| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 277, Issue 40, 36921-36930, October 4, 2002
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
§,§¶,,, and¶
From the Ontario Cancer Institute and
¶ Department of Medical Biophysics, University of Toronto,
Toronto, Ontario M5G 2M9, Canada
Received for publication, February 13, 2002, and in revised form, July 12, 2002
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
The c-myc proto-oncogene can direct a
diverse array of biological activities, including cell cycle
progression, apoptosis, and differentiation. It is believed that Myc
can affect this wide variety of activities by functioning as a
regulator of gene transcription, although few targets have been
identified to date. To delineate the molecular program regulated
downstream of Myc, we used a cDNA microarray approach and
identified 52 putative targets out of >6000 cDNAs analyzed. To
further distinguish the subset of genes whose regulation was dependent
upon Myc per se from those regulated in response to
activation of general mitogenic or apoptotic programs, the putative
cDNA targets were then screened by a series of assays. By this
approach 37 putative targets were ruled out and 15 Myc target genes
were uncovered. Interestingly, comparing our results with other high
throughput screens reveals that certain putative Myc targets
previously reported are shown not to be regulated downstream of Myc
(e.g. ribosomal proteins, HSP90 The c-myc oncogene has been implicated in both the
initiation and the progression of a wide variety of tumors. Indeed, it is estimated to contribute to one in seven cancer deaths, including breast, colon, and cervical carcinomas (reviewed in Ref. 1). Strikingly, 100% of Burkitt's lymphoma patients harbor a deregulated c-myc allele as a result of a translocation that juxtaposes
the c-myc gene with the regulatory region of an
immunoglobulin gene (reviewed in Ref. 2). This genetic rearrangement
leads to constitutively high expression levels of Myc protein within
the cell, as is commonly seen in other cancers harboring an activated
c-myc allele.
c-myc is an immediate early response gene following mitogen
stimulation, which encodes a nuclear phospho-protein that regulates numerous biological activities. Myc functions to drive cell cycle progression, induce apoptosis, block differentiation, and, when deregulated, promote cellular transformation (reviewed in Refs. 3-5).
This diversity of functions has been attributed to Myc's ability to
activate or repress the transcription of different target genes that
mediate these various activities. Myc activation of gene transcription
has been studied in depth for many years, yet few targets have been
discovered. These include ornithine decarboxylase, elongation
initiation factor 4E (eIF-4E), cdc25A, and
carbamoyl-phosphate synthase (glutamine hydrolyzing)/aspartate carbamoyltransferase/dihydroorotase
(cad)1 (6-9). The current
model of Myc activation involves Myc heterodimerization with its
partner protein Max forming a DNA binding domain. This Myc·Max
heterodimeric complex can then recognize and bind specific E-box
elements associated with the target gene to directly activate transcription. It is hypothesized that Myc can activate genes through
multiple regulatory events at the level of transcriptional initiation
and elongation (3, 10-16). In contrast, Myc repression of gene
expression is much less defined. Myc repressed genes include growth
arrest and DNA damage-inducible gene (gadd45 Numerous reports have been published recently that have employed a
variety of large scale gene expression approaches to uncover the subset
of genes regulated by Myc in an effort to understand Myc's biological
role (28-36). Interestingly, very few overlapping targets have been
identified in these array experiments, with the exception of many
ribosomal genes. This may be due to the different profile of cDNAs
analyzed in the various assays. However, it is likely that, in addition
to the identification of Myc-specific targets, genes that are regulated
as a consequence of Myc activity, but not dependent on Myc, may also be
captured by the array approach, thus obscuring the isolation of true
targets. This can lead to the study of false-positive gene targets as
well as misconceptions regarding the nature of the genes that are
regulated by Myc. For these reasons, the identification of target genes
that are specifically regulated in a Myc-dependent
manner is essential.
To this end, we have undertaken a microarray approach in combination
with a series of subsequent screening steps to identify genes that are
regulated downstream of Myc. By this approach we were able to rule out
37 of the original 52 putative target genes that were identified as
differentially regulated by the microarray analysis. We report the
identity of 15 genes that were regulated by Myc in each of our
experimental systems and fulfilled our criteria of a downstream target
of Myc.
Cell Culture--
Parental TGR-1 rat fibroblasts, Myc null
HO15.19 rat fibroblasts, HO15.19 cells infected with green fluorescent
protein (GFP) vector alone (HO15.19-GFP) and HO15.19 cells
infected with GFP-myc retrovirus (HO15.19-myc)
were described previously (18). They were maintained in 10% calf
serum-Dulbecco's modified Eagle's medium-H21 (DMEM H21, Invitrogen).
The media was supplemented with 100 µg of penicillin per milliliter
and 100 µg of streptomycin sulfate per milliliter. Quiescence was
achieved by maintaining the cells in 0.25% calf serum-DMEM H21 for
48 h, and serum stimulation was accomplished by replacing media
with 10% calf serum-DMEM H21 for the indicated time periods. Rat1
MycERTM cells have been described previously (18). They were maintained
in 10% fetal bovine serum- Retroviral Production and Infection--
To produce infectious
replication-deficient ecotropic retroviral particles, retroviral
constructs were transfected by the calcium phosphate method into the
Phoenix Eco packaging cell line, and viral supernatant was harvested
36-48 h later. This virus was then used immediately to infect target
cells for 3-18 h in the presence of 8 µg/ml Polybrene or frozen at
Microarray Analysis--
Approximately 4 × 107
subconfluent, proliferating HO15.19-GFP and HO15.19-myc
cells were harvested and frozen, and the cell pellets were forwarded
for microarray analysis in duplicate (Synteni, Fremont, CA) on a
chip containing 6355 mouse cDNAs and expressed sequence tags.
