From the Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, New Hampshire 03824
Received for publication, October 5, 2000, and in revised form, December 1, 2000
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ABSTRACT |
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The CCR4-NOT transcriptional regulatory complex
affects transcription both positively and negatively and consists of
the following two complexes: a core 1 × 106
dalton (1 MDa) complex consisting of CCR4, CAF1, and the five NOT
proteins and a larger, less defined 1.9-MDa complex. We report here the
identification of two new factors that associate with the CCR4-NOT
proteins as follows: CAF4, a WD40-containing protein, and CAF16, a
putative ABC ATPase. Whereas neither CAF4 nor CAF16 was part of the
core CCR4-NOT complex, both CAF16 and CAF4 appeared to be present in
the 1.9-MDa complex. CAF4 also displayed physical interactions with
multiple CCR4-NOT components and with DBF2, a likely component of the
1.9-MDa complex. In addition, both CAF4 and CAF16 were found to
interact in a CCR4-dependent manner with SRB9, a component
of the SRB complex that is part of the yeast RNA polymerase II
holoenzyme. The three related SRB proteins, SRB9, SRB10, and SRB11,
were found to interact with and to coimmunoprecipitate DBF2, CAF4,
CCR4, NOT2, and NOT1. Defects in SRB9 and SRB10 also affected processes
at the ADH2 locus known to be controlled by components of
the CCR4-NOT complex; an srb9 mutation was shown to reduce
ADH2 derepression and either an srb9 or
srb10 allele suppressed spt10-enhanced
expression of ADH2. In addition, srb9 and
srb10 alleles increased
ADR1c-dependent ADH2
expression; not4 and not5 deletions are the
only other known defects that elicit this phenotype. These results suggest a close physical and functional association between components of the CCR4-NOT complexes and the SRB9, -10, and -11 components of the holoenzyme.
The CCR4-NOT complex is one of several large groups of proteins
involved in transcription (1). It consists of at least two complexes,
1.9 and 1.0 MDa in size, that are distinct from other large,
transcriptionally important groups of proteins, such as the
SNF/SWI complex, the SAGA complex, TFIID, and the RNA polymerase II holoenzyme (1-4).1 The
smaller of the CCR4-NOT complexes contains CCR4, CAF1 (POP2), the five
NOT proteins (NOT1-5), and two other proteins (1, 3, 4).1
These proteins can all be coimmunoprecipitated with antibody specific
to either CCR4, CAF1, or NOT proteins (1, 4). All of the components of
the 1-MDa CCR4-NOT complex also comigrate at 1.9 MDa following gel
filtration analysis (1, 4),1 and mutations in individual
components of the CCR4-NOT complex destroy the ability of these
components to migrate at 1.9 MDa (4).1 Although antibody
directed against the core CCR4-NOT proteins does not
coimmunoprecipitate any other proteins, a number of other proteins that
do not coimmunoprecipitate at their physiological concentrations with
CCR4 antibody do interact with the CCR4-NOT complex genetically and can
be coimmunoprecipitated when overexpressed. These proteins may possibly
be components of the larger 1.9-MDa complex and include such proteins
as DBF2, a cell cycle-regulated protein kinase (5), MOB1, a protein
that binds DBF2 and is involved in cell cycle regulation (6), and DHH1,
a putative RNA helicase (7). Both DBF2 and MOB1 have also been observed to migrate at 1.9 MDa following gel filtration
analysis.1
The internal arrangement of factors in the 1-MDa CCR4-NOT complex has
been studied (4). NOT1 appears to be the core component of the complex.
CAF1 binds to the central region of NOT1 and links CCR4 to the rest of
the NOT proteins. The C terminus of NOT1, in turn, contacts NOT2, NOT5,
and NOT4. The physical separation of CCR4 and CAF1 from NOT2, NOT4, and
NOT5 agrees with several phenotypic differences between these proteins
(1, 4). Whereas CAF1 is absolutely required for CCR4 to associate with
the 1-MDa complex (1, 4), CCR4 can still associate in the 1.9-MDa complex in the absence of CAF1 (1). It is likely, therefore, that
within this larger complex CCR4 is making contacts to proteins other
than ones found in the core 1-MDa complex. Based on the observation
that CCR4 can only immunoprecipitate other components of the core 1-MDa
complex, these other interactions in the 1.9-MDa complex may be more
susceptible to disruption.
The CCR4-NOT proteins have been found to affect gene expression both
positively and negatively (1, 8-13). Their action as repressors are
likely to be the result of the NOT proteins restricting access of
TBP2 to noncanonical TATAAs
(8, 9). NOT1 has been shown to associate with TBP (14); NOT5 interacts
with TFIID (15, 47), and NOT2 has been shown to associate with ADA2, a
component of the SAGA complex (16). Consistent with these results,
deletions of upstream sequences do not apparently affect the ability of
CCR4 to affect ADH2 expression (17), and it has been shown
that CCR4 acts at a post-chromatin remodeling step in affecting
ADH2 derepression (18).
The large sizes of the CCR4-NOT complexes and the possible mode of
action of these proteins at or near the TATAA suggest that these
complexes would be likely to interact with other proteins acting to
control initiation of transcription. In this study we report the
identification of two additional factors, CAF4 and CAF16, that interact
with the CCR4-NOT proteins and that affect transcription both
positively and negatively. Neither CAF16 nor CAF4 was a component of
the 1-MDa CCR4-NOT complex, but both proteins were present in a 1.9-MDa
complex, and their presence in this complex was dependent on CCR4.
