A SUMOylation-dependent transcriptional subprogram is required for Myc-driven tumorigenesis.
ABSTRACT Myc is an oncogenic transcription factor frequently dysregulated in human cancer. To identify pathways supporting the Myc oncogenic program, we used a genome-wide RNA interference screen to search for Myc-synthetic lethal genes and uncovered a role for the SUMO-activating enzyme (SAE1/2). Loss of SAE1/2 enzymatic activity drives synthetic lethality with Myc. Inactivation of SAE2 leads to mitotic catastrophe and cell death upon Myc hyperactivation. Mechanistically, SAE2 inhibition switches a transcriptional subprogram of Myc from activated to repressed. A subset of these SUMOylation-dependent Myc switchers (SMS genes) is required for mitotic spindle function and to support the Myc oncogenic program. SAE2 is required for growth of Myc-dependent tumors in mice, and gene expression analyses of Myc-high human breast cancers suggest that low SAE1 and SAE2 abundance in the tumors correlates with longer metastasis-free survival of the patients. Thus, inhibition of SUMOylation may merit investigation as a possible therapy for Myc-driven human cancers.
- SourceAvailable from: Martine F Roussel[show abstract] [hide abstract]
ABSTRACT: Establishment of primary mouse embryo fibroblasts (MEFs) as continuously growing cell lines is normally accompanied by loss of the p53 or p19(ARF) tumor suppressors, which act in a common biochemical pathway. myc rapidly activates ARF and p53 gene expression in primary MEFs and triggers replicative crisis by inducing apoptosis. MEFs that survive myc overexpression sustain p53 mutation or ARF loss during the process of establishment and become immortal. MEFs lacking ARF or p53 exhibit an attenuated apoptotic response to myc ab initio and rapidly give rise to cell lines that proliferate in chemically defined medium lacking serum. Therefore, ARF regulates a p53-dependent checkpoint that safeguards cells against hyperproliferative, oncogenic signals.Genes & Development 09/1998; 12(15):2424-33. · 12.44 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Recently, a 76-gene prognostic signature able to predict distant metastases in lymph node-negative (N(-)) breast cancer patients was reported. The aims of this study conducted by TRANSBIG were to independently validate these results and to compare the outcome with clinical risk assessment. Gene expression profiling of frozen samples from 198 N(-) systemically untreated patients was done at the Bordet Institute, blinded to clinical data and independent of Veridex. Genomic risk was defined by Veridex, blinded to clinical data. Survival analyses, done by an independent statistician, were done with the genomic risk and adjusted for the clinical risk, defined by Adjuvant! Online. The actual 5- and 10-year time to distant metastasis were 98% (88-100%) and 94% (83-98%), respectively, for the good profile group and 76% (68-82%) and 73% (65-79%), respectively, for the poor profile group. The actual 5- and 10-year overall survival were 98% (88-100%) and 87% (73-94%), respectively, for the good profile group and 84% (77-89%) and 72% (63-78%), respectively, for the poor profile group. We observed a strong time dependence of this signature, leading to an adjusted hazard ratio of 13.58 (1.85-99.63) and 8.20 (1.10-60.90) at 5 years and 5.11 (1.57-16.67) and 2.55 (1.07-6.10) at 10 years for time to distant metastasis and overall survival, respectively. This independent validation confirmed the performance of the 76-gene signature and adds to the growing evidence that gene expression signatures are of clinical relevance, especially for identifying patients at high risk of early distant metastases.Clinical Cancer Research 07/2007; 13(11):3207-14. · 7.84 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Gene expression signatures encompassing dozens to hundreds of genes have been associated with many important parameters of cancer, but mechanisms of their control are largely unknown. Here we present a method based on genetic linkage that can prospectively identify functional regulators driving large-scale transcriptional signatures in cancer. Using this method we show that the wound response signature, a poor-prognosis expression pattern of 512 genes in breast cancer, is induced by coordinate amplifications of MYC and CSN5 (also known as JAB1 or COPS5). This information enabled experimental recapitulation, functional assessment and mechanistic elucidation of the wound signature in breast epithelial cells.Nature Genetics 05/2006; 38(4):421-30. · 35.21 Impact Factor
, 348 (2012);
, et al.Jessica D. Kessler
for Myc-Driven Tumorigenesis
A SUMOylation-Dependent Transcriptional Subprogram Is Required
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7. A. Pessino et al., J. Exp. Med. 188, 953 (1998).