Differential expression values, calculated as +P1/P2 where
P1Signal > P2Signal or Northern Blotting--
Total RNA isolated from the cell lines
using TRIzol (Invitrogen) was resolved by electrophoresis on a
formaldehyde-1.5% agarose gel, visualized by EtBr staining,
photographed, transferred to a nylon membrane (GeneScreen Plus,
Dupont), UV cross-linked, and baked. Blots were probed with
gel-purified cDNAs random prime-labeled with
[ RNase Protection Assay--
RNase protection assays were
conducted as previously described (17, 18, 21). Briefly, RNA harvested
from cell cultures (TRIzol, Invitrogen) was hybridized to a radioactive
32P-labeled riboprobe specific for the gene of interest
(pdgf To identify Myc target genes using a microarray approach, it was
important to employ a cellular system that would maximize sensitivity
of detection with respect to differential gene expression in the
presence or absence of Myc protein. To this end, we used the Myc-null
cell line HO15.19, established by homologous recombination of both
c-myc alleles of the parental Rat1 TGR-1 cells thereby replacing the coding region with drug-selectable markers (37). HO15.19
cells were infected with control retrovirus containing the cDNA for
green fluorescent protein (HO15.19-GFP) or retrovirus containing both
human c-myc and GFP cDNAs (HO15.19-myc). The
infected cells were isolated by fluorescence-activated cell sorting
(FACS) using GFP as the selectable marker. Immunoblot analysis of the pooled populations confirmed Myc protein expression was restricted to
the HO15.19-myc cells and not evident in the HO15.19-GFP
cells (data not shown). To determine whether Myc-regulated gene
expression was evident in this cell system, the expression of the known
Myc-activated gene cad and known Myc-repressed genes gadd45
), whereas others are
further supported by our analyses (e.g. pdgfr,
nucleolin). The identity of genes specifically regulated downstream of
Myc provides the critical tools required to understand the role Myc holds in the transformation process and to delineate how Myc functions as a regulator of gene transcription.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
), platelet-derived growth factor receptor (pdgfr), H-ferritin,
p15INK4b, and c-myc itself (17-21). Evidence
suggests that Myc may repress some genes through an initiator
element in the core promoter (20, 22-26). However, Myc can also
repress genes that lack initiator elements (17, 18), suggesting
multiple mechanisms of Myc-mediated repression. It is thought that
Myc's ability to repress certain genes occurs via interference with
transcription factors or enhancers that are required at these promoters
for gene activation (3, 20, 27). Identifying Myc target genes provides
the critical experimental tools to address both the biological role and
molecular mechanism of Myc action.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-modified Eagle's medium (MEM)
supplemented with 100 µg of penicillin per milliliter and 100 µg of
streptomycin sulfate per milliliter. Quiescence was achieved by
maintaining the cells in 0.25% fetal bovine serum-MEM H21 for
48 h, and MycERTM was activated by adding hydroxytamoxifen (OH-T)
to 100 nM (Sigma). Cycloheximide (CHX) was used at a
concentration of 10 µg/ml. Primary rat embryo fibroblasts and mouse
embryo fibroblasts were maintained in 10% fetal bovine serum-MEM.
The Phoenix Eco retroviral packaging cell line (American Type Culture
Collection) was maintained in 10% fetal bovine serum-DMEM.
70 °C for later use. Infected cells were isolated by
fluorescence-activated cell sorting (FACS) for the GFP marker expressed
from the bicistronic retroviral vector 2-3 days post-infection.
GFP-positive cells were isolated with a Becton Dickinson FACStarPLUS
cell sorter using a Coherent Enterprise laser emitting 175 milliwatts
of light at 488 nm; GFP fluorescence (emission) was collected through a 530/30 band pass dichroic filter. BDIS CellQuest software was used for
acquisition and analysis of data. GFP-positive cells from each
infection were pooled.
P2/P1 where P1Signal < P2Signal, were compared between the duplicate experiments. Clones differentially regulated in both experiments that had a significant ratio of P1 to P2
(defined by us as greater than the value of 2) were then PCR-amplified
and cloned into the TA cloning vector pCRII (Invitrogen).
-32P]dCTP (T7 Quick prime kit, Invitrogen).
Northern blots were prehybridized for 2 h at 42 °C in 50%
formamide, 5× saline/sodium phosphate/EDTA, 1% glycine, 5×
Denhardt's solution, and 100 µg/ml denatured, sheared salmon sperm
DNA for at least 2 h. Blots were then hybridized in 50%
formamide, 5× saline/sodium phosphate/EDTA, 1× Denhardt's, 0.3%
SDS, 100 mg/ml denatured, sheared salmon sperm DNA, 10% dextran sulfate, and 1 × 106 cpm/ml of denatured,
32P-radiolabeled cDNA. Hybridization was carried out at
42 °C for 16-20 h. Blots were washed three times for 15 min at room
temperature in 2× SSC, 0.1% SDS and then 2× at 60 °C in 0.2×
SSC, 0.1% SDS. Bands were visualized by autoradiography on Biomax MS
film (Kodak) using a Biomax Transcreen HE intensifying screen at
70 °C. Densitometry was accomplished using ImageQuaNT software and
normalized to a 36B4 loading control (15).
r, c-myc exon 1, gadd45, cad, or
glyceraldehyde-3-phosphate dehydrogenase). RNases were added to digest
single-stranded RNA molecules. Protected double-stranded RNA molecules
were then electrophoresed in a 6% polyacrylamide denaturing gel and
visualized by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
pdgfr, and c-myc were evaluated (Fig.
