cell proliferation, including the nasal process
(NP) and brain, and in the somites (S) and dorsal
(D) root ganglia. Imm, anti-NASP; Pre,
preimmune serum. B and D, control
sections show peroxidase-positive blood cells (D) but no
specific staining for NASP. E and F, ovary.
E, NASP is present in the germinal vesicle (nucleus,
GV) of an oocyte in a secondary follicle. F,
during the rapid proliferation of granulosa cells in growing follicles
(F), NASP is evident in the nuclei of the corona radiata
cells and in granulosa cells lining the antrum. No staining was seen in
control sections. G-I, spleen. H, NASP is
present in clustered groups of cells within germinal centers of the
white pulp (W) and in cells scattered throughout the splenic
cords of the red pulp (R). Areas of white pulp
(W) are outlined. The box indicates an
area of H seen at higher magnification in G. I, no staining was seen in control sections. G,
NASP nuclear localization is clearly evident in the nuclei of
proliferating lymphocytes.
View larger version (56K):
[in a new window]
Fig. 10.
Immunofluorescent localization of NASP in
mouse 3T3 fibroblasts and CV-1 cells. Mouse 3T3 fibroblasts were
cultured to 50% confluence (A-D), and CV-1 cells were
maintained as a confluent, contact-inhibited culture for 8 days
(E and F). The cells were stained with either
propidium iodide (B, D, and F) or rabbit
anti-recombinant NASP antibodies followed by a fluorescein
isothiocyanate-labeled goat anti-rabbit IgG (A, C, and
E). 3T3 cells show mainly nuclear staining (A and
C). All cells in the culture contain NASP (A-D).
In contrast, after 8 days, most CV-1 cells showed little or no
NASP (E and F), except when cells were in the
mitotic cycle (arrow, E and F).
View larger version (23K):
[in a new window]
Fig. 11.
H1 linker histones and mouse myeloma
NASP. A, reverse-phase HPLC separation and
identification of mouse histones bound to affinity-purified native
NASP. Total mouse myeloma H1 variants (gray trace) and NASP
bound H1 variants (black trace). B, precipitation
of complexes of biotin-labeled recombinant histone H1c and recombinant
NASP with streptavidin followed by Western blotting with anti-rNASP
antibodies. C, immunoprecipitation of biotin-labeled
recombinant histone H1c and rNASP. Pre-assembled complexes (+ or NASP) were immunoprecipitated with anti-rNASP antibodies.
Western blotting was performed using alkaline phosphatase-conjugated
avidin. Histone H1c was detected in the antibody-precipitated
fraction.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
Present address: Dept. of Immunobiology, Bulgarian Academy of
Sciences, Sofia 1113, Bulgaria.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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O. M. Alekseev, D. C. Bencic, R. T. Richardson, E. E. Widgren, and M. G. O'Rand Overexpression of the Linker Histone-binding Protein tNASP Affects Progression through the Cell Cycle J. Biol. Chem., February 28, 2003; 278(10): 8846 - 8852. [Abstract] [Full Text] [PDF]
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 M. L. Whitfield, G. Sherlock, A. J. Saldanha, J. I. Murray, C. A. Ball, K. E. Alexander, J. C. Matese, C. M. Perou, M. M. Hurt, P. O. Brown, et al. Identification of Genes Periodically Expressed in the Human Cell Cycle and Their Expression in Tumors Mol. Biol. Cell, June 1, 2002; 13(6): 1977 - 2000. [Abstract] [Full Text] [PDF]
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A. Abderrahmani, M. Steinmann, V. Plaisance, G. Niederhauser, J.-A. Haefliger, V. Mooser, C. Bonny, P. Nicod, and G. Waeber The Transcriptional Repressor REST Determines the Cell-Specific Expression of the Human MAPK8IP1 Gene Encoding IB1 (JIP-1) Mol. Cell. Biol., November 1, 2001; 21(21): 7256 - 7267. [Abstract] [Full Text] [PDF]
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