Cyclin E
Cyclin E is an essential regulator of S phase entry. Transcriptional regulation of Drosophila Cyclin E plays
an important role in the control of the G1 to S phase
transition during development. The first
comprehensive analysis of the transcriptional regulation
of a G1 phase cell cycle regulatory gene during
embryogenesis is presented here. Analysis of deficiencies, a genomic
transformant and reporter gene constructs all reveal
that Cyclin E transcription is controlled by a large and
complex cis-regulatory region containing tissue- and stage-specific
components. Separate regulatory elements for
transcription in epidermal cells during cell cycles 14-16,
central nervous system cells and peripheral nervous system
cells were found. An additional cis-regulatory element
drives transcription in thoracic epidermal cells that
undergo a 17th cell cycle when other epidermal cells have
arrested in G1 phase prior to terminal differentiation. The
complexity of Cyclin E transcriptional regulation argues
against a model in which Cyclin E transcription is regulated
simply and solely by G1 to S phase transcription regulators
such as RB, E2F and DP. Rather, this study demonstrates
that tissue-specific transcriptional regulatory mechanisms
are important components of the control of cyclin E
transcription and thus of cell proliferation in metazoans (Jones, 2000).
This analysis has revealed the presence of multiple
independent tissue-specific regulatory elements acting to
control DmcycE transcription during embryogenesis. These
include: (1) at least two different elements required for
expression in different cells of the peripheral nervous system;
(2) an element required for expression in the epidermal cells
and central nervous system, and (3) an element required for
expression in patches of thoracic epidermal cells that undergo
a 17th G1 phase-regulated cycle, in the PNS of the labial and
maxillary segments and in the posterior spiracle primordia.
These elements were identified by the presence or absence of
tissue-specific expression in a genomic transformant, in
animals carrying small regulatory region deletions generated
by P-element excisions and in genomic fragment-reporter gene
constructs. All these regions are detected upstream of the zygotic transcript, within the large region coding for three 5' exons and large introns of the maternal transcript. The results obtained with each of these different
approaches are consistent with the others in terms of the
location of the different elements. Members of one notable group of
elements that were not fully defined in this study are those
responsible for Cyclin E expression in endoreplicating tissues.
Only one element was identified that is responsible for
driving Cyclin E transcription in the central midgut.
Presumably elements responsible for regulating the remaining
patterns of DmcycE in embryonic endoreplicating tissues lie
further 5' or 3' of the genomic regions examined in this study.
The failure to define more than the one endoreplicative
regulatory sequence is surprising. It is possible that other
regulatory sequences lie outside the region studied, but it is also
possible that identification of these domains may be hampered
by the negative autoregulatory nature of Cyclin E expression in
these endoreplication domains. If the elements carrying the endoreplication
regulatory sequences also carry the autorepression sequences,
expression may be inhibited by the endogenous Cyclin E. It
should also be noted that additional complexity is likely to exist
in the regulation of Cyclin E transcription. For example,
Cyclin E was expressed in a subset of the epidermal thoracic
patch cells in Cyclin E 19L deficient embryos, indicating that
these patches may themselves be complex domains of Cyclin E
transcriptional regulation (Jones, 2000).
Although these analyses have identified separable tissue-specific
cis-acting elements in the Cyclin E regulatory region,
the factors that operate on these
elements to drive Cyclin E transcription remain to be elucidated. Some clues to the
trans-acting factors that could be regulating Cyclin E
transcription come from studies of cell cycle control
mechanisms in mammalian cells, where cyclin E
transcriptional regulation also plays an important role in
control of the G1 to S phase transition. A well-established model of
G1 phase transcriptional regulation in mammalian cells
postulates a cascade of events initiated by extracellular growth
factor signaling that leads to activation of the Cyclin D/Cdk4
complex, which in turn phosphorylates the tumor suppressor
Retinoblastoma (Rb), disassociating it from the S phase-specific
transcription factors E2F and DP and allowing
transcription of S phase-specific genes, such as cyclin E (Jones, 2000).
In Drosophila, where
G1 phase-regulatory events can be studied within a developmental context,
E2F and DP are required for embryonic Cyclin E
transcription in endoreplicating tissues, but are dispensable for
Cyclin E transcription in the CNS divisions that lack G1 and
G2 phases. It is not known whether E2F and DP
are required for Cyclin E expression in the early embryonic
PNS and epidermal divisions, because maternal products of
these genes may mask any requirement. Nonetheless, the fact
that these transcription factors are dispensable for Cyclin E
transcription in the CNS cells argues against the universality
of the mammalian model of cyclin E regulation by E2F. In
addition, the mammalian model predicts that E2F-dependent
transcriptional regulation of Cyclin E in endoreplicating tissues
should be mediated through an E2F/DP responsive element. In
the experiments described here, this model
is shown to be an oversimplification of Cyclin E regulation in Drosophila,
as only one element in the 16 kb analysed was found to drive
Cyclin E expression in a subset of endoreplicating tissues. Thus
a minimum of two regulatory elements are required for driving
Cyclin E expression in endoreplicating tissues. The fact that
expression of Cyclin E in these tissues has been shown to be
dependent on the E2F/DP complex appears at first sight to be
paradoxical. The paradox would be resolved if expression
requires activation by both E2F/DP and tissue-specific
activators. An absolute requirement for a developmental signal
cannot however exist, as ectopic expression of E2F and DP
together induces Cyclin E expression in all G1-arrested
epidermal cells. It remains possible that
the high levels of E2F and DP expressed following heat shock
induction of the respective transgenes override the tissue-specific
regulatory component (Jones, 2000).
Downregulation of Cyclin E transcription during cycle 16 is
essential for cycle 17 G1 arrest in epidermal cells prior to
differentiation. Significantly, the
regulatory element that drives constitutive Cyclin E expression
in the epidermal cells during cycles 14-16 shows
transcriptional downregulation, characteristic of wild-type
Cyclin E expression. If this downregulation requires active
repression of Cyclin E transcription, then the regulatory
sequences necessary for this repression must also be located in
the 1.0 kb regulatory element defined in this study. Alternatively, the
downregulation could be a consequence of the loss of
activation of Cyclin E transcription in the epidermis.
Cyclin E is known to be ectopically expressed in a subset of normally G1-arrested
epidermal cells in embryos that are deficient for RBF
(the Drosophila Rb homolog). However, Cyclin E transcription
is initially downregulated normally and a G1 cycle 17 arrest is
established in the absence of RBF. These data suggest that
Cyclin E is actively repressed in G1-arrested epidermal cells
and that RBF is required to maintain this repression. Although
dE2F or dDP do not result in ectopic Cyclin E transcription in
epidermal cells, a RBF/E2F/DP complex may still
mediate this repression if maternal E2F and DP are not
depleted in the respective mutant embryos at this stage. A
transcriptional repression mechanism of Cyclin E at this stage
may also be mediated by the second Drosophila E2F, E2F2,
which has been shown to act as a transcriptional repressor of
S phase genes in tissue culture cells.
Alternatively, E2F and/or DP may be needed to activate ectopic
Cyclin E expression in the absence of RBF. The initial
mechanism acting to downregulate Cyclin E transcription
remains to be determined. Further dissection of this regulatory
element may identify separate activation and repression
sequences (Jones, 2000).
The regulators of DmcycE transcription in other
developmental contexts may be more difficult to identify, since
many candidate genes may be involved. In some cases it is
possible to suggest the involvement of particular regulatory
genes. For example, the discrete expression of DmcycE in the
epidermal thoracic patches, which undergo a G1-regulated 17th
cell cycle, is much stronger in the first thoracic segment and is
absent in the abdominal segments. The products of the
homeotic genes of the Bithorax and Antennapedia Complexes,
which are key components of anterior/posterior patterning
are therefore candidate upstream regulators of Cyclin E
transcription in this tissue (Jones, 2000).
Understanding of cell cycle regulation has primarily
derived from the single cell yeast, from cultured cells and from
oocytes or very early embryos in which the complex patterning
of embryogenesis has not yet begun. In contrast, cell cycles in
the embryos of a multicellular organism respond to a variety
of developmental cues that give different tissue types and
different cell cycle kinetics. The significance of the relationship
between embryo patterning and cell cycle control is evident
from the pioneering work that has shown that regulation of the cycles that occur during gastrulation and germ band extension in Drosophila embryos is
mediated by the transcriptional regulation of the string
mitotic activator gene. This transcriptional regulation is
mediated by the patterning genes active during these stages.
The stg mitotic inducer gene exhibits a remarkably complex
transcriptional regulatory region that responds to a variety of
patterning genes to control cell cycle progression during early
embryogenesis. The
analysis of Cyclin E regulation offers an
interesting parallel with stg in that both genes exhibit
unexpected complexity in transcriptional regulation. It is
striking that both Cyclin E and stg, the first cell cycle regulators
to have their transcriptional regulation examined in a
developmental context, contain tissue-specific regulatory
elements. This indicates that transcriptional regulation is
mediated differently in distinct developmental contexts, such
as during the complex events of gastrulation when cycles 14-16 occur or during the 17th cycle in cells of the epidermal
thoracic patches (Jones, 2000).
Why is DmcycE transcriptional regulation so complex? One
clue comes from the observation that the G1 regulators
cyclin D, cyclin E, Rb and E2F/DP are highly conserved
between insects and mammals, but have no specific orthologs
in yeast. Perhaps these genes represent a
specialized mechanism that evolved to deal with G1 phase
regulation in multicellular organisms. Complex transcriptional
regulation of cell cycle genes such as Cyclin E may be part of
such a mechanism, which permitted the universal eukaryotic
cell cycle regulatory genes to be brought under the influence
of the more recently evolved transcriptional regulatory
mechanisms operating in different tissues and at different
developmental stages (Jones, 2000).
Cell proliferation and cell type specification are coordinately regulated during normal development. Cyclin E, a key G1/S cell cycle regulator, is regulated by multiple tissue-specific enhancers resulting in dynamic expression during Drosophila development. This study further characterized the enhancer that regulates cyclin E expression in the developing peripheral nervous system (PNS) and shows that multiple sequence elements are required for the full cyclin E PNS enhancer activity. Wg signaling is important for the expression of cyclin E in the sensory organ precursor (SOP) cells through two conserved TCF binding sites. Blocking Wg signaling does not completely block SOP cell formation but does completely block SOP cell proliferation as well as the subsequent differentiation (Deb, 2008).
The results reveal that cyclin E expression in developing PNS precursor cells is regulated by a large enhancer containing multiple sequence elements, including two TCF-binding sites that mediate the regulation by Wg signaling. While these TCF-binding elements are essential for the activity of the PNS enhancer, proximal and distal elements in the 4.6-PNS sequence appear to be important for full activity. The importance of Wg in the regulation of the PNS expression of cyclin E is supported by the fact that wg mutant embryos displayed decreased cyclin E expression in the developing PNS cells. This reduction in cyclin E expression in wg mutant embryos was accompanied by an inhibition of BrdU incorporation in the developing PNS, and an inhibition of the determination of the Pros and Elav expression cells in the developing PNS. It is possible that the block in differentiation into the Pros and Elav positive cells is a consequence of the inhibition of cyclin E expression or perturbations to the cell proliferation. However it is also possible that the observed differentiation block in PNS is due to a function of Wg that is independent of PNS cell proliferation. Further studies will be needed to resolve this issue (Deb, 2008).
In addition to wg, a number of other mutations such as achaete/scute (ac/sc) complex and da have also been reported to block PNS precursor proliferation and affect the expression of several cell cycle genes. Ac/Sc complex proteins and Da are bHLH proteins that are important in all aspects of es-PNS precursor differentiation while bHLH protein Atonal (ato) and Da are required for all aspects of ch-PNS precursor development. Recent studies of the expression of the Cdk inhibitor Dap during cell type specification revealed that Dap expression is directly regulated by the same developmental mechanisms that control the differentiation of these cell types. Therefore it will be interesting to test if bHLH proteins such as Da also directly regulate cyclin E expression in the developing PNS cells (Deb, 2008).
cyclin E expression at G1-S requires E2F, activation of E2F without Cyclin E is not sufficient for the induction of S phase. Early in G1, ectopic expression of Cyclin E alone can bypass E2F and induce S phase. Cyclin E is the downstream gene that couples E2F activity to G1 control. However it appears that S phase has a requirement for Cyclin E that is independent of transcription. However, not all embryonic cycles are similarly coupled to E2F activation. The rapidly proliferating CNS cells that exhibit no obvious G1 express Cyclin E constitutively and independently of E2F. Instead, Cyclin E expression activates E2F in the CNS. Thus, this tissue-specific E2F-independent transcription of Cyclin E reverses the hierarchical relationship between Cyclin E and E2F. Both hierarchies activate expression of the full complement of replication functions controlled by E2F with this caveat: whereas inactivation of E2F can produce a G1 when Cyclin E is downstream of E2F, an E2F-independent source of Cyclin E eliminates G1 (Duronio, 1995b).
The E2F transcription factor, a heterodimer of E2F and DP subunits, is capable of driving the G1-S
transition of the cell cycle. However, mice in which the E2F-1 gene has been disrupted develop
tumors, suggesting a negative role for E2F in controlling cell proliferation in some tissues. The
consequences of disrupting the DP genes have not been reported. A screen was carried out for mutations that
disrupt G1-S transcription late in Drosophila embryogenesis and five mutations in the dDP
gene were identified. Sequencing of dDP reveals the presence of
several important motifs, including the DNA-binding region, the DEF box that is predicted to be
required for DP/E2F heterodimerization, and three other highly homologous regions named
DP-conserved box 1 (DCB1), DCB2, and negatively charged box (NCB). Although mutations in dDP or dE2F nearly eliminate E2F-dependent G1-S transcription, S-phase
still occurs. Cyclin E has been shown to be essential for S-phase in late embryogenesis, but in dDP and
dE2F mutants the peaks of G1-S transcription of cyclin E are missing. Thus, greatly reduced levels of
cyclin E transcript suffice for DNA replication until late in development. Both dDP and dE2F are
necessary for viability, and mutations in the genes cause lethality at the late larval/pupal stage. The
mutant phenotypes reveal that both genes promote progression of the cell cycle (Royzman, 1997).