1A). Regulation of the
endogenous c-myc promoter was determined by analyzing levels
of c-myc exon 1, which remained intact after the knockout
procedure. RNase protection analysis was carried out on RNA extracted
from asynchronously growing TGR-1, HO15.19, HO15.19-GFP, and
HO15.19-myc cells. All genes displayed the expected pattern
of regulation in this system with the observed difference in signal
intensity clearly evident when wild-type parental TGR-1 and Myc-null
HO15.19 cells were compared, and strikingly evident between the
HO15.19-GFP and HO15.19-myc cells. As expected, HO15.19 and
the HO15.19-GFP control cell lines displayed a similar level of control
gene expression suggesting that retroviral infection did not alter
cellular gene expression. These results also indicate that
these target genes were regulated by both the endogenous Myc in the
parental TGR-1 cells and the exogenous constitutively expressed Myc in
the reconstituted HO15.19 cells. Regulation of these target genes was
more pronounced in the HO15.19-myc cells, which is
consistent with previous observations showing Myc regulates gene
expression in a dose-dependent manner (18, 21). Taken together, these data indicate that the
HO15.19-GFP/HO15.19-myc cell system serves as a valuable
tool for identify target gene regulation by Myc with maximum
sensitivity of differential gene expression.
View larger version (44K):
[in a new window]
Fig. 1.
Regulation of target genes in the
HO15.19/TGR-1 system. A, the expression of known Myc
target genes c-myc, pdgf r, gadd45, and cad were
analyzed by RNase protection assay using 10 µg of RNA harvested from
asynchronous, proliferating TGR-1, HO15.19, HO15.19-GFP, and
HO15.19-myc cells. B, primary screen of putative
Myc target genes identified by microarray analysis as differentially
expressed. Northern blot analysis was performed with 10 µg of RNA
harvested from the cells as in A using cDNA probes
identified in the microarray analysis. Representative blots of
"Activated," "Repressed," and clones that were "Not Myc
Regulated" are shown.
Identifying Differentially Expressed Genes Using a cDNA
Microarray Approach--
Fluorescently labeled cDNA representing
mRNA from asynchronously growing HO15.19-GFP and
HO15.19-myc cells was used to probe 6.3K cDNA
microarrays in duplicate (Synteni). A cDNA was considered a
putative Myc-regulated target if the hybridization signal was detected
as a greater than 2-fold difference in signal intensity consistent
across the two separate array experiments. These candidates were
validated by visual confirmation of a corresponding signal pattern of
uniform spots on the microarray. This stringent analysis identified 63 reproducible gene expression differences, representing 52 different
genes of which 27 appeared to be activated and 25 were repressed. Of
those genes, 5 were independently scored multiple times (ribosomal
proteins S23 and S17, ef1
, HSP90, and a novel gene). Importantly,
a gene previously identified as a c-Myc target, pdgfr (18), was also
detected as differentially expressed, validating both the cell system
and the microarray analysis. From our initial analysis, it was
impossible to determine whether the identified differentially regulated
genes were true Myc targets or if they were regulated as a consequence
of Myc activity. To distinguish between these two groups, the 52 genes
identified as differentially regulated by the microarray screen were
subsequently investigated in four separate screens, each evaluating a
specific aspect of Myc regulation. Our stringent criteria to identify
genes that are regulated in a Myc-dependent manner include
those that were regulated in both immortalized and primary cells and by
both exogenous and endogenous Myc molecules. The results of these
further screens are described below and summarized in Tables
I and
II.
|
|
Primary Selection Criteria: Confirmation of Differential Gene Expression by Northern Blot-- To evaluate the results obtained from the microarray screen, the cDNAs spotted on the microarray were amplified by PCR, and their identities were verified by nucleotide sequence analysis. These cDNAs were radiolabeled and used as probes to analyze the expression patterns of the putative Myc-regulated genes in asynchronously growing TGR-1, HO15.19, HO15.19-GFP, and HO15.19-myc cells. Northern blot analysis confirmed that the expression of the majority of genes corresponded to that assessed by the microarray analysis. Of the original 52 putative target genes, 5 were eliminated due to their lack of regulation or peculiar expression patterns. Of the remaining 47 targets, 25 were induced (Table I) and 22 were repressed (Table II) in the presence of Myc. Representative blots of this first level screen of the microarray results are shown in Fig. 1B. This screen was not only useful in verifying the microarray results, it was also informative in determining whether the presence of Myc at low endogenous levels (TGR-1) and/or deregulated exogenous levels (HO15.19-myc) affected the basal level expression of these targets in asynchronous cells. Those 47 genes identified as potential Myc-regulated targets in this first level screen were then further investigated.
Secondary Selection Criteria: Regulation in Primary Cells in
Response to Exogenous Myc--
To evaluate whether the differential
expression of the identified genes may have been a consequence of the
immortalized TGR-1/HO15.19 cell system, target gene regulation was
assessed in primary mouse or rat embryonic fibroblasts (EF). These
early passage mouse or rat EFs were infected with control retrovirus
containing GFP alone (Myc) or with retrovirus containing both human
c-myc and GFP (+Myc). Infected cells were then isolated by
FACS and pooled, and RNA was isolated from asynchronously growing
cultures and used for Northern blotting. In this second level screen,
12 of the 25 putative activated genes were not regulated in
embryonic fibroblasts, 12 of the genes were up-regulated, and the data
for 1 gene was not informative. 2 of the 22 putative Myc-repressed targets were not regulated in response to Myc expression in embryonic fibroblasts, whereas the remainder showed a down-regulation of expression (see Tables I and II). Representative results of this screen
are shown in Fig. 2.
|
Tertiary Selection Criteria: Regulation by the Inducible
MycERTM Protein in Serum-deprived Cells--
To determine
whether the putative target genes could be regulated in the absence of
mitogen in response to Myc activation, we used the inducible MycERTM
fusion protein system (38, 39) in Rat1 fibroblasts (18). This fusion
protein, of Myc and the regulatory region of the estrogen receptor
(ER), is constitutively expressed in the cells but maintained in an
inactive conformation. Upon the addition of hydroxytamoxifen (OH-T) Myc
is rapidly activated. Serum-deprived quiescent MycERTM cells that have
been exposed to OH-T progress from the G0 to S phase
of the cell cycle. In this third level screen, genes that are
downstream targets of Myc should exhibit regulation upon Myc induction.
