REVIEW ARTICLE
The central role of CDE/CHR promoter elements in the
regulation of cell cycle-dependent gene transcription
Gerd A. Mu
¨
ller and Kurt Engeland
Molecular Oncology, Department of Obstetrics and Gynecology, University of Leipzig, Germany
Introduction
The cell division cycle is a fundamental process. It is
regulated at different molecular levels. One central
modification controlling the cell cycle is phosphoryla-
tion by complexes of cyclin-dependent kinases (cdks)
and their corresponding cyclins. A prominent example
of such a pair is cyclin B and cyclin-dependent
kinase 1 (cdk1 ⁄ Cdc2) controlling the checkpoint
between G
2
phase and mitosis (Fig. 1).
Cyclins were discovered by their cyclic appearance
during the cell cycle [1]. In particular, the abrupt disap-
pearance of the proteins was noticed in early reports
and described to be regulated by ubiquitin-mediated
proteolysis. Much later control of cyclin synthesis was
investigated in more detail [2]. In mammals, two B-type
cyclins form complexes with cdk1 ⁄ Cdc2. Synthesis of
proteins encoded by cyclin B1 and cyclin B2 genes is
Keywords
cell cycle; cell cycle genes homology region
(CHR); cell cycle-dependent element (CDE);
DREAM complex; E2F
Correspondence

transcriptional regulation. In addition, the expression of genes regulated by
CDE ⁄ CHR elements is mostly achieved through CCAAT-boxes, which
bind heterotrimeric NF-Y proteins as well as the histone acetyltransferase
p300. Importantly, many CDE ⁄ CHR promoters are downregulated by the
tumor suppressor p53. In this review, we define criteria for CDE ⁄ CHR-
regulated promoters and propose to distinguish two classes of
CDE ⁄ CHR-regulated genes. The regulation through transcription factors
potentially binding to the CDE ⁄ CHR is discussed, and recently discov-
ered links to central pathways regulated by E2F, the pRB family and p53
are highlighted.
Abbreviations
CDE, cell cycle-dependent element; cdk, cyclin-dependent kinase; ChIP, chromatin immunoprecipitation; CHR, cell cycle genes homology
region; cIAP2, DRS, downstream repression site; EMSA, electrophoretic mobility shift assay; MEF, mouse embryonic fibroblast; SV40,
simian virus 40.
FEBS Journal 277 (2010) 877–893 ª 2009 The Authors Journal compilation ª 2009 FEBS 877
mostly regulated at the transcriptional level [3]. We and
others then observed that transcription from both cy-
clin B genes is controlled by combinations of tandem
sites called the cell cycle-dependent element (CDE) and
the cell cycle genes homology region (CHR) [4–7].
The CDE was first observed in the Cdc25C pro-
moter by in vivo footprinting as being protected in G
0
cells. The Cdc25C gene is not expressed in resting cells
or in cells in G
1
phase. Only in G
2
phase can strong
transcription of the gene be detected. Mutation of the

cells [10].
Genes cannot be regulated solely by repression: the
activation of promoters is also required. To this end,
CDE ⁄ CHR repressor sites are usually found in con-
junction with two or three CCAAT-box elements
through which NF-Y transcription factors activate the
Fig. 1. CDE ⁄ CHR-regulated genes controlling G
2
⁄ M progression. The expression of many central players appearing in G
2
phase and mitosis
was shown to be regulated at the transcriptional level by CDE ⁄ CHR tandem elements. Tightly controlled gene expression, as well as rapid
protein degradation, is required for cell cycle progression. Regulatory circuits also include control through p53. Cell cycle arrest can be medi-
ated by p53 downregulating the transcription of central cell cycle regulators such as cyclin B, Cks1, Cdc2 and Cdc25C.
CDE ⁄ CHR-dependent cell cycle-gene transcription G. A. Mu
¨
ller and K. Engeland
878 FEBS Journal 277 (2010) 877–893 ª 2009 The Authors Journal compilation ª 2009 FEBS
promoters. Activation by NF-Y generally contributes
the largest part to promoter activity, which is then
repressed through the CDE ⁄ CHR sites in the early
phases of the cell cycle [11,12].
Other proteins binding to CDE ⁄ CHR promoters are
E2F family members. It has been shown that CDEs
are related to E2F sites and can, at least in some cases,
also bind members of the E2F transcription factor
family [13]. Since the discovery of the first three genes
regulated by CDE ⁄ CHR tandem sites, many other
important cell cycle-regulator genes have been reported
to be controlled by this class of elements.

CHR. Consistent with our original description of the
first CDE ⁄ CHR promoters [10], the CDE in the human
cyclin A promoter was also identified as a variant E2F
site [15]. Cell cycle-dependent protection of the CDE in
Table 1. Class I and class II genes with their functions in the cell cycle.
Gene symbol Gene name Function
AURKA aurora kinase A Protein kinase, regulates microtubule formation and stabilization at the spindle pole
during chromosome segregation
AURKB aurora kinase B Protein kinase, key regulator of cytokinesis, mediates attachement of the mitotic
spindle to the centromere, phosphorylates histone H3 during mitosis
B-MYB ⁄ MYBL2 v-myb myeloblastosis viral
oncogene homolog
(avian)-like 2
Transcription factor, involved in cell cycle progression, possesses both activator
and repressor activities
CCNA cyclin A Regulatory subunit of CDC2 or CDK2 kinases, promotes both G
1
⁄ S and G
2
⁄ M
transitions
CCNB1 cyclin B1 Regulatory subunit of mitosis promoting factor (MPF), regulates G
2
⁄ M phase
transition, co-localizes with microtubules
CCNB2 cyclin B2 Regulatory subunit of mitosis promoting factor (MPF), regulates G
2
⁄ M phase
transition, co-localizes with Golgi region
CDC2 ⁄ CDK1 cell division cycle 2 ⁄

