CCR4/NOT complex associates with the proteasome and regulates histone methylation.
ABSTRACT The proteasome regulates histone lysine methylation and gene transcription, but how it does so is poorly understood. To better understand this process, we used the epistatic miniarray profile (E-MAP) approach to identify factors that genetically interact with proteasomal subunits. In addition to members of the Set1 complex that mediate histone H3 lysine 4 methylation (H3K4me), we found that deleting members of the CCR4/NOT mRNA processing complex exhibit synthetic phenotypes when combined with proteasome mutants. Further biochemical analyses revealed physical associations between CCR4/NOT and the proteasome in vivo. Consistent with the genetic and biochemical interactions linking CCR4/NOT with proteasome and Set1-mediated methylation, we find that loss of Not4 decreases global and gene-specific H3K4 trimethylation (H3K4me3) and decreases 19S proteasome recruitment to the PMA1 gene. Similar to proteasome regulation of histone methylation, loss of CCR4/NOT members does not affect ubiquitinated H2B. Mapping of Not4 identified the RING finger domain as essential for H3K4me3, suggesting a role for ubiquitin in this process. Consistent with this idea, loss of the Not4-interacting protein Ubc4, a known ubiquitin-conjugating enzyme, decreases H3K4me3. These studies implicate CCR4/NOT in the regulation of H3K4me3 through a ubiquitin-dependent pathway that likely involves the proteasome.
- SourceAvailable from: Tania M Roberts[show abstract] [hide abstract]
ABSTRACT: A genetic interaction network containing�1000 genes and�4000 interactions was mapped by crossing mutations in 132 different query genes into a set of �4700 viable gene yeast deletion mutants and scoring the double mutant progeny for fitness defects.Network connectivity was predictive of function because interactions often occurred among functionally related genes, and similar patterns of interactions tended to identify components of the same pathway.The genetic network exhibited dense local neighborhoods; therefore, the position of a gene on a partially mapped network is predictive of other genetic interactions.Because digenic interactions are common in yeast, similar networks may underlie the complex genetics associated with inherited phenotypes in other organisms.Science 02/2004; 303:808. · 31.20 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: We present a strategy for generating and analyzing comprehensive genetic-interaction maps, termed E-MAPs (epistatic miniarray profiles), comprising quantitative measures of aggravating or alleviating interactions between gene pairs. Crucial to the interpretation of E-MAPs is their high-density nature made possible by focusing on logically connected gene subsets and including essential genes. Described here is the analysis of an E-MAP of genes acting in the yeast early secretory pathway. Hierarchical clustering, together with novel analytical strategies and experimental verification, revealed or clarified the role of many proteins involved in extensively studied processes such as sphingolipid metabolism and retention of HDEL proteins. At a broader level, analysis of the E-MAP delineated pathway organization and components of physical complexes and illustrated the interconnection between the various secretory processes. Extension of this strategy to other logically connected gene subsets in yeast and higher eukaryotes should provide critical insights into the functional/organizational principles of biological systems.Cell 12/2005; 123(3):507-19. · 31.96 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Promoter recruitment of the Saccharomyces cerevisiae SAGA histone acetyltransferase complex is required for RNA polymerase II-dependent transcription of several genes. SAGA is targeted to promoters through interactions with sequence-specific DNA binding transcriptional activators and facilitates preinitiation-complex assembly and transcription. Here, we show that the 19S proteasome regulatory particle (19S RP) alters SAGA to stimulate its interaction with transcriptional activators. The ATPase components of the 19S RP are required for stimulation of SAGA/activator interactions and enhance SAGA recruitment to promoters. Proteasomal ATPases genetically interact with SAGA, and their inhibition reduces global histone H3 acetylation levels and SAGA recruitment to target promoters in vivo. These results indicate that the 19S RP modulates SAGA complex using its ATPase components, thereby facilitating subsequent transcription events at promoters.Cell 12/2005; 123(3):423-36. · 31.96 Impact Factor
