Two different E2F6 proteins generated by alternative splicing
and internal translation initiation
Tillman Dahme
1
, Jason Wood
1
, David M. Livingston
1
and Stefan Gaubatz
2
1
Dana Farber Cancer Institute and Harvard Medical School, Boston, MA, USA;
2
Institute for Molecular Biology and Tumor Research
(IMT), Philipps-University Marburg, Germany
E2F transcription factors play an important role in the
regulation of cell cycle progression. E2F6, the most recently
identified member of the E2F family, is a retinoblastoma-
protein-independent transcriptional repressor that is
required for developmental patterning of the axial skeleton.
It has recently been shown that the E2f6 locus produces two
different mRNAs, E2F6 and E2F6b. The E2F6b mRNA
contains an additional exon that is inserted by alternative
splicing. This exon contains an in-frame stop-codon and an
in-frame translation initiation codon. However, whether a
protein is translated from the E2F6b mRNA has not yet
been addressed. We now show that internal translation
initiation gives rise to E2F6b, an amino-terminal truncated
E2F6 protein. We also show that E2F6 and E2F6b mRNAs
are ubiquitously expressed in primary mouse tissues. During
the cell cycle, the highest expression of both forms is found at

domain, and is a pocket protein independent transcriptional
repressor [3–6]. We have recently shown that, in mice, E2F6
is required for developmental patterning of the axial skeleton
[7]. E2f6 deficient animals display homeotic transformations
of the skeleton that are strikingly similar to the transforma-
tions of certain polycomb deficient mice [7]. These observa-
tions are consistent with the recent finding that E2F6
associates with members of the mammalian polycomb
complex [8,9]. Taken together, these observations suggest
that one function of E2F6 is to recruit polycomb multipro-
tein complexes to target promoters during development.
It has been recently shown, that the E2f6 locus produces
two distinct mRNAs, E2F6 and E2F6b [10]. The E2F6b
mRNA contains the newly discovered exon 2. It has been
noted that this exon introduces an in frame termination
codon as well as an AUG codon on its 3¢ extremity, which
could potentially serve as a translation initiation codon.
However, no evidence for the generation of a protein has
been given.
In this study, we now demonstrate that an amino-terminal
truncated E2F6 protein is generated by internal translation
initiation of E2F6b. In addition, we show that the 5¢ untransl-
ated region of the E2F6 mRNA is unusually long, and that
they contain several upstream AUG codons followed by
short reading frames, features that impair normal CAP-
dependent translation initiation. E2F6 and E2F6b mRNAs
are widely expressed in primary mouse tissues. We propose
that regulated translation initiation can produce distinct
E2F6 isoforms under different physiological conditions.
MATERIALS AND METHODS

We first modified the sequence around the translation
initiation of the luciferase cDNA in pGL2-basic (Promega)
by introducing an AvrII-site and thereby abolishing the
initiating ATG, generating pGL2-Avr. A fragment enco-
ding for the first eight codons of E2F6 and partial 5¢UTR
was amplified by PCR with primers SG108 and SG92 using
a genomic E2f6 clone as a template. The resulting product
was digested with BglII and XbaI and inserted into pGL2-
Avr digested with BglII and AvrII. In a second step, a 2.4-kb
genomic SacI–BglII fragment containing the remaining
5¢UTR and 2.2 kb of the E2F6 promoter was inserted to
generate Exon1-luc. A similar strategy was used to generate
Exon3-luc and Exon2-luc. We first used RT-PCR of total
RNA with primers SG92 and SG122 to amplify part of the
coding regions and 5¢ UTRs of E2F6 and E2F6b mRNAs.
PCR-products were digested with BglII and XbaIand
inserted into pGL2-Avr. Secondly, the 2.4 kb genomic
SacI–BglII fragment was inserted to generate Exon2-luc
and Exon3-luc. In Exon2-mut-luc, the translation initiation
codon was modified by PCR with primers SG92 and SG142
and Exon2-luc as a template. The resulting PCR product
was digested with BglII and XbaI and inserted into pGL2-
Avr. Finally, the 2.4 kb genomic promoter fragment was
inserted as described above. An expression plasmid for
E2F6b was generated by PCR with primers SG109 and
SG35. The PCR product was digested with BamHI and
EcoRI and inserted into pcDNA3. All constructs were
confirmed by DNA sequencing. Other plasmids have been
described previously: E1B-luc [11], E2F1-luc [12].
Polyclonal antibodies and immunoprecipitations

