From the Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, New York, New York 10021
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ABSTRACT |
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Mammalian RNA polymerase II holoenzymes are large
complexes that have been reported to contain, in addition to RNA
polymerase II, homologues of several yeast SRBs, various general
transcription factors, and other polypeptides. On the basis of its
copurification with an SRB-containing RNA polymerase II complex by
conventional chromatography procedures, we have identified a human
homologue of Drosophila TRF-proximal protein, designated
hTRFP, and isolated its cognate cDNA. Antibody specific for SRB7
can immunoprecipitate hTRFP and RNA polymerase II and, reciprocally,
antibody specific for hTRFP can immunoprecipitate RNA polymerase II and
SRB7. These data indicate that hTRFP is an integral component of an RNA
polymerase II-SRB complex. Whereas the precise function of hTRFP
remains to be determined, the hTRFP-containing RNA polymerase II-SRB
complex supports basal level transcription and, relative to RNA
polymerase II alone, enhances transcriptional activation by Gal4-VP16
in the presence of cofactor PC4. Thus, hTRFP may regulate transcription of class II genes through association with the RNA polymerase II-SRB complex.
Eukaryotic RNA polymerase II holoenzymes have been purified from
yeast and mammalian cells by a variety of procedures (reviewed in Refs.
1 and 2). The eukaryotic RNA polymerase II holoenzyme (3) was first
isolated from yeast extract as a multicomponent complex containing RNA
polymerase II, TFIIB, TFIIF, TFIIH, and a group of cofactors (SRBs)
originally identified by genetic analysis (4). Another yeast holoenzyme
lacking all general initiation factors except TFIIF was found to
contain RNA polymerase II in association with a subset of SRBs, the
regulatory factors GAL11, ROX3, SIN4, and RGR1, and a novel group of
MED proteins (5-10). These RNA polymerase II-associated proteins form
a stable complex, designated mediator, that can be dissociated from RNA
polymerase II by anti-CTD1
antibodies and that supports transcriptional activation with RNA
polymerase II (5). Two mediator subcomplexes have been described, one
consisting of RGR1, GAL11, SIN4, SRB7, MED1, MED3, MED4, MED7, MED8,
and MED9, and another consisting of MED6, ROX3, SRB2, SRB4, SRB5, and
SRB6 (10, 11).
The function of SRBs in transcription was initially indicated by the
isolation, in yeast, of SRB mutants that suppressed an RNA polymerase
II mutant containing only a portion of the C-terminal heptapeptide
repeat domain of the largest subunit (4, 12). SRB4 is required for
expression of most class II gene in yeast, suggesting that it has a
general function in regulating transcription (13, 14). On the other
hand, SRB2 and SRB5 are dispensable for cell viability, indicating that
they have specific functions in the regulation of certain nonessential
genes (13, 15). Similarly, certain MED proteins are necessary for
transcription of most genes whereas others are required only for
specific genes (10, 11). Therefore, the yeast SRB-mediator complex may
play a number of important roles in transcriptional regulation.
Dependent upon the specific purification procedures, RNA polymerase II
holoenzymes from mammalian cells vary in size from 2 to 4 MDa (reviewed
in Refs. 1 and 2). In general, these holoenzyme preparations have been
found to variably contain various SRBs and general transcription
factors (16-18), as well as a variety of other proteins that include
elongation factor SII (19), RNA processing factors (20), RNA helicase
(21), factors involved in DNA repair and recombination (18), histone
acetyltransferases CBP and PCAF (22), Tat cofactors (23, 24),
components of the SWI-SNF complex (25), and the tumor suppressor BRCA1
(26). This has led to the proposal that the regulation of transcription during growth and differentiation of mammalian cells may involve multiple RNA polymerase II holoenzymes. Apart from the above mentioned proteins, a number of unidentified polypeptides also co-purified with
mammalian holoenzymes. Here we describe the purification of a human RNA
polymerase II holoenzyme by a combination of conventional chromatographic steps and affinity chromatography with an antibody specific for SRB7. We identified a component of the holoenzyme as a
homologue of Drosophila TRF-proximal protein (27),
indicating that TRF proximal protein (hTRFP) may be involved in the
regulation of transcription via association with RNA polymerase II holoenzyme.
