(Received for publication, November 9, 1994)
From the
The /immediate early genes of herpes simplex virus are
regulated by the specific assembly of a multiprotein enhancer complex
containing the Oct-1 POU domain protein, the viral
-transinduction
factor
TIF, (VP16, ICP25), and the C1 cellular factor. The C1
factor from mammalian cells is a heterogeneous but related set of
polypeptides that interact directly with the
-transinduction
factor to form a heteromeric protein complex. The isolation of cDNAs
encoding the polypeptides of the C1 factor suggests that these proteins
are proteolytic products of a novel precursor. The sequence of the
amino termini of these polypeptide products indicate that the proteins
are generated by site-specific cleavages within a reiterated 20-amino
acid sequence. Although the C1 factor appears to be ubiquitously
expressed, it is localized to subnuclear structures in specific cell
types.
The regulation of the expression of the herpes simplex virus
/immediate early genes by multiprotein assemblies has provided a
model system for analysis of DNA-protein and protein-protein
interactions involved in the modulation of homeodomain function. The
enhancer elements of these genes nucleate the assembly of
transcriptional regulatory complexes (C1 and C2 complexes) containing
Oct-1, a member of the POU domain family,
TIF (
)(VP16,
ICP25, VMW65), the HSV-encoded
gene transactivator, and C1 (HCF),
a novel cellular
factor(1, 2, 3, 4, 5, 6, 7, 8, 9) .
The POU-specific and Pou-homeo subdomains of the Oct-1 protein
cooperatively recognize a homolog of the octamer element (ATGCAAAT) in
the 5` domain of the gene enhancer element (a response element,
ATGCTAATGATATTCTTTGG)(9, 10, 11) . This
protein has also been implicated in the regulation of the expression of
small nuclear RNA
genes(12, 13, 14, 15, 16, 17, 18, 19) ,
the cell cycle expression of the histone H1 and H2B
genes(20, 21, 22) , the expression of
lymphoid-specific
genes(23, 24, 25, 26, 27, 28, 29, 30, 31) ,
and the stimulation of DNA
replication(32, 33, 34) . Although the
promoters of these genes bind the Oct-1 protein, they are each
regulated in a distinct manner. Therefore, the specificity of the
transcriptional regulation is likely to be determined by the combined
interactions of assembled regulatory complexes in a manner that is
analogous to the formation of the HSV-specific C1 complex.
The HSV
transactivator,
TIF, contributes to the specificity of the
C1 complex in two manners. As a DNA-binding protein, it recognizes
sequences in the 3` domain of the
response element (9, 35) and is stabilized by a cooperative DNA-binding
interaction with the Oct-1
homeodomain(9, 35, 36) . In addition,
TIF discriminates between the homeodomains of Oct-1 and the highly
related Oct-2 through selective protein-protein
interactions(36, 37) . However, the interactions of
the Oct-1 homeodomain and
TIF are insufficient to form a stable
regulatory complex, thus requiring the presence of an additional
cellular factor (C1 factor; Refs. 2, 6, 9, 38, and 39).
The highly
conserved C1 factor activity has been detected in insect and mammalian
cells (6, 9) and interacts with high affinity with
TIF to form a heteromeric protein
complex(9, 39) . The C1 factor does not appear to bind
DNA independently, but rather, interacts with the components of the C1
regulatory complex(9, 38, 39) . Purification
of this activity from mammalian cells has shown that it is a
heterogeneous set of related polypeptides, ranging from 100 to 230
kDa(40) . The presence of stoichiometric quantities of the 100-
and 123-135-kDa proteins in the purified preparations suggested
that these polypeptides were subunits of the factor(40) . In
addition, a subset of the purified proteins specifically interacted
with
TIF in a phosphorylation-dependent manner(40) .
cDNAs have been isolated which encode the polypeptides of the C1 factor. Furthermore, the use of specific antiseras has shown that these proteins are components of the C1 factor and are present in the assembled C1 complex. The analyses of the cDNAs as well as amino-terminal sequencing of the purified proteins indicate that a novel 230-kDa precursor is proteolytically processed to yield the family of C1 factor polypeptides.
cDNA clones H10 and H40 were isolated from an oligo(dT)
and random primed HeLa cell cDNA library in gt11 (Clontech).