CCR4/NOT complex associates with the proteasome
and regulates histone methylation
R. Nicholas Laribee*, Yoichiro Shibata*, Douglas P. Mersman†, Sean R. Collins‡§, Patrick Kemmeren‡, Assen Roguev‡,
Jonathan S. Weissman‡§, Scott D. Briggs†, Nevan J. Krogan‡¶, and Brian D. Strahl*¶
*Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599;†Department of Biochemistry
and Cancer Center, Purdue University, West Lafayette, IN 47907; and‡Department of Cellular and Molecular Pharmacology and California Institute
for Quantitative Biomedical Research, University of California and§Howard Hughes Medical Institute, San Francisco, CA 94143
Edited by Mark T. Groudine, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved January 17, 2007 (received for review September 13, 2006)
The proteasome regulates histone lysine methylation and gene
transcription, but how it does so is poorly understood. To better
understand this process, we used the epistatic miniarray profile
(E-MAP) approach to identify factors that genetically interact with
proteasomal subunits. In addition to members of the Set1 complex
that mediate histone H3 lysine 4 methylation (H3K4me), we found
exhibit synthetic phenotypes when combined with proteasome
mutants. Further biochemical analyses revealed physical associa-
tions between CCR4/NOT and the proteasome in vivo. Consistent
with the genetic and biochemical interactions linking CCR4/NOT
of Not4 decreases global and gene-specific H3K4 trimethylation
(H3K4me3) and decreases 19S proteasome recruitment to the
PMA1 gene. Similar to proteasome regulation of histone methyl-
ation, loss of CCR4/NOT members does not affect ubiquitinated
H2B. Mapping of Not4 identified the RING finger domain as
essential for H3K4me3, suggesting a role for ubiquitin in this
process. Consistent with this idea, loss of the Not4-interacting
protein Ubc4, a known ubiquitin-conjugating enzyme, decreases
H3K4me3. These studies implicate CCR4/NOT in the regulation of
H3K4me3 through a ubiquitin-dependent pathway that likely in-
volves the proteasome.
19S proteasome ? COMPASS ? transcription
(1). A large body of work now shows that histone lysine
proteins that contain a variety of methyllysine binding domains
(2, 3). Because histone lysine residues can receive up to three
methyl groups, lysine methylation has the potential to create
differential biological outputs that depend on the methyl state of
the residue (i.e., mono-, di-, or trimethylation). These attributes
make lysine methylation an important contributor to the ‘‘his-
tone code,’’ which has been postulated to govern epigenetic
Of the known sites of histone methylation, one of the best
characterized is histone H3 lysine 4 methylation (H3K4me) (5–7).
Chromatin immunoprecipitation (ChIP) coupled with whole-
genome microarray (ChIP-chip) analysis has revealed that the
mono-, di-, and trimethylated H3K4 residues segregate differen-
tially along genes (8, 9). In particular, H3K4 trimethylation
(H3K4me3) is localized specifically to the promoter and 5? ends of
genes (10). The enzyme complex responsible for H3K4me,
COMPASS, contains the Set1 methyltransferase and a number of
other protein subunits that contribute to methylation (5, 6, 11). In
subunit of COMPASS both contribute specifically to the establish-
ment of H3K4me3, whereas other COMPASS subunits control the
occurrence of the individual H3K4me states (12–14). The ability of
establishment of mono-ubiquitinated histone H2B (ubH2B) in a
istone methylation plays a significant role in chromatin
organization, gene transcription, and epigenetic regulation
‘‘trans-tail’’ regulatory pathway that is poorly understood but also
is known to regulate histone H3 lysine 79 (H3K79) methylation
mediated by Dot1 (15–17). The ubiquitin-conjugating E2 enzyme,
Rad6, and its E3 ubiquitin ligase partner, Bre1, are recruited to
chromatin in a mechanism that depends on the PAF transcription
elongation complex (18, 19). Once the Rad6/Bre1 complex is
localized to chromatin, it mono-ubiquitinates H2B and promotes
COMPASS-mediated H3K4me2 and H3K4me3 (15, 16, 20).
