Tài liệu Báo cáo khoa học: The central role of CDE/CHR promoter elements in the regulation of cell cycle-dependent gene transcription pdf

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

K. Engeland, Molecular Oncology, University
of Leipzig, Semmelweisstr. 14, D-04103
Leipzig, Germany
Fax: +49 341 9723475
Tel.: +49 341 9725900
E-mail:
(Received 18 September 2009, revised 9
November 2009, accepted 19 November
2009)
doi:10.1111/j.1742-4658.2009.07508.x
The cell cycle-dependent element (CDE) and the cell cycle genes homology
region (CHR) control the transcription of genes with maximum expression
in G
2
phase and in mitosis. Promoters of these genes are repressed by pro-
teins binding to CDE ⁄ CHR elements in G
0
and G
1
phases. Relief from
repression begins in S phase and continues into G
2
phase and mitosis. Gen-
erally, CDE sites are located four nucleotides upstream of CHR elements
in TATA-less promoters of genes such as Cdc25C, Cdc2 and cyclin A.
However, expression of some other genes, such as human cyclin B1 and
cyclin B2, has been shown to be controlled only by a CHR lacking a func-
tional CDE. To date, it is not fully understood which proteins bind to and
control CDE⁄ CHR-containing promoters. Recently, components of the
DREAM complex were shown to be involved in CDE ⁄ CHR-dependent

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

CDE in the Cdc25C promoter and analysis in reporter
assays shows that this element is responsible for cell
cycle-dependent expression of the gene. Surprisingly,
deregulation of the promoter does not lead to loss of
its activity but causes activation in resting cells and
in G
1
cells. Therefore, transcriptional repression is
responsible for regulation through the CDE [8].
Shortly after this initial description, another report
confirmed the CDE in the Cdc2 promoter as being dif-
ferentially occupied by protein complexes during the
cell cycle [9].
Mutation of nucleotides close to the CDE in the
Cdc25C promoter, and analysis in reporter assays,
yielded the first hints that there is another site rele-
vant for cell cycle-dependent repression of this gene.
Sequence comparison of the cyclin A and Cdc2 pro-
moters with that of the Cdc25C gene, followed by pro-
moter mutations analysed in reporter assays led us to
identify a new type of site downstream from the CDE.
Because of the high sequence conservation of this site
among the three promoters, we named this type of ele-
ment the CHR [10]. Transcriptional regulation through
this new site appeared to be functionally identical to
that of the CDE, with repression in resting cells and
relief from downregulation later in the cell cycle.
Mutation of the CHR led to derepression of transcrip-
tion in G
0

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.

Promoters regulated by CDE and CHR
sites
Genes regulated by CDE and CHR elements in their
promoters generally encode proteins with functions in
S, G
2
or M phases (Table 1). In quiescent cells these
genes are not expressed. CDE ⁄ CHR promoters usually
lack a TATA-box and employ multiple transcriptional
start sites [10,14]. Mutation of either a CDE or a
CHR in a promoter leads to the activation of tran-
scription in quiescent cells. A narrowly defined
sequence consensus for CDEs, from which functional
conclusions can be drawn, has not evolved.
After the initial description of CDE ⁄ CHR-depen-
dent gene regulation, many promoters were described
as being controlled by CDE or CHR sites [10]. How-
ever, one conclusion from these numerous reports is
that sequence comparison alone does not suffice for
genes to be designated as regulated by CDE and ⁄ or
CHR elements. We would like to derive, from the
many publications, functional requirements, sequence
similarities and characteristics of general promoter
structure for cell cycle-regulating sites to be regarded
as bona fide CDE ⁄ CHR elements (Table 2).
CDE sites represent special E2F-binding elements
and thereby display sequence similarity to these sites.
However, a requirement for functional CDEs, distin-
guishing them from E2F elements, is that they must be
positioned with a four-nucleotide spacer upstream of a