8. E. Cagnano et al., J. Invest. Dermatol. 128, 972 (2008).
9. R. Gazit et al., Nat. Immunol. 7, 517 (2006).
10. C. Gur et al., Nat. Immunol. 11, 121 (2010).
11. G. G. Halfteck et al., J. Immunol. 182, 2221 (2009).
12. E. Narni-Mancinelli et al., Proc. Natl. Acad. Sci. U.S.A.
108, 18324 (2011).
13. C. Levy et al., Mol. Cell 40, 841 (2010).
14. L. Chiossone et al., Blood 113, 5488 (2009).
15. S. Dovat et al., J. Immunol. 175, 3508 (2005).
16. S. H. Lee, K. S. Kim, N. Fodil-Cornu, S. M. Vidal, C. A. Biron,
J. Exp. Med. 206, 2235 (2009).
17. S. N. Waggoner, M. Cornberg, L. K. Selin, R. M. Welsh,
Nature 10.1038/nature10624 (2011).
18. D. M. Andrews et al., J. Exp. Med. 207, 1333 (2010).
19. K. Soderquest et al., J. Immunol. 186, 3304 (2011).
20. C. S. Tripp, S. F. Wolf, E. R. Unanue, Proc. Natl. Acad. Sci.
U.S.A. 90, 3725 (1993).
21. S. Kim et al., Nature 436, 709 (2005).
22. N. C. Fernandez et al., Blood 105, 4416 (2005).
23. N. Anfossi et al., Immunity 25, 331 (2006).
24. P. Brodin, T. Lakshmikanth, S. Johansson, K. Kärre,
P. Höglund, Blood 113, 2434 (2009).
25. D. H.Raulet, R. E. Vance, Nat.Rev. Immunol. 6, 520(2006).
26. W. M. Yokoyama, S. Kim, Immunity 24, 249 (2006).
27. M. T. Orr, L. L. Lanier, Cell 142, 847 (2010).
28. D. E. Oppenheim et al., Nat. Immunol. 6, 928 (2005).
29. J. C. Sun, L. L. Lanier, J. Exp. Med. 205, 1819 (2008).
30. S. K. Tripathy et al., J. Exp. Med. 205, 1829 (2008).
31. B. Zafirova et al., Immunity 31, 270 (2009).
32. O. Mandelboim et al., Nature 409, 1055 (2001).
Acknowledgments: We thank J. Ewbank, M. Bléry, J.-C. Andrau
and P. Kruse for advice; D. Dilg for bioinformatics analysis;
M. Dalod for MCMV reagents; M. C. Carroll for H1N1; G. Lauvau
for Lm-OVA; M. Roger for mice handling; and the Centre
d'Immunologie de Marseille-Luminy (CIML) mouse house
and cytometry core facilities. This work was supported
by an European Research Council advanced grant (E.V.
and S.U.); Functional Genomics in Mutant Mouse Models as
Tools to Investigate the Complexity of Human Immunological
Disease (MUGEN), Network of Excellence and Mechanisms
to Attack Steering Effectors of Rheumatoid Syndromes with
Innovated Therapy Choices (MASTERSWITCH), Integrating
Project from European Union (B.M. and M.M.); University of
Manitoba Dean of Medicine Strategic Fund and Natural
Sciences and Engineering Research Council (S.K.); Agence
Nationale de la Recherche (E.V. and S.U.); Equipe labellisée
“La Ligue,” Ligue Nationale contre le Cancer (E.V. and S.U.);
Agence pour la Recherche sur le Cancer (E.M.N.); Axa
research fund (B.N.J.); and institutional grants from INSERM,
CNRS, and Aix-Marseille University to the CIML. E.V. is a
cofounder of and shareholder in Innate-Pharma. E.N.M.,
E.V., and S.U. designed, analyzed the experiments, and wrote
the paper; C.B. identified Noé mouse during ENU screen;
E.N.M. and A.F. performed and analyzed the experiments;
S.K. and S.M. provided lentiviral vectors and protocols to
transduce NK cells; A.D.G. generated Helios-expressing
vectors; B.N.J., F.V., M.G., I.G.G., J.E., and S.C.H. performed
the sequencing and the bioinformatics analysis; L.N.G.
generated the model for NKp46W32R; M.M. and B.M. initiated
and conducted the early phases of the ENU screen; and
B.B. and E.B. helped in the ENU screen. The data reported
in this paper are tabulated in the main paper and in the
Supporting Online Material. Microarray data have been
deposited at the National Center for Biotechnology
Information GEO repository under accession no. GSE13229
and have been reported elsewhere (13). Ensembl accession
number for Ikzf2 is ENSMUST00000027146. Material
Transfer Agreements are required for use of the following
reagents: Noé mice, NKp46-specific mAb, NKp46iCre/iCre
mice, Helios short hairpin RNA, and control lentiviral
vectors. The invention (U.S. Patent Application 61/499,485;
NKp46-mediated NK cell tuning; E.N.M., S.U., and E.V.) relates
to compounds that inhibit NKp46.