the mouse cyclin A promoter was confirmed by in vivo
footprinting and named CCRE [16]. Earlier, the CDE ⁄
CHR region from the human Cdc2 gene had been
found to be responsible for 12-O-tetradecanoylphorbol-
13-acetate (TPA)-dependent transcriptional repression
and was termed the R box [17].
The best studied E2F site, located with a four-nucle-
otide spacer upstream from a CHR, is found in the
B-Myb gene. This site was first identified without rec-
ognizing the adjacent element comprising CHR func-
tion. However, it was observed that the E2F element
downregulates B-Myb transcription in G
0
and that its
mutation leads to derepression because it is observed
with CDE sites [18]. Repressive protein complexes
appear to occupy the CDE-related E2F site in G
0
and
G
1
cells, as determined by in vivo footprinting. Site
occupation during the cell cycle is lost precisely at the
time when B-Myb becomes expressed [19]. After the
E2F site was well established as regulating B-Myb
expression, a CHR-like element, named the down-
stream repression site (DRS), was identified to regulate
cell cycle-dependent transcription together with the
E2F site [20,21]. The DRS ⁄ CHR in the B-Myb pro-
moter deviates most from other CHR sequences with

different with the mouse Cdc25C promoter. The timing
of cell cycle-dependent expression from mouse and
human promoters is identical. Also, essentially all
promoter elements are conserved in the two genes
except for the CDE. Mutational analysis of the region
four nucleotides upstream from the CHR in mouse
Cdc25C promoter-reporter assays leads to only a small
Table 2. Criteria for promoters controlled by CDE ⁄ CHR sites.
Class I
Genes not expressed in G
0
and G
1
cells
Genes encode proteins with functions in S, G
2
or M phases
CHR consensus similar to 5¢-TTTGAA-3¢
CDE is a site rich in G and C found upstream of a CHR
CDE positioned with a four-nucleotide spacer upstream of a
CHR
Orientation with CHR proximal to the coding region
Only one CDE ⁄ CHR per promoter
TATA-less promoters, multiple transcriptional start sites
Protein binding to the elements in G
0
and G
1
cells as monitored
by in vivo footprinting

ments were shown using the mouse Cdc25C gene as an
example. The CHR in this promoter naturally lacking
a CDE can cooperate with bona fide CDE, E2F or
Sp1 ⁄ 3 sites introduced upstream of it, at least when
tested in reporter assays [27].
Many other genes were initially reported to be con-
trolled by both CDE and CHR elements. Examples
are present within the cyclin B family. In mammals,
three B-type cyclins are known. For the most recently
discovered family member, mammalian cyclin B3, the
exact function and kinase association partners are not
known [28,29]. By contrast, cyclin B1 and cyclin B2
are central to the regulation of progress through the
cell cycle (Fig. 1). Cyclins B1 and B2 appear in S phase
and accumulate in G
2
and mitosis before disappearing
at the transition from metaphase to anaphase. Synthe-
sis is controlled at the level of gene transcription [3].
Interest in control mechanisms of cyclin B1 and cyclin
B2 cell cycle-dependent transcription began early
[3,30–33]. When investigating the regulation of human
cyclin B1 transcription, a potential CDE was tested
and found to play only a limited role in cell cycle-
dependent transcription [4]. Later, this finding on the
CDE was confirmed and the major cell cycle-depen-
dent regulation was attributed to a novel type of CHR
site just next to the CDE. This CHR holds a change
of one nucleotide compared with other elements of this
type, which mostly follow the consensus 5¢-TTTGAA-

cyclin B2 promoters, can function without a CDE.
Such differences in sequence with identities in func-
tion are often found between mouse and human pro-
moters. However, regulation through CDE ⁄ CHR sites
found in the human Cdc25C, cyclin B1 and cyclin B2
promoters is fully conserved in nucleotide sequence
and function in closely related organisms such as chim-
panzee, orangutan and human [25].
Timing of gene expression during the cell cycle has
been believed to be dependent on the exact nucleotide
sequence of the CDE ⁄ CHR site. Expression from a
cyclin A reporter usually precedes that of cyclin B2,as
expected from the chromosomal expression [6]. In order
to test whether this solely depends on the CDE ⁄ CHR,
the CHR region and the element upstream from it were
replaced in the human cyclin B2 promoter with the
well-characterized CDE ⁄ CHR sites from the human
Cdc25C and cyclin A promoters [10] and expression
from the altered reporters was tested during the cell
cycle compared with expression from the wild-type con-
struct [7]. Timing of expression from the three promot-
ers was similar, without a significant shift between cell
cycle phases. This indicates that a promoter does not
simply adopt the timing of expression from the other
promoter as a result of replacing the cell cycle-regula-
tory elements. Thus, it is likely that cell cycle-dependent
timing of expression is also determined by elements
outside the CDE ⁄ CHR elements [7].
Furthermore, the effect of DNA methylation on
CDE ⁄ CHR-dependent transcriptional regulation, pos-