SG109: 5¢-GGGGATCCATGCCATCAAAAATAAGGA
TTAAT-3¢
SG142: 5¢-CCTCTAGATTAATCCTTATTTTTGATGG
CCCCCTTCTGTCTCTGCCTCCCAAGGACTGGC-3¢
SG35: 5¢-CGGAATTCCCCGTGCTGGAGGCGACT
CG-3¢
SG122: 5¢-CGGACGGCGCGGAGAC 3¢
b-actin fw: 5¢-TGTGATGGTGGGAATGGGTCAG-3¢
b-actin bw: 5¢-TTTGATGTCACGCACGATTTCC-3¢
RT-PCR
RT-PCR was performed with the Superscript One-Step RT-
PCR kit (Invitrogen) with 100 ng of total RNA. For the
detection of E2F6 mRNA with primers SG122 and SG24,
the following conditions were used: 1 cycle: 30 min, 50 °C; 1
cycle: 2 min, 94 °C; 35 cycles: 30 s, 94 °C, 30 s, 55 °C;
1min,72°C, followed by 1 cycle for 10 min at 72 °C. For
the detection of b-actin, the number of cycles was reduced to
32. Products were separated on 1.4% agarose gels.
Transient transfections and reporter assays
Cells (2 · 10
4
) were plated per each well of a 24 well cell
culture dish. 24 h later, cells were transfected in triplicate
using the indicated amount of luciferase fusion construct or
empty vector and 3–5 lL of Fugene (Roche) diluted in
100 lL Dulbecco’s modified Eagle’s medium per triplicate
reaction. To analyze the transcriptional properties of
E2F6b, U2-OS cells were transfected with 0.200 lgof
E2F-dependet luciferase reporter construct (E2F1-luc or
E1B-luc), 0.050 lgofCMV-bGal (to monitor transfection

,1lL0.25
M
dithiothreitol,
3.33 lL2m
M
dNTP mixture, 22.67 lLH
2
O, 1 lL1:20
diluted Superscript II reverse transcriptase (Invitrogen)].
RNA was subsequently degraded by incubating with
105 lLRNase(100lgÆmL
)1
sonicatedsalmonsperm
Ó FEBS 2002 E2F6b, an alternatively spliced E2F6 isoform (Eur. J. Biochem. 269) 5031
DNA/20 lgÆmL
)1
RNaseA)for15minat37°C. A
sequencing reaction was performed with the same SG50
32
P-end-labeled primer using Sequenase 2.0 (United States
Biochemical) and the pBS-Not/Xho4.5 template. The
sequencing products and the primer extension product were
separated on a QuickPoint gel (Novex) and visualized by
autoradiography.
RESULTS
Two distinct mRNAs are produced from the E2f6 locus
It has recently been shown that the E2f6 locus produces two
alternatively spliced isoforms [10] (see Fig. 1). The larger
splice variant is generated by insertion of an additional
exon 2. Exon 2 contains a stop codon, in-frame with the

variant. Black boxes represent coding exons. The open box represents
the 5¢ untranslated region of exon 1 (see Fig. 3). The sequence of
exon 2 is shown below. Translation termination and initiation codons
are indicated.
Fig. 2. Mouse E2F6 promoter and 5¢ untranslated region (5¢UTR). (A) Identification of the E2F6 transcriptional start site by primer extension
analysis. Primer extension analysis was performed with radiolabelled primer SG50 (right lane). A genomic clone was sequenced with the same
labelled primer, and the reaction was resolved on the same gel together with the primer extension reaction (left lanes). For primer location see (B).
(B) Sequence of the mouse E2F6 promoter and 5¢UTR. The 650 bp nucleotide sequence 5¢ of the transcriptional start site (+1) is shown. The
translation initiation codon is at +457. Upstream AUG codons are boxed. The location of the SG50 primer used in the primer extension reaction is
shown. (C) Schematic representation of upstream initiation codons and of corresponding open reading frames in E2F6 and E2F6b. Black lines
represent open reading frames with AUG codons in a context that favors translation initiation. Grey lines represent other open reading frames.
Translation initiation of E2F6 is at +457. Translation of E2F6b is initiated at +628. Numbers represent location of initiation and termination
codons relative to the transcription initiation site.
5032 T. Dahme et al. (Eur. J. Biochem. 269) Ó FEBS 2002
200 base pairs upstream of the E2F6 open reading frame,
suggesting a minimum length of 200 nucleotides for the
5¢UTR of E2F6 (not shown). For primer extension analysis,
we chose a backward primer in the untranslated region that
anneals close to the beginning of the known EST clones (see
Fig. 2B, primer SG50). Total RNA was isolated from
primary MEFs, then radiolabelled primer SG50 was
hybridized to the RNA and extended with reverse tran-
scriptase. Sequencing reactions with the same radiolabelled
primer and the subcloned genomic template were resolved
on the same gel with the primer extension reaction. We
observed a single band in the primer extension reaction
corresponding to a transcriptional start site about 457 base
pairs upstream of the translation start in Exon 1 (Fig. 2A).
The presence of a single primer extension product indicates
that transcription of E2F6 and E2F6b is initiated at the