Supporting Online Material
Materials and Methods
Figs. S1 to S15
21 October 2011; accepted 12 December 2011
Transcriptional Subprogram Is Required
for Myc-Driven Tumorigenesis
Jessica D. Kessler,1,2Kristopher T. Kahle,3,4Tingting Sun,1Kristen L. Meerbrey,1,2
Michael R. Schlabach,3Earlene M. Schmitt,1,2Samuel O. Skinner,1,5Qikai Xu,3Mamie Z. Li,3
Zachary C. Hartman,6Mitchell Rao,2Peng Yu,2Rocio Dominguez-Vidana,1,2Anthony C. Liang,3
Nicole L. Solimini,3Ronald J. Bernardi,7Bing Yu,8Tiffany Hsu,1,2Ido Golding,1,5Ji Luo,8
C. Kent Osborne,9,10,11,12Chad J. Creighton,9,13Susan G. Hilsenbeck,9,10,13
Rachel Schiff,9,10,11,12Chad A. Shaw,2Stephen J. Elledge,3* Thomas F. Westbrook1,2,7,9*
Myc is an oncogenic transcription factor frequently dysregulated in human cancer. To identify
pathways supporting the Myc oncogenic program, we used a genome-wide RNA interference
screen to search for Myc–synthetic lethal genes and uncovered a role for the SUMO-activating
enzyme (SAE1/2). Loss of SAE1/2 enzymatic activity drives synthetic lethality with Myc. Inactivation
of SAE2 leads to mitotic catastrophe and cell death upon Myc hyperactivation. Mechanistically,
SAE2 inhibition switches a transcriptional subprogram of Myc from activated to repressed. A subset
of these SUMOylation-dependent Myc switchers (SMS genes) is required for mitotic spindle function
and to support the Myc oncogenic program. SAE2 is required for growth of Myc-dependent tumors
in mice, and gene expression analyses of Myc-high human breast cancers suggest that low SAE1
and SAE2 abundance in the tumors correlates with longer metastasis-free survival of the patients.
Thus, inhibition of SUMOylation may merit investigation as a possible therapy for Myc-driven
suppressor genes. Removing oncogene function
can often reverse the tumorigenic phenotype, a
phenomenon referred to as “oncogene addiction”
(1, 2), and cancer researchers have focused on
difficult to inhibit pharmacologically, highlighting
ancers are driven by genomic alterations
that result in the activation of proto-
oncogenes and the inactivation of tumor
mutations confer dependencies on cellular pro-
The genes and signaling pathways underlying
such oncogenic support processes are largely un-
explored, and because these genes are not them-
selves oncogenes or otherwise mutatedincancer,
they cannot be identified through direct analyses
of cancer genomes and epigenomes.
The NOA pathways supporting the classical
c-Myc oncogene (referred to herein as Myc) are
poorly understood. The Myc gene, which codes
for a basic helix-loop-helix zipper transcription
factor, is frequently dysregulated in cancer cells
tein stabilization (7). Amplification or overex-
(8–11) and are associated with poor prognosis
(12). Genetic experiments have shown that Myc
is required for tumor maintenance and progres-
Oncogenic activation of Myc promotes a del-
icate balance in cells, conferring both pro- and
these opposing properties could be influenced by
inhibiting Myc oncogenic support pathways. To
1Verna and Marrs McLean Department of Biochemistry and
Molecular Biology, Baylor College of Medicine, Houston, TX
77030, USA.2Department of Molecular and Human Genetics,
Hughes Medical Institute, Department of Genetics, Harvard
Medical School, Division of Genetics, Brigham and Women’s
Hospital, Boston, MA 02115, USA.4Department of Neurosur-
Cancer Center, Houston, TX 77030, USA.7Department of Pe-
diatrics, Baylor College of Medicine, Houston, TX 77030, USA.