becomes maximally expressed in G
2
(Fig. 1). Human
and mouse Tome-1 promoters were tested by mutating
putative CDE and CHR sites separately in promoter
assays. Both sites are required for cell cycle-dependent
transcription. However, as in most other CDE ⁄ CHR
promoters, mutation of the CHR results in a smaller
remaining cell cycle-regulation than alteration of the
CDE [37]. Interestingly, the core of the human Tome-1
promoter CDE⁄ CHR has a sequence identical to the
tandem element in the human Cdc25C promoter [10].
Recently, the mitosis-related genes Ect2, MgcRac-
GAP and MKLP1 were shown to be transcriptionally
regulated during the cell cycle, being weakly expressed
in G
1
and strongly expressed in G
2
⁄ M. Promoters
became derepressed in the cell cycle when the CHRs
were mutated and assayed in the interleukin-2-depen-
dent Kit 225 T cells. Also, the interleukin-2-dependent
derepression, usually seen in this system, was dere-
pressed upon CHR mutation. The effects were very
strong with the MKLP1 promoter. The MgcRacGAP
CHR has the sequence ‘5-TTTCAA-3¢ and thereby a
reverse orientation to canonical CHRs. This may
explain why the effect in this promoter is particularly
small [38]. All three CHRs may be class II, although

ner, with low expression in G
1
and reaching peak lev-
els in G
2
⁄ M. Mutational analysis of the promoter in
HeLa cells synchronized by double-thymidine or noco-
dazole block showed that a CHR is responsible for
the cell cycle-dependent expression. The sequence
upstream of the CHR does not match any of the pub-
lished CDEs. However, alteration of a putative CDE,
which is more distant from the CHR than the usual
four nucleotides, in addition to the CHR, yielded a
further decrease in regulation. The CDE alone was not
tested [41]. The CDE mutation that was assayed would
also alter a putative CDE site with the standard dis-
tance of four nucleotides to the CHR. With the data
presented it is not quite clear where exactly the CDE is
located and what its contribution to cell cycle-depen-
dent regulation is. Possibly the CHR constitutes a class
II regulatory site.
Over the years numerous additional genes were
reported to be regulated by CDE ⁄ CHR sites. Often
sites were postulated only based on sequence similarity.
Generally, functional assays are required to define rele-
vant elements. Sometimes reported experiments do not
yield a consistent picture. Survivin, also named Birc5,
API4 or IAP4, functions as an apoptosis inhibitor and
is expressed in G
2

human survivin gene. The article tries to correlate muta-
tions or polymorphisms found in the survivin promoter
to regulation through several possible CDE and CHR
sites. When mutated the sites led only to a moderate
deregulation of cell cycle-dependent transcription of
the reporter. According to the results from this report,
possible protein binding to the putative CDE appears
stronger in G
2
⁄ M than in G
1
[44]. This contradicts
repression through a complex in G
1
and in vivo foot-
printing results in the original definition of the sites
[10]. Taken together, the sites in the survivin promoter
do not display properties of bona fide CDE ⁄ CHR ele-
ments. This notion is confirmed in a later report
describing transforming growth factor-b responsiveness
of the survivin promoter. In the experiments the puta-
tive CHR does not contribute to regulation [45].
Also, the BUB1B gene was implicated as a CDE ⁄
CHR-regulated gene. BubR1 is a protein important
for spindle checkpoint activation. Expression of the
BUB1B gene coding for BubR1 is undetectable in G
1
,
but peaks in G
2

regulate the CDC20 promoter through a site just
downstream of the E2F-responsive part of SIRF [48].
The results of Kidokoro and colleagues have been put
into question by a very recent report identifying a p53-
binding element further upstream in the CDC20 pro-
moter as the major regulatory site [49]. According to
Banerjee et al., [49] p21
WAF1 ⁄ CIP1
, the putative
CDE ⁄ CHR and CCAAT-boxes suggested by Kidokoro
et al. as relevant for p53-dependent downregulation,
are not required when p53 is expressed at physiological
levels. Another report suggests that the human and
mouse RB2 (p130) genes are controlled by a
CDE ⁄ CHR-like site. The element is occupied by pro-
tein, as measured by in vivo footprinting. Mutation of
this site leads to derepression of the promoter in repor-
ter assays. However, p130 expression does not oscillate
significantly during the cell cycle. Therefore, its regula-
tion may be related to, but appears to be different
from, cell cycle-controlled CDE ⁄ CHR-dependent
expression. E2F family proteins did not bind to the
CDE-related site [50].
In addition, some more genes were postulated to
be regulated through CDE and CHR sites during
the cell cycle without experimental verification. Based
on the mRNA expression pattern and a promoter
sequence comparison, the centromeric histone H3
homolog CENP-A gene was postulated to contain a
CDE ⁄ CHR site [51]. The gene coding for the kine-

CHR positioned downstream with a spacer of four nu-
cleotides (Fig. 2).
NF-Y is the main activator, and the
distance between two CCAAT-boxes is
31, 32, or 33 bp
Already with the discovery of the first CDE ⁄ CHR
genes it was recognized that these promoters were acti-
vated through CCAAT-boxes binding the transcrip-
tional activator NF-Y.
Functional CCAAT-boxes are found in both orien-
tations. Interestingly, from the first publications on
NF-Y binding to cell cycle promoters it appeared that
the protein complex is constitutively bound to the
CCAAT-elements throughout the cell cycle when
assayed by in vivo footprinting [10,54]. However, based
on chromatin immunoprecipitation (ChIP) assays, a
more recent report indicates that NF-Y is only bound
to DNA when the promoter is activated [55]. As the
identity of proteins occupying DNA cannot be solved
by in vivo footprints, it has not been ruled out that the
CCAAT-boxes are bound by other proteins in G
0
and
G
1
with a shift to NF-Y in later cell cycle phases
(Fig. 3).
Many cell cycle genes were found to contain two or
three CCAAT-boxes essential for promoter activity
(e.g. the mouse cyclin B1 and cyclin B2 genes) [56,57].