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 ⁄

cyclin-dependent
kinase 1
Serine ⁄ threonine kinase, catalytic subunit of the mitosis promoting factor (MPF),
controls G
1
⁄ S and G
2
⁄ M phase transitions
CDC25C cell division cycle 25
homolog C
Tyrosine phosphatase, triggers entry into mitosis
CKS1 CDC28 protein kinase
regulatory subunit 1
Binds to the catalytic subunit of cyclin-dependent kinases (CDK), essential for their
biological function
MKLP1 ⁄ KIF23 mitotic kinesin-like
protein 1 ⁄ kinesin
family member 23
Kinesin-like protein, motor enzyme that moves antiparallel microtubules, localizes to
the interzone of mitotic spindles
PLK polo-like kinase 1 Protein kinase, multiple function in cell cycle, activates CDC25, interacts with
anaphase-promoting complex (APC)
TOME-1 ⁄ CDCA3 trigger of mitotic entry ⁄ cell
division cycle associated 3
F-box like protein, required for degradation of the CDK1 inhibitory tyrosine kinase
WEE1, triggers entry into mitosis
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 879

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

its two-nucleotide exchange from the CHR consensus
(Fig. 2). Changing the distance between E2F and DRS
sites in reporter constructs by the insertion of two or
four nucleotides leads to derepression in G
0
cells in
reporter assays [22]. With only one nucleotide
exchange compared to the mouse sequence, the E2F
and DRS ⁄ CHR segment in the human B-Myb pro-
moter is well conserved [23]. Recently, the E2F site of
the B-Myb promoter was mutated in mice. Homozy-
gous mutation of the element was found to lead to
derepression of the B-Myb promoter in mouse embryo-
nic fibroblasts (MEFs) derived from the animals. Fur-
thermore, elevated expression of B-Myb mRNA,
indicating a deregulation, is observed in brain cells car-
rying the mutant E2F site compared with the wild-type
mice [24].
In the human Cdc25C gene, CDE and CHR cooper-
ate in cell cycle-dependent repression. They are of simi-
lar importance because their mutation leads to a
comparable derepression in the cell cycle [10,25]. We
designate such genes as class I CDE ⁄ CHR genes
(Fig. 2). Moreover, orientation of the CDE ⁄ CHR in
the general context of a promoter appears to be rele-
vant because inversion of the site in the human
Cdc25C promoter resulted in a deregulation of cell
cycle-dependent transcription. Deregulation is also
observed when the CDE alone is inverted [26].
Interestingly, regulation through the CDE ⁄ CHR is

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

Mutation of either CDE or CHR leads to a substantial
deregulation of repression in G
0
Two or three CCAAT-boxes, spaced 31–33 bp apart, which bind
heterotrimeric NF-Y proteins
NF-Y is the main activator of the genes
Class II
The same as for class I, but no functional CDE site four
nucleotides upstream of a functional CHR
Fig. 2. Experimentally validated CDE ⁄ CHR sites. Two classes of
promoters can be distinguished. Class I genes require both sites
for cell cycle-dependent repression. In contrast, class II genes do
not have a functional CDE and are only regulated through a well-
conserved CHR. Interestingly, some ortholog genes from mouse
and human, such as cyclin B2 and Cdc25C, can be members of
class I or class II depending on the species origin. The tandem ele-
ment in the mouse B-myb promoter is an E2F site in combination
with an element named the ‘downstream repression site’ (DRS or
CHR).
CDE ⁄ CHR-dependent cell cycle-gene transcription G. A. Mu
¨
ller and K. Engeland
880 FEBS Journal 277 (2010) 877–893 ª 2009 The Authors Journal compilation ª 2009 FEBS
deregulation when compared with changes in the CHR
[27]. We suggest referring to genes that have a func-
tional CHR but lack a site four nucleotides upstream
from the CHR, which, when mutated, does not lead to
any or to only a minor deregulation, as class II genes
(Fig. 2).
Furthermore, some other properties of CHR ele-