The proteasome, in particular the 19S regulatory particle, has
been implicated in transcriptional initiation and elongation (21,
22). Recent data also has established the 19S particle as a
regulator of nucleosomal histone modifications. In particular,
inhibition of 19S function revealed that this complex controls
H3K4 and H3K79 methylation at a step after the establishment
of H2B ubiquitination (23, 24). How the proteasome regulates
chromatin modifications and gene transcription is poorly under-
stood. In this article, we present data that define a previously
uncharacterized genetic and biochemical link between the pro-
teasome and the evolutionarily conserved CCR4/NOT complex
that may connect these two complexes to the selective regulation
Recent studies have shown that components of the 19S regula-
tory particle contribute to transcriptional regulation, at least in
part, by altering nucleosomal histone modifications. Specifically,
H3K4 and H3K79 methylation were shown to depend on 19S
function at a step after the establishment of histone H2B
ubiquitination (23). To further define this regulatory pathway,
we used synthetic genetic array (SGA) technology in high-
density epistatic miniarray profile (E-MAP) format (see Mate-
rials and Methods for a detailed description) to identify factors
that genetically interacted with genes coding for proteasome
subunits. As shown in Fig. 1A and consistent with previous
results, we found strong growth defects when mutations of
proteasomal subunits were combined with deletions of subunits
of the H3K4 methyltransferase complex (25). Unexpectedly,
components of the CCR4/NOT complex also were identified as
having genetic interactions with proteasome mutants (Fig. 1A).
Author contributions: R.N.L., S.D.B., N.J.K., and B.D.S. designed research; R.N.L., Y.S.,
reagents/analytic tools; R.N.L., Y.S., D.P.M., S.R.C., P.K., J.S.W., S.D.B., N.J.K., and B.D.S.
analyzed data; and R.N.L., N.J.K., and B.D.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Abbreviations: E-MAP, epistatic miniarray profile; H3K4me, histone H3 lysine 4 methyl-
ation; H3K4me3, histone H3 lysine 4 trimethylation; H3K36, histone H3 lysine 36; H3K79,
histone H3 lysine 79; RRM, RNA recognition motif; Pol II, RNA polymerase II; WCE, whole-
¶To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
April 3, 2007 ?
vol. 104 ?
This complex is known to regulate multiple cellular processes,
including mRNA deadenylation and decapping, protein ubiq-
uitination, and transcription (26, 27). Given its link to transcrip-
tional control, we tested whether components of the CCR4/NOT
complex also affected H3K4me. We screened yeast deletion
mutants of all of the core factors, excluding not1? and not2?
(which were either inviable or extremely sick) and tested these
strains for effects on H3K4me3. We found that not4? and not5?
showed significant reductions in the global levels of H3K4me3,
whereas other members had either minor effects (ccr4? and
caf1?) or no detectable changes (caf40? and caf130?; Fig. 1B).
Because Not5, but not Not4, controls overall stability of the
CCR4/NOT complex and the protein levels of its members (28),
we focused our studies on the mechanism by which Not4
Because H3K4 can be mono-, di-, or trimethylated, we next
asked whether loss of Not4 had similar effects on all three
H3K4me states. Interestingly, we found not4? reduced only
To determine whether gene-specific H3K4me3 was decreased,
we used ChIP to examine nucleosomal histones in vivo. In
agreement with our results in Fig. 1C, we found dramatic
reductions in the levels of H3K4me3, but not other H3K4me
forms or H3K36 or H3K79 methylation, on the PMA1 and
FMP27 genes (Fig. 1D and data not shown). Importantly, loss of
H3K4me3 was not caused by disruption of the COMPASS
complex or decreased levels of Set1 mRNA because we found
that the stoichiometry of COMPASS is unaffected in not4? cells,
and Set1 mRNA levels remain normal [supporting information
(SI) Fig. 5 A and B]. These data show that the CCR4/NOT
complex selectively regulates H3K4me3, a histone modification
intimately linked to transcriptional activation, without affecting
the integrity of COMPASS.
Our genetic and biochemical analyses suggested a link among
COMPASS, the CCR4/NOT complex, and the proteasome (see
Fig. 1A). To pursue whether CCR4/NOT was involved in
proteasome control of histone methylation, we confirmed that
inactivation of the 19S regulatory particle decreased H3K4me
(23, 24). Using mutant alleles of two different 19S components,
CIM3 (RPT6) and CIM5 (RPT1), we analyzed global H3K4me at
either the permissive (24°C) or the nonpermissive (37°C) tem-
perature. Consistent with other reports, we observed a signifi-
cant decrease in H3K4me3 in the cim3-1 or cim5-1 strains
relative to wild type at the nonpermissive temperature (SI Fig.