CAF16 and CAF4, in turn, were found to interact with SRB9, -10, and
-11, components of the RNA polymerase II holoenzyme (19-23). SRB10 and
-11 are a cyclin-dependent protein kinase/cyclin pair that
are capable of phosphorylating the CTD of RNA polymerase II (21, 24,
25). Whereas none of these three SRB genes are essential,
the SRB9-11 proteins have been found to play both positive and
negative roles in transcription (20, 26-28), although they appear to
be more predominantly involved in repressing transcription (21-23). We
found that the SRB9, 10, and 11 proteins can coimmunoprecipitate the
CCR4 and NOT proteins, and defects in these SRB proteins affect
expression at the ADH2 locus in a manner similar to that
observed for defects in CCR4 complex components. These results indicate
a close physical and functional link between CCR4-NOT components and
the SRB9, -10, and -11 proteins.
Yeast Strains--
Yeast strains are listed in Table
I.
DNA Sequencing and Analysis--
CAF16 was sequenced
on each strand by double-stranded sequencing using Sequenase (U. S.
Biochemical Corp.). Sequence comparison analysis was performed at the
National Center for Biotechnology Information, using the BLAST network
service MegAlign version 1.05 (BLAST version, 1.8.1). Alignments were
performed by using the Clustal method available in the DNASTAR package
(DNA Star Inc.).
Gel Filtration Chromatography and Immunoprecipitation--
A
Superose 6 column HR10/30 was used according to the manufacturer's
instructions (Amersham Pharmacia Biotech). Two hundred µl of yeast
extract at a concentration of about 10 mg/ml was placed over the column
following clarification by centrifugation as described previously (1).
Running conditions were as described (1), and the standards were eluted
as follows: blue dextran (2000 kDa) at 10 ml; thyroglobulin (669 kDa)
at 15 ml; bovine serum albumin (66 kDa) at 17.5 ml.
Immunoprecipitations were carried out as described previously (3, 5).
Western analyses were conducted as described (1). Antibody to CAF16 was
raised against its C-terminal peptide, CCKRDNQIPDKEIGI, whereas
antibody to CAF4 was against CAF4 tagged with three copies of the HA1
epitope at its C terminus and integrated at the CAF4 locus
as described (46).
Gene Disruptions and Plasmid Constructions--
The
caf4::URA3 disruption plasmid was created by
replacing the BclI fragment of CAF4 (base pairs
851-2162) with a BamHI fragment of URA3. The
CAF16 disruption plasmid was constructed by removing nucleotides 368-706 base pairs of CAF16 following cutting
with NdeI, blunt ending with the large subunit of
Escherichia coli DNA polymerase (Klenow), and replacing it
with a HindIII fragment of URA3 (blunt-ended with
Klenow). The resultant plasmid JP7 was cut with M1uI and
SstI prior to transformation to replace the chromosomal copy
of CAF16.
LexA-CAF16 (pJp1) was constructed by placing an EcoRI
fragment, 2 kilobase pairs, from pRS316-CAF16 into the EcoRI
polylinker of pLexA87- (9). LexA-CAF4 was formed by ligating the
EcoRI fragment of CAF4 from ML4-3 (2 kilobase
pairs EcoRV piece of CAF4 cloned into the
SmaI site of pSP72) into LexA-202-2 (9) cut with
EcoRI.
Enzyme Assays and Genetic Manipulations--
ADH and
CCR4 Interacts with Two Novel Proteins CAF4 and CAF16--
A yeast
two-hybrid screen using LexA-CCR4 as a bait was conducted with a
library of yeast sequences fused to the B42 activator to identify
additional components of the multisubunit CCR4-NOT complexes. In
addition to identifying CAF1 and DBF2 (5, 13) as components or
associated factors of the CCR4-NOT complex, two additional proteins
(designated CAF4 and CAF16) were found to interact with LexA-CCR4 and
not with LexA alone (Table II, lines 2 and 3 compared with line 1). Deletion analysis indicated that an intact
leucine-rich repeat (residues 345-470) of CCR4 was required for its
interaction with both CAF4 and CAF16 (Table II, lines 7 and 8) but not
the N-terminal region of CCR4 (lines 5 and 6); the leucine-rich repeat
was also required for CCR4 interaction with CAF1, NOT1, and DBF2 (1, 3,
5, 13). Sequencing of CAF4 revealed it to encode a novel
protein (yeast protein YKR036c) containing seven WD40 repeats in its C
terminus (residues 320-659). CAF16 when sequenced in its
entirety was found to encode a protein (now designated YFL028c) that
shares significant homology to the ABC ATPase family of proteins (31,
32). ABC ATPases are principally found to play roles in transport
across membranes and as membrane receptors (33). CAF16 differs from
most of the eucaryotic ABC ATPase proteins in that it lacks the
transmembrane domains characteristic of this family. One other
eucaryotic ABC ATPase, EF3, involved in translational elongation also
lacks these signature transmembrane domains (34). CAF16 also contains
only one ABC ATPase domain, whereas most other eucaryotic ABC ATPases
contain two domains, suggesting that CAF16 may interact with itself, as
was confirmed by two-hybrid analysis (Table II, line 14).