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-

3¢ [5]. As the putative CDE has, in contrast to the
CHR, only a modest impact on cell cycle-dependent
transcription, the human cyclin B1 gene is class II
(Fig. 2).
Analysis of cyclin B2 cell cycle-dependent transcrip-
tion offers some insights into the variability of CDEs
regarding sequence and function. Initially, mouse cy-
clin B2 expression was shown to be regulated by a
CDE ⁄ CHR tandem site and was therefore considered
to be a class I promoter; however, the CDE in this
promoter leads to a smaller deregulation than the
CHR when mutated [6]. By contrast, the human cyclin
B2 promoter does not require a CDE for cell cycle-
dependent transcription. Mutation of the site in the
human promoter that is equivalent to the CDE from
the mouse cyclin B2 promoter does not result in a
deregulation [7]. Therefore, human cyclin B2 is clearly
a class II gene (Fig. 2). In a comparison of nucleotide
sequences from both promoters, nine homologous
regions stand out. Only one of them, the CDE in the
mouse cyclin B2 promoter, is not perfectly conserved.
A one-nucleotide change is found in the human pro-
moter. The alteration appears to be sufficient to render
the human cyclin B2 promoter resistant to deregulation
through mutation of this region. Nevertheless, changes
in the CHR lead to a complete deregulation of expres-
sion from the human cyclin B2 promoter [7,25]. It
remains unclear why one CHR requires a CDE four
nucleotides upstream, whereas another CHR, particu-
larly in a very similar context as exemplified in the

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-

sibly through mediating protein binding to the
elements, was investigated. The CpG sites of the CDE
in the cyclin B2 promoter were found to be partially
methylated. However, quantitative methylation analy-
sis did not show any alterations during the cell cycle,
making it unlikely that protein binding to the
CDE ⁄ CHR is affected by change in DNA methylation
during different phases of cell division [34].
Another cell cycle gene, also relevant for mitosis,
codes for the serine ⁄ threonine-specific Polo-like kinase
1 protein. A mutation in the CHR deregulates cell
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 881
cycle-dependent transcription from the Plk1 promoter.
Changing a putative CDE four nucleotides upstream
from the CHR had almost no effect on the cell cycle-
dependent regulation of the promoter [35]. Therefore,
Plk1 was considered to be a class II gene (Fig. 2).
Also, Cks1, a member of the cyclin-dependent kinase
subunit family, reaches peak expression in S ⁄ G
2
phases
of the cell cycle. This expression pattern is dependent
on transcriptional repression through both a CDE and
a CHR in the Cks1 promoter [36].
Moreover, Tome-1 was reported as a CDE ⁄ CHR
gene. Tome-1 mediates destruction of the mitosis-
inhibitory kinase Wee1 via the E3 ligase SCF and

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

regions upstream from them were not tested for func-
tional E2F or CDE sites (Fig. 2).
The gene for Aurora A, a serine-threonine kinase
whose expression peaks in G
2
⁄ M, was found to be reg-
ulated by a CDE ⁄ CHR site. The CHR has the unusual
sequence 5¢-CTTAAA-3¢. In order to yield a high
sequence similarity for the CDE with sites published at
the time, the CDE and CHR were postulated to be
located next to each other without a spacer [39]. How-
ever, considering the great variability in CDE nucleo-
tide sequences, and the fact that functional assays
with just one mutant promoter could not pinpoint this
site exactly, we suggest that the site shifted upstream
by five nucleotides is the CDE. The functional data
would allow such a change in interpretation. The shift
in exact position of the CDE would yield a spacer
essential to define these elements as a CDE ⁄ CHR tan-
dem site [10]. One other member of the Aurora kinase
family was also found to be regulated by a typical
CDE ⁄ CHR site. The Aurora B promoter is controlled
by CDE and CHR sites separated by four nucleotides.
As in many similar promoters, mutation of the CHR
leads to a more pronounced deregulation than the
alteration of the CDE [40].
Another gene tested for its cell cycle regulation is
the cellular inhibitor of apoptosis protein 2 gene
(cIAP2). It is induced by nuclear factor-jB and was
shown to be expressed in a cell cycle-dependent man-