6A) (23, 24). These results further substantiate a critical role for
the 19S proteasome in regulating H3K4me3.
We next tested whether components of the CCR4/NOT
complex also regulated H2B mono-ubiquitination because this
modification is a prerequisite for the establishment of H3K4me
but is not controlled by proteasome. Deletion of either CCR4 or
NOT4 had no effect on the ubiquitination levels of histone H2B
(Fig. 2A). Instead, we consistently detected a slight, but repro-
ducible, increase in the level of ubH2B in the not4? strain (Fig.
2A and Z.-W. Sun, personal communication). Because the PAF
complex is required for H2B ubiquitylation and has been linked
biochemically to the CCR4/NOT complex (29), we examined its
localization on genes by using ChIP in wild-type, ccr4?, or not4?
cells. Consistent with results showing that the CCR4/NOT
complex had no effect on H2B ubiquitination (Fig. 2A), we saw
no difference in either the relative amount or the distribution of
2B). These results indicate that recruitment of PAF to tran-
scribed genes does not depend on the CCR4/NOT complex.
Furthermore, these results show that the CCR4/NOT complex
regulates H3K4me3 at a step after establishment of H2B ubiq-
uitylation, analogous to that seen for the proteasome.
Recently, two large-scale protein–protein interaction maps
were generated in Saccharomyces cerevisiae by using a similar
affinity-tagging and purification strategy (30, 31). We recently
combined these two data sets and, with an algorithm, provided
and regulates H3K4me3. (A) The proteasome has genetic interactions with
COMPASS and CCR4/NOT complex members. E-MAP and synthetic genetic
of proteasome subunits with an array of deletion strains to create sets of Natr
Kanrhaploid double mutants. Growth rates were assessed as described in ref.
41. Lines connect genes with negative (synthetic sick/lethal) genetic interac-
to the strengths of the indicated genetic interactions. (B) Loss of CCR4/NOT
complex members decreases H3K4me3. Individual CCR4/NOT complex dele-
tion mutants in mid-log phase were screened for effects on H3K4me3 as
described in SI Methods. (C) Global H3K4me3 is selectively regulated by
CCR4/NOT complex. Wild-type (WT) strain (9XMyc-Set1) and not4? (YNL031)
strains were grown to mid-log phase, and whole-cell extracts (WCEs) were
prepared. Samples were prepared and analyzed by immunoblot analysis.
Antibodies used are specified. (D) Nucleosomal H3K4me3, but not other
histone methylation marks, are decreased in not4?. ChIP was performed with
is shown at the top. Data are normalized to histone H3 levels and are the
average and SEM of three independent experiments.
The CCR4/NOT complex genetically interacts with the proteasome
Laribee et al.PNAS ?
April 3, 2007 ?
vol. 104 ?
no. 14 ?
confidence scores for individual interactions (32). Using several
independent metrics, we have shown that this recently derived
protein–protein interaction data set is of higher quality than
those reported previously. In an attempt to generate an accurate
portrait of the physical interactome that can be navigated easily,
we subjected these data to hierarchical clustering. This clustering
analysis accurately recapitulates stable, stoichiometric protein
complexes along the diagonal of the clustergram, whereas off-
diagonal interactions potentially represent either shared sub-
units of stable complexes or weaker, possibly transient associa-
tions between protein complexes (32) (SI Fig. 7 A and B). We
observed one such connection between Not1 and several sub-
units of the 19S proteasome (Fig. 3A). To confirm this obser-
vation, we immunoprecipitated either a 3XHA-tagged version of
Not1 or a 9XMyc-tagged version of Ccr4 or Not4 and immuno-
blotted the precipitates for the presence of the 19S subunit Rpt6
(Fig. 3 B and C). Reciprocal coimmunoprecipitations using an
?-Rpt6 antibody pulled down both the Ccr4 and Not4 subunits
(Fig. 3D). These experiments confirmed the association of the
19S particle with multiple members of the CCR4/NOT complex.
