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RESEARCH COMMUNICATION
The ATM-related domain
of TRRAP is required for histone
acetyltransferase recruitment
and Myc-dependent oncogenesis
Jeonghyeon Park,
1
Sudeesha Kunjibettu,
2
Steven B. McMahon,
2
and Michael D. Cole
1,3
1
Department of Molecular Biology, Lewis Thomas Laboratory,
Princeton University, Princeton, New Jersey 08544-1014,
USA;
2
The Wistar Institute, Philadelphia,
Pennsylvania 19104-4268, USA
The ATM-related TRRAP protein is a component of sev-
eral different histone acetyltransferase (HAT) complexes
but lacks the kinase activity characteristic of other ATM
family members. We identified a novel function for this
evolutionarily conserved domain in its requirement for
the assembly of a functional HAT complex. Ectopic ex-
pression of TRRAP protein with a mutation in the ATM-
related domain inhibits Myc-mediated oncogenic trans-
formation. The Myc-binding region of TRRAP maps to a
separable domain, and ectopic expression of this domain
inhibits cell growth. These findings demonstrate that
the ATM-related domain of TRRAP forms a structural
core for the assembly and recruitment of HAT com-
plexes by transcriptional activators.
Received March 30, 2001; revised version accepted May 14,
2001.
Members of the ATM family of proteins function in hu-
mans, yeast, and other organisms as critical sensors of
genomic integrity or growth conditions. In humans, the
ATM family includes ATM, ATR, FRAP, and DNA-
PKcs, and homologs of these proteins are found in yeast,
flies, and other organisms (e.g., Mec1p, Tel1p, RAD3p,
TOR1p, and Mei-41; Keith and Schreiber 1995). The de-
fining features of the proteins in the ATM family are
their general large size (2500–4000 amino acids) and a
300-amino-acid motif in their C terminus that resembles
the catalytic domain of PI3-kinases. However, most of
these proteins have been shown to phosphorylate other
proteins rather than lipids. More extended alignment of
the ATM family members identifies two other conserved
domains in addition to the PI3-kinase-like domain (Bo-
sotti et al. 2000). The PI3-kinase-like domain is flanked
by the FATC domain of 35 amino acids at the extreme C
terminus and the FAT domain, which comprises ∼500
amino acids amino terminal of the catalytic domain. All
existing evidence suggests that the integrity of the ki-
nase domain is essential for the function of ATM family
proteins, but the functions of the FATC and FAT do-
mains remain unknown. However, our recent identifica-
tion of the novel ATM-related TRRAP proteins has
raised new questions about the function of these con-
served domains. The TRRAP protein family was identi-
fied as transcriptional cofactors that mediate the recruit-
ment of large multiprotein histone acetyltransferase
(HAT) complexes to sequence-specific activators (Grant
et al. 1998; McMahon et al. 1998; Saleh et al. 1998; Vas-
silev et al. 1998). We initially identified TRRAP or-
thologs in humans, Saccharomyces cerevisiae,Schizo-
saccharomyces pombe, and Caenorhabditis elegans, and
more recently in Arabidopsis thaliana and Drosophila
melanogaster. The TRRAP proteins are true members of
the ATM family because they possess the FAT, kinase-
related, and FATC domains. However, the kinase-related
domain is conserved in sequence but not in function as
the specific residues mediating phosphate transfer are
absent (McMahon et al. 1998). This lack of catalytic ac-
tivity has been supported by biochemical studies on both
the human and yeast TRRAP proteins (Saleh et al. 1998).
The paradox of sequence conservation in the absence of
kinase activity prompted us to explore a noncatalytic
role for this conserved domain in the recruitment of
HAT complexes to transcriptional activation domains.
Results
To investigate the function of the ATM-related region of
TRRAP, a progressive series of C-terminal deletions (1–
3760, 1–3713, and 1–3087) was constructed from a FLAG
epitope-tagged full-length (1–3830) TRRAP cDNA ex-
pression vector (Fig. 1A). An internal deletion (⌬2108–
2403) within the TRRAP cDNA was also constructed.