The striking observation from
the Drosophila dDP and dE2F mutants is that although cyclic transcription of cyclin E, PCNA, and
ribonucleotide reductase 2 (RNR2) is not detectable, S phase still occurs. Although the possibility that cyclic
transcription of these genes occurs at a low level driven by maternal pools of dDP and dE2F cannot be excluded, the
bursts of transcription that normally precede S phase are not essential for the G1-S transition. In these
mutants the cell cycle may be driven by basal levels of transcripts and post-transcriptional regulation.
The maternal pools of components of the replication machinery can persist until late in development, as
evidenced by the fact that mutations in PCNA and MCM2 cause late larval lethality (Royzman, 1997 and references).
E2F is a heterogenous transcription factor and its role in cell
cycle control results from the integrated activities of many different
E2F family members. Unlike mammalian cells, which have a large number of
E2F-related genes, the Drosophila genome encodes just two E2F
genes, de2f1 and E2F transcription factor 2 (de2f2). de2f1
and de2f2 provide different elements of E2F regulation, and
they have opposing functions during Drosophila< development.
dE2F1 and dE2F2 both heterodimerize with dDP and bind to the promoters
of E2F-regulated genes in vivo. dE2F1 is a potent activator of
transcription, and the loss of de2f1 results in the reduced
expression of E2F-regulated genes. In contrast, dE2F2 represses the
transcription of E2F reporters and the loss of de2f2 function
results in increased and expanded patterns of gene expression. The loss
of de2f1 function has previously been reported to compromise
cell proliferation. de2f1 mutant embryos have reduced
expression of E2F-regulated genes, low levels of DNA synthesis, and
hatch to give slow-growing larvae. These defects are due
in large part to the unchecked activity of dE2F2, since they can be
suppressed by mutation of de2f2. Examination of eye discs from
de2f1;de2f2 double-mutant animals reveals that relatively normal patterns of DNA synthesis can occur in the absence of
both E2F proteins. This study shows how repressor and activator E2Fs
are used to pattern transcription and how the net effect of E2F on cell
proliferation results from the interplay between two types of E2F
complexes that have antagonistic functions (Frolov, 2001).
The relatively normal patterns of cell proliferation in
de2f1;de2f2 mutants are, at first glance, difficult
to reconcile with the idea that E2F is a critical regulator of gene
expression and cell proliferation. The expression
of de2f1-regulated genes was examined in de2f2 and de2f1;
de2f2-mutant animals. Third instar eye discs were chosen for
this analysis to avoid the possible contribution of maternally supplied
products. Because Cyclin E is one of the best-known targets of E2F and
is rate limiting for S-phase entry the pattern of Cyclin E transcription was examined in the de2f2;de2f1 double-mutant animals. The normal pattern of Cyclin E expression is not altered in de2f2 mutant discs and a similar pattern is also evident in de2f2;de2f1 double
mutants. However in the absence of both dE2F1 and dE2F2, the variations
in Cyclin E expression are reduced and the pattern of
expression is less distinct. Northern analysis shows that
the steady-state level of Cyclin E transcripts is not
decreased in the absence of E2F proteins. These results are
consistent with evidence that de2f1 contributes to the pattern
of Cyclin E expression but is not required for Cyclin E transcription. The finding that E2F target genes are expressed in de2f2;de2f1 double mutants may explain, at least in part, why normal cell proliferation is possible in the absence of E2F proteins (Frolov, 2001).
Neurons and glia are often derived from common multipotent stem cells. In Drosophila, neural identity appears to be the default fate of these precursors. Stem cells that generate either neurons or glia transiently express neural stem
cell-specific markers. Further development as glia requires the activation of glial-specific regulators. However, this
must be accompanied by simultaneous repression of the alternate neural fate. The Drosophila transcriptional repressor Tramtrack is a key repressor of neuronal fates. It is expressed at high levels in all mature
glia of the embryonic central nervous system. Analysis of the temporal profile of Tramtrack expression in glia shows that it follows that of existing
glial markers. When expressed ectopically before neural stem cell formation, Tramtrack represses the neural stem cell-specific genes asense and
deadpan. Surprisingly, Tramtrack protein levels oscillate in a cell cycle-dependent manner in proliferating glia, with expression dropping before
replication, but re-initiating after S phase. Overexpression of Tramtrack blocks glial development by inhibiting S-phase and repressing expression of
the S-phase cyclin, cyclin E. Conversely, in tramtrack mutant embryos, glia are disrupted and undergo additional rounds of replication. It is proposed that
Tramtrack ensures stable mature glial identity by both repressing neuroblast-specific genes and controlling glial cell proliferation (Badenhorst, 2001).
The absence of Ttk69 from replicating glia implies that ectopic expression of Ttk69 may block normal glial development by inhibiting cell cycle progression. Whether ectopic expression of Ttk69 blocks entry into S-phase was examined. BrdU incorporation is inhibited by ectopic expression of Ttk69 using the Kr-Gal4 driver. In Ttk69-expressing segments of a Stage 10 embryo, the normal BrdU incorporation in the ventral neuroectoderm is inhibited. In the embryonic nervous system, entry into S phase is driven by bursts of transcription of S-phase cyclins -- specifically cyclin E. Heat-shock induced overexpression of Ttk69 blocks zygotic transcription of cyclin E. At earlier stages, maternally deposited cyclin E transcripts are unaffected by Ttk69 overexpression, indicating that Ttk69 affects transcript synthesis rather than stability, consistent with its role as a transcription repressor. Ectopic expression of Ttk69 was induced by a 1 hour heat-shock, after which embryos were processed immediately for in situ hybridization. The rapidity of repression of cyclin E and the presence of multiple Ttk69 consensus recognition sites in the cyclin E promoters both suggest that repression is direct (Badenhorst, 2001).
It is clear from the current study that, at least in some glial populations, Ttk69 has the ability to regulate proliferation. In the Drosophila glioblasts Ttk69 is expressed at low levels soon after glial specification, blocking neural genes. This expression is not constant, though, but oscillates during the cell cycle. Significantly, Ttk69 can not be detected when glia enter S phase and commence DNA replication. Like neuroblasts, glioblasts appear to delaminate in G2 of the cell cycle. The timing of BrdU incorporation indicates that immediately after mitosis they enter S phase and undergo DNA replication. Although Ttk69 is expressed in glia in G2, Ttk69 is not detected in glia that incorporate BrdU. Since Ttk69 can repress cyclin E expression, the absence of Ttk69 allows replication to occur. Once replication is complete, Ttk69 is expressed again. The BrdU incorporation experiments indicate that the LGB undergoes three synchronous cell divisions to produce eight longitudinal glia. This agrees well with a recent estimate of between 7-9 longitudinal glia obtained by DiI labeling of the longitudinal glioblast. After the third mitosis, longitudinal glia do not undergo replication but instead express higher levels of Ttk. By inhibiting cyclin E, high levels of Ttk69 would keep glia in G1 of the cell cycle. Similarly, differentiation of oligodendrocytes and Müller glia is accompanied by high levels of the cyclin-dependent kinase inhibitor p27, blocking re-entry into the cell cycle (Badenhorst, 2001).
Cell proliferation in the developing renal tubules of Drosophila is strikingly patterned, occurring in two phases to
generate a consistent number of tubule cells. The later phase of cell division is promoted by EGF receptor signaling
from a specialized subset of tubule cells, the tip cells, which express the protease Rhomboid and are thus able to
secrete the EGF ligand, Spitz. The response to EGF signaling, and in consequence cell division, is patterned by the specification of a second cell type in the tubules. These cells are primed to respond to EGF signaling
by the transcription of two pathway effectors, PointedP2, which is phosphorylated on pathway activation, and Seven up. While expression of
pointedP2 is induced by Wingless signaling, seven up is initiated in a subset of the PointedP2 cells through the activity of the proneural genes. To understand how the proneural and neurogenic genes pattern the response to EGFR activation, the expression of genes involved in transduction of the pathway was analyzed. The orphan nuclear-receptor svp functions downstream of the EGF receptor to promote cell divisions in the tubules. In the absence of Svp function, cycE and stg transcription is abolished, with a consequent reduction in EGFR-driven cell divisions. These late divisions in the tubules of stage 12 wild-type embryos were followed and it was found that BrdU incorporation (and hence, cell division) is confined within the svp-lacZ domain. These results define the svp domain of expression as including those cells which will divide in response to Egfr activation. However, the expression of svp-lacZ is initiated in a group of cells surrounding the tip mother cell, before the birth of the TC. This early onset of svp expression occurs before the late divisions start (cycle 17 onwards), when neither Svp function nor Egfr activation is required for cell proliferation. The pattern of gene expression observed suggests that the Svp-positive cells surrounding the tip mother cell derive from the proneural cluster (Sudarsan, 2002).
So far, relatively few mechanisms have been shown to be capable of regulating both cell proliferation and cell death in a coordinated manner. In a screen for Drosophila mutations that result in tissue overgrowth, salvador (sav), a gene that promotes both cell cycle exit and cell death was identified. Elevated Cyclin E and DIAP1 levels are found in mutant cells, resulting in delayed cell cycle exit and impaired apoptosis. Salvador contains two WW domains and binds to the Warts protein kinase. The human ortholog of salvador (hWW45) is mutated in several cancer cell lines. Thus, salvador restricts cell numbers in vivo by functioning as a dual regulator of cell proliferation and apoptosis (Tapon, 2002).
Elevated levels of Cyclin E protein are found in the basal nuclei of sav clones posterior to the MF. These are the nuclei of the undifferentiated cells that continue to proliferate in sav clones. Such discs were examined for levels of cyclin E RNA. When sav clones are generated using eyFLP, a large proportion of cells in third instar discs are mutant, and these discs contain large patches of mutant tissue. In wild-type discs, cyclin E RNA is expressed in a narrow stripe immediately posterior to the morphogenetic furrow. In discs containing sav clones, the stripe of expression is broader and more intense, indicating that cyclin E RNA levels are elevated in these discs. Thus, the increased level of Cyclin E protein is likely to result, at least in part, from an increase in cyclin E RNA levels (Tapon, 2002).
Although mutations that activate the Hedgehog (Hh) signaling pathway have been linked to several types of cancer, the molecular and cellular basis of Hh's ability to induce tumor formation is not well understood. A mutation in patched (ptc), an inhibitor of Hh signaling, was identified in a genetic screen for regulators of the Retinoblastoma (Rb) pathway in Drosophila. Hh signaling promotes transcription of Cyclin E and Cyclin D, two inhibitors of Rb, and principal regulators of the cell cycle during development in Drosophila. Upregulation of Cyclin E expression, accomplished through binding of Cubitus interruptus (Ci) to the Cyclin E promoter, mediates the ability of Hh to induce DNA replication. Upregulation of Cyclin D expression by Hh mediates the distinct ability of Hh to promote cellular growth. The discovery of a direct connection between Hh signaling and principal cell-cycle regulators provides insight into the mechanism by which deregulated Hh signaling promotes tumor formation (Duman-Scheel, 2002).
During eye development in Drosophila, initiation of neural differentiation, marked by an indentation referred to as the morphogenetic furrow, begins at the posterior end of the disc and passes anteriorly. Cells within the furrow arrest in G1 phase before differentiating. Cells located just posterior to the furrow exit G1 arrest and enter a synchronous S phase referred to as the second mitotic wave. Overexpression of the Drosophila Retinoblastoma-family gene (Rbf), an inhibitor of the S phase promoting transcription factor E2F, produces a 'rough' adult eye phenotype, characterized by loss of bristles and fusion of ommatidia. This phenotype results from delay of S phase progression in cells of the second mitotic wave as a consequence of inhibited E2F target gene expression. Loss of one copy of the ptc gene suppresses this rough eye phenotype and restores E2F target gene expression. The observed genetic interaction between Rbf and ptc suggests that the Hh signaling pathway might regulate the cell cycle during eye development (Duman-Scheel, 2002).
Hh is secreted from differentiating neurons located just posterior to cells entering S phase in the second mitotic wave. This expression pattern is consistent with the idea that reception of the Hh signal might be required for S phase entry in the second mitotic wave. To test this hypothesis, the effect of blocking Hh signaling during eye development was assessed. Cells with a mutated smoothened (smo) gene cannot respond to the Hh signal and fail to enter S phase in the second mitotic wave. Conversely, when Ci, the transcription factor that mediates Hh signaling, is overexpressed in the furrow, cells normally arrested in G1 enter S phase. Ectopic expression of Ci can also promote S phase in G1-arrested cells located in the wing margin and in the brain. Thus, Ci can induce S phase in a variety of tissues (Duman-Scheel, 2002).
Cyclin E is a principal regulator of S phase during Drosophila development. Although Cyclin E is an inhibitor of Rb, it also has additional Rb/E2F-independent cell-cycle roles. The initiation of high levels of Cyclin E expression in cells of the second mitotic wave that is located just anterior to neurons secreting Hh protein, is consistent with the idea that Hh signaling may regulate Cyclin E expression in the eye. Indeed, loss of smo results in reduction of Cyclin E levels in cells entering the second mitotic wave. Conversely, when Ci is overexpressed in furrow cells, high levels of Cyclin E transcript and protein can be detected within these cells. In the eye, the ability of Ci to induce high levels of Cyclin E expression is limited to the furrow region, where it is capable of inducing S phase. Overexpression of Ci is also capable of inducing high levels of Cyclin E transcript and protein expression in the wing. Although Cyclin E is an E2F target gene, the ability of Ci to promote Cyclin E expression in the presence of RBF-280 indicates that Hh can induce Cyclin E expression independently of E2F. Therefore, in addition to promoting expression of Cyclin D and activating E2F, Hh signaling also promotes expression of Cyclin E independently of E2F. The ability of the Cyclin E-dependent kinase inhibitor Dacapo (Dap) to inhibit the ability of Ci to induce S phase in the furrow and wing margin indicates that Cyclin E is the principal mediator of the ability of Hh to promote S phase (Duman-Scheel, 2002).