Consistent with a recent report, this proteomic analysis also
identified interactions between Not1 and two DNA helicases,
Rvb1 and Rvb2, which are known to exist in multiple chromatin
remodeling and histone deposition complexes (30, 33). We
tested the potential involvement of Rvb1 and Rvb2 in regulating
H3K4me but found that strains harboring DamP alleles (de-
creased alleles by mRNA perturbation) (34) of these genes did
not affect any H3K4me state (data not shown).
To address whether CCR4/NOT affects proteasome function,
we used antisera against the 19S subunit Rpt6 in ChIP to
examine the localization pattern of the 19S particle on PMA1. In
wild-type cells, the 19S showed enrichment at both the 5? and 3?
ends of the PMA1 gene (Fig. 3E). Compared with the wild-type
strain, deletion of NOT4 had reduced, but not abolished, re-
cruitment of the 19S across the entire length of the gene (Fig.
3E). The 19S proteasome regulates RNA polymerase II (Pol II)
transcription elongation and termination (22, 35). Because
CCR4/NOT also interacts with Pol II, we tested whether the loss
of Not4 affected the relative amounts and distribution of Pol II
on the PMA1 gene as a way to explain the decreased Rpt6
between the wild-type and not4? strains on PMA1 (Fig. 3F).
Consistent with a lack of effect on Pol II recruitment and or
distribution, neither not4? nor ccr4? affected PMA1 mRNA levels
(SI Fig. 5B). This effect on 19S recruitment was not because of
protein levels for these factors are equivalent to wild-type cells (SI
Fig. 6B). Although these results reveal 19S recruitment to be
tail pathway. (A) CCR4/NOT complex does not regulate histone H2B mono-
ubiquitination. Wild-type (YZS276), K123R (YZS277), ccr4? (YNL015), and
not4? (YNL019) cells were grown to mid-log phase, and H2B ubiquitination
was analyzed as described in ref. 25. The lower arrow denotes histone Flag-
H2B, and the upper arrow indicates mono-ubiquitinated histone Flag-H2B
(ubH2B). (B) The PAF elongation complex is not disrupted on genes in CCR4/
NOT complex mutants. Wild-type (Rtf1-TAP), ccr4? (YNL032), and not4?
Data are the average and SEM of two independent experiments.
CCR4/NOT regulates H3K4me3 independently of the histone trans-
its recruitment to genes. (A) Summary of off-diagonal connections between
the 19S proteasome and the NotI component of the CCR4/NOT complex
(see SI Fig. 7 A and B). Not1 also shares off-diagonal connections with two
helicases, Rvb1 and Rvb2, involved in various aspects of chromatin remodel-
ing. The intensity of the yellow corresponds to the confidence of the protein–
protein interaction, and black signifies no detected interaction (32). (B) IP of
Not1 coprecipitates the 19S proteasome subunit, Rpt6. One milligram of WCE
from wild-type (W303) and NotI-3XHA-tagged strains (YNL047) were used in
IP experiments that included 3 ?l of the appropriate antibody. IP samples and
2% of starting material (Input) were resolved by either SDS/8% PAGE (for
Not1-3XHA detection) or SDS/10% PAGE (for Rpt6 detection), and samples
were processed for immunoblot analysis by using either anti-Rpt6 or anti-HA
antibodies. (C) IP of Ccr4 or Not4 coprecipitates the 19S proteasome. WCEs (1
mg) from wild-type (BY4741), Ccr4–9XMyc (YNL038), and Not4–9XMyc
(YNL039) were immunoprecipitated with anti-Myc antibody, resolved by SDS/
For the input samples, 5% (50 mg) of WCEs from each sample was examined.
(D) IP of the 19S coimmunoprecipitates the CCR4/NOT complex. Experiment
was performed as described in C except that anti-Rpt6 antibodies were used
F) 19S proteasome recruitment to the PMA1 gene is disrupted in the not4?
strain. Anti-Rpt6 (E) or anti-pol II (F) antibodies were used in ChIP analysis.
Primers are as described in Fig. 1D. Samples are the average and SEM of three
The CCR4/NOT complex interacts with the proteasome and regulates
www.pnas.org?cgi?doi?10.1073?pnas.0607996104Laribee et al.
defective on PMA1, we note that another gene examined (ADH1)
did not show a significant reduction in 19S association (data not
19S particle to a subset of Pol II-transcribed loci.