In addition to two-hybrid interactions observed between CAF4 or CAF16
with CCR4, LexA-CAF4 was shown to interact specifically with B42-NOT1
and B42-DBF2 (Table II, lines 11 and 12). It should be noted that
LexA-CAF4 was capable of activating transcription weakly by itself
(line 13), a phenotype also observed with LexA-CCR4, LexA-CAF1, and
several LexA-NOT proteins (3, 9, 13, 11).
CAF16 and CAF4 Physically Associate with CCR4-NOT Components in
Vivo--
To determine if the observed two-hybrid interactions were
the result of in vivo physical association of CCR4 with
CAF16 and CAF4, coimmunoprecipitation analysis was conducted.
Immunoprecipitating CCR4 with anti-CCR4 antibody failed, however, to
coimmunoprecipitate specifically B42-CAF16 or B42-CAF4 or their cognate
unfused proteins (data not shown). These data suggest that CAF16 and
CAF4 are not components of the 1-MDa CCR4-NOT complex since all of the
NOT proteins and CAF1 can be immunoprecipitated with CCR4 in this core
complex (1, 3, 4, 13).1 Moreover, neither CAF16 nor CAF4
was found to be present in a purified 1-MDa CCR4-NOT
complex.1
To determine whether the CAF16 and CAF4 proteins associated in the
1.9-MDa CCR4-NOT complex, gel filtration analysis was conducted. Following Superose 6 chromatography a subset of the CAF16 protein was
found to migrate at 1.9 MDa, coincident with the size of the 1.9-MDa
CCR4-NOT complex (Fig. 1, top
panel). A similar analysis with CAF4 protein tagged with the HA1
epitope showed that CAF4-HA migrated at 1.9 MDa (Fig. 1, 2nd
from top panel). Deletion of CCR4 was found to
remove effectively CAF16 and CAF4 from the 1.9-MDa complex (Fig.
1, top two panels). A ccr4 deletion did not have this effect on CAF1 or CAF40 (another component of the 1-MDa CCR4-NOT complex) (Fig. 1, bottom two panels), nor did it have this
effect on DBF2, which is also a presumed component of the 1.9-MDa
CCR4-NOT complex (Fig. 1, middle panel). The effect of the
ccr4 deletion on CAF16 and CAF4 migration at 1.9 MDa
supports the physical presence of CAF16 and CAF4 in the larger CCR4-NOT
complex and that CCR4 is required for these proteins to associate in
this complex. The observation that a majority of the CAF16 in the cell
is not apparently in the 1.9-MDa complex suggests that CAF16 may have
other functions than those dealing with CCR4 or that the CAF16
association with CCR4 is unstable.
The above results suggest that CCR4 interacts with CAF16 and CAF4 but
that CAF16 and CAF4 are external to the core 1-MDa CCR4-NOT complex.
Since CAF1 is absolutely required for CCR4 association in the 1-MDa
CCR4-NOT complex, we subsequently tested whether CCR4 interaction with
CAF16 and CAF4 was dependent on CAF1. In a caf1 deletion
strain, LexA-CCR4-(1-837) interacted with B42-CAF16 (280 units/mg
Although CAF4 was not in the 1-MDa CCR4-NOT complex, we analyzed
whether CAF4 when overexpressed was capable of immunoprecipitating with
DBF2 and NOT1 based on their strong two-hybrid interactions (Table II,
lines 11 and 12). Immunoprecipitating LexA-CAF4 with anti-LexA antibody
was capable of coimmunoprecipitating B42-DBF2 (Fig.
2, lane 9) and B42-NOT1
(lane 10). As a control the comparably expressed B42-SIP1
(lane 8), a non-CCR4 or DBF2-associated factor (31), did not
immunoprecipitate with LexA-CAF4, indicating the B42 moiety was not
responsible for interacting with LexA-CAF4. In addition,
immunoprecipitating LexA alone did not bring down B42-DBF2 (Fig. 2,
lane 7) or B42-NOT1 (1, 5) demonstrating that it was the
CAF4 moiety that coimmunoprecipitated with DBF2 and NOT1. These
combined results demonstrate that CAF4 and CAF16 can associate in
vivo with components of the CCR4-NOT complex and confirms the
two-hybrid interactions between CCR4, CAF16, CAF4, NOT1, and DBF2.
Phenotypic Effects of caf4 and caf16 Alleles--
Disruption of
the chromosomal loci of CAF4 and CAF16 was
conducted, and the resultant phenotypic effects were analyzed. Unlike ccr4 or caf1 alleles neither caf4 nor
caf16 affected ADH2 expression, suppressed
spt10-enhanced ADH2 expression, resulted in
caffeine sensitivity, produced high or low temperature sensitivity,
reduced growth in the presence of metal ions, or displayed defects on growth in nonfermentative carbon sources (data not shown). Both caf4 and caf16 alleles, however, did result in
increased ADH1 gene expression as did the ccr4-
and caf1-mutated alleles (Table III).
The caf16 and caf4 alleles also affected the
expression from several lacZ reporters (1). Deleting
caf16 reduced the function of HO-lacZ and
FKS1-lacZ reporters by 3-4-fold while having no effect on
GAL1-lacZ (data not shown). A caf4 deletion, in
turn, increased GAL1-lacZ expression by 3-fold and reduced
the expression of the HO-lacZ and FKS1-lacZ
reporters by 2-fold (data not shown). These data confirm that CAF16 and
CAF4 are required for full or proper gene expression in certain
promoter contexts.