To investigate the mechanism by which Hh signaling induces Cyclin E transcription, the Cyclin E promoter was examined. Several sequences with homology to the consensus Ci-binding site were identified within the 5' regulatory region of Cyclin E. Ci was found to bind to three Ci-binding sites (A, B and C in gel shift competition experiments). To examine whether this interaction occurs in vivo, chromatin immunoprecipitation (ChIP) experiments were carried out. These assays demonstrate that Ci-binding sites A-C are occupied by Ci protein in vivo. Several lines of evidence indicate that the observed ability of Ci to bind to the Cyclin E promoter is important for regulation of Cyclin E expression in the developing eye. First, normal upregulation of Cyclin E expression in the second mitotic wave is disrupted in Cyclin EJP mutant flies, which bear a P element inserted adjacent to Ci-binding sites A-C. Also, flies carrying the 16.4 Cyclin E lacZ13 reporter, which contains all three Ci-binding sites, show upregulation of ß-galactosidase (ß-gal) expression in the second mitotic wave; this pattern resembles the endogenous pattern of Cyclin E expression. By contrast, upregulated ß-gal levels in the second mitotic wave are not observed in flies carrying reporter 13.2 Cyclin E lacZ13, which lacks Ci-binding sites A and B. Furthermore, overexpression of Ci in the furrow can drive ectopic ß-gal expression from reporter 16.4, but not from reporter 13.2. These experiments suggest that the presence of sites A and B is required for normal Hh-mediated upregulation of Cyclin E expression in the second mitotic wave. Taken together, these results provide strong evidence that Hh signaling promotes S phase through direct induction of Cyclin E expression by the transcription factor Ci (Duman-Scheel, 2002).
The investigation demonstrates that Hh signaling has a distinct ability to promote cellular growth, which is mediated by Cyclin D. In addition, Hh signaling can induce proliferation during development by promoting expression of Cyclin D and Cyclin E. This study reveals a direct connection between Hh signaling and induction of Cyclin E expression, which is accomplished through binding of Ci to the Cyclin E promoter. Upregulation of murine cyclin D1, D2 and E in response to Hh signaling has been observed. It is therefore likely that the mechanism for Cyclin E induction by Hh described here is conserved in mammals. Furthermore, because both overexpression of Ptc-1 or mutation of cyclin D1 produces a small mouse phenotype, it is likely that the ability of Hh to promote cellular growth through upregulation of D-type cyclins is also conserved in mice. Thus, constitutive Hh signaling (which promotes deregulated expression of G1/S cyclins that have been associated with diverse forms of human cancer) would promote both cell proliferation and growth in tumors. In contrast, during development, cell growth and proliferation must be carefully regulated and coordinated with the processes of cell patterning and differentiation. These same processes are also regulated by Hh signaling. This delicate balance is probably maintained by tight control of the temporal and spatial expression patterns of Hh targets and the molecules that regulate them (Duman-Scheel, 2002).
Cancer is a multistep process involving cooperation between oncogenic or tumor suppressor mutations and interactions between the tumor and surrounding normal tissue. This study is the first description of cooperative tumorigenesis in Drosophila, and uses a system that mimics the development of tumors in mammals. The MARCM system was used to generate mutant clones of the apical-basal cell polarity tumor suppressor gene, scribbled, in the context of normal tissue. scribbled mutant clones in the eye disc exhibit ectopic expression of cyclin E and ectopic cell cycles, but do not overgrow due to increased cell death mediated by the JNK pathway and the surrounding wild-type tissue. In contrast, when oncogenic Ras or Notch is expressed within the scribbled mutant clones, cell death is prevented and neoplastic tumors develop. This demonstrates that, in Drosophila, activated alleles of Ras and Notch can act as cooperating oncogenes in the development of epithelial tumors, and highlights the importance of epithelial polarity regulators in restraining oncogenes and preventing tumor formation (Brumby, 2003).
A clonal approach, more closely resembling the clonal nature of mammalian cancer, was used to analyze the effects of removing Scrib function on tumor formation. This analysis indicates that Drosophila scrib- tumors: (1) lose tissue architecture, including apical-basal cell polarity; (2) fail to differentiate properly; (3) exert non-cell-autonomous effects upon the surrounding wild-type tissue; (4) upregulate cyclin E and undergo excessive cell proliferation; (5) are restrained from overgrowing by the surrounding wild-type tissue via a JNK-dependent apoptotic response, and (6) show strong cooperation with oncogenic alleles of Ras and Notch to produce large amorphous tumors. These conclusions are summarized in a model for tumor development in Drosophila. It is suggested that the role of epithelial cell polarity regulators in restraining oncogenes is likely to be of general significance in mammalian tumorigenesis (Brumby, 2003).
The model suggests that the wild-type larval eye disc is a monolayered columnar epithelium, in which cell proliferation is tightly regulated. Cell architecture is maintained by the formation of adherens junctions, the apical localization of Scribbled, and adhesion to the basement membrane. Mutation of scrib results in loss of apical-basal polarity, leading to multilayering and rounding up of cells. scrib- tissue also shows impaired differentiation, and ectopic cyclin E expression (by an unknown mechanism) leads to ectopic cell proliferation. Unrestrained overgrowth and tumor formation of scrib- cells is held in check by compensatory JNK-mediated apoptosis, dependent upon the presence of surrounding wild-type cells. Secondary mutations are required to avoid this apoptotic fate. If JNK activity is blocked within scrib- cells, by expressing a dominant-negative form of JNK, apoptosis is prevented, resulting in tissue overgrowth and lethality. Even more aggressive overgrowth results from the addition of activating oncogenic alleles of Ras or Notch. In addition to promoting cell survival, these oncogenes must also promote tumor cell proliferation; however, it is proposed that other downstream effectors of these oncogenes are likely also to be important, since it was not possible to mimic the cooperative overgrowth effects of RasACT or NACT on scrib- tissue by simply blocking apoptosis and enhancing cell proliferation (Brumby, 2003).
scrib- clones ectopically express cyclin E and undergo ectopic S phases and mitoses. Since cyclin E is rate limiting for cell cycle progression in the developing eye, it is likely that upregulation of cyclin E in scrib- clones is critical for the ectopic cell proliferation. Indeed, alleles of scrib and lgl were originally isolated as dominant suppressors of a hypomorphic cyclin E allele, DmcycEJP, suggesting that these cell polarity genes normally play a critical role in limiting cyclin E expression. Currently being investigated is which signaling pathways are altered in scrib, dlg or lgl mutants that could be responsible for cyclin E upregulation. A recent study in human lung epithelial cells shows that disrupting cell polarity allows mixing of the heregulin-alpha ligand and the erbB2-4 receptor, which are normally physically separated, resulting in activation of the pathway and cell proliferation. Further studies are required to determine whether the ectopic expression of cyclin E observed in the absence of Scrib is simply a consequence of the tissue disorganization induced by disrupting cell polarity, or if Scrib has a direct role in limiting cell proliferation independent of cell polarity. Interestingly, the rounding up of cells in the absence of Scrib appears to be predominantly a cell-autonomous effect, yet clearly non-cell-autonomous defects are also apparent, including the upregulation of cyclin E. This would suggest that altered cell-cell interactions between wild-type and mutant cells can also alter signaling pathways within wild-type cells, and that the loss of apical-basal polarity and collapse of the columnar epithelium is not intrinsically responsible for the deregulated expression of cyclin E. A deeper understanding of the relationship between epithelial cell polarization and cell proliferation is clearly important for understanding the development of cancer, since a loss of cell polarity often accompanies tumor progression and metastasis (Brumby, 2003).
In Drosophila, activated Ras exerts its oncogenic effects through Raf and the MAPK pathway. Downstream targets of MAPK in the eye disc promote differentiation, cell survival and cell proliferation. This work also demonstrates that Ras can increase cyclin E protein levels in the eye disc. In combination with scrib-, the differentiation output of RasACT signaling appears to be attenuated, and the proliferative and anti-apoptotic responses prevail (Brumby, 2003).
This study has described a novel multi-hit model of tumorigenesis in Drosophila. Furthermore, although it has been suspected that disruptions to cell polarity could potentiate tumor progression and metastasis, this work demonstrates for the first time how the oncogenic effects of activated Ras and Notch are unleashed in the absence of epithelial polarity regulators. It is predicted that in mammals also, defects in apical-basal polarity could cooperate with oncogenes during neoplastic development. This approach in Drosophila can now be used to screen for novel oncogenes that, when specifically overexpressed in scrib- clones, are capable of inducing cooperative tumorigenesis, and can also be extended to identify cooperative interactions between other tumor suppressors and oncogenes within a whole animal context (Brumby, 2003).
E2F transcription factors can activate or actively repress transcription of their target genes. The role of active repression during normal development has not been analyzed in detail. dE2F1su89 is a novel allele of Drosophila E2f that disrupts E2f's association with RBF Drosophila retinoblastoma protein (Rb) homolog but retains its transcription activation function. Interestingly, the dE2F1su89 mutant, which has E2f activation by dE2F1su89 and active repression by E2f2, is viable and fertile with no gross developmental defects. In contrast, complete removal of active repression in de2f2;dE2F1su89 mutants results in severe developmental defects in macrochaetae and salivary glands, tissues with extensive endocycles, but not in tissues derived from mitotic cycles. The endoreplication defect results from a failure to downregulate the level of cyclin E during the gap phase of the endocycling cells. Importantly, reducing the gene dosage of cyclin E partially suppresses all the phenotypes associated with the endoreplication defect. These observations point to an important role for E2f-Rb complexes in the downregulation of cyclin E during the gap phase of endocycling cells in Drosophila development (Weng, 2003).
A novel allele of E2f1, dE2F1su89, was identified from a genetic screen for suppressors of the Rbf overexpression phenotype. Sequence analysis revealed that dE2F1su89 contains a single base pair mutation in the conserved Rb-binding domain that converts the conserved amino acid leucine at position 786 to glutamine. To test whether this mutation disrupts the interaction between Rbf and E2f, a yeast two-hybrid interaction assay was performed. E2F1su89 is unable to bind to Rbf. To demonstrate further the effect of this mutation with endogenous proteins, a co-immunoprecipitation experiment was carried out. While both E2f and Dp co-immunoprecipitate with Rb from wild-type embryo extracts, no E2f co-immunoprecipitates with Rb from the dE2F1su89 embryo extracts, even though similar levels of E2f protein are present in the two extracts. These results indicate that the endogenous dE2F1su89 and Rb proteins do not form a complex. Interestingly, Dp still co-immunoprecipitates with Rb from the dE2F1su89 embryo extracts, indicating that Dp can still form a complex with Rb in the dE2F1su89 mutant background, probably through the other Drosophila E2F protein, E2f2 (Weng, 2003).
The decreased number of endocycles could be due to a lengthening of the S phase or a lengthening of the gap phase. Lengthening of the S phase would lead to an increased number of cells that are in the S phase, while lengthening of the gap phase would decrease the number of cells that are in S phase at any given time. A decreased number of S phase nuclei was observed in e2f2;dE2F1su89 salivary glands compared with that in wild-type salivary glands. Thus the gap phase of the endocycles in the e2f2;dE2F1su89 mutants is significantly lengthened. e2f2;dE2F1su89 but not wild-type salivary gland cells accumulate high levels of cyclin E in some gap phase cells (cells that are not incorporating BrdU). Since downregulation of cyclin E levels is required for continuous endoreplication, the failure to downregulate cyclin E levels properly in these gap phase cells probably inhibits endoreplication and leads to severe defects in tissues that require extensive endoreplication during development. The observation that decreasing the gene dosage of cyclin E partially suppresses the e2f2;dE2F1su89 phenotypes such as salivary gland endoreplication defects, macrochaetae defects and lethality provides strong support for the idea that the failure to downregulate cyclin E levels in these gap phase cells is a cause for the observed defects in e2f2;dE2F1su89 endocycle tissues (Weng, 2003).
Although previous results established that cyclin E oscillation is critical for continuous endoreplication, it is not clear how cyclin E oscillation in endocycle cells is achieved. No cyclin E oscillation defect is observed in salivary gland cells in the dE2F1su89 mutants, suggesting that active repression by the E2f2-Rb complexes is sufficient to downregulate the level of cyclin E during the gap phase, even in the presence of the unregulated dE2F1su89. In contrast, removal of the dE2F2-Rb complexes in the dE2F1su89 background results in extensive defects in endocycle tissues and defective cyclin E downregulation in the gap phase of endocycling cells. These results argue strongly that the E2f-Rb complexes are required for the normal downregulation of cyclin E in the gap phase of endocycling cells. These results, in conjunction with the observation that E2F activity is required for cyclin E expression and S phase progression of endocycle cells, suggests a model in which E2f activation is required for S phase of the endocycles and active repression by E2f-Rb complexes is required during gap phase. It is interesting to note that even in the complete absence of Rb-E2f active repression, there are still significant levels of endoreplication, suggesting that the oscillation of cyclin E activity, although defective, can still occur to some extent in e2f2;dE2F1su89 mutants. It is possible that additional mechanisms such as protein degradation or binding to inhibitor proteins such as Dacapo can also contribute to the downregulation of cyclin E activity (Weng, 2003).
Tissue growth during animal development is tightly controlled so that the organism can develop harmoniously. The salvador gene, which encodes a scaffold protein, restricts cell number by coordinating cell-cycle exit and apoptosis during Drosophila development. Hippo (Hpo), the Drosophila ortholog of the mammalian MST1 and MST2 serine/threonine kinases, is a partner of Sav. Hippo was described in five publications that appeared simutaneously: Pantalacci (2003) identified Hippo in a yeast two-hybrid screen in a search for Salvador interacting proteins, Udan (2003) identifed and positionally cloned hippo in a mutagenesis screen for genes that regulate tissue growth, and Harvey (2003), Jia (2003) and Wu (2003) identified hippo in screens for genes that restrict growth and cell number. Loss of hpo function leads to sav-like phenotypes, whereas gain of hpo function results in the opposite phenotype. Whereas Sav and Hpo normally restrict cellular quantities of the Drosophila inhibitor of apoptosis protein DIAP1 (Thread), overexpression of Hpo destabilizes DIAP1 in cell culture. DIAP1 is phosphorylated in a Hpo-dependent manner in S2 cells and that Hpo can phosphorylate DIAP1 in vitro. Thus, Hpo may promote
apoptosis by reducing cellular amounts of DIAP1. In addition, Sav is an unstable protein that is stabilized by Hpo. It is
proposed that Hpo and Sav function together to restrict tissue growth in vivo (Pantalacci, 2003; Harvey, 2003; Jia, 2003; Udan, 2003 and Wu, 2003).