Protein Purification--
100 ml of HeLa nuclear extract in
BC100 were subjected to chromatography on phosphocellulose (P11) as
described (29). 40 ml of the 0.5 M KCl fraction were loaded
onto a hydroxylapatite column (15 ml), which was washed with 0.1 M phosphate buffer (pH 7.9) and step eluted first with 0.2, 0.3, and 1 M phosphate buffers (pH 7.9) and then with 0.5 M phosphate buffer (pH 6). The 0.5 M phosphate
buffer (pH 6) fraction was then subjected to chromatography on a
Sephacryl S-500 column (140 ml). The peak fraction was then immunoprecipitated with anti-SRB7 and anti-RPB6 antibodies coupled to
Protein A beads (40 µl) (33).
For large scale purification for polypeptide sequence analysis, 100 ml
of nuclear extract in BC300 were loaded directly onto an affinity
column (2 ml) containing immobilized anti-SRB7 antibody. The column was
washed with BC300 and BC1000 and subsequently eluted with 0.1 M glycine buffer (pH 2.5). The eluates were concentrated by
trichloroacetic acid precipitation, and proteins were separated by
SDS-PAGE and transferred onto a PVDF membrane. The proteins on the
membrane were visualized by Ponceau S staining. The 28-kDa polypeptide
was excised and digested with endoproteinase Lys-C. Peptides were
isolated by high pressure liquid chromatography and subjected to amino
acid sequence analysis. Derived sequences were used to identify an EST
clone that was sequenced by the Rockefeller University core facility.
Antibody Preparation--
Expression plasmids pRSET-his-hTRFP,
pRSET-his-SRB7, and pRSET-his-CDK8 were constructed by inserting the
full-length cDNA (amplified by polymerase chain reaction) into the
plasmid pREST-6his (33). This created, in each case, an NdeI
site at the N terminus and a BamHI (BglII for
CDK8) site at the C terminus. His-tagged hTRFP, His-tagged SRB7, and
His-tagged CDK8 were used to prepare antibody as described previously
(33). All antibody affinity columns were prepared with antigen-purified
antibodies as described (33). Anti-RPB6 was provided by Dr. Zhengxin Wang.
In Vitro Transcription--
Reactions were carried out as
described (29). TFIIA, TFIID (33), TFIIB, TFIIE/F/H fraction, core RNA
polymerase II, and His-tagged Gal4-VP16 were purified as described
(29).
Purification of a Human RNA Polymerase II-SRB Complex--
A
HeLa nuclear extract was fractionated by conventional chromatographic
steps according the scheme in Fig.
1A, with the holoenzyme being
monitored by immunoblot with antibodies against RNA polymerase II and
the previously described SRB7 component (17, 18) of human holoenzyme.