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

⁄ M. In an initial study, cell cycle-
dependent regulation of about three-fold had been
described for G
2
⁄ M expression of the wild-type pro-
moter-reporter compared with the expression level in
G
1
. One site designated a CDE led to a partial deregu-
lation when mutated. However, a putative CHR led to
a deregulation upon mutation, although it is not part of
a CDE ⁄ CHR tandem site. Furthermore, one experi-
ment suggested a strong deregulation when an upstream
Sp1 site is mutated [42]. Objections to most of these
results were raised by a later study. A stronger cell
cycle-dependent regulation of the wild-type construct
was observed than in the first study. Although numer-
ous CDEs were also postulated, the experiments finally
yielded only one functional CDE close to the CHR [43].
In this report an alignment of CDE ⁄ CHR sites is
displayed in which the CDE is moved downstream by
CDE ⁄ CHR-dependent cell cycle-gene transcription G. A. Mu
¨
ller and K. Engeland
882 FEBS Journal 277 (2010) 877–893 ª 2009 The Authors Journal compilation ª 2009 FEBS
two nucleotides to yield a better consensus with other
CDEs. However, just one mutation, with a single
nucleotide change, was analyzed. The mutant does not
dictate such a shift [43]. The picture becomes even more
complicated when considering another report on the

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

⁄ M. Recently, the regulation of the
BUB1B promoter was tested. The transcription factor
hStaf ⁄ ZNF143 was found to be the main activator of
the promoter. Furthermore, cell cycle-dependent regu-
lation depends on two sites with similarity to CHRs
and CDEs. Interestingly, in the BUB1B promoter the
CHR is located upstream of the CDE-like site [46].
These observations and the fact that activation does
not rely on CCAAT-boxes, NF-Y or Sp1 proteins,
leave open the question of whether the BUB1B pro-
moter represents a canonical CDE ⁄ CHR-regulated
promoter.
Cell cycle-dependent transcription of the human
CDC20 ⁄ p55CDC ⁄ Fizzy promoter was reported to
depend on a new element named SIRF (Cell-Cycle
Site-Regulating p55Cdc ⁄ Fizzy-Transcription). E2F
proteins are able to bind the promoter as analyzed by
chromatin immunoprecipitations and can activate tran-
scription of the promoter in transient transfection
assays through the upstream part of the SIRF element.
Mutational analysis of a putative CDE ⁄ CHR site in
the human CDC20 promoter showed that this element
has no significant impact on promoter cell cycle regu-
lation [47]. Without reference to this earlier report,
Kidokoro and coworkers postulated a CDE ⁄ CHR in a
recent paper. They observed that CDC20 expression is
downregulated when p53 is active. The mechanism was
suggested to require p21
WAF1 ⁄ CIP1
, which appears to

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-

sin-like protein RB6K was observed to be expressed
similarly to cyclin B with RB6K lagging a little
behind cyclin B expression. In the RB6K promoter a
tandem element with similarity to known CDE ⁄ CHR
sites was observed, but not assayed functionally [52].
Furthermore, numerous CDE and CHR sites were
postulated for the human, mouse and rat cyclin A
genes; however, without experimental verification
[53].
In summary, all examples described here contribute
to the definition of which sites can be regarded as
CDE ⁄ CHR sites. They also help to define class I and
class II genes. One clear conclusion from the studies is
that just scanning a promoter for CDE- or CHR-like
sequences is not sufficient to identify functional sites.
Generally, sequence alignments yield numerous hits,
among them many false positives, particularly when
considering the not-very-restrictive consensus for
CDEs. Therefore, functional analyses are required
before naming a site a CDE or a CHR. One can
conclude from the many experimentally confirmed
CDEs, that this class of sites, in contrast to the CHRs,
is much more variable in its sequence. The consensus
for a CDE may just be a site rich in G and C
found upstream of a CHR with a distance of four
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 883
nucleotides. Additionally, CDEs always require a

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].