However, it remains possible that a subset of Myc-regulated targets,
which are regulated in collaboration with other mitogen-stimulated
factors, may be ruled out by this criteria. RNA extracted from
serum-deprived cells at various times following MycERTM induction was
analyzed by Northern blot. Fig. 3 depicts
the expression of representative activated and repressed targets and
those not regulated by Myc in this system. Of the 25 putative activated
targets, 12 were up-regulated in response to MycERTM activation with
OH-T, 12 were not regulated, and the data for 1 gene were not
informative (Table I). Of 22 potential down-regulated targets, 15 targets were repressed in this system, 6 were not regulated, and the
data for 1 gene were not informative (Table II). This system also
allows us to assess the kinetics of regulation upon Myc activation.
Induction of the majority of the activated genes was first evident at
1, 3, or 6 h after MycERTM activation in response to OH-T.
Interestingly, most genes were observed to respond maximally by 9 h as seen with many Myc-regulated genes (9, 15). The kinetics of
repression were more variable, which may be due to the specific
half-lives of the RNA in question.
|
Quaternary Selection Criteria: Lack of Regulation in
Serum-stimulated HO15.19 Cells--
To assess the
Myc-dependent regulation of putative target genes, we
compared gene expression in Myc-null HO15.19 cells and parental TGR-1
cells. Putative Myc target gene regulation was assessed following serum
stimulation of quiescent HO15.19 and TGR-1 cells. RNase protection
analysis showed that endogenous c-myc mRNA levels are
very low in serum deprived cells and increased quickly after the
addition of serum (data not shown). This comparison allows a fourth
level screen to determine whether Myc is essential for
target gene regulation in response to serum exposure. It is expected
that those genes whose regulation is solely dependent on Myc would not
be responsive to mitogen stimulation in the HO15.19 cells, but would be
regulated in the parental TGR-1 cells, which express endogenous c-Myc
upon serum stimulation. This stringent criterion may exclude a subset
of genes whose regulation is not solely dependent on Myc and can also
be modulated by serum-regulated factors in the presence or absence of
Myc. In addition, genetic alterations may have occurred during the
knock-out procedure that allow the cells to continue cycling in the
absence of Myc and may mask the requirement for Myc in the regulation
of some targets. Northern blots were prepared with RNA extracted from
serum-deprived HO15.19 and TGR-1 cells at various times after serum
stimulation and analyzed with probes generated from the cDNAs
identified from the original microarray. Importantly, the length of
time in which the HO15.19 cells were analyzed was extended to ~2.5
times that of the TGR-1 to reflect the differences in the cell-cycle
time between these cell lines (37). The time points chosen represent equivalent stages in the cell cycle between these cell lines, as
determined by DNA content assessed by flow cytometry (data not shown).
In this system 5 of the 25 activated targets were dependent on Myc for
their regulation, whereas 20 were not Myc-regulated (Table I). In
addition, of the 22 Myc-repressed targets 11 were found to be dependent
on Myc for their regulation in this cell system whereas 11 genes did
not appear to be regulated solely by Myc (Table II). Representative
blots of Myc-activated targets, Myc-repressed targets, and targets not
regulated by Myc are shown in Fig. 4. A
representative blot probed with 36B4 as a loading control is also
shown. In addition, quantification of signal is shown relative to the
zero time points and normalized to 36B4 for nucleolin and cathepsin B,
which possess expression patterns similar to that of the other
activated and repressed targets, respectively, as well as
CDT1, PAI-1, and Nop56, three targets that were
not regulated by Myc and show distinct patterns of regulation. Those
genes classified as "not Myc-regulated" in this system displayed one of two patterns of regulation. These include genes whose expression levels remained unchanged throughout the cell cycle and were not regulated as a result of serum stimulation (8 genes) as well as those
that were serum-regulated in both the HO15.19 and TGR-1 cells (23 genes). Genes that were regulated by Myc in each of the previously
described systems are strong candidates as Myc targets. These genes are
listed in Table III and include 5 activated and 10 repressed targets.
|
|
Regulation in the Absence of de Novo Protein Synthesis--
To
distinguish whether the identified Myc target genes are regulated in a
proximal or distal manner downstream of Myc, we evaluated whether
de novo protein synthesis was required for regulation. To
this end, the inducible MycERTM system and the translational inhibitor
cycloheximide (CHX) were employed. It would be expected that, if
de novo protein synthesis was not necessary, regulation of
the gene by activated MycERTM would be observed in the presence of CHX.
This would indicate that a gene was regulated proximally downstream of
Myc. Serum-deprived Rat1-MycERTM cells were exposed to CHX, CHX plus
OH-T, or OH-T alone for up to 12 h, and RNA was harvested for
Northern blot analysis. From this analysis we determined that
KIAA0664 is directly activated by Myc (Fig.
5A). KIAA0664 levels were up-regulated in response to OH-T in MycER cells, as expected. This up-regulation was not observed in cells treated with CHX
alone but occurred when Myc was activated in the presence of CHX. This
indicates that de novo protein synthesis is not necessary for the regulation of this gene. Similarly, a gene with similarity to
serine carboxypeptidase 1 was directly repressed downstream of Myc
(Table III). The results strongly suggest that these two genes are
regulated immediately downstream of Myc. A caveat to the use of CHX to
identify proximal gene regulatory events is that these experiments are
not informative for the majority of genes, because exposure to CHX
alone often effects target gene expression and precludes further
interpretation of the results. This was observed in the analysis of the
Myc-activated gene, APEX (Fig. 5B). The levels of
APEX were up-regulated in response to Myc alone. However,
RNA levels decreased in response to CHX alone and in the presence of
both CHX and OH-T. Unfortunately, CHX had an effect on the expression
of the majority of the genes that were assessed (Table III). This
experiment was also accomplished with the cells under asynchronous
growing conditions with similar results obtained (data not shown).