Each protein was transiently expressed in HEK293 cells,
immunoprecipitated with anti-FLAG antibodies, eluted
from the beads with an excess of FLAG peptide, and sub-
jected to histone acetyltransferase assays using core his-
tones as substrates. The reaction products were resolved
by graded porosity SDS-PAGE and transferred onto ni-
trocellulose membranes. The upper section of the mem-
brane was probed with antibodies for FLAG and hGCN5
and the bottom section was subjected to fluorography
after Ponceau-S staining (Fig. 1B). We found that the full-
length TRRAP protein bound to the endogenous hGCN5
protein and that the immunoprecipitates had readily as-
sayed histone acetyltransferase activity. In contrast, all
of the C-terminal deletion mutations abolished TRRAP
binding to hGCN5 and the recruitment of histone acet-
yltransferase activity, including the TRRAP(1–3760) pro-
tein with only a 70-amino-acid deletion including the
FATC domain. In addition, an internal 20-amino-acid
deletion (⌬3692–3713) of a highly conserved sequence
within the ATM-related domain also abolished the in-
teraction with hGCN5 (data not shown). An internal de-
letion (⌬2108–2403), distant from the ATM-related do-
main, showed little or no difference in hGCN5 binding
or HAT activity compared with full-length TRRAP.
These data establish that the ATM-related domain of
TRRAP is necessary for HAT recruitment. To determine
whether the ATM-related domain of TRRAP was suffi-
cient for HAT recruitment, the C terminus of TRRAP
(3404–3830) was expressed with a FLAG epitope tag (Fig.
1B, lane 7). The smaller C-terminal domain was ex-
pressed at a much higher level than full-length TRRAP,
[Key Words: TRRAP; histone acetyltransferase; Myc; ATM domain; on-
cogenesis]
3
Corresponding author.
E-MAIL mcole@molbio.princeton.edu; FAX (609) 258-4575.
Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/
gad.900101.
GENES & DEVELOPMENT 15:1619–1624 © 2001 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/01 $5.00; www.genesdev.org 1619
but this segment was inactive in binding to hGCN5 or in
the recruitment of histone acetyltransferase activity.
Thus, the ATM/PI-3 kinase homology domain is neces-
sary but not sufficient for the assembly of a functional
HAT enzyme complex with TRRAP.
The recruitment of hGCN5 by TRRAP is expected to
create an enzyme complex with predominantly H3-spe-
cific HAT activity (Yang et al. 1996; Wang et al. 1997),
but the TRRAP immunoprecipitates showed an equal
preference for H3 and H4 acetylation. The identity of the
H4-specific histone acetyltransferase in association with
TRRAP requires further characterization but could de-
rive from human MYST family proteins TIP60, MOZ, or
MORF (Sterner and Berger 2000).
Functional TRRAP and hGCN5 proteins are required
for cellular transformation by the c-Myc oncoprotein
(McMahon et al. 1998, 2000). The involvement of the
TRRAP ATM-related homology domain in the recruit-
ment of histone acetyltransferase activities prompted us
to investigate its physiological role in the cell cycle and
cellular transformation. Expression vectors for full-
length TRRAP or the TRRAP(1–3760) truncation mutant
were cotransfected with expression vectors for c-Myc
and/or oncogenic H-rasG12V into early-passage rat em-
bryo fibroblasts (Fig. 2A). Neither TRRAP itself nor the
truncation mutant had intrinsic transforming activity in
conjunction with the H-rasG12V oncogene. Cotransfec-
tion of c-myc and H-rasG12V leads to focus formation,
and the addition of the wild-type TRRAP expression vec-
Figure 2. Expression of a TRRAP mutant defective in HAT
recruitment inhibits c-Myc oncogenic activity. (A) Primary rat
embryo fibroblasts were transfected with expression vectors for
c-Myc, H-RasG12V, wild-type full-length TRRAP, and the
HAT-defective TRRAP(1–3760) truncation mutant in the differ-
ent combinations indicated. (yaxis) Number of transformed
foci. Three plates were assayed for each bar. (B) TRRAP and
TRRAP mutants do not inhibit colony growth. Primary rat em-
bryo fibroblasts were transfected as indicated along with pSG5
puromycin
R
plasmid. Puromycin
R
colonies were counted after
14 d. Two plates were assayed for each bar in the graph.