8Medical Oncology Branch, National Cancer Institute, Center
Drive, Bethesda, MD 20892, USA.9Dan L. Duncan Cancer
Center, Baylor College of Medicine, Houston, TX 77030, USA.
10The Lester and Sue Smith Breast Center, Baylor College of
ment of Molecular and Cellular Biology, Baylor College of
Medicine, One Baylor Plaza, Houston, TX 77030, USA.13Divi-
sion of Biostatistics, Baylor College of Medicine, Houston, TX
*To whom correspondence should be addressed. E-mail:
firstname.lastname@example.org (T.F.W.); email@example.com.
20 JANUARY 2012VOL 335
on January 24, 2012
search for pathways required for cells to tolerate
genetic screen for Myc–synthetic lethal (MySL)
short hairpin RNAs (shRNAs) in human mam-
mary epithelial cells (HMECs) engineered with
gene (Myc-ER HMECs) (fig. S1A). Induction
resulted in increased expression of known Myc
proliferation rate (fig. S1C). HMECs are ER-
negative, and in the absence of Myc-ER, do not
we screened for shRNAs that alter cell fitness
only in the presence of aberrant Myc signaling
(screen design in Fig. 1A).
To identify MySL shRNAs, we transduced
shRNAs targeting 32,293 unique transcripts in
three independent replicates. Transduced Myc-ER
HMECs were propagated in the absence or pres-
in shRNA-barcode abundance was measured in
both cell states (Myc-off and Myc-on). We iden-
tified 403 MySL shRNAs exhibiting more than a
twofold decrease in abundance in the Myc-on
1B, fig. S3, and table S1). Content analysis [Gene
Ontology (GO)] indicated that these candidates
were highly enriched for ion channels and en-
gation (including SUMOylation) (P = 0.002).
Components of the mitotic spindle were also
Analysis of the MySL candidates using Human
Protein Reference Database (HPRD) revealed a
highly connected protein-protein interaction net-
work, with many components of this network
playing a role in the mitotic spindle (Fig. 1D;
protein labels shown in fig. S4), suggesting that
er mitotic progression (17).
Among MySL candidates were several genes
previously implicated in the survival of Myc hy-
peractivated cells (GSK3b, FBXW7, and PTK2;
fig.S5A)(18–20).In addition,shRNAs targeting
MDM2 exhibited Mycsyntheticlethality,where-
as p53-targeting shRNAs enhanced proliferation
Myc promotes p53 activation (2, 21–23), and
p53 defects in promoting Myc-induced tumori-
genesis (2, 21, 22).
We also identified many candidates with pre-
viously unknown roles in Myc biology. To pri-
oritize these, we rank ordered MySL genes using
a modified two-way analysis of variance that we
for a given gene. Using this method, we identi-
with a P ≤ 0.001 (table S2). The most signifi-
cant candidate from both of these analyses was
the SUMO-activating enzyme (SAE) subunit 2
(SAE2/UBA2) (P < 0.00001), a critical compo-
nent of the sole SUMO-activating enzyme nec-
essary for SUMO conjugation to proteins (24).
Multiple SAE2-shRNAs exhibited Myc synthet-
ic lethality in the primary screen (fig. S5C). The
primary screen also identified SAE1, the hetero-
dimeric partner of SAE2 (table S2). Thus, we
interaction between SUMOylation and Myc.
To explore the physiological importance of
the Myc-SAE2 synthetic lethal interaction, we
transduced Myc-ER HMECs with two indepen-
and measured the effect of Myc activation on
pleted SAE2 protein (fig. S6A) and profoundly
Fig. 1. Genome-wide screen for Myc–synthetic lethal
(MySL) candidates. (A) Identification of MySL genes.
Myc-ER HMECs were transduced with a genome-wide library of
retroviral shRNAs in triplicate. At population doubling 0 (PD0),
cells were cultured with or without Myc-ER induction for 12
population doublings (PD12). To identify MySL candidates, we compared
relative barcode abundance from both conditions to that of initial PD0
samples via barcode microarrays. (B) Identification of MySL shRNAs. The Myc-
selective effect of all shRNAs from the genome-wide library are graphed
[y axis represents median difference between Myc-ER-on and Myc-ER-off
groups (log 2)], with a ratio < –1.0 indicating a decrease of at least twofold.
shRNAs are shown on the x axis (rank ordered by MySL effect). (C) Multiple
cellular processes are required to tolerate the Myc oncogenic state. MySL
components and processes). (D) MySL proteins engage in a highly connected
interaction network that regulates the mitotic spindle. Protein-protein in-
teractions between the top 100 MySL proteins were analyzed via HPRD.