A dominant-negative variant of the NF-YA subunit of
the heterotrimeric complex NF-Y was instrumental in
showing that the activating protein on multiple
CCAAT-boxes is indeed NF-Y [27,57].
The multiple sites synergize. Individual mutations
lead to a large drop in promoter activation, indicating
cooperation between the two or three CCAAT-boxes
of a gene [54,57]. Conspicuously, the distance between
two functionally important CCAAT-boxes is always
31, 32 or 33 bp. Comparison of nucleotide sequences
in promoters of ortholog genes from different organ-
isms shows that not only the CCAAT-boxes them-
selves, but also their distance, is conserved [7,25,58].
The particular distance with approximately three turns
of the DNA double helix yields binding of the two or
three NF-Y complexes on the same side of the DNA.
Conservation of spacing is required for optimal pro-
moter activity because changing the distance leads to a
loss of activation [58].
One reason for the specific spacing between CCAAT-
boxes may be binding of the p300 histone acetyltrans-
ferase (HAT) to NF-Y heterotrimers. Association of
NF-Y with HAT activity had been observed earlier.
A complex consisting of the three NF-Y subunits and
other proteins has been shown to possess histone ace-
tyltransferase activity through physical association
with the related GCN5 and P ⁄ CAF enzymes [59]. The
p300 HAT enzyme was observed to bind to the human
cyclin B1 promoter in vivo and is able to increase
expression from the promoter when overexpressed [5].

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

1
phases, transcription of these
cell cycle genes is repressed by a complex of inhibitory proteins at
the CDE ⁄ CHR. It was shown that E2F4 binds to the CDE and that
Lin-54 binds to the CHR in the Cdc2 promoter in G
0
. It is probable
that these proteins constitute part of the DREAM complex on
these promoters because Lin-54 is a constitutive member of
DREAM. Furthermore, in later cell cycle phases DREAM appears to
activate promoters, whereas Lin-54 may be bound to sites other
than the CHR. In order to activate in S ⁄ G
2
⁄ M phases, the composi-
tion of DREAM is altered by replacing E2F4 and p107 ⁄ p130 with
B-Myb. Because in vivo footprints provided evidence that the
CDE ⁄ CHR in G
2
⁄ M cells is devoid of proteins, but DREAM compo-
nents were detected at CDE ⁄ CHR promoters in G
0
⁄ G
1
as well as
in S ⁄ G
2
⁄ M phases, it is likely that the complex is able to bind alter-
native recognition sites outside the CDE ⁄ CHR. It still has to be
established which proteins contact DNA at which sites during late
phases of the cell cycle.

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-

vestigated whether another NF-Y-binding site at a
specific distance may be required in conjunction with
this first CCAAT-box. Furthermore, ChIP assays
demonstrated that NF-Y can bind to the Tome-1
promoter in vivo [37].
Also, the Aurora B promoter had been established
as being CDE ⁄ CHR-regulated. Similarly to other
CDE ⁄ CHR promoters, the Aurora B gene does not
contain a TATA-box, but two CCAAT-boxes with a
distance of 33 bp to each other were found upstream
of the CDE ⁄ CHR [40]. However, the two sites were
not tested functionally.
Generally, to our knowledge, all promoters contain-
ing functional CDE ⁄ CHRs that were also tested for
CCAAT-boxes were indeed found to be activated
through their CCAAT-boxes. However, many
CDE ⁄ CHR genes have not been assayed for CCAAT-
box-dependent activation. Therefore, it appears likely,
but is not proven, that CCAAT-boxes are required to
activate CDE ⁄ CHR promoters.
p53 and repression through the
CDE/CHR
Conspicuously, many CDE ⁄ CHR genes, such as Cdc2,
cyclin B1, cyclin B2 and Cdc25C, are downregulated
by the tumor suppressor p53 [27,62–66]. However,
there are also many examples of genes repressed by
p53 that are not regulated by CDE ⁄ CHR sites but
have been tested for such elements (e.g. Cdc25A and
Cks2) [67,68].
Evidence exists that DNA damage-dependent down-