p300 binding requires all three CCAAT-boxes and
association of NF-Y with these elements for optimal
transcriptional activation of the mouse cyclin B2 pro-
moter. Changing the distance of the CCAAT-sites
Fig. 3. Possible protein occupation on class I CDE ⁄ CHR promoters.
The model for regulation is primarily based on results obtained
using the Cdc2 and Cdc25C promoters. In G
0
, proteins appear to
bind to the CDE ⁄ CHR, as monitored by in vivo footprinting. Accord-
ing to these early experiments all binding is lost in G
2
⁄ M. In con-
trast, constitutive binding to the CCAAT-boxes is observed.
Trimeric NF-Y binds to the CCAAT-boxes and stimulates gene
expression in cooperation with the histone acetyltransferase p300
in S ⁄ G
2
⁄ M phases. Nevertheless, CCAAT-boxes are occupied by
proteins, as suggested by in vivo footprinting in G
0
and G
1
. How-
ever, these proteins are probably different from NF-Y and p300. For
efficient activation of the promoters, the distance between the
CCAAT-boxes has to be 31 to 33 bp, probably to allow binding of
the p300 co-activator. In G
0
and G

CDE ⁄ CHR-dependent cell cycle-gene transcription G. A. Mu
¨
ller and K. Engeland
884 FEBS Journal 277 (2010) 877–893 ª 2009 The Authors Journal compilation ª 2009 FEBS
reduces p300-dependent activation [58]. Recruitment of
p300 HAT on the cyclin A and Cdc2 promoters may
also be in accordance with activation and histone H3
and H4 acetylation beginning in late G
1
, as observed
by ChIP experiments [60].
Interestingly, NF-Y appears to form interactions
also with other activating factors. The results of
employing plasmid-based ChIP assays on the Cdc2
promoter indicate that E2F3 binding to the distal acti-
vating E2F site may require an intact CCAAT-box
occupied by NF-Y [61]. NF-Y proteins bind to the
human Cdc2, cyclin B1 and cyclin A2 promoters
throughout the cell cycle, as determined using ChIP
assays [61]. This is consistent with earlier genomic
footprinting observations [9,10,54].
In addition to the few CDE ⁄ CHR promoters ana-
lyzed in detail to determine a role of NF-Y in their
control, a number of such genes were implicated to
be regulated through CCAAT-boxes. Cotransfection
of dominant-negative NF-YA demonstrated that
most of the Tome-1 promoter activity depends on
NF-Y. One CCAAT-box had been tested for its role
in activation by reporter assays comparing wild-type
with mutant promoters. It was not further in-

regulation of Cdc2 transcription relies on intact CDE
and CHR elements. A report implied p53 and
p21
WAF1 ⁄ CIP1
in this downregulation by employing
p53-positive or p53-negative cell lines [69]. For the
Plk1 gene, coding for Polo-like kinase 1, downregula-
tion by p21
WAF1 ⁄ CIP1
was also postulated to be con-
trolled through CDE and CHR elements [70].
Experiments confirmed that p53 and p21
WAF1 ⁄ CIP1
reg-
ulate, in part, through the CDE and CHR sites. How-
ever, mutation of the CDE ⁄ CHR did not completely
abrogate p53-dependent downregulation [71]. Earlier,
it had been shown that the CHR in the Plk1 promoter
was more relevant for cell cycle-dependent transcrip-
tion than the CDE [35]. Furthermore, the topoisomer-
ase IIa gene is downregulated by overexpression of
p21
WAF1 ⁄ CIP1
. Like the Plk1 gene, topoisomerase IIa
was presented as downregulated through CDE ⁄ CHR
sites upon p21
WAF1 ⁄ CIP1
overexpression [70]. However,
a combination of a CDE and an adjacent CHR had
been postulated only by sequence comparison [72] but

G. A. Mu
¨
ller and K. Engeland CDE ⁄ CHR-dependent cell cycle-gene transcription
FEBS Journal 277 (2010) 877–893 ª 2009 The Authors Journal compilation ª 2009 FEBS 885
deleted [66]. In a later report the CDE ⁄ CHR was
implicated to be responsible, at least in part, for
p53-dependent downregulation of the Cdc25C pro-
moter. The results were based on transient expression
experiments employing a 76 bp CDE ⁄ CHR-containing
promoter fragment cloned upstream of a minimal
adenovirus E1b promoter-driven luciferase reporter
[74]. The same reporter system yielded activation
through a putative p53-binding site that was later
implicated in downregulation of Cdc25C by the same
group [74,75]. This short Cdc25C promoter fragment
lacks the CCAAT-boxes originally found to be
responsible for most of the promoter activity [10,54].
Therefore, the promoter exerts an artificially low activ-
ity. Further downregulation measured with this short
fragment may be unnatural. The partial downregula-
tion, observed in this experimental system, through the
Cdc25C CDE ⁄ CHR stands in contrast to earlier
results. In these experiments, deletion of the Cdc25C
CDE ⁄ CHR in the full promoter context had almost no
effect on p53-dependent repression after overexpressing
p53 in transient transfection assays [66]. Furthermore,
no binding of p53 protein to the segment was observed
for the proposed CDE ⁄ CHR-dependent repression
mechanism [74].
By contrast, protein binding to promoters of genes

