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.. Since the discovery of the first three gene
Trang 1The 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 G2phase 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: engeland@medizin.uni-leipzig.de
(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 G2 phase and in mitosis Promoters of these genes are repressed by pro-teins binding to CDE⁄ CHR elements in G0 and G1 phases Relief from repression begins in S phase and continues into G2phase 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.
Trang 2mostly 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 G0
cells The Cdc25C gene is not expressed in resting cells
or in cells in G1 phase Only in G2 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 G1 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 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 G0cells [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.
Trang 3promoters 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, G2 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 Apromoter 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.
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 G1⁄ S and G 2 ⁄ M
transitions CCNB1 cyclin B1 Regulatory subunit of mitosis promoting factor (MPF), regulates G2⁄ 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
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
Trang 4the 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 G0 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 G0 and
G1 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 G0 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 Cdc25Cpromoter-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, G2or 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 G0
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).
Trang 5deregulation 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 G2 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 B2promoters, 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
Trang 6cycle-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⁄ G2phases
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 G2 (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 G1 and strongly expressed in G2⁄ 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-depeninterleukin-2-depen-dent
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 G2⁄ 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 G1 and reaching peak lev-els in G2⁄ 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
IIregulatory 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 G2⁄ M In an initial study, cell cycle-dependent regulation of about three-fold had been described for G2⁄ M expression of the wild-type pro-moter-reporter compared with the expression level in
G1 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
Trang 7two 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 G2⁄ M than in G1 [44] This contradicts
repression through a complex in G1 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 G1,
but peaks in G2⁄ 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 p21WAF1⁄ 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] p21WAF1 ⁄ 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
Trang 8nucleotides 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 G0 and
G1 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 G0, proteins appear to bind to the CDE ⁄ CHR, as monitored by in vivo footprinting Accord-ing to these early experiments all bindAccord-ing 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 G0and G1 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.
Trang 9reduces 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 G1, 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 p21WAF1 ⁄ 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 p21WAF1⁄ CIP1 was also postulated to be con-trolled through CDE and CHR elements [70] Experiments confirmed that p53 and p21WAF1⁄ 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 p21WAF1⁄ CIP1 Like the Plk1 gene, topoisomerase IIa was presented as downregulated through CDE⁄ CHR sites upon p21WAF1⁄ 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
Trang 10deleted [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 G0 and G1 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
G0⁄ G1 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]