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

was not confirmed by experiments [73].
The mouse cyclin B2 and human Cdc25C promoters
are downregulated by p53 [65,66]. In order to pinpoint
the site responsible for the repression, numerous cyclin
B2 promoter mutants were tested. p53-dependent
downregulation does not appear to be dependent on
the CDE and CHR sites. Also, other regions or spe-
cific sites could not be conclusively established asre-
sponsible for repression. One challenge investigating
downregulation of the cyclin B2 promoter is that after
deletion or mutation of constitutively activating sites,
such as the destruction of CCAAT-boxes, small levels
of reporter activity remain to analyze further repres-
sion through the CDE or CHR elements [25,65].
The CDE ⁄ CHR in the human Cdc25C promoter
was implicated in the p53-dependent downregulation
of the gene [74]. Initially, a potential p53-binding site
was observed in the human Cdc25C promoter, which
is able to bind p53 in electrophoretic mobility shift
assays (EMSAs). When fused to a minimal promoter,
the p53 site can function as a transcriptional activator
from a reporter construct [75]. However, Cdc25C is
repressed by p53. Also, mouse Cdc25C is downregulat-
ed by p53 but lacks the putative p53 site in its pro-
moter [27]. By contrast, downregulation needs the
CCAAT-boxes in the promoter and functional NF-Y
transcription factors [66,76]. p53-dependent repression
of the human Cdc25C promoter does not require the
putative p53 site implicated earlier [75], or the
CDE ⁄ CHR, but is lost when three CCAAT-boxes are

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

such as cyclin B1 had been demonstrated upon p53
induction. Mannefeld et al. [77] describe, in a recent
report, that in the presence of p53 the DREAM com-
plex switches from containing activating B-Myb to
repressing E2F4 ⁄ p130. Although the report does not
specify binding sites, other reports imply CDE ⁄ CHR
sites for binding of DREAM (please see the later dis-
cussion on the binding of DREAM proteins to
CDE ⁄ CHR elements). However, because for some
CDE ⁄ CHR genes, such as mouse cyclin B2 and human
Cdc25C, a function of the CDE and CHR sites in p53-
dependent downregulation appeared unlikely, it will be
of interest to establish the promoter sites to which
DREAM complex components bind to participate in
p53-dependent transcriptional repression.
Influence of viral proteins on CDE/CHR
regulation
As it had been noted that CDE ⁄ CHR sites are related
to E2F elements, it is a pertinent question whether
viral proteins disturb regulation through the tandem
element in a manner similar to viral oncoproteins
interfering with the control by E2F and pRB-related
pocket proteins. One example of a gene deregulated by
viral proteins is Cdc2. The human Cdc2 promoter is
upregulated upon the expression of simian virus 40
(SV40) T antigen. However, CCAAT-boxes were made
responsible for the transcriptional activation by the
viral protein, whereas interaction of T antigen with
p53 or pRB did not appear to be essential [78].
Similarly, expression of the SV40 T oncogene