AUG in exon 2, it will give rise to an amino-terminal
truncated protein lacking the first 36 amino acids of E2F6.
This protein would be predicted to be of a smaller molecular
mass than the previously described E2F6 protein. To
identify a potential N-terminal truncated E2F6 protein, we
used two different polyclonal antisera directed against
oligopeptides derived from the C-terminus (C10) [7] and
from the amino-terminus (N14) of murine E2F6 (see
Fig. 3A). C10 is predicted to recognize both forms of
E2F6, while N14 will only recognize the full-length E2F6
protein. The specificity of C10 and N14 antisera was
confirmed by immunoprecipitation-Western experiments
with in vitro translated proteins (Fig. 3B). Interestingly,
lysates of MEFs derived from E2F6 deficient mice lacked
two E2F6 specific bands, as compared with lysates of
wildtype MEFs in a Western blot probed with C10
antiserum (data not shown, but see [7]). In these experi-
ments, a second, slightly faster migrating protein was
detected in wildtype MEFs but not in E2F6 deficient MEFs
[7]. Unfortunately, neither Western blots, nor immuno-
precipitation-Western blots of MEF lysates, probed with
N14 antiserum, revealed any E2F6 specific signal that could
be unambiguously distinguished from background (data
not shown). We therefore employed an approach that
turned out to be of higher sensitivity and specificity, and
immunoprecipitated E2F6 from lysates of metabolically
labelled MEFs. In these experiments, a common band
corresponding in size to E2F6 was detected by the C10 and
N14 antisera (Fig. 3C). Importantly, an additional, faster
migrating protein was immunoprecipitated by C10, but not

When E2F6b was in vitro translated, two E2F6b bands of slightly
different mobility were observed. (C) E2F6 was immunoprecipitated
from lysates of metabolically labelled MEFs with the affinity purified
E2F6 specific antisera C10 and N14, as indicated. C10 precipitates two
proteins that correspond in size to E2F6 and E2F6b, while N14
recognizes only one protein that corresponds to E2F6. Because of the
lower affinity of the N14 antiserum, we cannot completely exclude the
possibility that the band in the C10 immunoprecipitation that corres-
ponds in size to E2F6b is a proteolytic breakdown product.
Ó FEBS 2002 E2F6b, an alternatively spliced E2F6 isoform (Eur. J. Biochem. 269) 5033
this construct contains exon 1, exon 2, the first seven triplets
of exon 3, and thereafter the luciferase coding sequence
lacking only the initiating AUG. Two constructs that lack
exon 2 served as controls. In Exon3-luc, the luciferase
coding sequence was introduced after the seventh codon of
exon 3, which is the same fusion point as in Exon2-luc.
Since there is the possibility that this rather long amino-
terminal E2F6 sequence fused to the luciferase will influence
its activity, a second control that only contained the first
seven codons of exon 1 (Exon1-luc) was used. Importantly,
all luciferase constructs contain the complete E2F6 5¢UTR,
and their transcription is driven by a 2.2-kb fragment of the
E2F6 promoter (see Fig. 4A). Transient transfection assays
in U2-OS cells revealed dose-dependent activity of
Exon2-luc which was two to three times lower than that
of Exon1-luc and Exon3-luc (Fig. 4B). However, activity of
Exon2-luc was still up to more than 1000 times higher than
the activity of the empty vector, pGL2-Avr (Fig. 4B, right
lanes). Similar results were found in NIH-3T3 cells (data not
shown). We therefore concluded that a protein is expressed

To analyze whether the expression of E2F6 and E2F6b
is cell cycle dependent, MEFs were serum starved for 48 h,
and then released from starvation by the addition of
serum. Total RNA was isolated at three-hour intervals. In
addition, we isolated RNA from asynchronously growing
cells, and from confluent, contact inhibited cells. E2F6 and
E2F6b expression was again analyzed by RT-PCR with
Fig. 5. Expression of E2F6 and E2F6b. (A)ExpressionofE2F6and
E2F6b in primary mouse tissues was analyzed by RT-PCR with
primers SG122 and SG24 (top). RT-PCR with b-actin specific primers
was used as a control. (B) Expression of E2F6 and E2F6b during the
cell cycle. Mouse primary fibroblasts were brought to quiescence by
incubationfor48hinserumfreemedium,andthenreleasedintothe
cell cycle by the addition of 10% serum. RNA was isolated at the
indicated times after the addition of serum, and E2F6 and E2F6b
expression was analyzed by RT-PCR with primers SG122 and SG24.
RT-PCR with b-actin specific primers was used as a control. Expres-
sion in confluent (confl.) and asynchronously growing cells (asynchr.)
was also analyzed. The percentage of cells in the G0/G1, S, and G2/M
phases of the cell cycle at each time point was determined by FACS
analysis and is shown at the bottom.
Fig. 4. Translation of E2F6b is initiated at an internal initiation codon.
(A) Schematic representation of the E2F6-luciferase fusion constructs.
The SacIsiteat)2.2 kb was used for cloning of the E2F6 promoter
(see Fig. 1). Transcription initiation (+ 1) is indicated by a right arrow
(see Fig. 2). (B) Activity of Exon2-luc (Ex2-luc), Exon1-luc (Ex1-luc),
and Exon3-luc (Ex3-luc), compared to the activity of the empty vector
(pGL2-Avr). Plasmids were transiently expressed in U2-OS cells, and
luciferase activity was determined. Plasmid inputs (ng DNA) are
indicated. Activity of pGL2-Avr at 100 ng was set to 1. (C) Activity of