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
Use of microarrays to identify genes regulated by various stimuli and/or transcriptional regulators is an efficient tool for examining thousands of potential target genes in a relatively short period of time. During the course of our work, various high throughput assays have been reported that identify several hundred putative Myc-regulated genes (28-35). Although some of these genes are undoubtedly downstream targets of Myc, many of the observed changes in gene regulation may be an indirect consequence of Myc action. It is of enormous importance to delineate the subset of genes whose regulation is dependent upon Myc and to prevent false positives as well as misconceptions regarding Myc's true activities. These Myc-dependent targets are identified by additional experimentation to isolate genes that meet criteria consistent with genes regulated downstream of Myc. In this study a series of screens were designed to distinguish Myc-regulated genes from a panel of putative targets first identified by microarray analysis. This was accomplished using a variety of systems involving both primary and immortalized cells; inducible, constitutive, or endogenous Myc molecules; and asynchronous and synchronous cell growth conditions. We conducted each of these experiments in fibroblast cell systems to provide a homogeneous environment in which any additional variables that may be introduced due to cell type differences would not confound the primary goal of identifying the gene targets that are regulated by Myc. Those genes that were regulated in each of these conditions are strong candidates as universally regulated Myc targets. The identification of these regulated targets will equip us to determine how Myc can elicit its many biological functions in a wide variety of systems and, in addition, provide the critical experimental tools to investigate the mechanism(s) by which Myc can activate and repress gene transcription.
In our analysis, the genes that appear to be targets of c-Myc fall under a diverse array of functional categories (Table III). Many of the repressed targets that we identified had largely unknown functions, whereas others were involved in protein catabolism, growth responsiveness, gene regulation, integrin signaling, and cytoskeletal organization. The activated targets have functions pertaining to DNA repair, centrosome duplication, and/or ribosome assembly as well as unknown functions. This wide variety of targets is consistent with the ability of Myc to influence a wide variety of activities. Indeed, the Myc targets identified to date indicate that Myc regulates a diverse group of genes involved in many cellular processes (reviewed in Refs. 3 and 5). To determine the specific significance of Myc regulation of each of these genes, it will be imperative to study them further on a gene by gene basis in an Myc-specific manner. Ultimately the goal is to identify the functional consequences of these regulatory events and how they relate to the many biological activities of Myc.
Many of the genes identified by gene expression profiling by other
groups were also identified by our initial microarray analysis. Interestingly, these genes were subsequently segregated on the basis of
our selection criteria and either eliminated or verified as an
Myc-regulated gene. Those eliminated included all of the ribosomal
proteins that we analyzed (RPS17, RPS23,
RPL4, and RPL6). These and other ribosomal genes
have been identified in multiple screens for Myc target genes leading
to the hypothesis that Myc may be involved in regulating protein
synthesis at this level (28, 31, 32). Our results suggest ribosomal
genes are not necessarily Myc targets; however, further evaluation to
determine if the other identified ribosomal proteins are truly
Myc-regulated genes is required. In addition to ribosomal proteins,
eukaryotic elongation factor 1 and 1
(32), HSP90 (31, 32, 35) MIF (31), and DANCE (31) have been identified by
others as Myc targets but do not appear to be specifically regulated by Myc upon further investigation by our criteria. This indicates the
extreme importance of further screening to identify Myc target genes
identified by high throughput assays. Indeed, many of the genes that we
identified as true Myc targets have also been identified in other
screens or studies. These included pdgfr (18), nucleophosmin (31,
35, 40), nucleolin (30, 35, 41), fibronectin (30), and APEX
(31). Thus, it is important to distinguish Myc-specific targets from
the false positives that can be identified using a high throughput
approach, and a thorough analysis of each putative target is necessary
to determine whether it is truly a Myc-regulated gene. Indeed, one
method to assess direct Myc targets is to examine gene regulation in
response to Myc in the presence of the translational inhibitor
cycloheximide (CHX). Unfortunately, in our analysis CHX had a dominant
effect on the regulation of most gene targets on its own, thus we could
not assess direct regulation by Myc using this approach (Table
III).
An outstanding question that exists in the field of oncogenic research
is whether an endogenous cellular proto-oncogene has the same or
different function as the activated oncogene. This issue has been
brought to light most recently by Guo et al. (31) in
which a microarray analysis indicated that exogenous and endogenous Myc
might regulate different subsets of target genes. Interestingly, although we employed a similar cellular system, we did not observe such
a trend. Indeed, all 15 verified Myc downstream genes were regulated by
constitutive and inducible exogenous Myc expression in both immortal
and primary cell systems as well as in response to endogenous Myc
expression following mitogen stimulation of TGR-1 cells. Importantly,
the genes that were not regulated by endogenous Myc in the latter
system were also not regulated by exogenous Myc in at least one of the
cellular systems expressing ectopic Myc (HO15.19-myc,
primary, and MycERTM). These include BTF3, CDA02,
CDT1, eukaryotic elongation factor 1,
microphthalmia-associated transcription factor, and three novel genes
clones 16, 38, and IX. Taken together, our results strongly suggest
conclusions drawn directly from microarray screens must be verified by
an independent measure. It will be important to further investigate
each of the targets identified by Guo and colleagues (31) to
determine if the observed differences are indeed the result of
differential gene regulation by endogenous and exogenous Myc molecules.
These additional levels of analyses show the Myc target genes
identified and verified in the present report are regulated by both
endogenous and exogenous Myc expression.
