Molecular Cell, Vol. 11, 267–274, January, 2003, Copyright 2003 by Cell Press
Short ArticleBre1, an E3 Ubiquitin Ligase
Required for Recruitment and Substrate
Selection of Rad6 at a Promoter
methylated by COMPASS and Dot1, histone H2B must
be ubiquitinated on lysine 123, and this is catalyzed by
the Rad6 ubiquitin-conjugating enzyme (Dover et al.,
2002; Sun and Allis, 2002; Ng et al., 2002b; Robzyk et
al., 2000). Since Rad6 is involved in diverse biological
et al., 1994; Hishida et al., 2002; Kupiec and Simchen,
1986; Kang et al., 1992; Huang et al., 1997), different
ubiquitin-protein isopeptide ligases (E3 enzymes) are
ities. Thus, the E3 enzyme that directs Rad6 to ubiquiti-
nate histone H2B is expected to be a key determinant
of transcriptional regulation.
In this manuscript, we identify Bre1 as a RING finger-
containing protein that is: (1) found in a macromolecular
complex with Rad6; (2) required for the ubiquitination
of histone H2B in vivo, which seems to be a signal for
methylation of histone H3 at its fourth and seventy-ninth
lysines; (3) required for telomeric silencing; (4) recruited
of Rad6 to chromatin at a promoter; and (6) dedicated
to the transcriptional regulatory role of Rad6. Taken to-
that Bre1 is the likely E3 ubiquitin ligase for Rad6 in its
role in regulating chromatin structure and transcription.
Adam Wood,1,6Nevan J. Krogan,2,6
Jim Dover,3Jessica Schneider,1
Jonathan Heidt,1Marry Ann Boateng,1
Kimberly Dean,1Ashkan Golshani,1
Yi Zhang,4Jack F. Greenblatt,2
Mark Johnston,3and Ali Shilatifard1,5,*
1Department of Biochemistry
Saint Louis University School of Medicine
1402 South Grand Boulevard
St. Louis, Missouri 63104
2Banting and Best Department of Medical Research
Department of Molecular and Medical Genetics
University of Toronto
Toronto, Ontario M5G 1L6
3Department of Genetics
Washington University School of Medicine
St. Louis, Missouri 63110
4Department of Biochemistry and Biophysics
University of North Carolina at Chapel Hill
Chapel Hill, North Carolina 27599
5Saint Louis University Cancer Center
Saint Louis University School of Medicine
St. Louis, Missouri 63104
Results and Discussion
Ubiquitination of histone H2B catalyzed by Rad6 is
required for methylation of histone H3 by COMPASS.
We identified Bre1 as the probable E3 for Rad6’s role
in transcription. Bre1 contains a C3HC4 (RING) finger
of Bre1 is required for ubiquitination of histone H2B,
methylation of lysine 4 and 79 of H3 and for telomeric
silencing. Chromatin immunoprecipitation experiments
indicated that both Rad6 and Bre1 are recruited to a
and is dedicated to the transcriptional pathway of
Rad6. These results suggest that Bre1 is the likely E3
enzyme that directs Rad6 to modify chromatin and
ultimately to affect gene expression.
Bre1 Is Required for the Proper Histone H3
Methylation by COMPASS
To identify the E3 enzyme that directs Rad6 to modify
histone H2B, we tested each of the ?4800 nonessential
yeast gene deletion mutants for methylation of lysine 4
of histone H3. We discovered that BRE1 (YDL074c) is
required for the methylation of the fourth lysine of his-
tone H3 (Figure 1A) as described in the Experimental
Procedures. BRE1 was initially identified based on hy-
persensitivity of the bre1 mutants to brefeldin A, a drug
that affects intracellular protein transport (Oyen et al.,
zinc finger (Figure 1B), a motif found in several other
proteins, including Rad18, Rad16, Rad5, BRCA1, and
Snf2L (Figure 1B) (Conaway et al., 2002; Hochstrasser,
2000; Ulrich and Jentsch, 2000). RING finger proteins
contain a characteristic C3HC4 or C3H2C3 motif (Figure
1C) that binds a zinc ion and appear to act as E3 en-
zymes that play important roles in many processes,
including transcriptional regulation, cell cycle progres-
sion, oncogenesis, signal transduction, and develop-
ment (Kamura et al., 1999; Swanson et al., 2001; Lu et
al., 2002). Two independently generated bre1 mutants
were found to be defective for K4 methylation of histone
H3 (Figure 1D). Methylation of H3 was restored by intro-
duction into these strains of a plasmid containing wild-
type BRE1 but not a plasmid carrying point mutations
affecting key amino acids of the RING finger domain of
Bre1 (Figure 1E).