We had previously shown that caf1 and ccr4 reduce
the transcriptional function of several different LexA-activators by
2-4-fold (3, 13). We similarly found that the ability of a similar set
of LexA activators to activate transcription was diminished 2-4-fold
by caf4 and caf16 alleles (data not shown). These
results confirm a role for CAF4 and CAF16 in activated transcription
and are similar in degree and nature to the effects of ccr4
or caf1 disruptions on LexA activator function (3, 13).
Moreover, the caf4 and caf16 effects on LexA
activator functions appeared to be independent of the type of activator
used, suggesting they are affecting a function common to the core
transcriptional process.
CAF16 and CAF4 Interact with SRBs and CCR4 Is Required for This
Interaction--
To further our understanding of CAF16 function,
LexA-CAF16 was used as a bait in a two-hybrid screen. Only two B42
fusion proteins were identified that interacted specifically with
LexA-CAF16 as follows: B42-CAF16 (data not shown; see also Table II,
line 14) and B42-SRB9 (Table IV, line 1).
SRB9 is a component of the RNA polymerase II holoenzyme, and various
genetic and biochemical data have suggested that SRB9, -10, and -11 function closely together in a subcomplex within the holoenzyme (20,
22, 23). B42-SRB9 failed to interact with LexA alone (Table IV, line 3)
and a number of other LexA fusions tested (data not shown), indicating
that its interaction with LexA-CAF16 was specific to the CAF16 moiety. Surprisingly, B42-SRB9 interacted with several other known components of the 1-MDa CCR4-NOT complex as follows: LexA-CAF1 (line 4), LexA-CCR4-(496-837, line 8), and LexA-NOT2 (line 10). In addition, LexA-CAF4 (line 6) displayed a very strong interaction with B42-SRB9. Moreover, B42-DBF2, -NOT1, -NOT2, and -CAF4 interacted with LexA-SRB10 (Table IV, lines 13-16). The above two-hybrid results indicated a
cluster of interactions in which, for example, CCR4 interacted with
CAF16, CAF16 with SRB9, SRB9 with SRB10, and SRB10 with NOT1 and NOT2.
Such clusters have been shown to have biological relevance and to be
common in or indicative of multisubunit complexes and signaling and
development pathways (35, 36).
Because we had shown that CCR4 is required for CAF16 association in the
1.9-MDa complex, we tested whether CCR4 was required for CAF16 and CAF4
association with SRB9. A ccr4 disruption completely abrogated the two-hybrid interaction between B42-SRB9 and either LexA-CAF4 or LexA-CAF16 (Table V).
Deleting ccr4 did not in general have this effect on
two-hybrid interactions (3). For instance, LexA-CAF1 interactions with
B42-DBF2 were only affected 2-fold by a ccr4 disruption
(Table V), an extent expected for ccr4 effects on general
transcriptional activator function (2-3-fold, Ref. 3). In contrast, a
caf1 deletion did not have a similar effect on LexA-CAF4 or
LexA-CAF16 interactions with B42-SRB9 (Table V). Since a
caf1 deletion does not completely remove CCR4 from the 1.9-MDa complex (1) and is not required for CCR4 association with
either CAF4 or CAF16, these results suggest that the CAF16 and CAF4
interactions with SRB9 are occurring through the larger CCR4 complex
and are mediated by or require CCR4.
CCR4 Complex Components Can Be Immunoprecipitated by SRB9, -10, and
11--
We subsequently examined the physical interaction between
proteins associated with the CCR4 complex and the SRB9, -10, and -11 proteins using coimmunoprecipitation. Initially, to confirm the
two-hybrid interactions between LexA-CAF4 and B42-SRB9, yeast extracts
expressing LexA-CAF4 and either B42-SRB9 or B42-SIP1 were treated with
LexA antibody, and the resulting immunoprecipitates were analyzed by
Western analysis using HA1 antibody. B42-SRB9 was found to
immunoprecipitate with LexA-CAF4 (Fig.
3a, lane 4), whereas B42-SIP1
failed to coimmunoprecipitate (Fig. 2, lane 7). When the
immunoprecipitation was conducted from extracts expressing just LexA
alone and B42-SRB9, no B42-SRB9 was coimmunoprecipitated with LexA
(Fig. 3a, lane 6). Similarly, LexA-CCR4-(496-837) and B42-SRB9 coimmunoprecipitated after extracts containing these fusions
were treated with LexA antibody (Fig. 3a, lane 5).
The above results suggest that CAF4 and CCR4 when overexpressed can
associate physically with SRB9. Because immunoprecipitating any of the
components of the 1-MDa CCR4-NOT complex components when expressed at
their normal physiological concentrations does not coimmunoprecipitate
the SRB proteins, it is likely that the interactions between SRB
proteins and CCR4-associated factors are weak and may only be observed
under some immunoprecipitation conditions. We therefore explored
whether LexA-SRB fusions could coimmunoprecipitate CCR4-associated
components. Immunoprecipitating LexA-SRB10 with anti-LexA antibody
coimmunoprecipitated B42-DBF2 (Fig. 2, lane 10; Fig.