To identify true Myc target genes we systematically assessed the
regulation of each gene identified as differentially regulated in our
microarray screen. Our first criterion was to employ the same cell
system in which we conducted our microarray experiment to verify these
results. This primary screen ruled out 5 of 53 possible target genes,
leaving 48 candidate up- or down-regulated genes (summarized in Tables
I and II). Our secondary selection criteria was to evaluate whether Myc
regulation of the target was intact in primary cells and to ensure that
the observed regulatory events were not a consequence of cellular
immortalization or unique to the TGR-1/HO15.19 system used in the
initial microarray analysis. This screen identified 14 of 48 putative
targets that were not regulated in response to ectopic Myc expression
in asynchronous primary embryonic fibroblasts. Interestingly, these 14 clones also failed to meet other criteria (see below), and there were no instances in which a potential target was ruled out solely on the
basis of this screen in primary cells. This indicates that genes
regulated in the Myc-null HO15.19 immortalized cell system in response
to exogenous Myc expression is similar to that in primary cells, and
the former cell system can be successfully employed to identify Myc
target genes. Our third and fourth selection criteria involved the
Rat1-MycERTM cells and the HO15.19/TGR-1 system, respectively. The
MycERTM system allows for the identification and elimination of those
genes that are regulated as a result of full mitogenic stimulation and
not in response to the induction of Myc expression alone. By analyzing
gene expression in quiescent HO15.19 and TGR-1 cells following mitogen
stimulation, we could address whether the regulation of the potential
target gene was dependent or independent of c-Myc expression during the
transition of the G0/G1 to S phase of the cell
cycle. Analysis of the results of these two screens in combination was
very instructive and lead to four patterns of response. The first
pattern shows the putative target gene is serum responsive in the
absence (HO15.19 cells) or presence (TGR-1 cells) of Myc expression and
not regulated in response to MycERTM. These genes are likely regulated
as a consequence of cell cycle progression by factors other than Myc
and represent 17 out of 48 genes. The second pattern reveals a gene
that is serum-responsive in HO15.19 and TGR-1 cells as well as
responsive in MycERTM. This indicated that the target may be regulated
by Myc, but its cell cycle regulation is not dependent upon Myc and can
be orchestrated by other factors. Of the 48 genes entered into the
screen, 11 showed this pattern of response. The third pattern of
response is a target gene that is dependent on Myc expression in the
HO15.19 and TGR-1 cells and is not regulated in MycERTM. This gene is
potentially dependent on Myc expression but requires other
mitogen-stimulated factors for expression as well. 1 out of 48 genes
exhibited this pattern of regulation. The fourth pattern is a target
gene that is serum-regulated in the TGR-1 cells, but not in the HO15.19
cells, and is regulated as a result of Myc activation in the MycERTM
cells. Clearly, the latter pattern reveals a target that is cell
cycle-regulated in an Myc-dependent manner. Our last screen
involving the HO15.19/TGR-1 cell system was by far the most stringent
of our criteria; 31 out of 48 genes were eliminated based on these
results. These combinations of analyses allowed us to identify 15 Myc-regulated targets that will be invaluable in identifying Myc's
role within the cell.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Tak Mak of the Amgen Institute for the opportunity to utilize the microarray and John Sedivy for HO15.19 cells and TGR-1 cells and Peggy Farnham, Bruno Amati, Martin Eilers, Bernhard Luscher, and Michael Cole for communicating results in advance of publication. We thank Andrew Hessel and Alexandra Ho for technical assistance and members of the Penn laboratory for helpful comments.
| |
FOOTNOTES |
|---|
* This work was supported by a grant from the National Cancer Institute of Canada (NCIC) with funds from the Canadian Cancer Society (to L. Z. P.), by scholarships from the NCIC with funds from the Terry Fox Foundation (to S. K. O.), and by the Canadian Institute for Health Research (to S. K. O. and M. S.).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.
§ Both authors contributed equally to this work.
To whom correspondence should be addressed: Division of
Cellular and Molecular Biology, Ontario Cancer Institute/Princess Margaret Hospital, 610 University Ave., Toronto, Ontario M5G 2M9, Canada. Tel.: 416-946-2276; Fax: 416-946-2840; E-mail:
lpenn@uhnres.utoronto.ca.
Published, JBC Papers in Press, July 26, 2002, DOI 10.1074/jbc.M201493200
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
cad, carbamoyltransferase/dihydroorotase;
DMEM, Dulbecco's modified
Eagle's medium;
MEM, -modified Eagle's medium;
FACS, fluorescence-activated cell sorting;
GFP, green fluorescent protein;
EF, embryonic fibroblast;
ER, estrogen receptor;
OH-T, hydroxytamoxifen;
pdgfr, platelet-derived growth factor receptor;
CHX, cycloheximide.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. |
Dang, C. V.
(1999)
Mol. Cell. Biol.
19,
1-11 |
| 2. | Nesbit, C. E., Tersak, J. M., and Prochownik, E. V. (1999) Oncogene 18, 3004-3016[CrossRef][Medline] [Order article via Infotrieve] |
| 3. | Oster, S. K., Ho, C. S., Soucie, E. L., and Penn, L. Z. (2002) Adv. Cancer Res. 84, 81-154[Medline] [Order article via Infotrieve] |
| 4. |
Facchini, L. M.,
and Penn, L. Z.
(1998)
FASEB J.