Mutations are described in hippo, which encodes a protein kinase most related to mammalian Mst1 and Mst2. Like wts and sav, hpo mutations result in increased tissue growth and impaired apoptosis characterized by elevated levels of the cell cycle regulator Cyclin E and apoptosis inhibitor DIAP1. Three alleles of hippo are lethal either
when homozygous or in trans to another allele. Eyes containing
hpo mutant clones and wild-type clones have an overrepresentation of mutant tissue when compared
to eyes containing clones of the wild-type parental chromosome suggesting
that the mutant tissue may have a relative growth advantage. Mutant
ommatidial facets are slightly larger than wild-type facets and
sometimes contain extra interommatidial bristles. When
homozygous clones of hpo were generated in other imaginal
discs using hsFLP, outgrowths of tissue were observed and portions of wings containing large hpo clones were larger
than the corresponding portion of a wild-type wing, indicating a
role for hpo in regulating organ size in tissues other than
the eye. Retinal sections of adult eyes containing hpo
clones reveal that mutant ommatidia appear to have the normal
complement and arrangement of photoreceptor cells. However,
hpo mutant ommatidia appear to have significantly more
tissue between adjacent ommatidia. Cell outlines are visualized more
readily in the pupal retina. In contrast to the single
layer of interommatidial cells observed in wild-type retinas, mutant
ommatidia have several additional interommatidial cells.
These phenotypic abnormalities are very similar to those observed in
sav or wts mutant clones (Harvey, 2003).
sav or wts mutant cells in the eye imaginal disc fail to exit
from the cell cycle at the appropriate time. The additional rounds of
cell division generate an excess of interommatidial cells. Elevated
levels of cyclin E protein detected in mutant cells may underlie the
delayed cell cycle exit (Harvey, 2003).
In a disc mosaic for
the wild-type parent chromosome, BrdU-incorporation was evident in
the anterior portion of the disc and in a narrow stripe, the second
mitotic wave (SMW), but not in the morphogenetic furrow (MF) or
posterior to the SMW where cells arrest in the G1 phase of the cell
cycle. In
hpo mosaic eye discs, the pattern of S phases was normal in
the anterior portion of the disc and in the SMW but in mutant
portions of the disc, BrdU-incorporating nuclei were observed
posterior to the SMW and also in the MF. Thus, hpo cells continue to
cycle when surrounding wild-type cells are arrested in
G1, indicating that hpo function is essential for timely
cell cycle exit. In discs containing clones of the wild-type parental
chromosome, mitoses, visualized with anti-phospho histone H3, were
observed in the anterior portion of the eye disc and in several rows
of developing ommatidia immediately posterior to the SMW. In discs
mosaic for hpo however, extra mitoses were seen in mutant
clones many ommatidial rows posterior to the SMW, indicating
that at least a subset of hpo mutant cells continue to
divide when wild-type cells are mitotically quiescent. These
abnormalities are similar to, though less severe than, those found in
sav and wts imaginal discs (Harvey, 2003).
Elevated levels of cyclin E were found in
hpo mutant clones immediately anterior to the MF, in the
SMW, and posterior to the SMW. Cyclin E expression appeared
normal in hpo clones in the most anterior portions of the
third instar larval eye-antennal disc. In contrast, cyclins A, B, and
D were expressed at normal levels throughout the disc. In wild-type discs cyclin E RNA is expressed in a narrow stripe corresponding to the SMW. In discs mosaic for hpo, the level of cyclin E RNA was elevated and the expression domain of cyclin E was broader.
Additionally, an increase in cyclin E mRNA was detected by
semiquantitative RT-PCR performed on eye imaginal discs composed
almost entirely of hpo mutant tissue.
This indicates that, at least in part, hpo regulates
cyclin E at the level of transcription or RNA stability but
additional posttranscriptional regulation is also possible. In
experiments where hpo function was reduced in S2 cells by
RNAi, the levels of cyclin E protein were increased without an
obvious change in RNA levels as assessed by Northern blotting. Thus,
hpo may be capable of regulating cyclin E levels at both
transcriptional and posttranscriptional levels (Harvey, 2003).
The cycling
properties of hpo mutant cells were analyzed by generating
clones of mutant cells in third instar larval wing discs. Larvae were
heat shocked 48 hr after egg deposition (AED) and wing discs were
dissected 120 hr AED. Following dissociation with trypsin and
staining with Hoechst, cells were subjected to flow cytometry. Cell
size, as gauged by forward scatter, and DNA content were measured and
found to be almost indistinguishable between wild-type and
hpo mutant cells (Harvey, 2003).
The number of cells in hpo mutant
clones is consistently larger than in wild-type sister clones. The median
population doubling time in hpo clones was 13.1 hr, which
is significantly shorter than that of the GFP-bearing tester
chromosome, which was 14.7 hr. By
comparison, clones derived from the parent FRT42D chromosome had a
population doubling time that was not significantly different from
the same tester chromosome. It is unlikely that the increased cell
numbers in hpo clones can be explained by a block in
apoptosis, since overexpression of the caspase inhibitor p35 in wing
disc cells at this stage of development does not appreciably alter
the population doubling time. Thus, cells appear to divide faster in
hpo clones. Since cell size is essentially unchanged, this
would imply that hpo cells have an increased rate of growth
(mass accumulation) and a commensurate increase in the rate of cell
division (Harvey, 2003).
In wild-type pupal retinas, excess interommatidial
cells are eliminated in a wave of apoptosis during the midpupal
stage. There is a defect in apoptosis
in sav and wts mutant tissue. As a result, the additional cells
generated by excess cell division in sav and wts
tissue are not eliminated and account for the increased number of
interommatidial cells (Harvey, 2003).
Studies in Drosophila have defined a new growth inhibitory pathway mediated by Fat (Ft), Merlin (Mer), Expanded (Ex), Hippo (Hpo), Salvador (Sav)/Shar-pei, Warts (Wts)/Large tumor suppressor (Lats), and Mob as tumor suppressor (Mats), which are all evolutionarily conserved in vertebrate animals. The Mob family protein Mats functions as a coactivator of Wts kinase. This study shows that mats is essential for early development and is required for proper chromosomal segregation in developing embryos. Mats is expressed at low levels ubiquitously, which is consistent with the role of Mats as a general growth regulator. Like mammalian Mats, Drosophila Mats colocalizes with Wts/Lats kinase and cyclin E proteins at the centrosome. This raises the possibility that Mats may function together with Wts/Lats to regulate cyclin E activity in the centrosome for mitotic control. While Hpo/Wts signaling has been implicated in the control of cyclin E and diap1 expression, this study found that it also modulates the expression of cyclin A and cyclin B. Although mats depletion leads to aberrant mitoses, this does not seem to be due to compromised mitotic spindle checkpoint function (Shimizu, 2008).
Mats is essential for normal development; mats mutants stop their growth at the second instar larval stage and eventually die. In fact, this growth retardation phenotype facilitated identification of matsroo and matse235 mutant larvae for DNA sequence analysis. Using matse235 allele and the P-element-induced allele matsPB, it has been shown that mats homozygotes and hemizygotes grow slowly and their imaginal discs are much smaller than that of wild-type larvae at the same age. mats mutant cells in mosaic tissues acquire growth advantage likely through comparison and competition with neighboring wild-type cells. In contrast, the absence of wild-type cells in homozygous mats mutant animals renders no competitive growth advantage to mutant cells. The mechanism by which mats mutants acquire growth advantage in the context of mosaic tissue still needs to be investigated. mats mutant embryos missing both maternal and zygotic mats functions failed to hatch, indicating that mats is essential for embryonic development. By analyzing mitotic cells, it was found that maternally mats-depleted embryos show aberrant DNA segregation such that uneven amounts of DNA are segregated toward opposing centrosomes. However, this does not appear to be due to the compromised function of mitotic spindle checkpoint, since mats mutant tissue still accumulate M-phase cells in response to inhibition of mitotic spindle formation by colcemid treatment. Thus, mats is not required for mitotic spindle checkpoint, unlike mps1 (Shimizu, 2008).
Cyclin E is a critical cell cycle regulator. Through a Cdk2-dependent mechanism, cyclin E-Cdk2 plays a critical role in accelerating G1-S transition in the cell cycle. As a general rule, cyclin E is tightly regulated during the cell cycle by Cdk2 and GSK-mediated phosphorylation and subsequent degradation. A nondegradable cyclin E mutant can cause extra rounds of DNA synthesis and polyploidy, and overexpression of cyclin E is frequently detected in tumor cells exhibiting polyploidy. Intriguingly, cyclin E is a centrosomal protein that functions to promote S-phase entry and DNA synthesis in a Cdk2-independent manner (Matsumoto, 2004). Loss of cyclin E expression in the centrosome inhibits DNA synthesis, whereas ectopic expression of cyclin E in the centrosome accelerates S-phase entry. Thus, the centrosome is an important subcellular organelle for cyclin E to regulate cell proliferation, and the level and activity of cyclin E in centrosomes must be tightly controlled. The fact that Mats and Wts colocalize with cyclin E at the centrosome raises the possibility that Mats may function together with Wts kinase to regulate cyclin E function in the centrosome for mitotic control. In support of this hypothesis, loss-of-function mutations in mats increase the levels of cyclin E protein and both gain- and loss-of-function mutant alleles of cyclin E modulate the eye phenotypes caused by Wts overexpression. Although Mats/Wts-mediated inhibition of cyclin E could occur through Yki to regulate cyclin E transcription, a direct control of cyclin E at the protein level would allow a rapid response to an upstream signal (Shimizu, 2008).
The fact that both Mats and Wts show a intracellular localization pattern very similar to that of their respective yeast relatives Mob1 and Dbf2 suggests that their function is conserved. This conservation may extend to mammals; human LATS1, LATS2, and MOB1A (MATS2) also localize at the centrosome. In addition, localization at the bud neck/midbody appears to be conserved in humans. Interestingly, such centrosomal localization of Mats and Wts does not seem to rely on Wts kinase activity as kinase-inactive Wts and Mats can be still localized at the centrosome. To examine whether endogenous Mats protein localizes at the centrosome, embryo immunostaining was done with Mats antibodies. As in larval tissues, expression of Mats protein in developing embryos does not exhibit any obvious pattern and Mats expression level is low and ubiquitous. Although centrosomal localization of endogenous Mats protein has not been shown, likely due to some technical problems, Mats (CG13852/Mob4) has been recently reported to be a centrosomal protein (Shimizu, 2008 and references therein).
Both loss- and gain-of-function analysis supports a model in which cyclin E and diap1 are critical downstream targets of Hpo/Wts signaling. Evidence in this report suggests that Hpo/Wts signaling may also target cyclin A and cyclin B. Consistent with this notion, elevated levels of cyclin B were found in ex mutant cells. In addition, wts has been shown to be required for a negative control of cyclin A but not cyclin B expression. In humans, LATS1 was shown to be a negative regulator of Cdc2/cyclin A and to function at the G2/M-phase transition, while LATS2 affects cyclin E/Cdk2 activity and regulates G1/S phase passage. Thus, the ability of Hpo/Wts signaling to target cyclin genes important for cell cycle progression appears to be evolutionarily conserved (Shimizu, 2008).
Little is known about how patterns of cell proliferation and arrest are generated during development, a time when tight regulation of the cell cycle is necessary. In this study, the mechanism by which the developmental signaling molecule Wingless generates G1 arrest in the presumptive Drosophila wing margin is examined in detail. Wg signaling promotes activity of the Drosophila retinoblastoma family (Rbf) protein, which is required for G1 arrest in the presumptive wing margin. Wg promotes Rbf function by repressing expression of the G1-S regulator Drosophila myc (dmyc). Ectopic expression of dMyc induces expression of Cyclin E, Cyclin D, and Cdk4, which can inhibit Rbf and promote G1-S progression. Thus, G1 arrest in the presumptive wing margin depends on the presence of Rbf, which is maintained by the ability of Wg signaling to repress dmyc expression in these cells. In addition to advancing the understanding of how patterned cell-cycle arrest is generated by the Wg signaling molecule during development, this study indicates that components of the Rbf/E2f pathway are targets of dMyc in Drosophila. Although Rbf/E2f pathway components mediate the ability of dMyc to promote G1 progression, dMyc appears to regulate growth independently of the RBF/E2f pathway (Duman-Scheel, 2004).
The results indicate why exclusion of dMyc from the ZNC is necessary for Rbf activity. Overexpression of dMyc leads to high levels of Cyclin E, Cyclin D, and Cdk4 transcripts. dMyc also regulates Cyclin E posttranscriptionally in Drosophila. G1-S Cyclins/Cdks function to phosphorylate and inhibit Rbf, suggesting that dMyc blocks Rbf activity through activation of G1-S Cyclins/Cdks. Thus, inhibition of dMyc by Wg helps to ensure that G1-S Cyclins/Cdks do not activate S phase. This idea is supported by the results that indicate that only a combination of both Dap and constitutively active Rbf (that cannot be regulated by Cyclins/Cdks) can restore G1 arrest when Wg signaling is blocked or when dMyc is expressed. These data suggest that either Cyclin D or Cyclin E activity can mediate the ability of dMyc to promote S phase in the ZNC. Coexpressing Dap alone with dMyc, which would block only Cyclin E/Cdk2 activity, does not restore G1 arrest. Furthermore, overexpression of dMyc in a cdk4 mutant background still results in ectopic S phases, suggesting that Cyclin E/Cdk2 also are sufficient to mediate dMyc's ability to promote G1 progression. Thus, either Cyclin D/Cdk4 or Cyclin E/Cdk2 is sufficient to mediate the ability of dMyc to promote G1 progression. The ability of Wg to inhibit dMyc expression is thus critical for RBF activation and G1 arrest in the ZNC. Still, it is possible that Wg promotes G1 arrest through other mechanisms that have not yet been uncovered. The observation that overexpression of a dominant-negative form of dTCF (dTCFDeltaN) with C96>Gal4 can promote S phase, even in a dmyc mutant background, supports this idea (Duman-Scheel, 2004).