Figure 1. The ATM/PI-3 kinase domain of TRRAP is critical
for the recruitment of histone acetyltransferase activity. (A)
Schematic representation of wild-type, full-length TRRAP (top)
with the ∼300 C-terminal amino acids homologous to the ATM/
PI-3 kinase family members indicated as ATM. Below is shown
a series of TRRAP internal fragments, C-terminal truncation
mutants, and internal deletion mutants with their respective
sizes at the right. (B) ATM-domain dependence for HAT recruit-
ment. Complexes containing transiently expressed FLAG-
tagged TRRAP or TRRAP mutants were isolated from HEK293
cells (McMahon et al. 1998). The FLAG–TRRAP or TRRAP mu-
tants were eluted from beads and assayed for HAT activity on
calf thymus core histones. Reaction mixtures were resolved on
an SDS-PAGE gradient gel (4%–15%) and transferred to a nitro-
cellulose membrane. The upper section was probed with anti-
FLAG and anti-hGCN5 antibodies. Cell lysate containing 100
µg of protein was included as a positive control for hGCN5 (lane
left of lane 1). The bottom portion of the membrane below the
30-kD molecular mass marker was stained with Ponceau-S and
subjected to fluorography for HAT activities. The 97-kD band
common to lanes 1–7in the hGCN5 panel is a nonspecific band
that arises from the anti-FLAG beads, but is not found with cell
lysates (lane left of lane 1).
Parketal.
1620 GENES & DEVELOPMENT
tor had no effect on this activity. In contrast, cotransfec-
tion of c-myc and H-rasG12V with the TRRAP(1–3760)
mutant that is defective for HAT-recruitment resulted in
a 50% inhibition of focus formation. The finding of only
a partial dominant-negative phenotype with the
TRRAP(1–3760) mutant may be due to the large size of
the TRRAP protein and relatively low level of ectopic
expression, which may not allow effective competition
with the endogenous wild-type TRRAP. In parallel with
the REF transformation assay, we tested for transfection
efficiency or any toxicity from the TRRAP expression
constructs by scoring for puromycin-resistant colonies
after cotransfection with the pSG5-Puromycin
R
plasmid
(Fig. 2B). Approximately equal numbers of puromycin-
resistant colonies were obtained with all plasmid com-
binations tested, indicating that the inhibitory effect of
the TRRAP(1–3760) truncation mutant on oncogenic
transformation did not result from cellular toxicity or
general growth inhibition.
In addition to TRRAP–Tra1p and GCN5–PCAF, the
SAGA–PCAF complexes contain SPT3, which has been
suggested to regulate transcription through interaction
with TBP (Dudley et al. 1999; Belotserkovskaya et al.
2000). SPT3 protein has been found in all TRRAP com-
plexes that contain hGCN5 (Brand et al. 1999). Because
mutations in the ATM-homology region abolished bind-
ing to hGCN5, we were interested in determining what
other components of the complex were dependent on
this domain. To assess whether hSPT3 binding to
TRRAP was dependent on the ATM-related domain, im-
munoprecipitates of FLAG-tagged full-length or a C-ter-
minal deletion of TRRAP were probed with anti-SPT3
antibodies (Fig. 3). Full-length TRRAP bound to hSPT3,
but binding was abolished by a small deletion in the
ATM-related domain.
The exclusive enrichment of TRRAP by the c-Myc N
terminus in the initial affinity purification strategy sug-
gested that the interaction between these two nuclear
proteins is direct and unlikely to require any associated
cofactors (McMahon et al. 1998). We were interested in
determining which domain or motif in TRRAP is impor-
tant for c-Myc binding. Progressive deletions and several
internal fragments in the TRRAP C terminus were used
to map the sequence for Myc binding (Fig. 4). Plasmids
expressing FLAG-tagged TRRAP (or mutants thereof)
were transiently cotransfected into HEK293 cells with
an expression vector for c-Myc, and then anti-FLAG im-
munoprecipitates isolated under nondenaturing condi-
tions were probed by Western blot analysis for c-Myc
protein (Fig. 4A, bottom). The expression levels and re-
covery of the TRRAP proteins were tested by anti-FLAG
Western blotting (Fig. 4A, top), showing that smaller
TRRAP mutants were generally expressed better than
the full-length protein. In the initial experiments, dele-
tions from the C terminus up to amino acid 3087 re-
tained the ability to coprecipitate with c-Myc, in con-
trast with the hGCN5 binding described above. Expres-
sion of only the N-terminal half of TRRAP, 1–1700,
coprecipitated a negligible amount of c-Myc despite the
fact that the expression level was significantly higher
than the larger TRRAP proteins. These data indicate that
the Myc-binding site maps between amino acids 1700
and 3087. To refine the mapping further, three FLAG-
tagged internal fragments of TRRAP (1899–2401, 2402–
2869, and 2869–3470) were transiently expressed, immu-
noprecipitated with anti-FLAG antibody, and assayed for
binding to endogenous c-Myc by Western blot (Fig. 4B,
top). In parallel, immunoprecipitates by anti-FLAG anti-
bodies were analyzed by Western blotting to assess the
expression level of FLAG-tagged TRRAP fragments (Fig.