12,
633-651 |
| 5. | Grandori, C., Cowley, S. M., James, L. P., and Eisenman, R. N. (2000) Annu. Rev. Cell Dev. Biol. 16, 653-699[CrossRef][Medline] [Order article via Infotrieve] |
| 6. |
Bello-Fernandez, C.,
Packham, G.,
and Cleveland, J. L.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
7804-7808 |
| 7. | Jones, R. M., Branda, J., Johnston, K. A., Polymenis, M., Gadd, M., Rustgi, A., Callanan, L., and Schmidt, E. V. (1996) Mol. Cell. Biol. 16, 4754-4764[Abstract] |
| 8. | Galaktionov, K., Chen, X., and Beach, D. (1996) Nature 382, 511-517[CrossRef][Medline] [Order article via Infotrieve] |
| 9. | Miltenberger, R. J., Sukow, K. A., and Farnham, P. J. (1995) Mol. Cell. Biol. 15, 2527-2535[Abstract] |
| 10. |
Eberhardy, S. R.,
and Farnham, P. J.
(2001)
J. Biol. Chem.
276,
48562-48571 |
| 11. |
Park, J.,
Kunjibettu, S.,
McMahon, S. B.,
and Cole, M. D.
(2001)
Genes Dev.
15,
1619-1624 |
| 12. |
Bouchard, C.,
Dittrich, O.,
Kiermaier, A.,
Dohmann, K.,
Menkel, A.,
Eilers, M.,
and Luscher, B.
(2001)
Genes Dev.
15,
2042-2047 |
| 13. | McMahon, S. B., Van Buskirk, H. A., Dugan, K. A., Copeland, T. D., and Cole, M. D. (1998) Cell 94, 363-374[CrossRef][Medline] [Order article via Infotrieve] |
| 14. |
McMahon, S. B.,
Wood, M. A.,
and Cole, M. D.
(2000)
Mol. Cell. Biol.
20,
556-562 |
| 15. |
Frank, S. R.,
Schroeder, M.,
Fernandez, P.,
Taubert, S.,
and Amati, B.
(2001)
Genes Dev.
15,
2069-2082 |
| 16. |
Eisenman, R. N.
(2001)
Genes Dev.
15,
2023-2030 |
| 17. | Marhin, W. W., Chen, S., Facchini, L. M., Fornace, A. J., Jr., and Penn, L. Z. (1997) Oncogene 14, 2825-2834[CrossRef][Medline] [Order article via Infotrieve] |
| 18. |
Oster, S. K.,
Marhin, W. W.,
Asker, C.,
Facchini, L. M.,
Dion, P. A.,
Funa, K.,
Post, M.,
Sedivy, J. M.,
and Penn, L. Z.
(2000)
Mol. Cell. Biol.
20,
6768-6778 |
| 19. |
Wu, K. J.,
Polack, A.,
and Dalla-Favera, R.
(1999)
Science
283,
676-679 |
| 20. | Staller, P., Peukert, K., Kiermaier, A., Seoane, J., Lukas, J., Karsunky, H., Moroy, T., Bartek, J., Massague, J., Hanel, F., and Eilers, M. (2001) Nat. Cell Biol. 3, 392-399[CrossRef][Medline] [Order article via Infotrieve] |
| 21. | Penn, L. J., Brooks, M. W., Laufer, E. M., and Land, H. (1990) EMBO J. 9, 1113-1121[Medline] [Order article via Infotrieve] |
| 22. | Li, L. H., Nerlov, C., Prendergast, G., MacGregor, D., and Ziff, E. B. (1994) EMBO J. 13, 4070-4079[Medline] [Order article via Infotrieve] |
| 23. |
Mai, S.,
and Martensson, I. L.
(1995)
Nucleic Acids Res.
23,
1-9 |
| 24. | Park, D. S., Razani, B., Lasorella, A., Schreiber-Agus, N., Pestell, R. G., Iavarone, A., and Lisanti, M. P. (2001) Biochemistry 40, 3354-3362[CrossRef][Medline] [Order article via Infotrieve] |
| 25. |
Wang, X.,
Peters, M. A.,
Utama, F. E.,
Wang, Y.,
and Taparowsky, E. J.
(1999)
Mol. Endocrinol.
13,
254-267 |
| 26. | Yang, W., Shen, J., Wu, M., Arsura, M., FitzGerald, M., Suldan, Z., Kim, D. W., Hofmann, C. S., Pianetti, S., Romieu-Mourez, R., Freedman, L. P., and Sonenshein, G. E. (2001) Oncogene 20, 1688-1702[CrossRef][Medline] [Order article via Infotrieve] |
| 27. | Izumi, H., Molander, C., Penn, L. Z., Ishisaki, A., Kohno, K., and Funa, K. (2001) J. Cell Sci. 114, 1533-1544[Abstract] |
| 28. |
Schuhmacher, M.,
Kohlhuber, F.,
Holzel, M.,
Kaiser, C.,
Burtscher, H.,
Jarsch, M.,
Bornkamm, G. W.,
Laux, G.,
Polack, A.,
Weidle, U. H.,
and Eick, D.
(2001)
Nucleic Acids Res.
29,
397-406 |
| 29. | O'Hagan, R. C., Schreiber-Agus, N., Chen, K., David, G., Engelman, J. A., Schwab, R., Alland, L., Thomson, C., Ronning, D. R., Sacchettini, J. C., Meltzer, P., and DePinho, R. A. (2000) Nat. Genet. 24, 113-119[CrossRef][Medline] [Order article via Infotrieve] |
| 30. |
Coller, H. A.,
Grandori, C.,
Tamayo, P.,
Colbert, T.,
Lander, E. S.,
Eisenman, R. N.,
and Golub, T. R.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
3260-3265 |
| 31. |
Guo, Q. M.,
Malek, R. L.,
Kim, S.,
Chiao, C., He, M.,
Ruffy, M.,
Sanka, K.,
Lee, N. H.,
Dang, C. V.,
and Liu, E. T.
(2000)
Cancer Res.