resulted in deregulation of the Cdc25C promoter by
destroying repression in G
0
and G
1
. Expression of
SV40 T antigen in promoter-reporter assays yielded
deregulation, which was dependent on the CDE of the
human Cdc25C promoter. Dimethyl sulfate footprint-
ing of the CDE in the presence of SV40 large T indi-
cated a loss of protein occupation on this site in vivo
[79].Elevated expression of cyclin A, cyclin B, Cdc25C
and Cdc2 had already been observed after expression
of SV40 T antigen, which led to the disruption of
mitotic checkpoints [80]. This information, combined
with the change of protein occupation on the Cdc25C
CDE, indicates that CDE ⁄ CHR sites which regulate
cyclin A, cyclin B and Cdc2 promoters may lose bind-
ing of their regulator proteins and repression in
G
0
⁄ G
1
.
Deregulation by viral proteins was also tested using
the mouse cyclin A promoter as an example. Polyoma-
virus T antigen has functions similar to those of ade-
novirus E1A, human papillomavirus E7 proteins and
simian virus T antigen regarding the dissociation of
pocket proteins from E2Fs [81]. Large T from polyo-

There are several hints for a functional connection of
E2F4 binding and CDE-dependent regulation. It was
shown, by EMSAs, that E2F4 and p130, but not
E2F1, E2F2, p107 or pRB, are able to bind to the
CDE in the Cdc2 promoter in vitro [9]. Later, when
ChIP was developed, binding of E2F4, p107 and p130
to the Cdc2 and B-Myb promoters was shown without
specifying the particular site [85].
Similarly, E2F4 ⁄ DP1 and p107 associate with the
mouse B-Myb E2F site when assayed by EMSAs. This
binding site is close to the CHR-related DRS element
[20]. Interestingly, the core of this E2F element dis-
plays an identical sequence to the CDE from the
human Cdc25C promoter (Fig. 2), but, in contrast to
the B-Myb site, the Cdc25C element does not bind any
E2F proteins in vitro [83]. Obviously, nucleotides out-
side the core sites are responsible for this differential
binding, possibly the different CHRs. Probably, the
distinct binding to the related elements, together with
the different DRS ⁄ CHR, are responsible also for
altered timing of gene expression as seen with B-Myb
mRNA appearing earlier in the cell cycle than Cdc25C
expression (Fig. 2). E2F binding-site occupation is
reduced in p107
) ⁄ )
p130
) ⁄ )
MEFs compared with
wild-type cells when assayed using in vivo footprinting.
Loss of the two pocket proteins leads to deregulation

Binding of the activating E2Fs during the cell cycle to
the promoter alternates with binding of E2F4 in quies-
cent cells. Mutation of the distal E2F site allows E2F4
to associate with the Cdc2 promoter also in G
2
cells,
suggesting that E2F1, -2 and -3, although they bind to
the distal E2F site, block binding of E2F4 to the CDE
[61].
For other CDE ⁄ CHR promoters, E2F and pocket
proteins were also implicated in binding. The CDE in
the human Aurora B promoter bound DP2, E2F1 and
E2F4, but not DP1, when HeLa nuclear extracts were
employed for biotin–streptavidin pull-down assays
followed by western blot analysis [40]. Furthermore, the
CDE in the human cyclin A promoter, also identified as
a ‘variant E2F site’, binds E2F complexes (including
DP1 and p107) in EMSAs from nuclear extracts. The
complexes appear to lack pRB, E2F1, E2F2 or E2F3
because antibodies directed against these proteins did
not recognize any of the protein complexes formed on
this site in the cyclin A promoter [10,15]. By contrast,
another set of experiments showed that the cyclin A
CDE can bind E2F1 and E2F3 from HeLa nuclear
extracts, as well as recombinant E2F1 and DP1 glutathi-
one S-transferase fusion-protein complexes in EMSAs.
However, E2F4 was not shifted with this probe
although the protein was present in the HeLa extracts
[83]. In a large study employing ChIP on cell cycle
promoters from human cells, the cyclin A promoter was

However, individual deletion of pocket proteins in
p130
) ⁄ )
or p107
) ⁄ )
MEFs does not lead to deregula-
tion, indicating that p107 and p130 can substitute for
G. A. Mu
¨
ller and K. Engeland CDE ⁄ CHR-dependent cell cycle-gene transcription
FEBS Journal 277 (2010) 877–893 ª 2009 The Authors Journal compilation ª 2009 FEBS 887
each other [88]. It is likely, although not proven, that
regulation by p107 and p130 is executed through the
E2F ⁄ CDE sites in these promoters. Interestingly,
Cdc25C, the gene with the first identified CDE ⁄ CHR
promoter, is not deregulated in Rb
) ⁄ )
or
p107
) ⁄ )
p130
) ⁄ )
MEFs [88]. This implies that Cdc25C
does not require pocket proteins for its regulation and
is regulated differently from cyclin A, B-Myb or Cdc2.
The DREAM complex: a possible role in
CDE/CHR-dependent regulation
E2F4 and the pocket protein p107 have been shown,
in vitro by EMSA, to bind to the B-Myb promoter
E2F site with its neighboring CHR-related DRS ele-