somes are important for proper regulation of gene ex-
pression (Workman and Kingston, 1998; Urnov and
Wolffe, 2001; Wolffe, 2001). Methylation of histone H3
on lysine 4 catalyzed by COMPASS (a protein complex
that includes Set1) (Krogan et al., 2002a; Miller et al.,
2001; Roguev et al., 2001; Nagy et al., 2002) and on
lysine 79 (catalyzed by the Dot1 protein) (van Leeuwen
ing of expression of genes located near the telomeres
of chromosomes in S. cerevisiae. For histone H3 to be
6These authors contributed equally to this work.
Figure 1. Surveying the S. Cerevisiae Genome Identified Bre1, a C3HC4 (RING) Finger-Containing Protein, as a Gene Required for Methylation
of Lysine 4 of Histone H3
(A) Extracts of S. cerevisiae mutants missing one of the approximately 4800 nonessential genes (Dover et al., 2002) were tested for the
presence of Lys4-methylated histone H3. One of the mutants lacking this histone modification is bre1 (row H, lane 5). Arrows at position d10
and h3 indicate empty wells as plate markers.
(B) Sequence comparison of several other proteins containing a C3HC4 domain.
(C) Structure of a C3HC4 (RING) finger domain.
(D) Extracts of wild-type strains deleted for set1 or two independently generated mutant strains (MATa and MAT?) deleted for bre1 were
tested for the presence of Lys4-methylated histone H3. The presence of histone H3 phosphorylated at its serine 10 was used as an indicator
of equivalent loading.
(E) The K4 methylation-deficient phenotype of the bre1? cells was complemented by either an episomal vector containing either full-length
cDNA for Bre1 or point mutated bre1 (C703S) or (H705A).
Recruitment of Rad6 to a Promoter via Bre1
Figure 2. Biochemical Characterization of the Bre1- and Rad6-Containing Complexes
(A) Tandem affinity purification (TAP) of Bre1 and Rad6. Silver stain analysis of TAP-tag affinity-purified Bre1 and Rad6 complex. Tagged-
Rad6 copurified with Bre1, Rad18, Ubr1, and Ubr2. The position of tagged and untagged Bre1 and Rad6 is indicated by the arrow.
(B) Histone methylation at K4 H3 was tested in either wild-type or Bre1-TAP strains.
The Ring Finger of Bre1 Is Required for Its Molecular
Function In Vivo
To identify proteins that physically associate with Bre1,
we attached an affinity tag (TAP) to the C termini of Bre1
and Rad6 and purified them by affinity purification as
described in the Experimental Procedures. Rad6-TAP
copurifies with Bre1, Rad18, Ubr1, and Ubr2. However,
Bre1-TAP was isolated as a single subunit with no trace
blot analysis using polyclonal antibodies specific to
Rad6 [data not shown]). Since the C3HC4 RING finger
nus of Bre1 disrupts its function. Indeed, the strain with
the chimeric Bre1 is defective in methylation of K4 of
histone H3 (Figure 2B), suggesting that C3HC4 RING
finger domain of Bre1 is required for interaction with
Rad6. When the purified Rad6-containing complex is
subjected to size exclusion chromatography, Rad6 co-
migrates with Bre1, Rad18, Ubr1, and Ubr2 (data not
shown). This observation is consistent with the idea that
Bre1 associates with Rad6. These experiments indicate
that Rad6 and Bre1 can physically interact and that
the RING finger of Bre1 is required for this biochemical
ubiquitination of histone H2B. In a strain whose only
copy of the H2B gene is fused to the FLAG epitope tag,
the unmodified form by SDS-PAGE and Western blot
analysis using either an antibody against the Flag epi-
tope or anti-ubiquitin specific antibodies (Figure 3A,
lanes 1–3); only the more slowly migrating form of H2B
panel, lanes 1–3). Deletion of RAD6 or BRE1 from this
strain results in the lossof the slowly migrating, ubiquiti-
nated H2B (Figure 3A, lanes 4–9).
Bre1 Is Essential for Methylation of Histone H3
on Lysine 4 and 79 and Is Dedicated
to the Transcriptional Role of Rad6
Since ubiquitination of K123 of H2B (Figure 3A) is re-
quired for methylation of K79 of H3, we tested whether
Bre1 is also required for this modification of histone H3.
Indeed, a strain lacking BRE1 has significantly reduced
levels of lysine 79-methylated histone H3 (Figure 3B),
indicating that Bre1plays the same rolein histone modi-
fications as does Rad6.