4B, bottom). Only the TRRAP(1899–2401) fragment co-
precipitates with endogenous c-Myc, suggesting that it
contains the primary c-Myc interaction domain in the
context of intact TRRAP.
Coprecipitation of c-Myc and TRRAP from whole cell
lysates could be mediated by another component of
SAGA or other intermediate protein. A more direct assay
of protein–protein interactions using the GST-pulldown
technique was used to map the Myc-binding domain on
TRRAP (Fig. 4C). To broaden the analysis, we tested the
binding of N-Myc to TRRAP as N-Myc binds with an
affinity equal to that of c-Myc (Wood et al. 2000). A se-
ries of TRRAP protein fragments was expressed to equal
levels in Escherichia coli and purified on beads. The
beads were then incubated with affinity-purified N-Myc
protein to assess binding. Five segments spanning 50%
of the 3830-amino-acid TRRAP protein were tested for
N-Myc binding, and only one segment (1591–2026) ex-
hibited significant binding compared with GST alone.
This segment overlaps the binding domain predicted
from mammalian expression studies above, suggesting
that the major site of Myc interaction with TRRAP maps
between amino acids 1899 and 2026.
To provide an in vivo test of the N-Myc–TRRAP in-
teraction domain, two FLAG–TRRAP protein segments
(1261–1579 and 1899–2401) were stably expressed at
equal levels in human neuroblastoma cells, which con-
tain an amplified N-myc gene (data not shown). Because
Myc is required for cell cycle progression, competition
between endogenous N-Myc and a critical cofactor
would be expected to impede cell growth. Consistent
with this model, expression of FLAG–TRRAP(1899–
2401) containing the Myc-binding domain increased the
Figure 3. The ATM/PI-3 kinase domain of TRRAP is critical
for interaction with hSPT3. HEK293 cells were transiently
transfected with expression vectors for FLAG-tagged TRRAP
fragment or its deletion mutant, TRRAP(1–3713). Cell lysates
were prepared and immunoprecipitated with anti-FLAG anti-
bodies. Each precipitate was resolved by SDS-PAGE and ana-
lyzed for the extent of its coprecipitation with endogenous
hSPT3. To check the relative expression levels of TRRAP frag-
ments and hGCN5 recruitment, the same membrane was
reprobed with anti-FLAG antibodies (top) and anti-hGCN5
(middle).
TRRAP interaction with HAT and Myc
GENES & DEVELOPMENT 1621
doubling time of IMR5 cells from 28 to 63 h, or 2.25-fold
(Table 1). Expression of another segment of TRRAP
(1261–1579) had no impact on growth rate compared
with control cells, even though the latter can inhibit
c-Myc and N-Myc transforming activity in primary fi-
broblasts (McMahon et al. 1998).
Discussion
This study provides the first functional mapping of do-
mains within the large ATM-related TRRAP protein that
is implicated in transcriptional regulation and chroma-
tin modification from yeast to man. We find that the
c-Myc-binding region is distinct from the ATM/PI-3 ki-
nase domain, and this region is conserved among TRRAP
orthologs from other species, but not conserved in other
ATM family members. Interestingly, this region over-
laps the direct binding site for transcriptional activators
on the yeast ortholog of TRRAP (Tra1p; J. Workman,
pers. comm.), suggesting that the function of TRRAP–
Tra1p as a mediator of HAT-activator interactions has
been highly conserved in evolution. Furthermore, this
same domain of Tra1p binds to Yng2p in yeast HAT
complexes and hence may play a major functional role in
all TRRAP–Tra1p complexes (Loewith et al. 2000).