Nytran (Schleicher and Schuell) filter lifts were prepared for
hybridization according to the manufacturer's recommendations.
The filters were subsequently irradiated in a Stratagene UV-Linker;
baked for 30 min at 80 °C; rinsed in 3
SSC (SSC; 150 mM NaCl, 17 mM Na citrate), 0.1% SDS at 25 °C for 45 min
and at 55 °C for 60 min; prehybridized in 6
SSC, 5
Denhardt's solution, 0.5% SDS, 0.05%
Na
HPO
, 100 µg/ml salmon sperm DNA at 55
°C for 20 h; and hybridized in 6
SSC, 1
Denhardt's solution, 0.05% Na
HPO
, 100
µg/ml yeast tRNA, 2
10
cpm/ml of C1.probe A at
55 °C for 20 h. Following hybridization, the filters were washed
twice in 6
SSC, 0.05% Na
HPO
at 25
°C for 1 h, once in 2
SSC, 0.05% SDS at 25 °C for 30
min, and twice in 6
SSC, 0.05% Na
HPO
at
55 °C for 30 min. Both strands of the cDNA inserts were sequenced
by the dideoxynucleotide method at 50 °C in reactions containing
either deoxyguanosine or deoxyinosine according to the
manufacturer's recommendations (Sequenase 2.0, Stratagene).
cDNA clones PB5c, PB6e, and PB7e were isolated from a subsequent
screening of nytran filter lifts (gift of J. Lees) prepared from a
human pre-B cell cDNA library (gift of A. Bernards). The filters were
rinsed as above, prehybridized in 5 SSC, 50% formamide, 5
Denhardt's solution, 0.5% SDS, 125 µg/ml salmon sperm
DNA at 42 °C for 12 h and hybridized in 5
SSC, 50%
formamide, 1
Denhardt's solution, 0.5% SDS, 125 µg/ml
salmon sperm DNA, 75 µg/ml yeast tRNA, 2.0
10
cpm/ml of H10 cDNA insert at 42 °C for 12-16 h. The
filters were subsequently washed twice in 2
SSC, 0.1% SDS at 25
°C for 30 min, and three times at 68 °C for 30 min. cDNA clones
H302 and H303 were isolated from a custom cDNA library (Stratagene)
produced by the specific priming of HeLa cell poly(A)
mRNA with a mixture of the oligonucleotides
(5`-GCAGCGGTGCTGACCGCATGG-3`) and (5`-GCACAGCAGTGCCTCCAGG-3`). cDNA
clones designated FR were isolated from a human fetal retina cDNA
library (gift of M. McDonald) while those designated FL were isolated
from a human fetal liver cDNA library (Clontech) using probes generated
from the 5`-termini of cDNAs H5c, H302, H303, and FL150.2 in successive
screenings. Both strands of the cDNA inserts were sequenced as
described above.
For the production
of antigen, the purified fusion proteins were further resolved by
preparative SDS-PAGE, visualized by staining with KCl, and
excised(47) . Peptide antigens (CPEELQVSPGPRQQLPPRQ (amino
acids 1501-1518) and CTSKDSSGTKPANKRPMS (amino acids
2003-2019)) were synthesized by J. Coligan in the NIAID
Biological Resources Branch and were conjugated to keyhole limpet
hemocyanin (Pierce) according to the manufacturer's
recommendations. New Zealand White rabbits were innoculated with
GST-PB5c (Ab2125), GST-PB5cT (Ab2131), or a mixture of the
conjugated peptides (Ab2126) according to standard procedures (48) .