It is likely that dMyc/dMax directly up-regulate transcription of Cyclin D and cdk4 in Drosophila. Myc/Max heterodimers regulate transcription by binding to various consensus sequences, such as the E box. Previous studies indicated that cMyc induces Cyclin D2 expression in mice by binding to two consensus E boxes in the Cyclin D2 promoter. cdk4 also was identified as a transcriptional target of c-Myc. Furthermore, it has been suggested that cdk4 is a transcriptional target of dMyc and Cyclin D is a transcriptional target of dMax. Although future studies should analyze the Drosophila Cyclin D and Cdk 4 regulatory regions in more detail, these results suggest that the observed ability of dMyc to induce Cyclin D and Cdk4 expression in the ZNC most likely occurs through transcriptional regulation of these proteins by dMyc/dMax. In contrast, Cyclin E was not identified as a target of dMyc or dMax. It is more likely that the ability of dMyc to induce growth in the wing indirectly leads to increased Cyclin E transcript levels (Duman-Scheel, 2004).
In the Drosophila CNS, neuroblasts undergo self-renewing
asymmetric divisions, whereas their progeny, ganglion mother cells (GMCs),
divide asymmetrically to generate terminal postmitotic neurons. It is not
known whether GMCs have the potential to undergo self-renewing asymmetric
divisions. It is also not known how precursor cells undergo self-renewing
asymmetric divisions. Maintaining high levels of Mitimere
or Nubbin, two POU proteins, in a GMC causes it to undergo self-renewing
asymmetric divisions. These asymmetric divisions are due to upregulation of Cyclin E in late GMC and its unequal distribution between two daughter cells. GMCs in an embryo overexpressing Cyclin E, or in an embryo mutant for archipelago, also undergo self-renewing asymmetric divisions.
Although the GMC self-renewal is independent of inscuteable and
numb, the fate of the differentiating daughter is
inscuteable and numb-dependent. These results reveal that
regulation of Cyclin E levels, and asymmetric distribution of Cyclin E and
other determinants, confer self-renewing asymmetric division potential to
precursor cells, and thus define a pathway that regulates such divisions.
These results add to understanding of maintenance and loss of
pluripotential stem cell identity (Bhat, 2004).
Maintenance of a self-renewing fate can be viewed as a state where
activities of certain genes maintain that state. Once the activity of such
genes is switched off, the cells become committed to a differentiation pathway. The results reported in this study indeed support this type of mechanism. That POU genes might be a class
of genes that maintain a self-renewing capacity is indicated by the fact that the Oct4 POU gene (Pou5f1 -- Mouse Genome Informatics), which is expressed in pluripotent stem cells of the mouse early embryo, is turned off when these cells begin to differentiate
(Rosner, 1990). Similarly, SCIP is expressed in the progenitors of oligodendrocytes, but it is downregulated when these cells are induced to differentiate (Collarini, 1992). The current results provide direct evidence that these genes can induce a cell that is
committed to a differentiation pathway to acquire a self-renewing capability in a lineage specific manner. Moreover, studies undertaken in the past several years using the Drosophila nervous system as a paradigm have revealed how asymmetry can be generated during cell division to produce two distinct postmitotic cells. However, there is very little information on how an asymmetric self-renewing division pattern is determined. In this paper, results are presented that provide insight into this particular process. (Bhat, 2004).
The strongest evidence that a GMC-1 undergoes a self-renewing type of
asymmetric division in embryos overexpressing miti/nub or
CycE, and in embryos mutant for ago, comes from the presence
of hemisegments with two sibs and one RP2. There are two ways the second sib cell can be generated: (1) a self-renewed GMC-1 generates another sib when it divides, and (2) some other cell is transformed into a sib. The following set of evidence indicates the former scenario: (1) the second sib cell always appears later in development, i.e. at ~8.5 hours of age (as opposed to in
wild type where the GMC-1 terminally divides by ~7.5 hours of age into an
RP2 and a sib); (2) the dynamics of Eve expression itself in the sib --
expression of eve is switched off in a sib during the asymmetric
division of GMC-1 and there is no de novo synthesis of Eve thereafter. If a postmitotic cell from an Eve-negative lineage transforms into a sib, it would be negative for Eve and would not be detected. The development of the other Eve-positive neuronal lineages is normal in these embryos, thus it is unlikely that a cell from those Eve-positive lineages is transformed into a sib. (3) The Eve and Spectrin staining of UAS-nub; ftz-GAL4 embryos provides more direct evidence for the self-renewal of GMC-1. In ~8. 5-hour-old UAS-nub; ftz GAL4 embryos, the larger GMC-1 (this Eve-positive cell is Zfh1 negative, indicating that it is indeed a GMC-1) can be observed undergoing asymmetric cytokinesis for the second time. From the heat-shock induction experiments of nub or miti mutant embryos, it can be argued that higher levels of these proteins in the parental NB4-2 cause later born GMCs to adopt a GMC-1 fate. However, the GMC-1 self-renewing phenotype observed following targeted expression of nub using the ftz-GAL driver makes this scenario unlikely. (4) The results obtained with the mitiP; insc and mitiP; nb double mutant embryos (P referring to prolonged expression), and the mis-localization of Insc in GMC-1 of these embryos, are also consistent with this conclusion. (Bhat, 2004).
These results indicate that the level, timing and duration of presence of Miti or Nub proteins determine the dynamics of the GMC-1 division pattern. For example, the asymmetric divisions (which generate the 3-cell phenotypes) and the symmetric divisions (which generate the 4-cell phenotype) were observed when the transgenes were induced for 20-25 minutes. However, the multiple cell-phenotype was observed only when the transgenes were induced for 90 minutes. Once the induction was stopped and the levels returned to normal, the two GMC-1s appeared to exit from the cell cycle to generate postmitotic cells. Similarly, when the transgene was induced with ftz-GAL4, only the 3-cell phenotypes, and not the 4-cell or multi-cell phenotypes were observed. Thus, the following picture emerges from these results. Although high levels of Miti and Nub proteins are required for the specification of GMC-1 identity, their level must be downregulated in order for the GMC-1 to divide asymmetrically into postmitotic RP2 and sib. Maintaining a high level of these proteins in GMC-1 commits that cell to adopt a self-renewing stem cell type of division pattern. The results described here also show that Miti and Nub prevent GMC-1 from exiting the cell cycle by upregulation of CycE (Bhat, 2004).
The results clearly show that upregulation of CycE in late GMC-1 is the
cause for the adoption of a self-renewing asymmetric division pattern. In
other words, presence of high levels of CycE in late GMC-1 and its unequal
distribution to one of the two daughter cells prevents this cell from exiting the cell cycle. Since this daughter cell still maintains the GMC-1 identity and
has sufficient CycE to divide again, a further asymmetric division(s) is
ensured. The cell that has lower amounts of CycE becomes committed to a
differentiation pathway (RP2 or sib) (Bhat, 2004).
What lines of evidence support this conclusion? (1) In contrast with
wild type, there is a significant amount of CycE present in a late GMC-1 in embryos overexpressing miti or nub. This CycE preferentially segregates to one of the two daughters of that GMC-1, usually the larger cell. When miti or nub genes are overexpressed only briefly, the level of CycE is downregulated after just one additional round of division, whereas with prolonged induction, the level is maintained at high levels in one or two cells of the multi-cell cluster for a prolonged duration of time (Bhat, 2004).
(2) Upregulation of CycE in a late GMC-1 is also observed in embryos mutant for ago, which is known to regulate CycE levels. In
ago mutants, the two daughter cells of such a GMC-1 have unequal CycE levels accompanied by a self-renewing asymmetric division phenotype. The CycE is always downregulated after one additional GMC-1 division, which is consistent with the finding that the self-renewal occurs only once in these embryos. Since penetrance in ago mutants is partial, and CycE is downregulated in this lineage after just one additional division, there must be additional factors that mediate the downregulation of CycE in this lineage (Bhat, 2004).
(3) Embryos expressing high levels of CycE from a CycE
transgene exhibit the same GMC-1 phenotypes as embryos expressing high levels of Miti or Nub. Thus, these results indicate that upregulation of CycE alone is sufficient for the GMC-1 to adopt a self-renewing type of division pattern. Finally, mitiP phenotypes are found to be dependent on CycE. That is, no multi-cell
clusters were observed in mitiP; CycE double mutant embryos (Bhat, 2004).
In wild type, the downregulation of CycE in GMCs appears to occur through switching off CycE transcription and degradation of the protein by factors such as Ago. At what level does Miti or Nub regulate CycE? Since
POU genes are thought to be transcriptional activators, they can regulate
transcription of CycE either directly or indirectly. However, this
does not seem to be the case since expressing high levels of miti does not have a discernible effect on the levels of CycE mRNA in GMC-1, as assessed by whole-mount RNA in situ hybridization. In
addition, the putative promoter/enhancer region of CycE gene does not contain any consensus POU protein-binding sites. Therefore, it seems likely that Miti and Nub regulate factors that are involved in the degradation of CycE in late GMC-1 (Bhat, 2004).
The question arises as to how only one cell has a high level of CycE. There are several ways this can happen. There might be an asymmetric degradation of CycE. This scenario seems unlikely since there is only one of two daughter cells with high levels of CycE in ago mutants. Given that Ago downregulates CycE via a protein degradation mechanism, if
there was an asymmetric degradation, in those hemisegments where the levels of CycE was elevated in GMC-1, it would initially be expected that both the daughter cells
would have high CycE levels. However, this was not the case. An asymmetric
transcription of the CycE gene also seems unlikely since the
transcription of CycE ceases prior to GMC-1 division, as judged by
whole-mount RNA in situ hybridization. The most likely
possibility is that CycE is unequally distributed between the two daughter
cells of GMC-1. The unequal distribution of CycE could be a passive process due to the size difference between daughter cells, especially in the GMC-1-->RP2/sib lineage. Moreover, no cytoplasmic
crescent of CycE was observed during mitosis. By contrast, it could also be an active process. For instance, the size difference between an aCC and a pCC (or between a GMC1-1a and an aCC) is very small, and the fact that GMC1-1a undergoes a self-renewing asymmetric division suggests that the segregation of CycE may not be entirely a passive process (Bhat, 2004).
Finally, the results indicate that while a GMC that does not normally
express Miti or Nub is insensitive to its ectopic expression (e.g., GMC1-1a of NB1-1; this GMC produces an aCC/pCC pair of neurons), a brief induction of CycE in the same GMC causes it to undergo self-renewing asymmetric division. Therefore, CycE can confer a stem cell type of division potential to more than one GMC. Another important conclusion one can draw from this result
is that the segregation of CycE may be an active process. In the case of
GMC1-->RP2/sib lineage, the cytokinesis of GMC-1 is asymmetric, and the
size difference between an RP2 and a sib is significant. Thus, CycE can be
asymmetrically segregated because of this size difference. However, the size difference between an aCC and a pCC (or between a GMC1-1a and an aCC) is very small, and the fact that GMC1-1a undergoes a self-renewing asymmetric division suggests that the segregation of CycE may not be entirely a passive process. It is possible that the difference between the levels of CycE needed to retain a cell within the cell cycle and the levels that do not support maintaining the cell within the cell cycle are quite small. Thus, even a minor change in the amount that a cell receives during division might be sufficient to make a
difference. Thus, the segregation of CycE can still be a passive process.
Nonetheless, these results reveal how a cell can adopt a self-renewing
asymmetric division potential through CycE. (Bhat, 2004).
Pros has been implicated in inhibiting the ability of GMCs to
divide more than once by preventing continued expression of cell-cycle genes. The caveat of this study, however, is that none of the GMC lineage was examined using cell-specific markers to determine whether GMCs continue to divide in embryos mutant for pros. The conclusion that Pros inhibits GMC division was mainly based on the presence of additional BrdU-positive cells in
late stage (post 15-hours-old) pros mutant embryos. Pros is expressed in GMC-1 of the RP2/sib lineage and, in null alleles, this GMC-1 identity is not specified. In pros17, a
loss-of-function allele, ~5% of the hemisegments had an RP2/sib lineage
specified. In these hemisegments, the GMC-1 divides only once to generate an RP2 and a sib cell as in wild type. Moreover, specification of U and CQ lineages was observed in ~20% and ~13% of the hemisegments, respectively, and no additional
cell division appeared to occur in these lineages. A previous study found that the aCC/pCC neurons (from GMC1-1a) have an abnormal axon morphology, but it did not find any additional neurons in this lineage. Similarly,
NB6-4 of the thoracic segment produced the normal number of progeny in
pros mutant embryos. These results suggest that Pros does not regulate cell division in RP2/sib, U and CQ lineages, and possibly not in many other neuronal lineages, and therefore it is unlikely to function in the miti/nub pathway (Bhat, 2004).
How is the specification of identity of one of the two progeny, either as an RP2 or as a sib, from a self-renewing asymmetric division of GMC-1
regulated? (Specification of the other progeny as GMC-1 is by high levels of CycE.) The results indicate that specification of an RP2 versus a sib identity to this differentiating cell is through a combination of low levels of CycE and localization of Insc. This is indicated by the finding that overexpression of Miti and Nub causes localization of Insc to be non-asymmetric. Non-asymmetric Insc also causes non-asymmetric localization of Numb. The cell that has lower levels of CycE and also has Numb becomes an RP2. Whenever the cell with lower levels of CycE fails to inherit Numb (the effect of overexpression of Miti or Nub on the localization of Insc is partially penetrant) that cell will become a sib. That the generation of an RP2 during the asymmetric division of GMC-1 is tied to Numb is also indicated by the analysis of mitiP; numb embryos. Although the self-renewal of GMC-1 in mitiP embryos is
numb-independent, the commitment of a progeny to become a sib is
numb-dependent. Thus, in ~13-hour-old
mitiP; numb embryos, multiple cells
are observed adopting a sib fate. An often overlooked fact is that in insc mutants the GMC-1 division is normal in ~30% of the hemisegments despite having no insc. Similarly, the penetrance of the symmetrical division of GMC-1 in pins (where Insc localization is affected as in mitiP embryos) is also partial, indicating the presence of additional (partially redundant) pathways for Insc that mediate asymmetric fate specification. These very same additional pathways must also influence the choice between a sib and an RP2 when the GMC-1 in mitiP embryos undergoes a self-renewing type of asymmetric division (Bhat, 2004).