To further define the mechanism of CCR4/NOT proteasome
regulation of H3K4me3, we sought to determine which domain
of Not4 was responsible for regulating this modification. Not4
contains both a RING and a RRM domain, and although the
RRM domain has significant homology to domains in other
proteins known to bind RNA, its function in Not4 biology is
unknown (36). RING domains are known to mediate protein–
protein interactions, and some can act as E3 ubiquitin ligases
(37). A previous study has shown that Not4 can mediate ubiq-
uitin transfer to substrates in an in vitro ubiquitin conjugation
reaction, but the only known substrates in vivo are members of
the nascent polypeptide-associated complex (NAC), none of
which affect H3K4me3 (M. Collart, personal communication)
(38, 39). We made a series of N-terminal truncation mutants
lacking either the RING domain or both the RING and RRM
domains and transformed these constructs, or a full-length
NOT4 construct, individually into a not4? strain (Fig. 4A). As
shown in Fig. 4B, exogenous expression of full-length NOT4 fully
restored the H3K4me3 defect seen in not4? cells. Interestingly,
loss of the RING domain, or the RING and RRM domains,
failed to rescue H3K4me3. The inability to rescue H3K4me3 by
the deletion mutants, however, was not attributable to inade-
quate protein expression because each mutant was expressed to
comparable levels as the full-length construct (Fig. 4B). These
data reveal that the RING domain of Not4 is essential for
establishing wild-type levels of H3K4me3.
The requirement of the RING domain in Not4 for regulation of
H3K4me3 suggested that Not4-mediated ubiquitin transfer to one
or more substrates is critical in this process. To examine this idea
further, we again analyzed the recently generated E-MAP data to
identify links between Not4 or proteasome members and any
known ubiquitin-conjugating E2 enzymes. Because E-MAP anal-
ysis is quantitative, one can detect both negative (synthetic sick/
lethal) interactions and positive ones (where the double mutant
grows better than is expected from growth of the two single
mutants) (see SI Methods). We previously showed that these latter
interactions can identify cases in which genes are functioning in the
same pathway in vivo (40, 47). Interestingly, our E-MAP analysis
identified Ubc4, an E2 ubiquitin ligase, as having either positive or
negative genetic interactions with proteasomal subunits (RPN10,
RPN6, and UBP6) or chaperones (DOA1 and UMP1) (data not
shown), suggesting a strong functional link between Ubc4 and the
proteasome. These data are consistent with two previously pub-
in the yeast two-hybrid system (31, 38). We next tested whether
Ubc4 and a related E2 enzyme, Ubc5, regulate H3K4me3. Com-
paring single deletions of these two enzymes relative to the wild-
(Fig. 4C). We attempted to create a ubc4?ubc5? deletion in this
background but found that it was synthetically lethal because the
double mutant was not able to lose a UBC5 expression plasmid in
the ubiquitin-conjugating activity of Not4, perhaps in partnership
with Ubc4, regulates H3K4me3 (Fig. 4D).
is not understood. To further define how this complex functions,
we used genetic data from an E-MAP that focused on chromo-
some function and a recently generated physical interaction data
set to identify a physical and genetic connection among the
CCR4/NOT complex, COMPASS, and the proteasome (41). We
found CCR4/NOT specifically regulates H3K4me3 in a fashion
that does not alter the integrity of the COMPASS complex
because this complex remains intact upon tandem affinity
purification (TAP) (see Fig. 1C and SI Fig. 5). By testing
individual deletion mutants of CCR4/NOT, we show that the E3
ubiquitin ligase Not4 was the subunit of the complex critical for
establishing H3K4me3. Although we do not rule out a role for
other CCR4/NOT subunits in the regulation of H3K4 methyl-
ation (i.e., Ccr4 and Caf1), our studies identify Not4 as a key
regulator of this modification in the CCR4/NOT complex.