3b, lane 2) but not B42-SIP1 (Fig. 2, lane 9). As
a control, immunoprecipitating LexA alone with LexA antibody did not
coimmunoprecipitate B42-DBF2 (Fig. 2, lane 6; Fig. 3b, lane 1). In addition, immunoprecipitating LexA-SRB10
immunoprecipitated CCR4 (Fig. 3b, lane 2). Other SRB
components such as SRB9 and SRB4 also coimmunoprecipitated with
LexA-SRB10 (Fig. 3b, lane 2). Similarly, when LexA-SRB11 was
immunoprecipitated with anti-LexA antibody, NOT1, B42-NOT2 (Fig.
3c, lane 2), and CCR4 (data not shown) were found to
coimmunoprecipitate. Immunoprecipitating LexA alone failed to bring
down CCR4, SRB4, SRB9 (see Ref. 17; Fig. 3b, lane 1),
B42-NOT2, or NOT1 (Fig. 3c, lane 1) (31). We also found that
immunoprecipitating LexA-SRB9 coimmunoprecipitated NOT1 and lesser
amounts of B42-NOT2 and CCR4 (data not shown). This group of
experiments demonstrate that DBF2, CAF4, CCR4, NOT1, and NOT2 can
physically associate with several components of the SRB complex
components, although the strength of these associations depended on
which LexA-SRB protein was used to conduct the immunoprecipitation.
srb9 and srb10 Alleles Share Phenotypes at the ADH2 Locus
Associated with Defects in CCR4 Complex Components--
The
observation that components of CCR4 complex can contact the SRB9, -10, and -11 proteins suggests that they should behave similarly in
affecting some transcriptional events. CCR4, CAF1, and NOT proteins are
required for full ADH2 derepression. ccr4, caf1, not2, and dbf2 alleles
are also the only known suppressors of the enhanced ADH2
expression caused by an spt10 allele (1, 5, 10, 13, 17). We
examined, therefore, the effects of disrupting SRB9,
SRB10, and SRB11 on these two phenotypes. As shown in Table VI, line 2 compared with
line 1, an srb9 disruption reduced by 2-fold the ability of
ADH2 to derepress. Most importantly, srb9 reduced
by about 3-fold the ability of an spt10 allele to enhance
ADH2 expression under glucose-repressed conditions (compare lines 5 and 4). Whereas an srb10 disruption did not have an
effect on ADH2 derepression (data not shown), it did
suppress spt10-enhanced ADH2 expression (Table
VI, line 7 compared with line 6). An srb11 deletion did not
elicit these phenotypes (data not shown). These results suggest that
the mechanism by which CCR4 activates expression at ADH2 and
suppresses spt10 effects at the ADH2 locus is
shared by SRB9 and SRB10.
The SRB9, -10, and -11 proteins have been found to act as repressors in
certain promoter contexts (19, 20, 21, 37), as have the NOT, CCR4, and
CAF1 proteins (1, 9). Deletion of either the NOT4 gene or
the NOT5 gene caused a 2-3-fold increase in
ADR1c-activated ADH2 expression under
glucose growth conditions (Table VI, lines 11 and 12 compared with line
8). Both srb9 and srb10 defects resulted in
similar 2-fold increases in ADH2 expression in an
ADR1-5c-containing strain (Table VI, lines 9 and
10). This repressive effect displayed by NOT4, NOT5, SRB9, and SRB10 in
an ADR1-5c context is unique to these following
factors; deletion of SNF/SWI components (17), or SAGA components GCN5,
ADA2, and SPT3 (38) do not result in this phenotype.
Overproduction of CAF4 Specifically Impairs LexA-SRB11
Activation--
The above results indicate that the SRB9, -10, and -11 proteins can be physically and functionally associated with the
CCR4-NOT group of proteins. No synthetic phenotypes were observed,
however, when these SRB genes were deleted in combination
with either CCR4, CAF1, CAF4, or
CAF16 (data not shown). We did observe, however, that
overexpression of a C-terminal portion of CAF4 (residues 544-659)
specifically impaired the ability of LexA-SRB11 to activate the
LexA-LEU2 reporter (Table
VII). This CAF4 fragment had no effect on
the function of LexA-CAF1, LexA-SRB9, or other LexA activators (Table
VII; data not shown). Overexpression of larger fragments of CAF4 did
not result in this phenotype, suggesting that residues 545-659 of CAF4
were specifically blocking an interaction of LexA-SRB11 important to
its recruitment of the transcriptional machinery to the
LexA-LEU2 promoter (37, 38).