which luciferase expression is under the control of the
p14
ARF
and the E2F1 promoters. Both promoters contain
E2F sites and have previously been shown to be regulated in
an E2F-dependent manner [11,12]. p14
ARF
and E2F1
luciferase reporter plasmids were cotransfected with E2F6
and E2F6b expression plasmids. As expected, E2F6 reduced
activity of both promoters by about 50% to 60% (Fig.
6A,B). Surprisingly, E2F6b also reduced the activity of these
promoters, although it was slightly less efficient than E2F6.
To rule out the possibility that sequestration of the
dimerization partner DP is responsible for the reduction
in luciferase activity, we coexpressed DP2 together with
E2F6 and E2F6b (Fig. 6A,B, + DP2). Coexpression of
DP2 resulted in even further reduction of reporter activity,
indicating that repression by E2F6b is not a result of
titration of endogenous DP proteins. Taken together, these
results show that E2F6b is also a transcriptional repressor.
DISCUSSION
E2F6, the most recently identified E2F protein, is a
retinoblastoma-protein independent transcriptional repres-
sor. In mice, E2F6 is required for developmental patterning
of the axial skeleton [7]. Together with the recent finding
that E2F6 associates with polycomb proteins [8,9], these
observations suggest that E2F6 recruits polycomb com-
plexes to certain target promoters during development. It
has recently been reported that the E2f6 locus generates two

site of E2F6 was mapped to 256 and 241 nucleotides
upstream of the E2F6 AUG codon [10]. In contrast, we
found a single start at 457 nucleotides upstream of the
AUG. It is possible that the RNase-protection approach
used by Kherrouche et al. is more sensitive to extensive
mRNA secondary structure than our primer extension
strategy. Alternatively, it is possible that the transcription
start site of E2F6 is tissue dependent.
In agreement with an earlier report, we found that the
E2F6b mRNA is ubiquitously expressed in a wide variety of
tissues. The highest expression levels were found in heart
and skeletal muscle. In most tissues, except for skeletal
muscle and heart, E2F6 was more abundant than E2F6b.
However, in another study, for some of those tissues reverse
ratios between E2F6 and E2F6b were found [10]. It is
possible that this discrepancy is due to the different primers
used for the reverse transcription reaction. While we used a
Fig. 6. E2F6b is a transcriptional repressor. U2-OS cells were trans-
fected with 200 ng E2F-dependent reporter plasmids E2F1-luc (A)
[12], or with E1B-luc (with the p14
ARF
promoter) [11] (B), and with
200 ng E2F6, E2F6b, or DP2 expression plasmids, as indicated. 50 ng
CMV-b gal was cotransfected, and luciferase activity was normalized
to b-galactosidase activity. Basal activity of the reporter plasmid in
presence of empty expression vector was set to 1.
Ó FEBS 2002 E2F6b, an alternatively spliced E2F6 isoform (Eur. J. Biochem. 269) 5035
gene specific primer, an oligo(dT) primer was used in the
previous study. We also show that the ratio between the two
E2F6 mRNAs does not change significantly during the cell

5¢UTRs. Indeed, sequence analysis demonstrated that
mRNAs with complex 5¢UTRs often encode for proto-
oncogenes [21]. It remains to be shown whether E2F6 and/
or E2F6b play a role in tumorigenesis.
ACKNOWLEDGEMENTS
We wish to thank Stefanie Hauser and our laboratory and divisional
colleagues for many helpful conversations. We thank Kelly Farrenkopf
for proofreading and for helpful comments. We also thank Gordon
Peters and William Kaelin for the E1B-luc and E2F1-luc constructs,
respectively. This work was supported by fellowships from the
Leukemia and Lymphoma Society and the Volkswagenstiftung to
S. G.
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