Similar to what previously has been described for the protea-
some, we demonstrated that the CCR4/NOT complex does not
affect ubH2B (23, 24). These data suggest that CCR4/NOT and
the proteasome are connected to the regulation of H3K4me (see
Fig. 4D). Because the RING domain of Not4 and the E2
ubiquitin-conjugating enzyme Ubc4 are required for H3K4me3,
matic of full-length and N-terminal truncation constructs of Not4 used in this
study. (B) The RING domain of Not4 regulates H3K4me3. Wild-type (9XMyc-
Set1) cells were transformed with an empty ADH1 expression vector. not4?
sion constructs described in A. Cells were grown to mid-log phase in Sc-Ura
media and harvested, and WCEs were prepared, normalized to total H3, and
analyzed by SDS/15% PAGE. Red arrowheads indicate the occurrence of the
Not4 Flag-tagged proteins. (C) Loss of the E2 ubiquitin-conjugating enzyme,
Ubc4, decreases H3K4me3. Wild-type (9X-Myc-Set1), not4? (YNL031), ubc4?
(YNL040), and ubc5? (YNL041) strains were grown to mid-log phase, pro-
cessed into WCEs, and analyzed as described in B. (D) Hypothetical model
depicting how CCR4/NOT controls H3K4me3. In this model, CCR4/NOT regu-
lates H3K4me3 by recruiting Ubc4 to ubiquitylate an as-yet-unidentified
substrate (X) that directly affects 19S proteasome function/recruitment to
genes (see Fig. 3E). Although our studies suggest a role for the proteasome in
H3K4me3 regulation by Not4, we emphasize that this regulation will likely
include other mechanisms in addition to the proteasome (see Discussion).
A Not4 ubiquitin-dependent pathway regulates H3K4me3. (A) Sche-
Laribee et al.PNAS ?
April 3, 2007 ?
vol. 104 ?
no. 14 ?
they imply a role for Not4 in conjugating ubiquitin to an
undefined substrate that regulates this modification, perhaps by
altering proteasome localization or function (Fig. 4D).
Although CCR4/NOT is known to interact both genetically
and physically with other multiprotein complexes, such as Me-
diator, SAGA, and Pol II, this complex never has been func-
tionally connected to the proteasome or to chromatin regulation
(26, 36). In support of a role for CCR4/NOT in regulating 19S
proteasome, we found that loss of Not4 reduced overall levels of
19S proteasome on the PMA1 gene. However, reduced protea-
some recruitment cannot be the sole explanation for the de-
creased H3K4me3 because another test gene examined (ADH1)
showed no appreciable reduction in 19S recruitment. Further-
more, H3K79 methylation is unaffected in the not4? cells,
suggesting that proteasome localization to chromatin cannot be
disrupted globally. We speculate that ubiquitin transfer by Not4
partially may regulate 19S chaperone function and/or it may
ubiquitylate a factor that is required for full 19S chromatin
association and subsequent control of the H3K4me3 activity of
COMPASS (see Fig. 4D). Although the precise mechanism of
Not4 regulation of H3K4me3 is not known, understanding how
the CCR4/NOT complex regulates this mark will depend on
finding additional Not4 substrates.
In summary, we have discovered a functional interaction
between the CCR4/NOT complex and the proteasome that
appears critical for the selective establishment of histone
H3K4me3. The data presented directly link CCR4/NOT to both
the proteasome and to chromatin regulation. These studies open
the way for investigating other possible functions of the CCR4/
NOT complex (and potentially other complexes involved in
mRNA function) in chromatin biology.
Materials and Methods
Yeast Strains and Cloning. Yeast strains and their genotypes are
listed in SI Table 1. Strains unique to this study that are ccr4? or
not4? were made by amplifying the KanMX cassette from the
respective deletion strain (obtained from Open Biosystems, Hunts-
ville, AL) and then by using this integration cassette in a high-
factors was performed by using plasmids and techniques as de-
scribed in ref. 43. De novo deletions of UBC4 and UBC5 were
generated by using primers containing gene-specific sequences,
along with sequences specific for amplifying a KanMX2 cassette.
The full-length NOT4 ORF and truncation derivatives were
cloned by using the restriction sites XbaI and EcoRI as C-terminal
mono-Flag fusions into plasmid pN827, which contains an ADH1
promoter driving expression of the inserted sequence (44).