mitotic spindle function, red indicates a MySL protein with a known role in
spindle function, and gray indicates a protein that interacts with a MySL
VOL 335 20 JANUARY 2012
on January 24, 2012
increased doubling time upon Myc induction
panels). Similar results were observed when a
constitutive Myc transgene was expressed to-
gether with shSAE2, indicating that these obser-
vations are not an artifact of the Myc-ER fusion
shRNAs elicited a Myc–synthetic lethal pheno-
type (Fig. 2A), and restoration of SAE2 protein
abundance with a SAE2 wild-type cDNA sup-
2, C and D; described below), indicating that the
Myc-SAE2 synthetic phenotype is not due to an
RNA interference (RNAi) off-target effect. Fur-
thermore, multiple shRNAs targeting SAE1 and
the downstream SUMO E2-conjugating enzyme
UBE2I (UBC9) (fig. S6, B and C) were also
synthetically lethal with Myc hyperactivation
(Fig.2A,middle and right graphs), demonstrat-
ing that SUMOylation interference is syntheti-
cally lethal with hyperactivated Myc.
We next investigated whether SUMOylation
is required for cells to tolerate aberrant Myc ac-
tivation. Depletion of SAE2 decreased abundance
of SUMO1- or SUMO2/3-modified proteins (Fig.
2B), indicating global impairment of SUMOyla-
tion in these cells. To determine whether SAE2
enzymatic activity is required to support Myc, we
engineered Myc-ER HMECs with an inducible
SAE2-shRNA (pINDUCER11-shSAE2) (25) to-
gether with constitutive shRNA-resistant cDNAs
encoding wild-type (WT) SAE2, catalytically in-
active SAE2-C173S, or control enhanced green
fluorescent protein (eGFP). SAE2 WT and mu-
tant cDNAs restored SAE2 to endogenous SAE2
levels (Fig. 2C). Restoration of WT SAE2 sup-
pressed the MySL phenotype of SAE2 shRNA
(Fig. 2D). However, SAE2-C173S failed to sup-
press the synthetic lethality of SAE2-shRNA (Fig.
2D), indicating that SAE2 enzymatic activity is
required to prevent the Myc-SAE2 synthetic le-
thal interaction. Together, these data suggest that
SUMOylation is required for HMECs to tolerate
aberrant Myc signaling.
A key question is how SAE2 depletion in the
presence of Myc hyperactivation impairs prolif-
eration. This could be due to changes in the cell
cycle and/or cell death, so we examined the ef-
on these processes. In SAE2-depleted cells, Myc
induction increased the number of cells with a
content (Fig. 3A and fig. S8A). These cell cycle
defects were followed by a significant apoptotic
response (Fig. 3B and fig. S8, B and C). The
increase in G2/M and >2N DNA content is char-
acteristic of mitotic defects known to cause mi-
genes are enriched for genes involved in the mi-
totic spindle (Fig. 1D), suggesting that Myc hy-
peractive cells might experience mitotic stress.
To explore this possibility, we examined mitotic
spindles in Myc hyperactive cells in the presence
or absence of SAE2 depletion. As hypothesized,
nificantly more spindle defects (defects in >25%
alone (Fig. 3, C and D; P = 7 × 10−7). These de-
fects, which included abnormal spindle number
and lagging chromosomes, might explain the ex-
tensive aneuploidy and apoptosis observed. Col-
lectively, these data suggest that the Myc-SAE2
genetic interaction results in dysregulation of the
mitotic spindle, which may in turn contribute to
We next investigated how Myc hyperactiva-
tion and SAE2 depletion result in the mitotic
aberrations. Myc hyperactivation induces differ-
ent cellular consequences (e.g., proliferation,
apoptosis, senescence) depending on the genetic
and epigenetic context. If this is due to the ability
of Myc to regulate distinct transcriptional pro-
grams, loss of SAE2 may lead to mitotic dys-
Therefore, we used gene expression profiling to
define the transcriptional effects of Myc with or
without SAE2 inactivation. Myc activation alone
in HMECs led to significant changes in the level
Fig. 2. HMECs require the E1 SAE to tolerate on-
cogenic Myc. (A) Inactivation of SAE2, SAE1, or
UBE2I is synthetically lethal with Myc hyperac-
tivation. Myc-ER HMECs were infected with control,
SAE2, SAE1, or UBE2I-targeting shRNAs and cul-
tured in the absence or presence of Myc-ER induc-
tion. Cell number was quantified after 6 days. Data
versus Myc-off states and normalized to control shRNA (aver-
images demonstrating altered morphology and decreased cell number in Myc
and shSAE2 cells are shown on the right. (B) Total protein SUMOylation is
decreased uponSAE2knockdown.Myc-ERHMECs infected withcontrolorSAE2-
ing control) protein abundance. (C and D) SAE2 catalytic activity is required to
tolerate Myc hyperactivation. Myc-ER HMECs transduced with a doxycycline
SAE2 UTR-eGFP) were subsequently infected with a virus expressing GFP, SAE2
WT, or SAE2 C173S cDNAs. Western blots were performed to confirm depletion
of SAE2 (C). Cells were cultured with or without Dox and in the absence or
presence of Myc-ER induction, and cell number was quantified after 8 days.