If Bre1 is indeed the E3 enzyme for Rad6, it should
also be required for silencing of genes located near
chromosome telomeres (van Leeuwen et al., 2002; Kro-
gan et al., 2002a; Dover et al., 2002; Huang et al., 1997).
Indeed, BRE1, like RAD6, appears to be required for
normal silencing of a URA3 gene located near the telo-
mere of chromosome VII (Figure 3C). However, unlike
rad6 mutants, a bre1 mutant is not as sensitive to DNA-
damaging agents such as UV irradiation (Heude et al.,
1995) (Figure 3F), or to MMS (Ulrich, 2001; Ramotar and
Masson, 1996) (Figure 3D) and to phleomycin (He et al.,
Bre1 Is Required for Monoubiquitination
of Histone H2B In Vivo
Rad6-catalyzed ubiquitination of histone H2B (Robzyk
et al., 2000) is required for efficient methylation of lysine
4 and lysine 79 of histone H3 (Dover et al., 2002; Sun
and Allis, 2002; Briggs et al., 2002; Ng et al., 2002b).
Therefore, we tested whether Bre1 is involved in the
Figure 3. The Role of Bre1 in Histone H2B Ubiquitination, Histone H3 Lysine 79 Methylation, and Telomeric Silencing
(A) Bre1 is required for the ubiquitination of histone H2B. Increasing concentrations of highly purified acid extracted histones from wild-type
strains (strains containing FLAG-tagged H2B as the only source of histone H2B) or strains deleted for Rad6 or Bre1 in the same background,
were analyzed by Western blotting using (upper panel) monoclonal antibodies against the FLAG epitope or (lower panel) polyclonal antibody
Recruitment of Rad6 to a Promoter via Bre1
1996) (Figure 3E). Thus, Bre1 seems to be dedicated to
the transcriptional regulatory function of Rad6.
promoter. Fifth, Bre1 is essential for the recruitment of
Rad6 to chromatin at a promoter. Finally, Bre1 seems
to be dedicated to the transcriptional regulatory role of
Rad6. Our results, taken together, suggest that Bre1 is
the E3 enzyme that directs Rad6 to its role in regulating
chromatin structure and transcription.
We are unable to detect autoubiquitination of recom-
binant Rad6 with Bre1 or even with its known E3 ligases
Ubr1/2 or Rad18. Autoubiquitination is an intrinsic prop-
erty of some E2s but not of others, and it appears that
Rad6 falls in the class of E2s that cannot be activated
to autoubiquitinate. Since we find that Rad6 is recruited
to nucleosomes at a promoter via Bre1 but not to the
body of the gene, we believe Rad6 must be recognizing
somethingthat isspecialaboutnucleosomes atthepro-
moters, such as acetylation, phosphorylation, or other
modifications. For this reason, defining the nucleosome
substrate of Rad6-Bre1 is difficult to isolate at this time.
A fundamental role for protein ubiquitin in the regula-
tion of transcription via RNA polymerase II is rapidly
becoming clear (Conaway et al., 2002). Identification of
the E3 enzymes involved in the regulation of transcrip-
tion by ubiquitination is essential for understanding the
specificity and regulation of any given pathway. Identifi-
cation of Bre1 as the likely E3 enzyme that links Rad6
to the transcription machinery hasnow opened the door
for the molecular dissection of the role of histone H2B
ubiquitination and transcriptional regulation.
Bre1 Is Recruited to a Promoter and Is Required
for the Recruitment of Rad6 to a Promoter
To further determine the role of Bre1 as the ligase for
Rad6 in chromatin modification by histone ubiquitina-
tion, we employed chromatin immunoprecipitation
(ChIP) as described in the Experimental Procedures. We
have used PMA1 gene for these studies because it is a
Rad6 is directed toward chromatin specifically at the
promoter (Figure 4A). To determine whether Bre1 can
direct Rad6 toward the promoter, ChIP experiment for
Rad6 was performed in cells lacking Bre1. As seen in
Figure 4B, Bre1 is essential for the recruitment of Rad6
to chromatin at a promoter, further supporting a role of
Bre1 as the E3 enzyme for Rad6 in transcription.