Identification of Two New Factors That Associate with CCR4--
We
have identified two novel factors that can physically interact with the
CCR4-NOT proteins, CAF4 and CAF16. CAF4 interacted in the two-hybrid
assay with CCR4 and NOT1, whereas CAF16 interacted with CCR4. These
interactions were shown to be the result of in vivo physical
interactions by two pieces of evidence. First, CAF4 immunoprecipitated
with NOT1. Second, the ability of CAF16 and CAF4 to associate in a
1.9-MDa complex following gel filtration analysis was dependent on the
presence of CCR4. These experiments suggest that CAF4 and CAF16 are
associated with or components of the CCR4-NOT complex. However, neither
CAF4 nor CAF16 was a component of the 1-MDa CCR4-NOT complex. In
contrast, the gel chromatography results suggest that CAF16 and CAF4
are components of the 1.9-MDa CCR4-NOT complex. The ability of CAF16
and CAF4 to interact with CCR4 in the two-hybrid assay in a
cafl deletion background confirms that CAF16 and CAF4 lie
outside of the core 1-MDa CCR4-NOT complex. CAF4, in turn, physically
interacted with DBF2 which is also known to associate with CCR4-NOT
proteins but not to be present in the 1-MDa CCR4-NOT complex. The
identification of DBF2, CAF4, and CAF16 in the 1.9-MDa CCR4-NOT complex
will require its purification and characterization of its constituents. Our previous analysis has shown that the 1.9-MDa CCR4-NOT complex is
not identical to the SRB holoenzyme (1), and others (13, 40) have not
identified CCR4-NOT components in the SRB polymerase II holoenzyme.
A number of CCR4-NOT phenotypes were analyzed using caf4 and
caf16 deletions, but in general caf4 and
caf16 did not affect processes controlled by the CCR4-NOT
proteins. We did observe, however, that deletions in both of these
genes resulted in enhanced ADH1 gene expression, a phenotype
shared by ccr4 and caf1 defects. HO-lacZ and FKS1-lacZ reporter gene expression
were also reduced 2-3-fold by caf4 and caf16
deletions similar to the 2-fold differences observed for
ccr4 and caf1 defects (1). These concurrences suggest that both CAF16 and CAF4 may share a subset of phenotypes with
CCR4-NOT proteins.
The SRB9, -10, and -11 Proteins Interact with Components of
the CCR4-NOT Complex and Associated Factors--
To extend our
understanding of CAF16, a two-hybrid search demonstrated that it could
interact with SRB9. CAF4, in turn, was found to associate with the SRB9
and SRB10 proteins, components of the yeast RNA polymerase II
holoenzyme. CCR4, CAF1, the NOT proteins, and DBF2 were subsequently
shown to interact with the SRB9, -10, and -11 proteins. These
interactions were indicated by two-hybrid analysis and by
coimmunoprecipitation studies. For example, LexA-CAF4 and LexA-CCR4
coimmunoprecipitated B42-SRB9, whereas immunoprecipitating LexA-SRB10
brought down CCR4 and B42-DBF2, and immunoprecipitating either
LexA-SRB11 or LexA-SRB9 coimmunoprecipitated NOT1, B42-NOT2, and CCR4.
The two-hybrid interactions between CAF4 and CAF16 and that of SRB9 was
further shown to be absolutely dependent on the presence of CCR4 in the
cell (Table VI). This CCR4 dependence could be explained by either a
CCR4 requirement for the integrity of the SRB9 interaction with CAF4
and CAF16 or CCR4 being an intermediary of this interaction. These
observations indicate that components of CCR4-NOT complex, CAF4, and
CAF16 can contact the SRB9, -10, and -11 proteins. Relatedly, the
ccr4 and srb8 to -11 mutations were
recently identified in causing increased expression from a defective
promoter, drawing an additional link between CCR4 and these SRB
proteins (39).
It is clear from these immunoprecipitation studies, however, that the
strength of the interactions between SRB9, -10, and -11 and that of
CCR4-NOT components and/or CAF16 and CAF4 is not strong. In most cases
the interactions were only observed when the proteins were
overexpressed. Interactions between large complexes such as the 1.9-MDa
CCR4-NOT complex and the yeast holoenzyme might be expected to be weak
and not as stable as interactions that are within the complex.
The SRB9, -10, and -11 Proteins Are Phenotypically Related to the
CCR4-NOT Complex and Associated Factors--
The physical interactions
described above suggest that the SRB9, -10, and -11 proteins and the
CCR4-NOT complex should be involved in regulating some similar
processes in the cell. Defects in the SRB9, -10, and -11 factors have
generally indicated that they act as repressors, although they have
been shown to function as activators in some cases (20, 25, 37).
Similarly, CCR4, CAF1, and the NOT proteins have both positive and
negative effects on transcription (1), although in most cases the NOT
proteins are considered as repressors (9). We found that at the
ADH2 locus, SRB9 is required for full derepression of
ADH2 as are CCR4, CAF1, and the NOTs (1, 10, 13). Components
of the core CCR4-NOT complex and DBF2 are also the only known
suppressors of spt10-enhanced ADH2 expression (1,
5, 3, 10, 13) and defects in either SRB9 or SRB10 elicited this same
phenotype. In these above cases the SRB9 or SRB10 proteins function as
activators, but their repressive function was also observed at the
ADH2 promoter. srb9, srb10,
not4, and not5 deletions all shared the phenotype of enhancing ADR1-5c activity under glucose growth conditions.
It should be noted, however, that many of the effects of
srb9, ccr4, caf16, and caf4
or various processes were weak (2-4-fold effects). Although it is
possible that these changes could arise from indirect effects, it
should be stressed that many of the processes involved in
activation/repression appear to occur through redundant mechanisms.
There are several activator and repressor complexes (e.g.
SAGA, TAFIIs, SRBs, SNF/SWI, and SSN6-TUP1), inactivating any one of which might only impair or augment transcription to a
limited extent (24, 41-43).