WCE Preparation, Coimmunoprecipitation, and Immunoblot Analysis.
For analysis of H3K4me3 and H3 protein levels (Figs. 1 B and
C and 4C), yeast WCEs were prepared as described in ref. 25.
After normalizing samples to total H3 content, samples were
fractionated by SDS/15% PAGE, transferred to PVDF, and
analyzed by immunoblotting. Anti-mono-, di-, and trimethyl
H3K4 antibodies, along with the anti-H3 antibodies, were ob-
tained from Upstate Biotechnology (Charlottesville, VA). Anti-
trimethyl H3K36 and anti-trimethyl H3K79 were from Abcam
WCEs were prepared for coimmunoprecipitation analysis by
growing cells to mid-log phase and then lysing the cells in IP
buffer (10 mM Tris, pH 8.0/150 mM NaCl/0.1% Nonidet P-40/
10% glycerol) containing protease and phosphatase inhibitors
and 1 mM DTT. IPs were performed in IP buffer with a total of
1 mg of WCEs. To these extracts, 3 ?l of respective antibody was
added and incubated overnight with rotation. To pellet immune
complexes, 10 ?l of a 50% slurry of Protein A Sepharose
(Amersham, Uppsala, Sweden) was added, and samples were
rotated for 1 h at 4°C. Samples were washed three times with 500
?l of IP buffer, resuspended in 10 ?l of 2? SDS sample buffer,
boiled, fractionated by SDS/8% or 10% PAGE, and immuno-
blotted with the appropriate antibody. Analysis of ubiquitinated
H2B was performed as described in ref. 25.
ChIP Analysis. ChIP assays were performed and quantitated as
previously described by using 3 ?l of the anti-protein A antibody
(Sigma–Aldrich, St. Louis, MO), anti-Rpb1 (sc-25758; Santa Cruz
Biotechnology, Santa Cruz, CA), or the anti-Rpt6 (i.e., Sug1)
antibody (a gift from Thomas Kodadek, University of Texas
Southwestern Medical Center, Dallas, TX) and 1 mg of WCE (25).
Multiplex PCR was performed by using primers specific to target
genes (i.e., PMA1, FMP27, and ADH1) and also to a region of
chromosome V devoid of ORFs (internal control). The histone
modification-specific ChIPs were normalized to histone H3 levels.
E-MAP and Protein–Protein Interaction Analysis. Synthetic genetic
array technology was used to generate a high-density, quantita-
tive E-MAP that focused on various aspects of chromosome
function (45–47). The systematic creation of double deletion
strains in a 768-colony arrayed format was carried out on a set
of 743 essential and nonessential genes involved in processes
such as transcriptional regulation, DNA repair, DNA replica-
tion, and chromosome segregation (45, 47). Images of the plates
containing the colonies corresponding to the double mutants
were analyzed with recently developed software designed for
E-MAP experiments (41). Briefly, quantitative values are gen-
strains. E-MAP analysis therefore can identify not only negative
interactions (synthetic sick/lethal pairs) but also positive ones in
which the double mutant grows no worse or better (suppression)
than the sickest single mutant. A protein–protein interaction
map for S. cerevisiae was derived (32) from the raw data of two
recent large-scale proteomic analyses (30, 31).
We thank Vincent Geli (Centre National de la Recherche Scientifique,
Marseille, France), Mark Hochstrasser (Yale University, New Haven,
CT), Stefan Jentsch (Max Planck Institute, Martinsried, Germany), and
Thomas Kodadek (University of Texas Southwestern Medical Center,
Dallas, TX) for generous gifts of antibody and yeast strains; Martine
Collart, Mary Ann Osley, Ali Shilatifard, Zu-Wen Sun, and Marc Timmers
for sharing unpublished data; and members of the Briggs, Krogan, and
Strahl laboratories for helpful discussions. This study was supported by
(to S.D.B.) and National Institutes of Health Postdoctoral Fellowship
Award GM71106-01A1 (to R.N.L.). J.S.W. is an Investigator of the
Howard Hughes Medical Institute, B.D.S. is a Pew Scholar in the
Biomedical Sciences, and N.J.K. is a Sandler Family Fellow.
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vol. 104 ?
no. 14 ?