They axisindicates the relativechangeingrowth ofshSAE2-expressing cells
eight replicates) (D). Error bars in (A) and (D) represent the SEM.
20 JANUARY 2012 VOL 335
on January 24, 2012
of 605 mRNAs (P < 0.05; Fig. 3E, left panel).
Surprisingly, 22.5% (86/383) of Myc-induced
transcripts are not induced or become repressed
in response to Myc when SAE2 is depleted (Fig.
3E, right panel), suggesting that a portion of the
Myc transcriptional response is “switched” de-
pending on the status of SAE2 function. Because
induced to Myc-repressed in a SAE2-dependent
manner, we termed these genes SUMOylation-
dependent Myc switchers (or SMS genes).
MESH analysis revealed that SMS genes
were significantly enriched for regulators of the
mitotic spindle (P < 4.9 × 10−12) (fig. S9A), and
mining of published literature revealed that 17 of
86 SMS genes have been shown genetically to
participate in the assembly or integrity of mitotic
spindles (26–31). Each of these spindle-related
and fig. S9B, blue bars) but exhibits a strong
SAE2-dependent switch in their Myc response
(Fig. 3F and fig. S9B, red bars). These observa-
tions highlight regulation of spindle assembly as
a key vulnerability in cells harboring the Myc-
active, SAE2-inactive state, and suggest that
SMS genes may be linchpins in the Myc-SAE2
synthetic lethal relationship. To test this hypo-
thesis, we examined whether SMS genes known
to play a role in the mitotic spindle are synthetic-
ally lethal with Myc. We found that three of the
top four SMS genes (CASC5, BARD1, CDC20)
Fig. 3. Inactivation of SAE2 switches the Myc transcriptional pro-
gram and dysregulates mitotic fidelity and cell viability. (A) Ectopic
and aberrant chromosomal content. Myc-ER HMECs transduced with
inducibleshSAE2 were cultured in the absenceor presenceof Myc-ER
analyzed for DNA content by flow cytometry (quantification of cells
with >2N DNA, right panel). (B) Depletion of SAE2 induces apoptosis
in cooperation with Myc hyperactivation. pINDUCER-mir-SAE2-eGFP
Myc-ER HMECs were cultured in the absence or presence of Myc-ER-
induction and with or without shSAE2 induction (48 hours). The cells
Myc-SAE2 genetic interaction leads to defects in the mitotic spindle.
Myc-ER HMECs transduced with inducible shRNA-SAE2 were cultured
and abnormal mitotic events (D). Data are represented as the percentage of abnormal mitoses (at least 100 mitotic events were counted per condition; P values
are from Fisher’s exact test). Scale bar, 5 mM. (E) Loss of SAE2 alters the transcriptional response to Myc. HMECs expressing Myc-ER and dox-inducible SAE2-
by Myc-ER induction (P < 0.05, twofold) are shown. The effect of Myc-ER induction on mRNA levels in the absence or presence of shRNA-SAE2 induction are
shown (left and right panels,respectively).mRNAs that change their response to Mycin thepresence orabsence of shSAE2are termed“sumoylation-dependent
Myc switchers,” or SMS genes. (F) Loss of SAE2 alters Myc control of spindle-regulatory genes. The effect of Myc in the absence or presence of shSAE2 (blue and
red bars, respectively) is shown for the top 4 of 17 SMS genes with known roles in spindle integrity and function (see fig. S9B for the list of 17 SMS genes). (G)
(F) are the SE.
VOL 335 20 JANUARY 2012
on January 24, 2012
Samuel O Skinner