CycE and Ago are part of a mechanism that converts a normal cell into a
cancer cell. In ago mutants, CycE protein is not degraded and a
number of cancer cell lines carry a mutation in ago. The current
results showing that these genes are also involved in a stem cell type of
division suggest a commonality between stem cells and cancer cells. These
results also provide a molecular mechanism of how self-renewing asymmetric
divisions are possible (Bhat, 2004).
During central nervous system development, glial cells need to be in the
correct number and location, at the correct time, to enable axon guidance and
neuropile formation. Repair of the injured or diseased central nervous system
will require the manipulation of glial precursors, so that the number of glial
cells is adjusted to that of neurons, enabling axonal tracts to be rebuilt,
remyelinated and functional. To a large extent, the molecular mechanisms controlling
glial precursor proliferative potential are unknown. This study shows that glial
proliferation is regulated by interactions with axons and that the
Drosophila gene prospero is required to maintain the mitotic
potential of glia. During growth cone guidance, Prospero positively regulates
cycE promoting cell proliferation. Neuronal Vein activates the MAPKinase
signalling pathway in the glia with highest Prospero levels, coupling axon
extension with glial proliferation. Later on, Prospero maintains glial
precursors in an undifferentiated state by activating Notch and antagonising the
p27/p21 homologue Dacapo. This enables prospero-expressing cells alone to
divide further upon elimination of neurons and to adjust glial number to axons
during development (Griffiths, 2004).
The longitudinal glia (LG) of the Drosophila CNS share some features with
vertebrate oligodendrocyte precursors. Like oligodendrocytes, LG are also
produced in excess and the excess cells are eliminated through apoptosis. The
survival of both oligodendrocytes and at least some of the LG
depends on contact with axons and on Neuregulin/Vein.
There is also suggestive evidence that LG proliferation may be under
non-autonomous control. The LG originate from the segmentally repeated
longitudinal glioblasts (LGBs). DiI labelling of the LGB produces a clone of
variable number of progeny cells, resulting in between 7 and 10 progeny cells.
There is apoptosis in up to three cells in this lineage
in normal embryos, meaning that the resulting progeny of
the LG lineage if they were all to survive may be around 12 cells. This suggests
that the mitotic profile of the LGB lineage is not simply symmetrical and/or
perhaps LG precursor divisions are under non-autonomous control (Griffiths, 2004).
This study analyzed the mechanisms that regulate proliferation of the LG as they
interact with pioneer axons. Proliferation of the LG is shown to be regulated
by neurons and prospero (pros) is shown to play a key role in linking glial
proliferation and axon guidance. Early on, Pros enables glial proliferation in
response to pioneer neurons. Once the axonal bundles are formed, Pros maintains
glial precursors in an arrested, immature state, enabling pros-expressing
cells alone to divide further upon elimination of neurons (Griffiths, 2004).
This study has found novel roles of Pros in promoting cell proliferation and
preventing cell cycle exit. The glia reach the extending
growth cones in clusters of four cells, when cell division halts for some time.
Normally, two of these LG then divide, resulting in a total of six, which then
divide again, but since some LG die the real final number ranges between 8 and
11 cells. In pros mutants, the glia contact the pioneer neurons in
clusters of eight cells rather than the normal four, suggesting that LG divided
faster than normally in the presence of maternal CycE, skipping a G1 phase. The
division of four LG into six is also missed, thus changing the mitotic pattern
from its normal 4-6-12 to 4-8 (Griffiths, 2004).
Loss of
pros function causes a reduction in LG proliferation, which is manifested
in three ways. (1) In pros mutants, the first division of the two
anterior LG with highest Pros levels is missed, because there is no dpERK. (2)
LG do not divide at the normal times during axon guidance and fasiculation is not produced in
pros mutants, because of the absence of CycE.
Thus, although LG divided earlier in pros mutants, these divisions are
uncoupled from axon guidance. Thus, Pros changes the mitotic profile in the LG
from a simple symmetric pattern to a pattern in which the LG respond to incoming
axons. (3) In the absence of Pros, LG do not have the potential to
overproliferate when neurons are ablated (Griffiths, 2004).
Pros protein is
present in all dividing LG and in LG that retain mitotic potential. During
growth cone guidance and axonal fasciculation, Pros promotes LG proliferation of
the two LG that are able to respond to Vein and activate the MAP kinase pathway.
Vein induces LG cell division as well as cell survival of the two EGFR-positive
LG. Knock-down of Vein function with targeted RNAi exclusively directed to the the MP2 neurons is
sufficient to cause LG apoptosis. Loss of Vein function in genetic null embryos
reduces mitosis, also when apoptosis is blocked. Thus, the EGFR/MAPKinase
signalling pathway controls both cell survival and cell proliferation in these
two LG. The EGFR also controls both cell survival and cell proliferation in the
retina, in response to the ligand Spitz.
Later on, when the axonal fascicles are formed, Pros maintains the mitotic
potential in the LG by preventing them from exiting the cell cycle. In fact,
only Pros-positive LG can enter S phase upon ectopic expression of cycE.
In this way, at the end of embryogenesis, the LG are divided into Pros-positive
G1-arrested LG and Pros-negative LG, which have exited the cell cycle and are in
G0. Pros maintains the LG in the G1-arrested undifferentiated, immature
precursor state by positively regulating Notch and by antagonising Dacapo (Griffiths, 2004).
These findings on the roles of Pros in the LG during axon guidance
differ from Pros' neuroblast functions. In neuroblast lineages, Pros protein is
located in a crescent and it is distributed asymmetrically to the daughter cell
upon the division of the neuroblast. In the ganglion mother cell, Pros is
internalised into the nucleus, where it determines cell fate and it restricts
cell division. However, the progeny of
the LGlioblast [from the time in which they contact the pioneer axons (four-cell
stage)] divide apparently symmetrically, although asynchronously. During these
divisions, Pros is present in the nuclei of all dividing LG, and not in
crescents. Upon cell division, Pros is segregated symmetrically to the two
daughter cells and it is downregulated after cell division, at the time that the posterior LG migrate with the axons. Finally, during axon guidance, pros mutations
cause a reduction in LG proliferation rather than an excess, meaning that
pros is necessary for cell division to proceed (Griffiths, 2004).
Pros and its vertebrate homologue Prox1 can inhibit cell proliferation and promote cell cycle exit. In fact,
both in pros and Prox1 mutants, cell proliferation and the
expression of cyclin increase, and both Prox1 and Pros can promote
p27/dap expression. In the LG, Pros promotes cell
proliferation and it prevents cell cycle exit by antagonising Dap. Therefore,
Pros controls cell cycle genes in different ways in different cellular contexts.
Moreover, temporal regulation is crucial and Pros can both promote and antagonise dap
expression at different time points. Upon ectopic pros expression
the LG divide less and do
not express cycE. However, in the LG this may not be due to the promotion
of cell cycle exit but to the earlier halt of precursors in cell cycle
arrest (Griffiths, 2004).
These findings also contrast with the roles of Pros in
mixed neuro-glioblast lineages, where Pros is segregated asymmetrically to the
daughter cell that will become a glial cell. The LG is a glial-only lineage. In the
LG, Pros may control the fate of the two LG with higher Pros levels, which
signal through MAPKinase/dpERK. The results show that during axon guidance Pros
plays a primary role in the maintenance of the proliferative and undetermined
state (Griffiths, 2004).
The current findings on the non-autonomous regulation of
glial proliferation contrast with previous work that envisioned a
cell-autonomous proliferation profile determined by lineage identity.
Accordingly, the LGB would divide in a straightforward symmetrical fashion, into
2-4-8 cells. This conclusion
was based on the finding that BrdU is incorporated in four Repo-positive cells.
The data show that the incorporation of BrdU into four LG represents a narrow
time window in the LG lineage, and not the final division. In fact,
mitosis is detected in up to five LG at the same stage (Griffiths, 2004).
The finding of a
different LG profile has important implications. It means that the final number
of LG is not fixed at eight cells, but variable between 8 and 11, depending on
how many LG die. A final fixed number of eight LG could be achieved faster
through simple symmetrical divisions without considerable influence on final
glial cell mass. In fact, in Pros mutants a final number of eight cells is
achieved at an earlier time point, and these eight cells stretch out to occupy
the whole length of the segmental neuropile. However, the sequential increase
and adjustment in LG number deploys a restricted number of LG at sequential
steps in axonal patterning. This enables glia to be in the correct number at
discrete time points to enable axon guidance and fasciculation (Griffiths, 2004).
The first event in growth cone guidance occurs at the four-cell
stage, when LG stop dividing for some time and wait for the pioneer growth cones
to extend. At this time, the LG are in the first G1 phase in the lineage. The G1
phase is a characteristic time in which cells respond to growth factors to
signal through ERK, and
in the retina axons approach selectively precursors that are in G1.
As the growth cones approach, the
two anterior LG (with higher Pros levels) of the four-cell clusters divide in
response to Vein. Vein is produced by the MP2 pioneer neurons, which require LG
for axon guidance. By regulating both cell survival
and cell proliferation, Vein ensures that LG are present in the correct number
to enable growth cone guidance. Pros regulates the zygotic expression of CycE in
LG, thus introducing the first G1-S transition, and the fate of the EGFR
signalling cells. In this way, Pros modulates the timing of the response of glia
to a neuronal signal to divide. Subsequently, the LG continue to divide at times
in which axons undergo fasciculation and defasciculation. In this way, LG are
deployed in restricted numbers to enable sorting out of axons through time (Griffiths, 2004).
Stem cells have the remarkable ability to give rise to both self-renewing and differentiating daughter cells. Drosophila neural stem cells segregate cell-fate determinants from the self-renewing cell to the differentiating daughter at each division. This study shows that one such determinant, the homeodomain transcription factor Prospero, regulates the choice between stem cell self-renewal and differentiation. The in vivo targets of Prospero have been identified throughout the entire genome. Prospero represses genes required for self-renewal, such as stem cell fate genes and cell-cycle genes. Surprisingly, Prospero is also required to activate genes for terminal differentiation. In the absence of Prospero, differentiating daughters revert to a stem cell-like fate: they express markers of self-renewal, exhibit increased proliferation, and fail to differentiate. These results define a blueprint for the transition from stem cell self-renewal to terminal differentiation (Choksi, 2006).
To identify sites within the Drosophila genome to which Prospero binds, use was made of an in vivo binding-site profiling technique, DamID. DamID is an established method of determining the binding sites of DNA- or chromatin-associated proteins. Target sites identified by DamID have been shown to match targets identified by chromatin immunoprecipitation (ChIP). DamID enables binding sites to be tagged in vivo and later identified on DNA microarrays. In brief, the DNA or chromatin-binding protein of interest is fused to an Escherichia coli adenine methyltransferase (Dam), and the fusion protein is expressed in vivo. The DNA-binding protein targets the fusion protein to its native binding sites, and the Dam methylates local adenine residues in the sequence GATC. The sequences near the protein-DNA interaction site are thereby marked with a unique methylation tag, over approximately 2-5 kilobase pairs (kb) from the binding site. The tagged sequences can be isolated after digestion with a methylation-sensitive restriction enzyme, such as DpnI (Choksi, 2006).
Dam was fused to the N terminus of Prospero, and transgenic flies were generated. The fusion protein is expressed from the uninduced minimal Hsp70 promoter of the UAS vector, pUAST, as high levels of expression of Dam can result in extensive nonspecific methylation and cell death. As a control for nonspecific Dam activity, animals expressing Dam alone were generated. To assess the sites to which Prospero binds during neurogenesis, genomic DNA was extracted from stage 10-11 embryos, approximately 4-7 hr after egg laying (AEL), expressing either the Dam-Prospero fusion protein or the Dam protein alone. The DNA was digested with DpnI and amplified by PCR. DNA from Dam-Prospero embryos was labeled with Cy3, and control DNA with Cy5. The samples were then cohybridized to genomic microarrays. Microarrays were designed that tile the entire euchromatic Drosophila genome. A 60 base oligonucleotide was printed for approximately every 300 bp of genomic DNA, resulting in roughly 375,000 probes on a single array (Choksi, 2006).
Log-transformed ratios from four biological replicates (two standard dye configurations plus two swapped dye configurations) were normalized and averaged. Regions of the genome with a greater than 1.4-fold log ratio (corresponding to approximately a 2.6-fold enrichment) of Dam-Prospero to the control over a minimum of four adjacent genomic probes were identified as in vivo Prospero binding sites. Using these parameters, a total of 1,602 in vivo Prospero binding sites were identified in the Drosophila genome. This work demonstrates that it is possible to map in vivo binding sites across the whole genome of a multicellular organism (Choksi, 2006).
Prospero is known to regulate the differentiation of photoreceptors in the adult eye, and recently sites have been characterized to which Prospero can bind upstream of two Rhodopsin genes, Rh5 and Rh6. A variant of the Prospero consensus sequence is found four times upstream of Rh5 and four times upstream of Rh6. Prospero was shown to bind this sequence in vitro, by band shift assay, and also by a 1-hybrid interaction assay in yeast. In addition, deletion analysis demonstrated that the consensus sequence is required for the Pros-DNA interaction both in vivo and in vitro. It was found that 67% of in vivo binding sites contain at least one Prospero binding motif. Combining in vivo binding-site data with searches for the Prospero consensus sequence reveals 1,066 distinct sites within the Drosophila genome to which Prospero binds during embryogenesis (Choksi, 2006).