On the phosphocellulose column most of RNA polymerase II and SRB7 were
detected in the 0.5 M KCl step fraction. On the subsequent
hydroxylapatite column, RNA polymerase II was detected in the 0.2, 0.3, and 1 M phosphate buffer (pH 7.9) fractions and in the 0.5 M phosphate buffer (pH 6) fraction. SRB7 and CDK8 were detected mainly in the 1 M phosphate (pH 7.9) and the 0.5 M phosphate buffer (pH 6) fractions. Because RNA polymerase
II and SRB7 were present in a more highly purified state in the 0.5 M phosphate buffer (pH 6) fraction, which contained only
about 5% of total input protein, this fraction was subjected to
chromatography on a gel filtration column. RNA polymerase II and SRB7
appeared to copurify in fractions corresponding to a size of about 2 MDa. The peak fractions of RNA polymerase II and SRB7 were pooled, and
equal portions were subjected to independent immunoprecipitations with
antibody specific for SRB7 and with antibody for the RPB6 subunit of
RNA polymerase II. Each antibody immunoprecipitated a common population
of about 30 polypeptides (Fig. 1B), and the presence of SRB7
and RNA polymerase II in both immunoprecipitates was verified by
immunoblot analysis with anti-SRB7 and anti-RPB1 antibodies (data not
shown). The fact that each antibody immunoprecipitated most of the
major polypeptides in the input gel filtration fraction reflects the
high degree of purification (about 400-fold) of SRB7 and RNA polymerase
II at this step, whereas the few input proteins that were not
precipitated indicate the specificity of the immunoprecipitation. A
direct comparison of the anti-SRB7 immunoprecipitate with highly purified core RNA polymerase II revealed several polypeptides, specifically immunoprecipitated with anti-SRB7, with the same molecular
weight as the subunits of RNA polymerase II (Fig. 1C). As a
further control to show that the major group of polypeptides was
specifically precipitated by anti-SRB7 antibody, control beads containing only protein A and beads containing protein A and bound anti-SRB7 antibody were used to immunoprecipitate proteins directly from HeLa nuclear extract. SRB7 and RNA polymerase II, as well as the
28-kDa protein (see below), were specifically detected in the anti-SRB7
immunoprecipitates by Western blot analysis and silver staining (data
not shown).
Identification of Human TRF-proximal Protein--
Because the
antibody against SRB7 was previously reported to be able to
immunoprecipitate the holoenzyme (17), an anti-SRB7 antibody affinity
column was used to purify the holoenzyme on a larger scale. The
polypeptides of the holoenzyme were resolved by SDS-PAGE and
transferred to PVDF membrane. A 28-kDa polypeptide (Fig. 1B)
was excised from the PVDF membrane and digested with protease. Three
polypeptide sequences, SVQQTVELLTR, QGTFCVDCETYHTAAS, and
XXQVPVAGIR, were obtained. These peptide sequences were used as queries to search the NCBI data base of expressed sequence tags with
the TBLASTN homology searching program. All three peptides were found
in an open reading frame encoded by an EST cDNA clone (accession
number 531746). This clone was obtained from I.M.A.G.E., and the DNA
sequence of the 0.8-kilobase pair insertion was determined. The
complete open reading frame encodes a 209-amino acid protein that has
66% sequence similarity and 44% sequence identity with Drosophila TRF-proximal protein, whose cognate cDNA was
found upstream of the TRF gene (Fig. 2)
(27).
Association of Human TRF-proximal Protein with an RNA Polymerase
II-SRB Complex--
To investigate the functional role of hTRFP, the
full-length protein was expressed in bacteria, purified, and used for
polyclonal antibody production. Antibody specific for hTRFP, antibody
specific for CDK8 (a holoenzyme component equivalent to yeast SRB10) as a positive control, and purified rabbit antibody (from preimmune serum)
as a negative control were used for immunoprecipitation. As shown in
Fig. 3A, the anti-CDK8
(lane 4) and anti-hTRFP (lane 3) antibody, but
not preimmune serum (lane 2), specifically precipitated both
RNA polymerase II (RPB1 subunit) and SRB7. This result, together with a
reciprocal experiment in which hTRFP was immunoprecipitated with
anti-SRB7 antibody (as evident from Fig. 1B and from the purification protocol that generated the hTRFP employed for direct sequence analysis (see above)), indicates that hTRFP is associated with
RNA polymerase II and SRBs in vivo.
To further determine whether hTRFP is present in a preassembled protein
complex containing RNA polymerase II and SRBs, the hydroxylapatite
fraction was subjected to gel filtration on Sephacryl S-500, and
derived fractions were immunoblotted with antibodies directed against
hTRFP, the RPB1 subunit of RNA polymerase II, SRB7, and MED7. As shown
in Fig. 3B, four polypeptides are colocalized in fractions
(peak fraction 33) corresponding to a size of about 2 MDa, consistent
with the idea that they are present in a large complex.