Although the ATM-related C-terminal domain of
TRRAP lacks kinase activity, we show here that this
domain is essential for the recruitment of HAT activity
by the complex, and similar findings have been made for
the yeast Tra1p (J. Workman, pers. comm.). Generally
protein–protein interactions are mediated by relatively
small motifs, and, therefore, it was unexpected that mu-
tations in the ATM-related domain abolished HAT re-
cruitment whereas the isolated ATM-related domain is
defective for binding. These results suggest that TRRAP
may adopt a complex three-dimensional structure that is
dependent on the ATM homology domain for the re-
cruitment of HATs and most other components of the
complex. A parallel can be drawn with the ATM-related
Rad3p in fission yeast, which requires an N-terminal
protein domain for the activity of the C-terminal kinase
domain (Chapman et al. 1999). ATM/PI3 kinase family
proteins are frequently involved in checkpoint or sensing
functions within the cell, suggesting that the activity of
TRRAP itself may also be regulated.
Figure 4. TRRAP amino acids 1899–2026 mediate Myc bind-
ing. (A) In vivo coimmunoprecipitation. HEK293 cells were
transiently cotransfected with CMV-driven expression vectors
for c-Myc and FLAG-tagged full-length TRRAP or a series of
TRRAP C-terminal truncation mutants. Cell lysates were pre-
pared and the FLAG-tagged TRRAP protein or mutants were
immunoprecipitated with anti-FLAG antibodies. Precipitates
were resolved by SDS-PAGE and analyzed by Western blotting
for anti-c-Myc antibodies (bottom). The same membrane was
also probed with anti-FLAG antibodies to detect TRRAP expres-
sion (top). Apparent molecular masses in kilodaltons of protein
markers are indicated at the left of fluorogram. (B) Localization
of the c-Myc-binding domain. HEK 293 cells were transiently
transfected with expression vectors for FLAG-tagged TRRAP
fragment. Cell lysates were prepared and immunoprecipitated
with anti-FLAG antibodies. Each precipitate was resolved by
SDS-PAGE and analyzed for the extent of its coprecipitation
with endogenous c-Myc (top). Cell lysate from the c-Myc trans-
fection was included as a positive control (lane left of lane 1). To
check the relative expression levels of TRRAP fragments, the
same membrane was reprobed with anti-FLAG antibodies (bot-
tom). (C) An internal region of TRRAP (spanning amino acids
1591–2026) interacts with N-Myc. GST fusion proteins contain-
ing the indicated TRRAP regions were expressed in E. coli and
bound to glutathione beads. Equal amounts of bead-bound fu-
sion proteins were incubated with affinity-purified FLAG-
tagged N-Myc protein. After washing, interacting proteins were
subjected to SDS-PAGE analysis and immunoblotted with anti-
FLAG antibody. The FLAG-N-Myc starting material (20% of
input) was run with the bound material.
Table 1. Myc-interacting region of TRRAP has a dominant
negative effect on cell growth
Cell line TRRAP segment Doubling time (h)
IMR-5 — 26.7 ± 6.4
IMR-5 1261–1579 27.9 ± 5.3
IMR-5 1899–2401 63.1 ± 12.2
IMR-5 human neuroblastoma cells were stably transfected with
expression vectors for FLAG-tagged TRRAP fragments as indi-
cated. Cell lysates were immunoprecipitated with anti-FLAG
antibody followed by immunoblot analysis with the same anti-
body (data not shown). Both fragments were comparably ex-
pressed. Doubling times were calculated from three individual
plates from each line (± SE).
Parketal.
1622 GENES & DEVELOPMENT
A common mechanism of chromatin modification is
the acetylation of N-terminal lysine residues on his-
tones, which reduces histone–DNA interactions to pro-
mote a more open chromatin configuration (Grant et al.
1997). A key feature of the multiprotein HAT complexes
is the ability to acetylate nucleosomal histones, whereas
the HAT enzyme alone can only acetylate free histones
(Kuo et al. 1996; Grant et al. 1997). Therefore, one or
more components of these complexes must modify HAT
activity to facilitate nucleosome recognition, as well as
mediate binding to transcriptional activators. TRRAP is
remarkably conserved in evolution as a 3800 ± 100-
amino-acid polypeptide, implying that size and the ori-
entation of functional elements are critical. TRRAP–
Tra1p appears to be a direct, global mediator of transcrip-
tional activator–HAT interactions, and further
elucidation of its structure and function may provide
important insights into the dynamics of transcriptional
activation and chromatin modification.
Materials and methods
Plasmid constructs and transfections
Expression plasmids were created by standard methods in a CMV-pro-
moter driven vector containing a FLAG-epitope tag and verified by se-
quence analysis (McMahon et al. 1998). Details of individual constructs
are available on request.