with cell cycle phases. Lin proteins form a core
complex that associates with different proteins during
passage through the cell division cycle. It appears that
E2F4/p107/p130 form part of the complex in G
0
phase
and B-Myb constitutes a component of the complex in
S-phase. ChIP analyses have shown that DREAM
complex components are able to bind to CDE ⁄ CHR-
regulated promoters, mostly without specifying partic-
ular binding sites [20,22,61,90,92–96]. Very recently, it
has been shown that Lin-54 can bind to two sites in
the Cdc2 promoter in vitro. Lin-54 binds in EMSAs to
a region in the upstream part as well as to the CHR of
the Cdc2 promoter [86].
Cyclin A and human cyclin B1 are class I and class
II CDE ⁄ CHR-controlled genes, respectively (Fig. 2).
Both promoters become derepressed in G
0
cells when
CDE ⁄ CHR sites are mutated [5,10]. Supporting this
observation, Litovchick et al. [90] showed an upregula-
tion in the expression of cyclin B1 after knockdown of
DREAM complex components in G
0
-arrested cells.
Consistently, the opposite effect was shown with a
slowdown in cell cycle progression after serum restimu-
lation of G
0

an upregulation would indicate a function of Lin-9 in
repression. This indicates that Lin-9 is involved in the
activation of these genes in cycling cells.
In summary, it has been shown that the DREAM
complex controls expression of some CDE ⁄ CHR-
regulated genes. E2F4 can bind to certain CDE sites.
Lin-54 can bind to the CHR in the Cdc2 promoter
in vitro. Incorporating the results from different
reports, it appears that a change in the composition of
the DREAM complex shifts its properties from a
repressor complex in G
0
cells to an activator complex
in S ⁄ G
2
⁄ M cells. It is likely that these contrasting
features are mediated by the shift from E2F4 ⁄ p107 ⁄
p130 to B-Myb (Fig. 3). It still has to be shown which
CDE ⁄ CHR-dependent cell cycle-gene transcription G. A. Mu
¨
ller and K. Engeland
888 FEBS Journal 277 (2010) 877–893 ª 2009 The Authors Journal compilation ª 2009 FEBS
elements in the promoter are bound by the ‘activating
DREAM complex’ because the CDE ⁄ CHR tandem
element is not occupied by proteins in later cell cycle
phases and is not necessary for activation of the
promoters.
Conclusions and perspectives
What defines CDE and CHR elements? A CHR usu-
ally has the sequence 5¢-TTTGAA-3¢. However, this

How are class II promoters, which do not contain a
functional CDE, regulated? If the DREAM complex
is the main regulator of CDE ⁄ CHR cell cycle genes,
to which promoter sites does it bind during any par-
ticular phase of the cell cycle? What is the exact
mechanism of p53-dependent downregulation of
CDE ⁄ CHR genes? Is this mechanism the same for
all promoters?
More than a decade after the initial observation of
the tandem site, CDE ⁄ CHR-dependent regulation has
been established for many central genes involved in cell
cycle control. The CDE is demonstrated to be different
from classical E2F elements. Class II promoters have
been identified as controlled by just a CHR and to
lack a functional CDE. Links have been discovered
from CDE ⁄ CHR promoter control to pathways regu-
lated by p53, E2F and the pRB family.
Acknowledgements
G.A.M. was the recipient of a graduate fellowship
awarded by the Freistaat Sachsen. Research in our
group was supported by the Bundesministerium fu
¨
r
Bildung und Forschung (BMBF) through the Interdis-
ciplinary Center for Clinical Research (IZKF) at the
University of Leipzig and the Deutsche Forschungs-
gemeinschaft by grants SPP 314, EN 218 ⁄ 6-1 and 6-2
(to K. E.).
References
1 Evans T, Rosenthal ET, Youngblom J, Distel D &

7 Wasner M, Haugwitz U, Reinhard W, Tscho
¨
p K, Spies-
bach K, Lorenz J, Mo
¨
ssner J & Engeland K (2003)
Three CCAAT-boxes and a single cell cycle genes
homology region (CHR) are the major regulating sites
for transcription from the human cyclin B2 promoter.
Gene 312, 225–237.
8 Lucibello FC, Truss M, Zwicker J, Ehlert F, Beato M
&Mu
¨
ller R (1995) Periodic cdc25C transcription is
mediated by a novel cell cycle- regulated repressor ele-
ment (CDE). EMBO J 14, 132–142.
9 Tommasi S & Pfeifer GP (1995) In vivo structure of the
human cdc2 promoter: release of a p130-E2F-4
complex from sequences immediately upstream of the
G. A. Mu
¨
ller and K. Engeland CDE ⁄ CHR-dependent cell cycle-gene transcription
FEBS Journal 277 (2010) 877–893 ª 2009 The Authors Journal compilation ª 2009 FEBS 889
transcription initiation site coincides with induction of
cdc2 expression. Mol Cell Biol 15, 6901–6913.
10 Zwicker J, Lucibello FC, Wolfraim LA, Gross C, Truss
M, Engeland K & Mu
¨
ller R (1995) Cell cycle regulation
of the cyclin A, cdc25C and cdc2 genes is based on a