The ability of the srb9, -10, and -11 alleles to result in similar phenotypes as observed for defects in the
CCR4-NOT complex suggests that the physical interactions between these
protein groups represent shared regulatory interactions. The fact that overexpression of a C-terminal portion of CAF4 can specifically block
LexA-SRB11 ability to activate a LexA-LEU2 reporter further supports the connection of these complexes and their associated factors. It has been suggested that LexA-SRB11 can activate
transcription by its recruitment of the RNA polymerase II holoenzyme,
of which it is a part (37, 38). Overproduction of this fragment of CAF4
may interfere with this process. However, it is unlikely that CAF4 is a
direct intermediary in this interaction between SRB11 and the
holoenzyme. Deletion of CAF4 did not have a specific effect on
LexA-SRB11 activation of transcription, for it also reduced activation
by most LexA activators tested (data not shown). Conversely, deletion
of srb9 or srb10 did not impair LexA-CAF4, LexA-CCR4, or LexA-CAF1 in their ability to activate (data not shown).
These results suggest that in contrast to the SNF1 protein that appears
to act through SRB9, -10, and -11 in affecting transcription (44), the
functional interactions between the CCR4-NOT complex, CAF4, and CAF16
with the SRB 9, -10, and -11 proteins remain less clear.
Three possible models may explain the interactions between the CCR4-NOT
complex and the SRB9-11 proteins. In the first model, the SRB proteins
stabilize, recruit, or otherwise influence CCR4-NOT function. For
example, the CCR4-NOT proteins play a role in restricting TBP access to
noncanonical TATAAs (8, 15) and are involved in activated transcription
under some circumstances (1). The CCR4-NOT complex may be controlled by
the SRB9-11 proteins for both these activated and repressor functions,
suggesting a means by which TBP stabilization at certain promoters is
connected to holoenzyme function. However, deletions of the
SRB9-11 genes did not elicit phenotypes characteristic of
the not alleles at the HIS3 locus or of
ccr4 or caf1 defects in terms of caffeine or glycerol temperature sensitivity. It seems unlikely, therefore, that
the SRB9-11 proteins are required for CCR4-NOT function unless it were
restricted to only a few promoter contexts such as at the
ADH2 locus.
In the second model the CCR4-NOT proteins regulate SRB9-11 function.
This can be imagined in two ways. The activator or repressor function
of the CCR4-NOT complex may exert some of its effects by affecting the
SRB9-11 subcomplex activity. The SRB10 protein may become stimulated
or inhibited by the CCR4-NOT complex in its phosphorylation of its
target proteins (45, 48). For instance, SRB10 phosphorylation of the
CTD of RNA polymerase II may be enhanced and thereby prevent efficient
preinitiation complex formation as has been recently proposed (45).
Alternatively, the CCR4-NOT complex could aid in recruiting or
stabilizing the SRB9-11 proteins and thereby aid in bringing the
holoenzyme to the promoter. This could occur by virtue of CCR4-NOT
contacts to TFIID (15, 47). In this model the observation that
overexpression of a segment of CAF4 impairs SRB11 function would
suggest that CAF4 regulates or otherwise affects SRB11 contacts within
the holoenzyme. A more detailed analysis of the effects of CCR4-NOT
defects on SRB10 protein kinase activity or on the association of the
SRB9-11 proteins within the holoenzyme might illuminate these possible interactions.
A third model would suggest that the physical connections between the
CCR4-NOT complex and the SRB9-11 proteins derive from their proximity
at the promoter in shared regulatory events but may not be restricted
to one complex specifically controlling the function of the other.
Identification of the genes controlled by the CCR4-NOT proteins using
whole genome microarray analysis would be a step toward identifying the
genes controlled both by the CCR4-NOT complex and by the SRB9-11
proteins (49). If this set of genes is very small, then the two groups
of proteins may be in physical contact without functional implication.
In contrast, direct functional interactions would be supported by
shared control of a number of genes.
Although we favor the above described second and third models, it is
apparent that the ability of the CCR4-NOT complex to have multiple
contacts and functions is not unique to it. The SAGA complex, the RNA
polymerase II holoenzyme, TAFs, and the SNF/SWI complex all display
varied roles in the cell. Identifying the connections between these
large complexes of proteins should be one means of clarifying how
transcriptional processes are controlled.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Yeast strains
-galactosidase activities were determined as previously described
(29). The screen for proteins interacting with LexA-CCR4 (full-length)
was conducted as described previously using strain EGY188 containing
the p34 reporter plasmid (8 LexA operators upstream of the
lacZ gene). The B42 fusion proteins contain the HA1 epitope,
the B42 E. coli-derived activation domain, a nuclear
localization sequence, and fragments of the yeast genome fused to the C
terminus of B42 (30). The LexA activators were LexA-ADR1 containing
full-length ADR1, LexA-ADR1-TADIV containing residues 642-704 of ADR1,
LexA-B42 containing the E. coli-derived B42 activation
domain fused to LexA-(1-202) (29), LexA-SRB9, -10, and -11 (24)
containing full-length versions of the respective SRB proteins, and
LexA-CCR4-(1-344) containing residues 1-344 of CCR4 (5).
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
CCR4 interacts with CAF4 and CAF16
View larger version (30K):
[in a new window]
Fig. 1.
Gel filtration chromatography of CAF16 and
CAF4. Yeast extract was separated on a Superose 6 column, and the
resultant 0.5-ml fractions were subjected to immunoblot analysis using
CAF16, CAF40, CAF1, c-Myc (DBF2-6c-Myc), or HA1 (CAF4-HA1) antiserum.