Glutathione-Sepharose purified GST-PB5c,
GST-PB5cT, and control GST proteins were further purified by
Superose 12 gel filtration chromatography, dialyzed against 100 mM Hepes (pH 7.5), and coupled to Affi-Gel 10 or Affi-Gel 15 (5.0 mg
of protein/1.5 ml of matrix) according to the manufacturer's
recommendations. Ab2125 and Ab2131 seras were diluted 1:5 (v/v) with 10
mM Tris (pH 7.5), passed through the control GST protein
column, and adsorbed to the appropriate fusion protein matrix. The
matrices were washed sequentially with 15 ml each of 10 mM Tris (pH 7.5) and 10 mM Tris (pH 7.5), 500 mM NaCl. Antibodies were eluted from the columns with 15 ml of 100
mM glycine (pH 2.5). Ab2126 was similarly purified by direct
absorption to a peptide affinity matrix produced by the coupling of the
peptide antigens to Sulfolink (Pierce) according to the
manufacturer's recommendation.
Figure 1: The cDNAs encoding the polypeptides of the C1 factor. The structure of the assembled cDNA encoding the C1 factor polypeptides is schematically illustrated with the positions of the 5`-untranslated domain (nucleotides 1-349), C1 open reading frame (C1 ORF, nucleotides 350-6455), and the 3`-untranslated domain (nucleotides 6456-8210). The overlapping clones that were isolated in this study are positioned above the complete cDNA and are designated according to the cDNA library from which they were derived as follows: FL, human fetal liver; FR, human fetal retina; H, HeLa cell; and PB, human pre-B cell.
Surprisingly, the assembled cDNA sequence (Fig. 2) contains a single ORF which is predicted to encode a polypeptide of 209 kDa. All of the tryptic peptides derived from the 100- and 123-135-kDa are found within the carboxyl-terminal portion of the ORF, whereas the peptides derived from the 68-kDa protein are clustered within the amino-terminal sequences, suggesting that the final C1 factor polypeptides are processed from a larger precursor protein. The 155-180- and 230-kDa proteins which are detected in lower abundance in preparations of the C1 factor (40) are likely to be additional products or intermediates of the complete C1 factor ORF.
Figure 2: The amino acid sequence encoded by the complete C1 factor ORF. The complete amino acid sequence of the C1 factor ORF is listed. The sequence of the tryptic peptides derived from the 68 kDa (C-8, 22, and 17) and the 100 or 123-135 kDa (A-34, 50, 35, 11, and 15) polypeptides of the purified C1 factor are underlined. The bold type designates the core of the six C1 factor amino acid reiterations while the positions of the amino termini of the 100- and 123-135-kDa C1 factor polypeptides are indicated with closed or open triangles, respectively. The portions of the ORF used to produce the Ab2125 (amino acids 1176-1787) and Ab2131 (amino acids 1176-1488) antiseras are indicated by the arrows, while the peptides used to generate the Ab2126 antisera are denoted with hatched lines.
HeLa cell nuclear extract and chromatographic fractions containing purified C1 factor were resolved by SDS-PAGE (Fig. 3, center). Parallel aliquots were transferred to nitrocellulose and reacted with either preimmune (lanes 4 and 5) or immune seras (Ab2125 (ORF amino acids 1176-1787), lanes 6 and 7). As shown, the immune sera specifically reacted with the 100-, 123-135-, 155-180-, and 230-kDa polypeptides of the purified C1 factor (compare Fig. 3, left and center). Similarly, the sera specifically reacted with the corresponding proteins in the HeLa cell nuclear extract, indicating that the heterogeneity of the family of polypeptides was not a result of the purification protocol.
Figure 3: Western blot of the C1 factor polypeptides with anti-C1 seras. Left panel, a silver-stained SDS-denaturing gel shows the resolution of the polypeptides of the chromatographically purified mammalian C1 factor (lane 3, (40) ). MW1 and MW2 (lanes 1 and 2) are protein markers whose molecular weight, in thousands, are indicated at the left. Center and rightpanels, Parallel aliquots of HeLa cell nuclear extract (lanes 4, 6, and 8-10) and purified C1 factor (lanes 5 and 7) were resolved in an SDS-denaturing gel, transferred to nitrocellulose, and probed with the antisera indicated at the top of the lane. Ab2125 preimmune and immune indicate the use of seras while Ab2125, Ab2126, and Ab2131 represent affinity purified antibodies.