¨
ller
R (1996) Cell cycle regulation of E2F site occupation in
vivo. Science 271, 1595–1597.
20 Bennett JD, Farlie PG & Watson RJ (1996) E2F bind-
ing is required but not sufficient for repression of
B- myb transcription in quiescent fibroblasts. Oncogene
13, 1073–1082.
21 Liu N, Lucibello FC, Zwicker J, Engeland K & Mu
¨
ller
R (1996) Cell cycle-regulated repression of B-myb tran-
scription: cooperation of an E2F site with a contiguous
corepressor element. Nucleic Acids Res 24 , 2905–2910.
22 Catchpole S, Tavner F, Le CL, Sardet C & Watson RJ
(2002) A B-myb promoter corepressor site facilitates in
vivo occupation of the adjacent E2F site by p107 x E2F
and p130 x E2F complexes. J Biol Chem 277,
39015–39024.
23 Lam EW, Bennett JD & Watson RJ (1995) Cell-cycle
regulation of human B-myb transcription. Gene 160,
277–281.
24 Tavner F, Frampton J & Watson RJ (2007) Targeting
an E2F site in the mouse genome prevents promoter
silencing in quiescent and post-mitotic cells. Oncogene
26, 2727–2735.
25 Mu
¨
ller GA, Heissig F & Engeland K (2007) Chimpan-
zee, Orangutan, Mouse, and Human Cell Cycle Promot-

(1995) Cell cycle-dependent regulation of the cyclin B1
promoter. J Biol Chem 270, 28419–28424.
32 Piaggio G, Farina A, Perrotti D, Manni I, Fuschi P,
Sacchi A & Gaetano C (1995) Structure and growth-
dependent regulation of the human cyclin B1 promoter.
Exp Cell Res 216, 396–402.
33 Thatcher JD, McBride B & Katula KS (1995) Promoter
binding factors regulating cyclin B transcription in the
sea urchin embryo. DNA Cell Biol 14, 869–881.
34 Tscho
¨
p K & Engeland K (2007) Cell cycle-dependent
transcription of cyclin B2 is influenced by DNA methyl-
ation but is independent of methylation in the CDE
and CHR elements. FEBS J 274, 5235–5249.
35 Uchiumi T, Longo DL & Ferris DK (1997) Cell cycle
regulation of the human polo-like kinase (PLK)
promoter. J Biol Chem 272, 9166–9174.
36 Rother K, Li YY, Tscho
¨
p K, Kirschner R, Mu
¨
ller GA,
Mo
¨
ssner J & Engeland K (2007) Expression of Cyclin-
Dependent Kinase Subunit 1 (Cks1) is Regulated
During the Cell Cycle by a CDE ⁄ CHR Tandem
Element and is Downregulated by p53 but not by p63
or p73. Cell Cycle 6, 853–862.

regulation of TIAP ⁄ m-survivin expression. Biochim
Biophys Acta 1493, 188–194.
44 Xu Y, Fang F, Ludewig G, Jones G & Jones D (2004)
A mutation found in the promoter region of the human
survivin gene is correlated to overexpression of survivin
in cancer cells. DNA Cell Biol 23, 419–429.
45 Yang J, Song K, Krebs TL, Jackson MW & Danielpour
D (2008) Rb ⁄ E2F4 and Smad2 ⁄ 3 link survivin to TGF-
beta-induced apoptosis and tumor progression. Onco-
gene 27, 5326–5338.
46 Myslinski E, Gerard MA, Krol A & Carbon P (2007)
Transcription of the human cell cycle regulated BUB1B
gene requires hStaf ⁄ ZNF143. Nucleic Acids Res 35,
3453–3464.
47 Haugwitz U, Tscho
¨
p K & Engeland K (2004) SIRF–a
novel regulator element controlling transcription from
the p55Cdc ⁄ Fizzy promoter during the cell cycle.
Biochem Biophys Res Commun 320, 951–960.
48 Kidokoro T, Tanikawa C, Furukawa Y, Katagiri T,
Nakamura Y & Matsuda K (2008) CDC20, a potential
cancer therapeutic target, is negatively regulated by p53.
Oncogene 27, 1562–1571.
49 Banerjee T, Nath S & Roychoudhury S (2009) DNA
damage induced p53 downregulates Cdc20 by direct
binding to its promoter causing chromatin remodeling.
Nucleic Acids Res 37, 2688–2698.
50 Fajas L, Le Cam L, Polanowska J, Fabbrizio E, Ser-
vant N, Philips A, Carnac G & Sardet C (2000) A

G (1999) Down-regulation of cyclin B1 gene transcrip-
tion in terminally differentiated skeletal muscle cells is
associated with loss of functional CCAAT-binding
NF-Y complex. Oncogene 18, 2818–2827.
57 Bolognese F, Wasner M, Lange-zu Dohna C, Gurtner
A, Ronchi A, Muller H, Manni I, Mo
¨
ssner J, Piaggio
G, Mantovani R et al. (1999) The cyclin B2 promoter
depends on NF-Y, a trimer whose CCAAT- binding
activity is cell-cycle regulated. Oncogene 18, 1845–1853.
58 Salsi V, Caretti G, Wasner M, Reinhard W, Haugwitz
U, Engeland K & Mantovani R (2003) Interactions
between p300 and multiple NF-Y trimers govern Cyclin
B2 promoter function. J Biol Chem 278, 6642–6650.
59 Currie RA (1998) NF-Y is associated with the histone
acetyltransferases GCN5 and P ⁄ CAF. J Biol Chem 273,
1430–1434.
60 Takahashi Y, Rayman JB & Dynlacht BD (2000) Anal-
ysis of promoter binding by the E2F and pRB families
in vivo: distinct E2F proteins mediate activation and
repression. Genes Dev 14, 804–816.
61 Zhu W, Giangrande PH & Nevins JR (2004) E2Fs link
the control of G1 ⁄ S and G2 ⁄ M transcription. EMBO J
23, 4615–4626.
62 de Toledo SM, Azzam EI, Keng P, Laffrenier S & Lit-
tle JB (1998) Regulation by ionizing radiation of
CDC2, cyclin A, cyclin B, thymidine kinase, topoisom-
erase IIalpha, and RAD51 expression in normal human
diploid fibroblasts is dependent on p53 ⁄ p21Waf1. Cell