A total of 730 genes have one or more of the 1,066 Prospero binding sites located within 1 kb of their transcription unit. Statistical analyses to determine GO annotation enrichment on the members of the gene list that had some associated annotation (519) was performed by using a web-based set of tools, GOToolbox. Using Biological Process (GO: 0008150) as the broadest classification, a list was generated of overrepresented classes of genes (Choksi, 2006).
The three most significant classes of genes enriched in the list of putative Prospero targets are Cell Fate Commitment, Nervous System Development, and Regulation of Transcription. Utilizing GO annotation, it was found that nearly 41% of all annotated neuroblast fate genes (11 of 27) are located near Prospero binding sites and that approximately 9% of known cell-cycle genes are near Prospero binding sites. These include the neuroblast genes achaete (ac), scute (sc), asense (ase), aPKC, and mira and the cell-cycle regulators stg and CycE. In addition, it was found that the Drosophila homolog of the mammalian B lymphoma Mo-MLV insertion region 1 (Bmi-1) gene, Posterior sex combs, is located near a Prospero binding site. Bmi-1 is a transcription factor that has been shown to regulate the self-renewal of vertebrate hematopoetic stem cells. It is concluded that Prospero is likely to regulate neuroblast identity and self-renewal genes as well as cell-cycle genes directly, repressing their expression in the GMC (Choksi, 2006).
Prospero enters the nucleus of GMCs, and its expression is maintained in glial cells but not in neurons . Therefore the list of targets was searched for genes annotated as glial development genes. Prospero binds near 45% of genes involved in gliogenesis. Among the glial genes, it was found that the master regulator of glial development, glial cells missing (gcm), and gilgamesh (gish), a gene involved in glial cell migration, are both near Prospero binding sites and are likely directly activated by Prospero in glia (Choksi, 2006).
In summary, Prospero binds near, and is likely to regulate directly, genes required for the self-renewing neural stem cell fate such as cell-cycle genes. It was also found that Prospero binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh) and to genes required for glial cell fate. The in vivo binding-site mapping experiments are supportive of a role for Prospero in regulating the fate of Drosophila neural precursors by directly controlling their mitotic potential and capacity to self-renew (Choksi, 2006).
The Drosophila ventral nerve cord develops in layers, in a manner analogous to the mammalian cortex. The deepest (most dorsal) layer of the VNC comprises the mature neurons, while the superficial layer (most ventral) is made up of the mitotically active, self-renewing neuroblasts. Neuroblast cell-fate genes and cell-cycle genes are normally expressed only in the most ventral cells, while Prospero is found in the nucleus of the more dorsally lying GMCs. If in GMCs, Prospero normally acts to repress neuroblast cell-fate genes and cell-cycle genes, then in a prospero mutant, expression of those genes should expand dorsally. Conversely, ectopically expressed Prospero should repress gene expression in the neuroblast layer.
The neuroblast genes mira, ase, and insc and the cell cycle genes CycE and stg show little or no expression in differentiated cells of wild-type stage 14 nerve cords. Expression of these neuroblast-specific genes was examined in the differentiated cells layer of prospero embryos and it was found that they are derepressed throughout the nerve cord of mutant embryos. mira, ase, insc, CycE, and stg are all ectopically expressed deep into the normally differentiated cell layer of the VNC. To check whether Prospero is sufficient to repress these genes, Prospero was expressed with the sca-GAL4 driver, forcing Prospero into the nucleus of neuroblasts. Prospero expression is sufficient to repress mira, ase, insc, CycE, and stg in the undifferentiated cell layer of the VNC. These data, combined with the Prospero binding-site data, demonstrate that Prospero is both necessary and sufficient to directly repress neuroblast genes and cell-cycle genes in differentiated cells. This direct repression of gene expression is one mechanism by which Prospero initiates the differentiation of neural stem cells (Choksi, 2006).
Having shown that Prospero directly represses genes required for neural stem cell fate, it was asked whether Prospero also directly activates GMC-specific genes. Alternatively, Prospero might regulate a second tier of transcription factors, which are themselves responsible for the GMC fate. Of the few previously characterized GMC genes, it was found that Prospero binds to eve and fushi-tarazu (ftz). In the list of targets several more GMC genes were expected to be found, but not genes involved in neuronal differentiation, since Prospero is not expressed in neurons. Surprisingly, however, it was foudn 18.8% of neuronal differentiation genes are located near Prospero binding sites (Choksi, 2006).
To determine Prospero's role in regulating these neuronal differentiation genes, in situ hybridization was carried out on prospero mutant embryos. Prospero was found to be necessary for the expression of a subset of differentiation genes, such as the adhesion molecules FasciclinI (FasI) and FasciclinII (FasII), which have roles in axon guidance and/or fasciculation. Netrin-B, a secreted protein that guides axon outgrowth, and Encore, a negative regulator of mitosis, also both require Prospero for proper expression. Therefore, in addition to directly repressing genes required for neural stem cell self-renewal, Prospero binds and activates genes that direct differentiation. These data suggest that Prospero is a binary switch between the neural stem cell fate and the terminally differentiated neuronal fate (Choksi, 2006).
To test to what extent Prospero regulates the genes to which it binds, genome-wide expression profiling was carried out on wild-type and prospero mutant embryos. While the DamID approach identifies Prospero targets in all tissues of the embryo, in this instance genes regulated by Prospero were assayed in the developing central nervous system. Small groups of neural stem cells and their progeny (on the order of 100 cells) were isolated from the ventral nerve cords of living late stage 12 embryos with a glass capillary. The cells were expelled into lysis buffer, and cDNA libraries generated by reverse transcription and PCR amplification. cDNA libraries prepared from neural cells from six wild-type and six prospero null mutant embryos were hybridized to full genome oligonucleotide microarrays, together with a common reference sample. Wild-type and prospero mutant cells were compared indirectly through the common reference (Choksi, 2006).
In the group of Prospero target genes that contain a Prospero consensus sequence within 1 kb of the transcription unit, 91 show reproducible differences in gene expression in prospero mutants. Seventy-nine percent of these genes (72) exhibit at least a 2-fold change in levels of expression. Many of the known genes involved in neuroblast fate determination and cell-cycle regulation (e.g., asense, deadpan, miranda, inscuteable, CyclinE, and string) show increased levels in a prospero mutant background, consistent with their being repressed by Prospero. Genes to which Prospero binds, but which do not contain an obvious consensus sequence, are also regulated by Prospero: CyclinA and Bazooka show elevated mRNA levels in the absence of Prospero, as does Staufen, which encodes a dsRNA binding protein that binds to both Miranda and to prospero mRNA (Choksi, 2006).
Expression of genes required for neuronal differentiation is decreased in the prospero mutant cells, consistent with Prospero being required for their transcription. These include zfh1 and Lim1, which specify neuronal subtypes, and FasI and FasII, which regulate axon fasciculation and path finding (Choksi, 2006).
The stem cell-like division of neuroblasts generates two daughters: a self-renewing neuroblast and a differentiating GMC. Prospero represses stem cell self-renewal genes and activates differentiation genes in the newly born GMC. In the absence of prospero, therefore, neuroblasts should give rise to two self-renewing neuroblast-like cells (Choksi, 2006).
The division pattern of individual neuroblasts was studied in prospero mutant embryos by labeling with the lipophilic dye, DiI. Individual cells were labeled at stage 6, and the embryos allowed to develop until stage 17. S1 or S2 neuroblasts were examined, as determined by their time of delamination. Wild-type neuroblasts generate between 2 and 32 cells, producing an average of 16.2 cells. Most of the clones exhibit extensive axonal outgrowth. In contrast, prospero mutant neuroblasts generate between 8 and 51 cells, producing an average of 31.8 cells. Moreover, prospero mutant neural clones exhibit few if any projections, and the cells are smaller in size. Thus, prospero mutant neuroblasts produce much larger clones of cells with no axonal projections, suggesting that neural cells in prospero mutants undergo extra divisions and fail to differentiate (Choksi, 2006).
Recently it was shown, in the larval brain, that clones of cells lacking Prospero or Brat undergo extensive cell division to generate undifferentiated tumors. Given that Prospero is nuclear in the GMC but not in neuroblasts, the expanded neuroblast clones in prospero mutant embryos might arise from the overproliferation of GMCs: the GMCs lacking Prospero may divide like neuroblasts in a self-renewing manner. It is also possible, however, that neuroblasts divide more frequently in prospero mutant embryos, giving rise to supernumerary GMCs that each divide only once. To distinguish between these two possibilities, the division pattern of individual GMCs was followed in prospero mutant embryos (Choksi, 2006).
S1 or S2 neuroblasts were labeled with DiI as before. After the first cell division of each neuroblast, the neuroblast was mechanically ablated, leaving its first-born GMC. All further labeled progeny derive, therefore, from the GMC. Embryos were allowed to develop until stage 17, at which time the number of cells generated by a single GMC was determined (Choksi, 2006).
To determine whether mutant GMCs are transformed to a stem cell-like state, stage 14 embryos were stained for the three neuroblast markers: Miranda (Mira), Worniu (Wor), and Deadpan (Dpn). In wild-type embryos at stage 14, the most dorsal layer of cells in the VNC consists mostly of differentiated neurons. As a result, few or none of the cells in this layer express markers of self-renewal. Mira-, Wor-, and Dpn- expressing cells are found on the midline only or in lateral neuroblasts of the differentiated cell layer of wild-type nerve cords. In contrast, a majority of cells in the differentiated cell layer of stage 14 prospero mutant embryos express all three markers: Mira is found cortically localized in most cells of the dorsal layer of prospero nerve cords; Wor is nuclear in most cells of mutant VNCs; Dpn is ectopically expressed throughout the nerve cord of prospero mutants (Choksi, 2006).
Expression of neuroblast markers in the ventral-most layer of the nerve cord (the neuroblast layer), to exclude the possibility that a general disorganization of cells within the VNC contributes to the increased number of Mira-, Wor-, and Dpn-positive cells in the dorsal layer. The number of neuroblasts in a prospero mutant embryo is normal in stage 14 embryos, as assayed by Wor, Dpn, and Mira expression. Thus, the increased expression of neuroblast markers in prospero mutants is the result of an increase in the total number of cells expressing these markers in the differentiated cell layer. It is concluded that prospero mutant neuroblasts divide to give two stem cell-like daughters. GMCs, which would normally terminate cell division and differentiate, are transformed into self-renewing neural stem cells that generate undifferentiated clones or tumors (Choksi, 2006).
Therefore, Prospero directly represses the transcription of many neuroblast genes and binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh), which regulate the timing of cell-fate specification in neuroblast progeny. Prospero maintains hb expression in the GMC, and it has been suggested that this is through regulation of another gene, seven-up (svp). Prospero not only regulates svp expression directly but also maintains hb expression directly. In addition, Prospero maintains Kr expression and is likely to act in a similar fashion on other genes of the temporal cascade. Intriguingly, Prospero regulates several of the genes that direct asymmetric neuroblast division (baz, mira, insc, aPKC). aPKC has recently been shown to be involved in maintaining the self-renewing state of neuroblasts (Choksi, 2006).
Prospero initiates the expression of genes necessary for differentiation. This is particularly surprising since prospero is transcribed only in neuroblasts, not in GMCs or neurons. Prospero mRNA and protein are then segregated to the GMC. Prospero binds near eve and ftz, which have been shown to be downstream of Prospero, as well as to genes required for terminal neuronal differentiation, including the neural-cell-adhesion molecules FasI and FasII. Prospero protein is present in GMCs and not neurons, suggesting that Prospero initiates activation of neuronal genes in the GMC. The GMC may be a transition state between the neural stem cell and the differentiated neuron, providing a window during which Prospero functions to repress stem cell-specific genes and activate genes required for differentiation. There may be few, or no, genes exclusively expressed in GMCs (Choksi, 2006).
Prospero acts in a context-dependent manner, functioning alternately to repress or activate transcription. This implies that there are cofactors and/or chromatin remodeling factors that modulate Prospero's activity. In support of this, although Prospero is necessary and sufficient to repress neuroblast genes, forcing Prospero into the nuclei of neuroblasts is not sufficient to activate all of the differentiation genes to which it binds (Choksi, 2006).
Neuroblasts decrease in size with each division throughout embryogenesis. By the end of embryogenesis, they are similar in size to neurons. A subset of these embryonic neuroblasts becomes quiescent and is reactivated during larval life: they enlarge and resume stem cell divisions to generate the adult nervous system. Neuroblasts in prospero mutant embryos divide to produce two self-renewing daughters but still divide asymmetrically with respect to size, producing a large apical neuroblast and a smaller basal neuroblast-like cell. The daughter may be too small to undergo more than three additional rounds of division during embryogenesis. prospero mutant cells eventually stop dividing, and a small number occasionally differentiate. This suggests that there is an inherent size limitation on cell division. The segregation of Brat, or an additional cell fate determinant, to the daughter cell may also limit the potential of the prospero mutant cells to keep dividing (Choksi, 2006).
The Prox family of atypical homeodomain transcription factors has been implicated in initiating the differentiation of progenitor cells in contexts as varied as the vertebrate retina, forebrain, and lymphatic system. Prospero/Prox generally regulates the transition from a multipotent, mitotically active precursor to a differentiated, postmitotic cell. In most contexts, Prox1 acts in a similar fashion to Drosophila Prospero: to stop division and initiate differentiation (Choksi, 2006).
It is proposed that Prospero/Prox is a master regulator of the differentiation of progenitor cells. Many of the vertebrate homologs of the Drosophila Prospero targets identified in this study may also be targets of Prox1 in other developmental contexts. Prospero directly regulates several genes required for cell-cycle progression, and it is possible that Prox1 will regulate a similar set of cell-cycle genes during, for example, vertebrate retinal development. In addition, numerous Prospero target genes have been identified whose orthologs may be involved in the Prox-dependent differentiation of retina, lens, and forebrain precursors (Choksi, 2006).