60,
5922-5928 |
| 32. | Boon, K., Caron, H. N., van Asperen, R., Valentijn, L., Hermus, M. C., van Sluis, P., Roobeek, I., Weis, I., Voute, P. A., Schwab, M., and Versteeg, R. (2001) EMBO J. 20, 1383-1393[CrossRef][Medline] [Order article via Infotrieve] |
| 33. | Kim, S., Zeller, K., Dang, C. V., Sandgren, E. P., and Lee, L. A. (2001) Anal. Biochem. 288, 141-148[CrossRef][Medline] [Order article via Infotrieve] |
| 34. | Nesbit, C. E., Tersak, J. M., Grove, L. E., Drzal, A., Choi, H., and Prochownik, E. V. (2000) Oncogene 19, 3200-3212[CrossRef][Medline] [Order article via Infotrieve] |
| 35. |
Neiman, P. E.,
Ruddell, A.,
Jasoni, C.,
Loring, G.,
Thomas, S. J.,
Brandvold, K. A.,
Lee, R.,
Burnside, J.,
and Delrow, J.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
6378-6383 |
| 36. | Schuldiner, O., and Benvenisty, N. (2001) Oncogene 20, 4984-4994[CrossRef][Medline] [Order article via Infotrieve] |
| 37. | Mateyak, M. K., Obaya, A. J., Adachi, S., and Sedivy, J. M. (1997) Cell Growth Diff. 8, 1039-1048[Abstract] |
| 38. | Eilers, M., Picard, D., Yamamoto, K. R., and Bishop, J. M. (1989) Nature 340, 66-68[CrossRef][Medline] [Order article via Infotrieve] |
| 39. |
Littlewood, T. D.,
Hancock, D. C.,
Danielian, P. S.,
Parker, M. G.,
and Evan, G. I.
(1995)
Nucleic Acids Res.
23,
1686-1690 |
| 40. |
Zeller, K. I.,
Haggerty, T.,
Barrett, J. F.,
Guo, Q.,
Wonsey, D. R.,
and Dang, C. V.
(2001)
J. Biol. Chem.
276,
48285-48291 |
| 41. |
Greasley, P. J.,
Bonnard, C.,
and Amati, B.
(2000)
Nucleic Acids Res.
28,
446-453 |
This article has been cited by other articles:
|
|
 |
|
  M. Bunger, H. M. van den Bosch, J. van der Meijde, S. Kersten, G. J. E. J. Hooiveld, and M. Muller Genome-wide analysis of PPAR{alpha} activation in murine small intestine Physiol Genomics, July 18, 2007; 30(2): 192 - 204. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. I. Zeller, X. Zhao, C. W. H. Lee, K. P. Chiu, F. Yao, J. T. Yustein, H. S. Ooi, Y. L. Orlov, A. Shahab, H. C. Yong, et al. Global mapping of c-Myc binding sites and target gene networks in human B cells PNAS, November 21, 2006; 103(47): 17834 - 17839. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. H. Cowling, S. Chandriani, M. L. Whitfield, and M. D. Cole A Conserved Myc Protein Domain, MBIV, Regulates DNA Binding, Apoptosis, Transformation, and G2 Arrest. Mol. Cell. Biol., June 1, 2006; 26(11): 4226 - 4239. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Barsyte-Lovejoy, S. K. Lau, P. C. Boutros, F. Khosravi, I. Jurisica, I. L. Andrulis, M. S. Tsao, and L. Z. Penn The c-Myc Oncogene Directly Induces the H19 Noncoding RNA by Allele-Specific Binding to Potentiate Tumorigenesis. Cancer Res., May 15, 2006; 66(10): 5330 - 5337. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Bredel, C. Bredel, D. Juric, G. R. Harsh, H. Vogel, L. D. Recht, and B. I. Sikic Functional Network Analysis Reveals Extended Gliomagenesis Pathway Maps and Three Novel MYC-Interacting Genes in Human Gliomas Cancer Res., October 1, 2005; 65(19): 8679 - 8689. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Perini, D. Diolaiti, A. Porro, and G. Della Valle In vivo transcriptional regulation of N-Myc target genes is controlled by E-box methylation PNAS, August 23, 2005; 102(34): 12117 - 12122. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Li, Y. Wang, K. I. Zeller, J. J. Potter, D. R. Wonsey, K. A. O'Donnell, J.-w. Kim, J. T. Yustein, L. A. Lee, and C. V. Dang Myc Stimulates Nuclearly Encoded Mitochondrial Genes and Mitochondrial Biogenesis Mol. Cell. Biol., July 15, 2005; 25(14): 6225 - 6234. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Huang, C. S.W. Ho, R. Ponzielli, D. Barsyte-Lovejoy, E. Bouffet, D. Picard, C. E. Hawkins, and L. Z. Penn Identification of a Novel c-Myc Protein Interactor, JPO2, with Transforming Activity in Medulloblastoma Cells Cancer Res., July 1, 2005; 65(13): 5607 - 5619. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Liao, M. Patel, Y. Hu, S. Charles, D. J. Herrick, and G. Brewer Targeted Knockdown of the RNA-binding Protein CRD-BP Promotes Cell Proliferation via an Insulin-like Growth Factor II-dependent Pathway in Human K562 Leukemia Cells J. Biol. Chem., November 19, 2004; 279(47): 48716 - 48724. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Marinkovic, T. Marinkovic, E. Kokai, T. Barth, P. Moller, and T. Wirth Identification of novel Myc target genes with a potential role in lymphomagenesis Nucleic Acids Res., October 11, 2004; 32(18): 5368 - 5378. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-w. Kim, K. I. Zeller, Y. Wang, A. G. Jegga, B. J. Aronow, K. A. O'Donnell, and C. V. Dang Evaluation of Myc E-Box Phylogenetic Footprints in Glycolytic Genes by Chromatin Immunoprecipitation Assays Mol. Cell. Biol., July 1, 2004; 24(13): 5923 - 5936. [Abstract] [Full Text] [PDF]
|
|