Recent evidence suggests that ubiquitination of his-
tone H2B on its lysine 123, catalyzed by Rad6, directs
COMPASS and Dot1 to methylate Lys4 and Lys79 of
ical processes such as DNA repair, DNA damage-
induced mutagenesis, meiosis, transposition of retro-
transposons, and gene silencing (Jentsch et al., 1987;
Koken etal., 1991;Bailly etal., 1994;Hishida et al.,2002;
Kupiec and Simchen, 1986; Kang et al., 1992; Huang et
al., 1997) different ubiquitin-protein isopeptide ligases
(E3 enzymes) likely determine the specificity of Rad6
and regulate its function. Several pieces of evidence
reported here suggest that Bre1 is the E3 enzyme that
directs Rad6 to catalyze ubiquitination of lysine 123 of
tination of H2B (Figure 3A). Second, Bre1 possesses
a protein motif—the RING finger—characteristic of E3
enzymes. Third, Bre1 physically associates with Rad6
(Figure 2). Other proteins thatassociate with Rad6, such
as Rad18 (which also contains a RING finger), Ubr1,
and Ubr2, are not required for methylation of H3 and
therefore for ubiquitination of H2B and are unlikely to
be the E3 enzymes for Rad6 in this pathway (Dover et
linked to Rad6—Rex4 and Ubr1—are also dispensable
for ubiquitination of H2B (Dover et al., 2002). Fourth,
both Rad6 and Bre1 are recruited to chromatin at a
Functional Genomic Analyses of Histone
Modification by Methylation
Using a 96-well pinning device, the entire collection of 4800 yeast
nonessential gene deletion mutants were inoculated from ?80?C
stocks onto agar plates containing YPD ? 200 ?g/ml GENETICIN
(GIBCO) and allowed to grow 48 hr and used to inoculate 96-tube
PCR-plates filled with 100 ?l of YPD. After 48 hr of growth at 30?C,
the plates were centrifuged at 2000 g for 10 min. The medium was
removedby wrist-snapinversionand drainedinto absorbenttowels.
The plates were then covered and frozen at ?80?C for up to 1 week.
Cells were thawed at room temperature, resuspended in 30 ?l lysis
buffer (20 mM Tris [pH 7.5], 50 mM KCl, 1 mM EDTA, 1 mM DTT,
0.1%NP40, 1 mg/ml Zymolyase 100 T), and incubated at 37?C for
15 min. Ten microliters of 4? Laemmli loading buffer was added,
and the samples were vortexed briefly before heating at 100?C for
5 min. The lysates were subjected to SDS-PAGE, transferred to
raised against ubiquitin. As indicated by the arrows, Ubq.-H2B is the slower migrating form, and the unubiquitinated species of histone H2B
is the faster migrating form.
(B) To determine the role of Bre1 in methylation of K79 of histone H3, extracts of wild-type strains or strains deleted for rad6 and bre1 were
tested for the presence of ubiquitinated H2B and methylated K4 and K79 of histone H3.
(C) Deletion of Bre1 results in a defect in silencing of gene expression at telomeres. Either the wild-type parental strain (UCC1001, harboring
URA3 near the left telomere of chromosome 7 as a reporter of telomeric gene silencing [Nislow et al., 1997]) or UCC1001 deleted for set1,
rad6, or bre1 was tested for defect in silencing of gene expression at telomeres. Wild-type cells silence expression of the telomere-associated
URA3 gene and are therefore resistant to 5FOA. Cells defective for telomeric gene silencing have increased expression of URA3 and hence
are sensitive to 5FOA (Nislow et al., 1997). Two-fold serial dilutions of cultures (from about 5 ? 104to 50 cells) were spotted on minimal
glucose plates containing (left panel) or lacking (right panel) 5FOA. These plates were incubated at 30?C for 36 (?4) hr. (Far right panel) The
ability of each strain to methylate its histone H3 either on K4 or K79 was tested by Western analysis. In the same experiment, the addition
of plasmid containing wild-type Bre1 and not the (RING) finger-mutated bre1 complemented the silencing phenotype of bre1? strains (data
(D–F) To inquire whether Bre1 is dedicated to Rad6’s role in histone ubiquitination or is involved in some of the other functions in which Rad6
plays a role, we tested whether bre1 mutant is as sensitive to DNA damaging agents such as UV irradiation (254 nM) or to chemicals treatment
such as MMS or phleomycin. Rad6 mutant cells have been demonstrated to be sensitive to such treatments. As a control, we also tested
the sensitivity of yeast cells deleted for either cps50 subunit of COMPASS (involved in histone H3 Lys4 methylation) or dot1 (involved in
histone H3 Lys79 methylation).
Figure 4. Bre1 Is Required for the Recruit-
ment of Rad6 to a Promoter
Chromatin immunoprecipitation assays with
Rad6-TAP and Rad6-TAP bre1? were per-
formed as described previously. To monitor
the presence of either Rad6 along the PMA1
gene, chromatin was immunoprecipitated
with rabbit IgG-agarose from a strain con-
taining either Rad6-TAP or Rad6-TAP bre1?.