¨
ssner J
& Engeland K (2007) p53 downregulates expression of
the G(1) ⁄ S cell cycle phosphatase Cdc25A. Oncogene
26, 1949–1953.
68 Rother K, Dengl M, Lorenz J, Tscho
¨
p K, Kirschner R,
Mo
¨
ssner J & Engeland K (2007) Gene expression of cy-
clin-dependent kinase subunit Cks2 is repressed by the
tumor suppressor p53 but not by the related proteins
p63 or p73. FEBS Lett 581, 1166–1172.
69 Badie C, Itzhaki JE, Sullivan MJ, Carpenter AJ & Por-
ter AC (2000) Repression of CDK1 and other genes
with CDE and CHR promoter elements during DNA
damage-induced G(2) ⁄ M arrest in human cells. Mol
Cell Biol 20 , 2358–2366.
70 Zhu H, Chang BD, Uchiumi T & Roninson IB (2002)
Identification of promoter elements responsible for tran-
scriptional inhibition of polo-like kinase 1 and topo-
isomerase IIalpha genes by p21(WAF1 ⁄ CIP1 ⁄ SDI1).
Cell Cycle 1 , 59–66.
71 Jackson MW, Agarwal MK, Yang J, Bruss P, Uchiumi
T, Agarwal ML, Stark GR & Taylor WR (2005)
p130 ⁄ p107 ⁄ p105Rb-dependent transcriptional repression
during DNA-damage-induced cell-cycle exit at G2. J
Cell Sci 118, 1821–1832.
72 Isaacs RJ, Davies SL, Sandri MI, Redwood C, Wells

by inducing a CCAAT box binding factor. J Biol Chem
271, 13959–13967.
79 Zwicker J, Korner K & Muller R (1999) The SV40
large T oncoprotein disrupts DNA-binding of the cell
cycle-regulated transcriptional repressor CDF. Oncogene
18, 2023–2025.
80 Chang TH, Ray FA, Thompson DA & Schlegel R
(1997) Disregulation of mitotic checkpoints and regula-
tory proteins following acute expression of SV40 large
T antigen in diploid human cells. Oncogene 14,
2383–2393.
81 Schuchner S, Nemethova M, Belisova A, Klucky B,
Holnthoner W & Wintersberger E (2001) Transactiva-
tion of murine cyclin A by polyomavirus large and
small T antigens. J Virol 75, 6498–6507.
82 Liu N, Lucibello FC, Korner K, Wolfraim LA, Zwicker
J&Mu
¨
ller R (1997) CDF-1, a novel E2F-unrelated fac-
tor, interacts with cell cycle- regulated repressor ele-
ments in multiple promoters. Nucleic Acids Res 25 ,
4915–4920.
83 Liu N, Lucibello FC, Engeland K & Mu
¨
ller R (1998) A
new model of cell cycle-regulated transcription: repres-
sion of the cyclin A promoter by CDF-1 and anti-
repression by E2F. Oncogene 16, 2957–2963.
84 Philips A, Chambeyron S, Lamb N, Vie A & Blanchard
JM (1999) CHF: a novel factor binding to cyclin A

DeCaprio JA (2007) Evolutionarily conserved multisub-
unit RBL2 ⁄ p130 and E2F4 protein complex represses
human cell cycle-dependent genes in quiescence. Mol
Cell 26, 539–551.
91 Mauri P & Scigelova M (2009) Multidimensional pro-
tein identification technology for clinical proteomic
analysis. Clin Chem Lab Med 47, 636–646.
92 Schmit F, Korenjak M, Mannefeld M, Schmitt K,
Franke C, von EB, Gagrica S, Hanel F, Brehm A &
Gaubatz S (2007) LINC, a human complex that is
related to pRB-containing complexes in invertebrates
regulates the expression of G2 ⁄ M genes. Cell Cycle 6,
1903–1913.
93 Osterloh L, von EB, Schmit F, Rein L, Hubner D, Sa-
mans B, Hauser S & Gaubatz S (2007) The human syn-
Muv-like protein LIN-9 is required for transcription of
G2 ⁄ M genes and for entry into mitosis. EMBO J 26,
144–157.
94 Pilkinton M, Sandoval R, Song J, Ness SA & Colamo-
nici OR (2007) Mip ⁄ LIN-9 regulates the expression of
B-Myb and the induction of cyclin A, cyclin B, and
CDK1. J Biol Chem 282, 168–175.
95 Sandoval R, Pilkinton M & Colamonici OR (2009)
Deletion of the p107 ⁄ p130-binding domain of
Mip130 ⁄ LIN-9 bypasses the requirement for CDK4
activity for the dissociation of Mip130 ⁄ LIN-9 from
p107 ⁄ p130-E2F4 complex. Exp Cell Res 315,
2914–2920.
96 Knight AS, Notaridou M & Watson RJ (2009) A Lin-9
complex is recruited by B-Myb to activate transcription