Molecular weight markers were blue dextran (2.0 × 106
daltons), thyroglobulin (6.7 × 105 daltons), and
bovine serum albumin (66 kDa). For the top panel the strains
were CCR4 (EGY188) and ccr4 (EGY188-1); for the
2nd from the top and the bottom two
panels the strains were CCR4 (1588-2d) and
ccr4 (1588-2d-1a); and for the panel 3rd from the
top the strains were CCR4 (1637-2b) and
ccr4 (1637-2b-1a).
-galactosidase) as well as it did in a CAF1 strain (Table
II, line 3; 370 units/mg
-galactosidase). These data confirm that
CCR4-CAF16 interactions are separate from those that link CCR4 to the
1-MDa CCR4-NOT complex. A similar two-hybrid analysis with CAF4 showed
that B42-CAF4 interacted with LexA-CCR4 in a caf1 strain
(150 units/mg
-galactosidase) (compare with 54 units/mg
-galactosidase in the wild-type strain, Table II, line 2). The fact
that CAF4 interacted better with CCR4 in a caf1 background suggests that CAF1 interferes with CAF4 binding to CCR4.
View larger version (36K):
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Fig. 2.
CAF4 immunoprecipitates with DBF2 and
NOT1. Yeast extracts from diploid strain EGY188/EGY191 containing
LexA and B42 fusion proteins as indicated were immunoprecipitated with
anti-LexA antibody, and LexA and B42 fusion proteins were detected by
Western analysis following SDS-polyacrylamide gel electrophoresis using
LexA and HA1 antibodies, respectively. LexA-CAF4 contained residues
61-659, LexA-SRB10, and LexA-NOT1 contained full-length SRB10 and
NOT1, respectively, and B42-DBF2 and B42-SIP1 contained full-length
DBF2 and SIP1, respectively. Crude extracts are represented in
lanes 1-6, whereas the immunoprecipitates are depicted in
lanes 7-12. Similar volumes were loaded in the
SDS-polyacrylamide gel electrophoresis, but 5-fold more crude extract
(Ex.) was immunoprecipitated than was analyzed in the crude
extract lanes.
caf4 and caf16 alleles increase ADH1 gene expression
CCR4-NOT proteins interact in the two-hybrid with SRB9 and SRB10
-Galactosidase activities were determined as described in Table II
in diploid EGY188/EGY191. LexA-CAF16 contains residues 20-288 of
CAF16; LexA-CAF4 contains residues 61-659 of CAF4; and LexA-NOT2,
-CAF1, and -SRB10 contain full-length versions of NOT2, CAF1, and
SRB10, respectively. B42-SRB9 contains residues 395-655 of SRB9;
B42-CAF4 contains of residues 61-659 of CAF4; and B42-DBF2, -NOT1, and
-NOT2 contain full-length DBF2, NOT1, and NOT2, respectively. S.E. is
less than 25%.
CCR4 is required for CAF4 and CAF16 interactions with SRB9
-Galactosidase activities were conducted as described in Table II.
Wild type, strain EGY188; caf1, strain EGY188-c1-1 (FOA revertant of
EGY188-c1); and ccr4, strain EGY188-1-1 (FOA revertant of
EGY188-1); LexA and B42 fusions are the same as described in Table
IV. S.E. was less than 20%. ND, not determined.
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Fig. 3.
CCR4-NOT proteins coimmunoprecipitate with
SRB9, -10, and -11 proteins. Immunoprecipitations using LexA
antiserum and subsequent analyses were conducted as described in Fig.
2. a, CAF4 and CCR4 immunoprecipitate B42-SRB9. Protein
fusions were LexA-CAF4-(61-659), LexA-CCR4-(496-837), and
B42-SRB9-(395-655). Ex., extract. b, LexA-SRB10
immunoprecipitates (IP) CCR4 and DBF2. HA-DBF2 refers to
B42-DBF2 (full-length) and LexA-SRB10 contains full-length SRB10 fused
to LexA-(1-87). c, LexA-SRB11 immunoprecipitates NOT1 and
B42-NOT2. LexA-SRB11 contains full-length SRB11 fused to LexA-(1-87)
and B42-NOT2 contains full-length NOT2.
srb9 and srb10 defects affect ADH2 expression
Overexpression of CAF4 impairs LexA-SRB11 ability to activate
transcription
indicates no growth. B42-CAF4
contained residues 544-659 of CAF4, and the LexA fusions contained
full-length versions of the respective proteins.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank M. Carlson and R. Young for plasmids and strains related to the SRB proteins. The technical assistance of J. Farrell was greatly appreciated. We thank M. Hampsey and D. Stillman for comments regarding this manuscript.
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FOOTNOTES |
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* This research was supported by National Institutes of Health Grant GM41512. This is Scientific Contribution number 1995 from the New Hampshire Agricultural Experiment Station.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.
To whom correspondence should be addressed. Tel.:
603-862-2427; Fax: 603-862-4013.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M009112200
1 H.-Y. Liu, Y.-C. Chiang, J. Chen, V. Badarinarayana, and C. L. Denis, unpublished observations.
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ABBREVIATIONS |
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The abbreviation used is: TBP, TATA box protein.
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