PCR amplifications were carried out using
primer pairs recognizing promoter (1), coding
(2, 3, 4, and 5) and 3? untranslated (6) regions
for PMA1. Primer pairs are as follows:
PMA1?370 and PMA1?90 (1), PMA1168 and
PMA11010 and PMA11250 (4), PMA12018 and
PMA12290(5), and PMA13287and PMA13500(6).
Each PCR contained a second primer pair
that amplified a region of chromosome V de-
void of ORFs (*), thus providing an internal
control for background. The ratio of the ex-
itated DNA was divided by the ratio of the
experimental to the control signal for the in-
nitrocellulose membrane, and probed with anti-methylhistone anti-
sera at 1:1000 dilution, followed by detection of the bound antibody
with horseradish peroxidase-conjugated to anti-rabbit IgG second-
ary antibodies (1:10,000 dilution). Anti-methyl Lys4 histone H3 was
purchased from Boston Biochemical.
Rad6 were tagged by chromosomal integration via the C-terminal
domain following a previously published method (Miller et al., 2001).
Tagged complexes were purified essentially as described on IgG,
and calmodulin columns from extracts were obtained from 5 liter
yeast culturesgrown inYPD mediumto an OD600of 1.0–1.5as before
(Miller et al., 2001). The protein bands were reduced, alkylated, and
subjected to in-gel tryptic digestion, and the peptides were then
purified and identified by MALDI-TOF spectrometry using a PerSep-
tive DE STR (Miller et al., 2001). Selected mass values from the
MALDI-TOF experiments were taken to search the protein nonre-
dundant database (NR; NCBI, Bethesda, MD) using the Peptide-
Search algorithm. MS/MS spectra were inspected for y″ ion series
to compare with the computer-generated fragment ion series of the
predicted tryptic peptides.
Isolation of Yeast Extracts at Larger Quantities
To obtain yeast cell extracts in larger quantities, yeast cells were
grown to mid-log phase in YPD medium, pelleted, washed with
distilled water, pelleted, and resuspended in lysis buffer (20 mM
Tris [pH 7.5], 50 mM KCl, 1 mM EDTA, 0.1% NP40, 1 mMDTT)
and fresh protease and phosphatase inhibitors (1 ?g/ml aprotinin,
leupeptin, and pepstatin A; 1 mM PMSF; 1 ?M microcystin-LR; 2
mM p-chloromercuriphenylsulfonic acid). Cells were then disrupted
by vortexing with glass beads (0.5 mm; Biospec Products) for 15
min at 4?C. The bottoms of the microcentrifuge tubes were punc-
tured, and cell extracts were recovered into a larger tube by brief
centrifugation in a microfuge. The lysate was clarified by centrifuga-
tion at 20,000 g for 30 min, subjected to SDS-PAGE, transferred to
nitrocellulose membrane, and probed with either anti-K4 methylhis-
tone H3, anti-Flag, anti-ubiquitin, or anti-K79 methylhistone H3 anti-
sera at about 1:1000 dilution, followed by detection of the bound
antibody with horseradish peroxidase-conjugated to anti-rabbit IgG
secondary antibodies (1:10,000 dilution).
Size Exclusion Chromatography on Superose 6PC
The conductivity of TAP-purified complexes was adjusted to a con-
ductivity equivalent to that of 400 mM KCl in buffer A by dropwise
addition of buffer C containing 1 M KCl. Samples were centrifuged
at 14,000? g for 30 min and then applied to a Superose 6PC column
(Pharmacia) equilibrated in buffer A containing 400 mM KCl. The
column was eluted at 0.1 ml/min, and 100 ml fractions were col-
lected. Fractions were subjected to SDS-PAGE and developed by
In Vivo Analysis of Histone Ubiquitination
Strains expressing FLAG-tagged H2B as the only source of histone
H2B were obtained from Dr. Struhl’s laboratory (Ng et al., 2002a,
ing published methods. Highly purified acid extracted histones from
either wild-type strain or strains deficient for rad6 or bre1 were
subjected to 16% SDS-PAGE electrophoresis and transferred to
Biochemical Purification of Rad6 and Bre1
Purification of the Bre1 and Rad6 containing complexes was carried
out at 4?C. All purification steps were performed several times to
assure the identification of correct polypeptides. Fractions were
tested for Rad6 by Western analysis with anti-Rad6 polyclonal anti-
bodies. For the affinity purification of complexes, both Bre1 and
Recruitment of Rad6 to a Promoter via Bre1
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