Effect of the hTRFP- and SRB-containing RNA Polymerase II Complex
on in Vitro Transcription--
Because it was previously shown that
SRB-containing yeast holoenzyme and mediator complexes are able to
support transactivation (5) (reviewed in Ref. 1), we tested whether the
hTRFP-containing RNA polymerase II-SRB complex could support both basal
and activated transcription. Because our purified hTRFP- and
SRB-containing RNA polymerase II complex appears not to contain any
general transcription factors as judged by Western blot (data not
shown), an in vitro system reconstituted with ectopic
general transcription factors was used to assay transcription. To
compare the activity of the hTRFP-containing RNA polymerase II complex
with that of the 12-subunit core RNA polymerase II, the amount of the
complex assayed was adjusted to contain the same amount of RNA
polymerase II as the core RNA polymerase II preparation that was
assayed (based on quantitative immunoblot assays with anti-RPB1
antibody; data not shown). As shown in Fig.
4, the assay system has little or no basal transcriptional activity without RNA polymerase II (lanes 1 and 2), and the hTRFP-containing RNA polymerase
II-SRB complex can substitute for core RNA polymerase II in effecting
basal transcription from a minimal promoter (lanes 3 and
4). This complex could not support transcriptional
activation by Gal4-VP16 in the absence of the USA cofactor fraction
(28) or the recombinant PC4 (29) (lanes 3 and 4).
However, addition of either the USA cofactor fraction (lanes
7 and 8) or PC4 (lanes 5 and 6)
to the reactions enhanced activated transcription by Gal4-VP16. In
contrast, at the equivalent concentration tested, the core RNA
polymerase supported a low level of basal transcription (lanes
9 and 10) but was not able to support transcriptional
activation in the presence of recombinant PC4 (lanes 11 and
12). Consistent with previous data (28, 29), the core RNA
polymerase II did support transcriptional activation in the presence of
the USA cofactor fraction (lanes 13 and 14). This
latter result may reflect the presence in the partially purified USA
fraction not only of other co-activators, including PC2 (30), but also
substantial amounts of SRB7, CDK8, and hTRFP (data not shown). Thus,
SRB and mediator components in USA may synergize with other endogenous
coactivators (including PC4 and PC2), as observed for purified PC4 and
holoenzyme components, to effect high level activation in conjunction
with core RNA polymerase II. As observed previously (28, 29), the
overall increased level of transcription with USA relative to PC4
(lane 8 versus lane 6) reflects the effects of
USA components on basal transcription (lane 7 versus
lane 3 and lane 13 versus lane 9).
The present results demonstrate that hTRFP is a component of an RNA
polymerase II-SRB complex that can synergize with coactivators derived
from the USA fraction to enhance transcriptional activation. Our
results are also consistent with a recent report by Kornberg and
colleagues (31) of a mouse cell extract-derived mediator complex that
contains SRBs, MED proteins, and other polypeptides. The amino acid
sequence of a 28-kDa polypeptide in the mouse complex matched the amino
acid sequence of hTRFP, indicating that a mouse TRFP is present in the
mediator complex. Additionally, a human SRB- and MED-containing
cofactor complex that mediates activated transcription by RNA
polymerase II has recently been purified in our laboratory by affinity
methods (32), and this complex has also been found to contain
hTRFP2. The conservation of TRFP among Drosophila,
mouse, and man indicates that TRFP may have a specific role in
regulation of transcription. Because of
our inability to significantly deplete hTRFP from nuclear extract with
anti-TRFP antibody, the role of hTRFP in transcription remains unknown.
However, because deletion of the gene encoding TRFP in
Drosophila results in larval (but not embryonic) lethality (27), it is unlikely that TRFP is a general transcriptional cofactor
for transcriptional activation in Drosophila. Thus, it is
possible that hTRFP is involved in regulation of transcription of
specific gene(s) and that it functions via the RNA polymerase II-SRB complex.