HAT assays
FLAG-tagged TRRAP protein was transiently expressed in HEK293 cells
and isolated from whole-cell lysates by binding to anti-FLAG antibody
covalently conjugated to beads (Sigma). The complexes containing
FLAG–TRRAP or its mutants were released by addition of excess FLAG
peptide in HAT assay buffer (50 mM Tris at pH 8.0, 10% glycerol, 50 mM
KCl, 0.1 mM EDTA, 10 mM butyric acid, 1 mM DTT, 1 mM PMSF). Calf
thymus core histones (3 µg) along with
14
C-acetyl-CoA were incubated
with each eluate, and reaction mixtures were resolved on a SDS-PAGE
gradient gel (4%–15%; Ogryzko et al. 1996). Proteins were transfered
onto a nitrocellulose membrane, and the section above the 30-kD mo-
lecular mass marker was subjected to Western blot analysis and probed
with anti-FLAG and anti-hGCN5 antibodies. The bottom section of the
membrane was stained with Ponceau-S and subjected to fluorography for
HAT activities.
GST pulldown assay
TRRAP fragments were PCR amplified, cloned into pGXT4T1 (Amer-
sham Pharmacia Biotech) and expressed as fusion proteins in E. coli. For
large-scale purification, bacterial pellets were sonicated in STE buffer (10
mM Tris at pH 8.0, 150 mM NaCl, 1 mM EDTA, 5 mM DTT, 1.5%
Sarkosyl, PMSF–aprotinin). After addition of Triton X-100 to a final con-
centration of 2%, lysates were cleared by centrifugation, and the fusion
proteins were bound to glutathione–Sepharose 4B beads (Amersham
Pharmacia Biotech). Beads were analyzed for the fusion protein by SDS-
PAGE followed by Western analysis for GST.
FLAG–N-Myc was stably expressed from a CMV promoter in IMR-5
neuroblastoma cells after neo cotransfection and G418 selection. Cells
from three semiconfluent 15-cm plates were lysed in E1A lysis buffer
(McMahon et al. 1998), followed by overnight immunoprecipitation with
anti-FLAG antibody (Sigma) and Protein A/G beads (Santa Cruz). Bound
FLAG–N-Myc was eluted with FLAG peptide (Sigma). Equal amounts of
GST–TRRAP or control beads (based on the Western), were incubated
with 30–50 µL of the FLAG–N-Myc proteins in binding buffer for 3 h in
4°C. Beads were washed three times in the same buffer, and then bound
proteins were resolved by SDS-PAGE. N-Myc interaction was detected
with anti-FLAG antibody, and the same blot was reprobed with anti-GST
to assess the amount of protein on the beads.
Cell culture
Rat embryo fibroblasts were prepared by trypsinization of 15-d embryos
(Land 1995), and focus formation was assessed as described (McMahon et
al. 1998). Foci were scored after 18 d. Growth properties after transfection
of the same DNAs were assessed on duplicate plates of primary rat em-
bryo fibroblasts cotransfected as indicated along with 1 µg of pSG5 pu-
romycin
R
plasmid. Transfected cells were selected in 2.5 µg/mL puro-
mycin for 14 d, at which time the number of colonies per plate was
determined.
FLAG–TRRAP fragments were stably expressed in IMR-5 neuroblas-
toma cells by cotransfection with neo and selection with G418. Growth
rates and protein expression were assessed at early passage of G418-
resistant polyclonal populations. Equal numbers of viable cells were
plated in parallel for the parental IMR-5 line and the two TRRAP frag-
ment-expressing lines. Cells were counted 48 and 72 h after plating, and
the doubling time was calculated.
Acknowledgments
We thank John Maris for providing IMR-5 cells, and Christine Brown and
Jerry Workman for communicating results in advance of publication.
This work was supported by a grant from the National Cancer Institute
to MDC. This work was also supported by a Leukemia Society Special
Fellow award and grants from the Edward Mallinckrodt, Jr. Foundation,
the Mary L. Smith Charitable Lead Trust, the Gustavus and Louise
Pfeiffer Research Foundation, and the American Association for Cancer
Research to S.B.M.
The publication costs of this article were defrayed in part by payment
of page charges. This article must therefore be hereby marked “adver-
tisement” in accordance with 18 USC section 1734 solely to indicate this
fact.
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Parketal.
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