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-

mavirus was able to deregulate transcription, which
was dependent on the CDE in the mouse cyclin A pro-
moter. The CHR was not tested separately. However,
protein complexes did not change as one would expect
when pocket proteins dissociate from the complexes,
and free E2F would remain bound to the site [81].
Proteins binding to the CDE and CHR
elements
One central question stemming already from the early
days of CDE ⁄ CHR research is which protein regula-
tors bind to this tandem site. E2F proteins were impli-
cated early on to regulate through the CDE. Analysis
and identification of factors regulating through the
CHR is particularly important because class II pro-
moters lack functional E2F ⁄ CDE elements (Fig. 2).
Preliminary reports on proteins requiring a CHR for
binding include a factor named CDF-1, which was
observed to bind to the CDE ⁄ CHR elements in the
Cdc25C and cyclin A promoters [82,83]. However,
many attempts by several groups to clone and further
characterize this factor failed. A protein called CHF
has been observed to bind by EMSA to the CHR in
the mouse cyclin A promoter. However, also this
factor was not further characterized [84].
CDE ⁄ CHR-dependent cell cycle-gene transcription G. A. Mu
¨
ller and K. Engeland
886 FEBS Journal 277 (2010) 877–893 ª 2009 The Authors Journal compilation ª 2009 FEBS
More information is available on the binding of
E2F and pocket proteins to CDE ⁄ CHR promoters.

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

of the B-Myb promoter during the cell cycle. Re-intro-
duction of p107, and to some extent also p130, causes
repression of the B-Myb promoter reporter, which is
dependent on an intact E2F site. Regulation cannot be
restored by pRB. Another important aspect related to
protein binding to the E2F site in the B-Myb promoter
is that ChIP assays on stably transfected promoter
constructs reveal that in vivo binding of p107 and p130
to the B-Myb promoter is lost when the DRS is
mutated [22]. By analogy, one is tempted to speculate
that also bona fide CDE and CHR sites cooperate in
protein binding. Similar results on the cooperative reg-
ulation of putative CHR-binding proteins with an
E2F ⁄ CDE site were observed when studying Cdc2 pro-
moter regulation. Mutation of the CHR abolished the
interaction of E2F4 with the negative-regulating proxi-
mal E2F ⁄ CDE site, as measured by ChIP with trans-
fected mutant human Cdc2-promoter plasmids [10,61].
Later, this cooperation was confirmed by Lin-54 bind-
ing to the CHR, cooperating with E2F4 binding to the
CDE in EMSAs in vitro [86].
ChIP experiments on the Cdc2 gene also provided
insights into the association of other cell cycle proteins
with the promoter. E2F4 binding during the cell cycle
coincides with the binding of p107 or p130 [61,85].
Another E2F site distal to the E2F ⁄ CDE acts as an
activating element binding E2F1, -2 and -3. These acti-
vating E2Fs cooperate with NF-Y proteins binding to
CCAAT-boxes and with Myb proteins associating with
a distal Myb site in activating the Cdc2 promoter.

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

described to bind E2F4 and p130 in G
0
and early G
1
in vivo. In late G
1
, some E2F3 binding appears in the
ChIP assays [60]. Similarly, ChIP experiments on the
cyclin A, Cdc2 and cyclin B2 genes suggest that E2F4
and E2F6 bind to the promoters in G
0
and to some
extent also in G
1
[55]. Furthermore, E2F4 binds to the
promoters of cyclin B1 and cyclin B2 in human tissues
according to ChIP, followed by identification of genes
by genome-wide DNA-microarray hybridization [87].
However, the limitation of ChIP analyses is that the
exact binding location is not identified in the experi-
ments. Therefore, it is not clear whether the CDE ⁄ CHR
is involved in binding these factors.
Consistent with a function of p107 and p130 in the
control of CDE ⁄ CHR promoters is the loss of regula-
tion observed for the expression of cyclin A, B-Myb
and Cdc2 in p107
) ⁄ )
p130
) ⁄ )
double knockout cells.