The relationship of the various polypeptides of the purified C1 factor to the cloned C1 factor ORF was further investigated by Western blot of the purified factor with antisera produced against central and carboxyl-terminal domains of the C1 ORF. As illustrated in Fig. 3(right), antiseras Ab2125 and Ab2131 representing amino acids 1176-1787 and 1176-1487, respectively, reacted in a similar fashion with the 100-, 123-135-, 155-180-, and 230-kDa polypeptides of the C1 factor preparation (lanes 8 and 10). In contrast, Ab2126, produced against amino acids 1501-1518 and 2003-2119, reacted with a subset of the proteins which included the 100 and several of the 123-135-kDa polypeptides. In addition, this sera reacted weakly with the 230-kDa polypeptide (lane 9). Thus, the 100-, 123-135-, 155-180-, and 230-kDa polypeptides contain common determinants from the central region of the C1 factor ORF whereas the 100, a subset of the 123-135, and the 230-kDa proteins contain determinants derived from the carboxyl-terminal domain of the ORF. Furthermore, as expected, the antiseras did not react with the 68-kDa polypeptide as this protein is derived from the amino terminus of the ORF (Fig. 2, peptides C-8, C-22, and C-17) and thus would not contain determinants common to these antigens (Fig. 3, lanes 6-8).
Figure 4:
Supershift of the assembled C1 complex
with anti-C1 antibodies. Protein-DNA binding reactions were done as
described under ``Materials and Methods.'' The positions of
the Oct-1-DNA, C1, and C1-antibody complexes are indicated at the right. Reactions 1, 3, and 6 contained only
the Oct-1 protein while reactions 2, 4, 5, 7, and 8 contained Oct-1, TIF, and the purified C1 activity. In
addition, the reactions in lanes 3, 5, 6, and 8 contained 0.25 µg, while lanes 4 and 7 contained 0.1 µg of the affinity purified antibody that is
indicated at the top of the gel.
However, the most striking feature of the C1 factor ORF is the presence of 6 reiterations of a novel amino acid repeat ( Fig. 2and 5). As demonstrated below, this repeat represents a site of specific proteolytic processing of the C1 factor precursor to generate members of the family of C1 factor polypeptides.
Amino-terminal sequence of the 100-kDa polypeptide generated a mixture of two peptide sequences which were resolved to THETGTTHTATTVTSNM and PPPAASDQGEVE. These two positions correspond to sequences within and directly adjacent to the sixth amino acid repeat, respectively ( Fig. 2and Fig. 5). Similarly, amino-terminal analysis of the 123-135-kDa polypeptides generated peptide sequences containing TH(E/V)TGTT(H/N)TATTA(T/M)S and THETGTTHTATTATSNGG. These sequences represent a mixture of polypeptides whose amino termini lie within the second, third, and fifth repeat. Therefore, it is likely that these members of C1 factor polypeptides result from the specific proteolytic processing of the larger 230-kDa precursor precisely between positions 8 and 9 of the amino acid reiterations (Fig. 5).
Figure 5: The C1 factor amino acid repeats. The sequences of the six C1 factor amino acid repeats are aligned above the derived consensus. In each case, the highly conserved core sequence is set apart from the more divergent flanking residues. RPT-D represents an additional repeat, located between RPT-5 and RPT-6 in the C1 factor ORF, whose sequence diverges from the conserved core. The proposed site of the proteolytic cleavage(s) that generate the polypeptides of the C1 factor is indicated below the consensus (C1-PPS, C1 proteolytic processing site).
The 230- and 155-180-kDa
proteins which are detected in lower abundance in preparations of the
C1 factor probably represent full-length products and intermediates of
the complete ORF. In support of this, an mRNA of >7 kilobases,
sufficient to encode the precursor polypeptide, was detected in
Northern blot analyses of HeLa cell poly(A) mRNA (data
not shown).