INTRODUCTION
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Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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Fig. 1.
Purification of a SRB-containing RNA
polymerase II complex. A, purification scheme.
IP, immunoprecipitation. B, immunopurification of
an SRB-containing RNA polymerase II complex. Peak fractions (fractions
32, 33, and 34) containing both SRB7 and RNA polymerase II were pooled,
concentrated by Centricon-30 (Amicon), adjusted to BC300 buffer plus
0.1% Nonidet P-40, and used as input (lane 1) for
immunoprecipitation. Proteins precipitated by anti-SRB7 (lane
2) and anti-RPB6 (lane 3) antibodies were
resolved by SDS-PAGE and silver-stained. Arrow indicates
28-kDa protein. C, comparison of an SRB-containing RNA
polymerase II complex with core RNA polymerase II. The hydroxylapatite
fraction (pH 6.0) containing both SRB7 and RNA polymerase II was
concentrated by Centricon-30, adjusted to BC300 buffer plus 0.1%
Nonidet P-40, and used as input for immunoprecipitation. Proteins
precipitated by Protein A (lane 1) and anti-SRB7 (lane
2) and affinity-purified core RNA polymerase II (a gift from
Z. X. Wang) were resolved by SDS-PAGE and silver-stained.
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Fig. 2.
Sequence alignment of human TRFP and
Drosophila TRFP. Colons indicate
identities and dots similarities identified by the
MacVector.
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Fig. 3.
Western blot analysis of an hTRFP-containing
RNA polymerase II-SRB complex. A, coimmunoprecipitation
of hTRFP with RNA polymerase II. HeLa nuclear extract (lane
1) was incubated with beads containing protein A coupled with
preimmune serum IgG (lane 2), anti-hTRFP (lane
3), or anti-CDK8 antibody (lane 4). Immunoprecipitates
were resolved by SDS-PAGE and immunoblotted with anti-SRB7 and
anti-RPB1 antibodies as indicated. B, copurification of
hTRFP with RNA polymerase II and SRB7. Fractions derived from the
Sephacryl S-500 gel filtration column were resolved by SDS-PAGE and
immunoblotted with anti-hTRFP, anti-RPB1, anti-SRB7, and anti-MED7
antibodies as indicated.
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Fig. 4.
Analysis of an hTRFP- and SRB-containing RNA
polymerase II complex in an in vitro reconstituted
transcription system. Each reaction contained all basal
transcription factors and two DNA templates (30). As judged by Western
blot analysis with anti-RPB1 antibodies, the hTRFP- and SRB7-containing
RNA polymerase II and core RNA polymerase II preparations added to the
reaction contained equivalent amounts of RNA polymerase II. Reactions
were carried out in the presence or absence of activator Gal4-VP16 and
in the presence of PC4 or USA as indicated. Arrows indicate
the RNA transcripts from the specific DNA templates.
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ACKNOWLEDGEMENTS |
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We are grateful to our colleagues for discussions and Z. X. Wang for providing anti-RPB6 antibody and core RNA polymerase II. Peptide sequencing was performed by the Protein Sequencing Facility of The Rockefeller University.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AI37327 and CA42567 (to R. G. R.).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) AF097725.
These two authors contributed equally to this work.
§ Supported in part by a fellowship from the National Institutes of Health.
¶ Present address: DuPont Agricultural Products, Stine-Haskell Research Center, Bldg. 300, Newark, DE 19714.
To whom correspondence should be addressed. Tel.:
212-327-7600; Fax: 212-327-7949; E-mail:
roeder{at}rockvax.rockefeller.edu.
The abbreviations used are: CTD, C-terminal domain of the largest subunit of RNA polymerase II; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; EST, expressed sequence tag.
2 Y. Tao, W. Gu, H. Xiao, and R. G. Roeder, unpublished observation.
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REFERENCES |
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