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-

ment. Importantly, p107 and p130 binding, measured
using ChIP on stably transfected promoter constructs,
is lost when the DRS ⁄ CHR is mutated [20,22]. Fur-
thermore, E2F4 binds to the CDE in the Cdc2 gene
assayed using ChIP in cells transfected with promoter-
carrying plasmids. Importantly, this association also
requires an intact CHR, indicating cooperation
between proteins binding to both sites of the tandem
element [61].
In recent years, E2F4 has been identified as a
component of the DREAM complex. The ortholog
dREAM complex was first identified in Drosophila
[89]. Human DREAM was isolated by employing the
MudPIT method with overexpression of an HA-tagged
p130 protein followed by extraction via the tagged
protein, chromatography and identification by MS
[90,91]. In another approach the complex was affinity-
purified, employing a stably expressed flag-tagged
Lin-37 protein followed by MS. Composition of the
protein complex was later confirmed by purifying the
endogenous components by classical chromatography
monitored through Lin-37 western blotting [92]. The
complex contains mammalian homologs of Caenor-
habditis elegans synMuvB proteins and is composed of
p130, E2F4 ⁄ 5 and DP1 ⁄ 2 and a module containing the
MuvB proteins Lin-37, Lin-52, Lin-54 and chromatin-
associated Lin-9 and Lin-53 ⁄ RBBP4 ⁄ RbAp48.
Furthermore, A-Myb and B-Myb were present in
immunoprecipitations for Lin-9, Lin-37 and Lin-54
[90,92]. Composition of the DREAM complex changes

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

cells when p130, Lin-9, Lin-37 or Lin-54
are stably overexpressed.
In contrast to a possible function in cell cycle-depen-
dent repression mediated by CDE ⁄ CHR sites in the G
0
phase, Lin proteins contribute to activation of
CDE ⁄ CHR promoters in cycling cells. It was shown
that Lin-9 activates the cyclin A and cyclin B1 promot-
ers [94]. Interestingly, functional mapping of the cyclin
B1 promoter did not yield the class II CHR as the site
through which Lin-9 exerts its control [94]. In another
report, depletion of Lin-9, Lin-37, Lin-52 or Lin-54 by
RNAi also led to a repression of genes, such as cyclin
A and cyclin B1, in dividing cells [92]. Furthermore,
depletion of Lin-9 and B-Myb in p53
) ⁄ )
MEFs caused
lowered expression of cyclin A, cyclin B and Cdc2 [94].
In similar experiments, knockdown of Lin-9 or Lin-54
was responsible for strong shifts in cell cycle distribu-
tion. A large cell fraction is found in G
2
or appears to
have an 8n DNA content. Many cell cycle-regulated
genes are downregulated upon Lin-9 knockdown (e.g.
Polo-like kinase, Aurora kinase B, cyclin A, Cdc2, cyclin
B1 and cyclin B2) [93,96]. The shift in cell cycle-distri-
bution towards the later phases would favor elevated
expression of these genes. Without this effect, down-
regulation would be expected to be even lower, whereas

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

sequence appears too frequently in promoters to
clearly indicate a CHR with a role in cell cycle-depen-
dent transcriptional repression (Table 2). Therefore,
this site has to be confirmed in each case by functional
assays. CDEs follow a loose consensus similar to that
of E2F sites. What distinguishes CDEs from E2F ele-
ments? CDEs must always be located with a four-
nucleotide spacer upstream of a CHR. Both CDEs
and CHRs control cell cycle-dependent transcription
as repressor sites in G
0
cells. Classic E2F sites do not
appear next to CHR elements and can either activate
or repress transcription. In the genes analyzed to date,
any promoter appears to hold only one CDE ⁄ CHR
pair with the CDE upstream of the CHR. CDE ⁄ CHR
promoters employ multiple start sites, do not bear
functional TATA-boxes and are usually activated by
two or three CCAAT-boxes with certain spacing. Do
E2F proteins regulate CDE ⁄ CHR promoters? There is
considerable evidence that E2F4 is involved in their
control through the CDEs (Fig. 3).
Central questions still have to be addressed. Are
all CDE ⁄ CHR promoters regulated by the same pro-
tein complex? Will changes in the sequence of a
CDE ⁄ CHR tandem site lead to a recruitment of dif-
ferent proteins? Are class I and class II promoters
bound by different protein complexes? Why are some
promoters class I in mouse and class II in humans,
and vice versa, as demonstrated for cyclin B genes?

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.).
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