Figure 6: Immunofluorescent localization of the C1 factor. The preparation, fixation, and staining of cells was done as described under ``Materials and Methods.'' In each case, cells were stained with affinity purified Ab2125. The cells in panels A-D are as follows: A, HeLa; B, MRC-5; C, HepG2; and D, 143.
Surprisingly, in addition to the diffuse nuclear staining, HeLa cells specifically exhibited localization of the C1 factor to subnuclear punctate structures (Fig. 6, panel A). These structures were evident in all of the cells and were present in a relatively constant number per nucleus. Hep-2 cells, a clonal derivative of the HeLa line, exhibited an identical pattern while several other human papilloma virus positive and negative cervical carcinoma lines (C4II, SiHa, C33A, Caski, and ME180) exhibited only the typical diffuse nuclear fluorescence (data not shown). In addition, neither fixation of the cells with organic solvents nor infection of the cells with HSV-1(F) for 2 h altered the localization of the C1 factor in these cells (data not shown). Finally, identical patterns were obtained using affinity purified Ab2131 and Ab2126 antibodies. Therefore, although the C1 factor appears to be highly conserved and widely expressed in many cell types, the specific nuclear pattern observed in the HeLa cells suggests that the function(s) of this protein complex may differ in various cell types.
Interestingly, the amino termini of the 100- and 123-135-kDa polypeptides lie within or directly adjacent to one of the six C1 factor amino acid reiterations, strongly suggesting that this family of polypeptides are derived by specific proteolytic processing of the larger precursor protein. In support of this, Wilson et al.(50) have also purified the mammalian C1 factor (HCF) and have obtained cDNA clones which contain the identical C1/HCF ORF. Upon expression of an epitope-tagged form of the complete ORF in mammalian cells, the entire set of C1/HCF polypeptides were detected by immunoblot. These results suggest that the processing of the C1 (HCF) precursor occurs in vivo.
The proteolytic processing of nuclear proteins is
unusual. In the case of the C1 factor, the function of the cleavage is
not apparent as the resulting products appear to remain complexed. The
native factor has a gel permeation size of 10 daltons and
sediments at 4-5 S (50) , suggesting that it is of high
molecular mass with an extended structure. Thus, it is likely that
several of the proteolytic cleavage products of the 230-kDa precursor
remain associated with one another in a fibrous structure. Furthermore,
as the C1 factor was purified in the presence of 3 M urea, the
association of the polypeptide constituents is unusually
stable(40) . These characteristics are distinct from those of
other transcription factors which are proteolytically processed such as
the large polypeptide of the basal transcription factor IIA (51, 52, 53) and the sequence specific
factors NF-
B (54) and sterol response element-binding
protein(55, 56) . In the latter examples, the
precursor polypeptides are cleaved in response to cytoplasmic
regulatory signals before transport to the nucleus. Whether the site of
the C1 factor processing is cytoplasmic or nuclear is unclear. However,
it is interesting that this cleavage occurs within a sequence that is
reiterated six times in the precursor polypeptide. This repeated
structure may be important for the frequency and efficiency of the
cleavage, or alternatively, may be utilized to generate products which
have distinct specific activities.
The isolation of a cDNA
encoding the C1 proteins will permit further analyses of the
interactions which dictate the regulated assembly of a multiprotein
transcription complex. Specifically, the interaction of this protein
with the HSV immediate early gene transactivator (TIF, VP16,
ICP25, VMV65) probably represents a critical determinant in the
expression of the
/immediate early genes and the lytic/latent
cycle of HSV. In this respect, it is of interest that the
123-135-, 155-180-, and 230-kDa proteins of the purified C1
factor specifically interacted with
TIF in a
phosphorylation-dependent manner. In contrast, the 68-kDa protein which
is derived from the amino terminus and the 100 kDa which is derived
from the carboxyl terminus of the C1/HCF ORF did not participate in
this interaction(40) . Therefore, the specific processing,
modification, or selective representation of the various C1 factor
polypeptides may be critical determinants in mediating the function of
this factor. The characterization of the composition and subcellular
localization of the C1 polypeptides may provide further insights into
the proteolytic mechanisms of regulation.