Coordination between cell proliferation and cell death is essential to maintain homeostasis in multicellular organisms. In Drosophila, these two processes are regulated by a pathway involving the Ste20-like kinase Hippo (Hpo) and the NDR family kinase Warts (Wts; also called Lats). Hpo phosphorylates and activates Wts, which in turn, through unknown mechanisms, negatively regulates the transcription of cell-cycle and cell-death regulators such as cycE and diap1. Yorkie (Yki), the Drosophila ortholog of the mammalian transcriptional coactivator yes-associated protein (YAP), has been identified as a missing link between Wts and transcriptional regulation. Yki is required for normal tissue growth and diap1 transcription and is phosphorylated and inactivated by Wts. Overexpression of yki phenocopies loss-of-function mutations of hpo or wts, including elevated transcription of cycE and diap1, increased proliferation, defective apoptosis, and tissue overgrowth. Thus, Yki is a critical target of the Wts/Lats protein kinase and a potential oncogene (Huang, 2005).
Activation of yki leads to increased transcription of diap1 and CycE. The increased cell proliferation and decreased apoptosis resulting from yki overexpression are strikingly similar to those caused by loss of hpo, sav, or wts, suggesting that Yki functions in the Hpo pathway. To further explore this possibility, the transcription of cell-death inhibitor diap1 and cell-cycle regulator cycE, known targets of the Hpo pathway were examined. Elevated DIAP1 protein is detected in yki-overexpressing clones in the eye discs. This regulation is largely mediated at the level of diap1 transcription since the expression of thj5c8, a P[lacZ] enhancer trap reporter inserted into the diap1 locus, is similarly elevated in yki-overexpressing clones in a cell-autonomous manner. A cycE-lacZ reporter containing 16.4 kb of the 5′ regulatory sequence of cycE is also increased in yki-overexpressing clones, especially those close to the MF, although the effect is less profound than that observed with the diap1 reporter. Thus, like loss of hpo, sav, or wts, overexpression of yki results in increased transcription of diap1 and cycE. It is worth noting that previous analyses of hpo mutant clones also revealed a 'tighter' regulation of diap1: while diap1 transcription is elevated in all hpo mutant cells irrespective of their relative position to the MF, cycE transcription is only elevated in hpo mutant cells close to the MF (Wu, 2003). These observations suggest that diap1 might represent a more direct transcriptional target of the Hpo pathway (Huang, 2005).
Appropriate cell number and organ size in a multicellular organism are determined by coordinated cell growth, proliferation, and apoptosis. Disruption of these processes can cause cancer. Recent studies have identified the Large tumor suppressor (Lats)/Warts (Wts) protein kinase as a key component of a pathway that controls the coordination between cell proliferation and apoptosis. Growth inhibitory functions are described for a Mob superfamily protein, termed Mats (Mob as tumor suppressor), in Drosophila. Loss of Mats function results in increased cell proliferation, defective apoptosis, and induction of tissue overgrowth. Mats and Wts function in a common pathway. Mats physically associates with Wts to stimulate the catalytic activity of the Wts kinase. A human Mats ortholog (Mats1) can rescue the lethality associated with loss of Mats function in Drosophila. Since Mats1 is mutated in human tumors, Mats-mediated growth inhibition and tumor suppression is likely conserved in humans (Lai, 2005).
Cyclin E, a key regulator for the G1-S transition, is normally expressed in the second mitotic wave (SMW) of larval eye discs. In mats mosaic eye discs, Cyclin E levels are elevated in mutant clones located in the morphogenetic furrow (MF) and SMW regions. Moderate upregulation of Cyclin A and Cyclin B expression is also observed. Thus, an important mechanism for mats to control cell proliferation is to negatively regulate expression of key cell cycle regulators such as Cyclins. Interestingly, Cyclin E expression in mutant cells immediately anterior to the MF is much less elevated than that immediately posterior to the MF, suggesting that mats may use a different mechanism to restrict cell proliferation in cells anterior to the MF. The cell proliferation defects observed in mats mutants are similar to those caused by sav, wts, and hpo mutations (Lai, 2005).
A coordinate program of transcription of S-phase genes (DNA polymerase alpha, Proliferating cell nuclear antigen and the two ribonucleotide reductase subunits) can be induced by Cyclin E (the G1 cyclin). In Drosophila embryos, this program drives an intricate spatial and temporal pattern of gene expression that perfectly parallels the embryonic program of S-phase control. This dynamic pattern of expression is not disrupted by a string mutation that blocks the cell cycle. Thus, the transcriptional program is not a secondary consequence of cell cycle progression. It is likely that developmental signals control this transcriptional program and that its activation either directly or indirectly drives transition from G1 to S phase in the stereotyped embryonic pattern (Durano, 1995a).
The onset of cyclin expression in endoreduplicating tissues is not dependent on Cyclin E function, as Cyclin E expression is observed in CycE mutants. However, instead of being terminated rapidly, the expression is maintained in CycE mutants. Thus functional Cyclin E protein is required directly or indirectly to shut off its own expression. Consistent with this notion, it is found that overexpression of Cyclin E down-regulates the endogenous CycE transcription (Sauer, 1995).
To test if the cell cycle transcription of double parked (dup) is dependent on
E2F, embryos homozygous for a null allele of the dE2F1 subunit, dE2F91 were collected and hybridized with
dup riboprobes. The levels of dup transcript are
decreased in the endoreplicating gut and appear to be slightly
decreased in the CNS. A similar effect on dup
transcript is seen in embryos that are homozygous mutant for the
other subunit of the E2F transcription factor, of the genotype
dDPa2. Thus, dup is a downstream
target of the E2F transcription factor. Interestingly, yeast cdt1
transcription is also cell-cycle regulated. Expression of cdt1
is controlled by the G1-S transcription factor Cdc10 that, like E2F,
regulates transcription of many genes required for S phase (Hofmann, 1994). This suggests that cell cycle control of dup may
be conserved, and Dup may prove to be an important downstream target of
E2F in mammalian cells (Whittaker, 2000).
Cyclin E is required to regulate positively the transcription of S
phase genes in the nervous system and to downregulate these transcripts
in endo cycling cells.
The cyclinEl(2)305 and cyclin
EPZ5 mutations and a cyclin E deficiency,
Df(2L)TE35D1, have similar effects on double parked
transcripts. In these embryos, dup is not downregulated properly in the endoreplicating gut such that dup transcripts persist at higher levels than wild type in the anterior midgut, central midgut, and posterior midgut in later embryonic stages. In cyclin E mutant embryos dup
transcripts are reduced in the CNS, although not to as great an extent
as other S phase genes. Thus, dup expression also is regulated
by cyclin E (Whittaker, 2000).
The Cip/Kip family of cyclin-dependent kinase inhibitors (CKIs) has been implicated in mediating cell cycle arrest prior to terminal
differentiation. In many instances, increased expression of CKIs immediately precedes mitotic arrest. However, the mechanism that activates
CKI expression in cells that are about to stop dividing has remained elusive. This issue was addressed by investigating the
expression pattern of dacapo, a Cip/Kip CKI in Drosophila. The accumulation of Dacapo RNA and protein requires Cyclin
E and ectopic expression of Cyclin E can induce dacapo expression. The oscillation of the Cyclin E and Dacapo
proteins is tightly coupled during ovarian endocycles. These results argue for a mechanism where Cyclin E/Cdk activity induces Dacapo
expression but only within certain windows that are permissive for dacapo expression (de Nooij, 2000).
In a number of different tissues, expression of Dap occurs
precisely during the last mitotic division that cells undergo
before they terminally differentiate. For instance, in the
embryonic epidermis, a rapid accumulation of DAP mRNA
and Dap protein is detected only following S-phase 16,
just before these cells arrest in the G1-phase of cycle 17. Exit from the
cell cycle also requires alterations in the levels of Cyclins
and the activity of Cyclin-Cdk complexes. Is the
induction of dap directly regulated by the developmental
cues that dictate cell cycle exit? Or alternatively, is the
induction of DAP RNA and protein a response to an alteration in the activity of one of the other cell cycle regulators (de Nooij, 2000)?
To help distinguish between these possibilities, whether alterations in the levels of one of the
known cell cycle regulators would affect the normal pattern
of Dap expression was examined in different embryonic tissues. Embryos that were mutant for the G1-S regulators, cyclin E, E2F1 and DP and for the regulators that
mediate the G2-M transition, string (stg), cyclin A, rca1,
cyclin B and cdc2 were examined. With the notable exception of cyclin E mutants, no obvious abnormalities in the Dap expression
pattern could be observed in any of these mutants. Analysis of cyclin E (cycE) mutant embryos, however, shows significant differences in the
expression pattern of Dap. Dap protein levels are severely
reduced in the cells of the PNS and are almost absent in the
cells of the CNS of embryos homozygous or hemizygous for
the cycE null allele cycEAr95. The level of DAP
RNA is also reduced in the cells of the PNS and the CNS. This suggests that Cyclin E may regulate dap expression at least in part at the transcriptional level. Immunostaining of cycE mutant embryos with an anti-Elav antibody does not show significant abnormalities in the cellularity of the PNS and CNS,
suggesting that the loss of dap expression is not a secondary
consequence of a failure to form these tissues (de Nooij, 2000).
Interestingly, no reduction in Dap expression levels is
observed in the epidermis of cycE mutant embryos. However, cycE mutants have been shown to
complete all 16 epidermal divisions normally, possibly
due to the activity of residual maternal supplies of Cyclin
E. It is therefore likely that the residual supply of Cyclin E may also be sufficient to trigger Dap
expression in the epidermis. Alternatively, the apparently
normal expression of Dap in the cycE mutant epidermal
cells may reflect a difference between the mechanisms
that regulate Dap expression in the epidermis and in the
nervous system (de Nooij, 2000).
These results also show that normal Dap expression is not
contingent upon progression through the cell cycle.
Embryos mutant for string, which encodes a Cdc25-like
phosphatase, arrest in the G2 phase of cycle 14. Despite
the absence of S-phase progression and cell division in
string mutants, the onset of Dap expression occurs at the
appropriate developmental stage in both the epidermis and
the cells of the nervous system. These observations also show that Dap expression is not normally contingent upon cell cycle progression or on reaching cycle 16 in
the epidermis (de Nooij, 2000).
The reduced level of dap RNA in cycE mutant embryos
seems to indicate a role for Cyclin E in the transcriptional
regulation of dap. A potential mechanism for the transcriptional regulation of dap by Cyclin E could involve the E2F
transcription factor. It has been demonstrated previously
that E2F can mediate the activation of a 'G1-S transcriptional program' that, in the CNS, is dependent upon cyclin E
function. E2F can also activate stg expression during G2. However, while E2F1 and DP mutant embryos
clearly show a reduction in the RNA levels of the E2F
responsive genes PCNA and RNR2, Dap expression appears to be normal
in both E2F and DP mutant embryos (de Nooij, 2000).
Cyclin E is also expressed normally in the CNS in E2F
and DP mutant embryos. Thus, a redundant function for E2F in regulating dap expression cannot be ruled out, these experiments do not provide any evidence for E2F as a regulator of dap expression. Taken together, these results show that when Cyclin E activity is abolished in the nervous system, Dap expression is no longer observed thus indicating a requirement for Cyclin E activity in regulating Dap expression. This activity of Cyclin E does not appear to involve the activity of the E2F transcription factor. Moreover, Dap expression in either the epidermis or the nervous system is not contingent upon cell cycle progression (de Nooij, 2000).
In both eye and wing imaginal discs, ectopic Cyclin E can induce ectopic
expression of Dap. To determine whether the induction of Dap is due to an
increase in DAP RNA levels, RNA levels of
DAP were examined in whole-mount preparations by in situ hybridizations in
eye and wing discs in which Cyclin E was ectopically
expressed. In contrast to the dramatic increase in Dap
protein, relatively modest increases in the levels of DAP
RNA are observed in both the eye and wing discs. Nonetheless, these results are consistent with the notion that Cyclin E regulates DAP RNA levels, and furthermore, suggest that dap transcription may be responsive to
Cyclin E levels (de Nooij, 2000).
To test whether dap regulatory elements are responsive
to Cyclin E levels, several lines of transgenic flies were generated that contain a 2.7 kb fragment of the dap promoter region linked to a beta-galactosidase reporter. Immunohistochemical analysis of wing discs from flies
that contain the pCasdap2.7kb-lacZ transgene either
alone, or in the presence of the enGAL4 driver, shows no
significant lacZ staining in the posterior compartment of
the wing disc. In the presence of both the enGAL4 driver and a UAScycE transgene, increased beta-galactosidase activity is clearly detected in the region where Cyclin E is expressed. Thus Cyclin E can activate the expression of a reporter gene under the control of dap regulatory elements (de Nooij, 2000).
Since Cyclin E appears to be a requirement for the
expression of the Dap protein, Dap expression was examined
in the context of oscillating levels of Cyclin E in the nurse
cell nuclei in the ovary. Oogenesis normally starts with four
mitotic germ cell divisions which generate a 16-cell cyst.
Surrounded by somatically-derived follicle cells, each cyst
forms an individual egg chamber that ultimately gives rise
to a mature egg. One of the 16-germ cell nuclei arrests in the prophase of
meiosis I, becomes transcriptionally silent and is specified
as the oocyte nucleus. The remaining 15 cells, the nurse cells, proceed
through a series of endocycles. In wildtype ovaries, Dap expression is first detected in the germ cell nuclei in the germarium at a time when the four mitotic cyst cell divisions are about to be completed (region 2A of oogenesis). In the maturing egg chambers Dap is detected in the endocycling nurse cell nuclei at levels which, within an individual egg chamber, vary from very high to undetectable. This is likely to reflect an oscillation in the level of Dap protein during the endocycles, as has been postulated for Cyclin E. In contrast to the nurse cell nuclei, high levels of Dap are observed in the oocyte nucleus throughout oogenesis (de Nooij, 2000).
Consistent with the immunostaining, DAP RNA can be
observed in the germarium early in oogenesis, and subsequently, high levels of DAP RNA are found in the future oocyte throughout oogenesis. However, the
pattern of DAP RNA expression in the nurse cells differs
significantly from the pattern of Dap protein. In contrast
to the dramatic oscillation of Dap protein levels in the
nurse cell nuclei, no oscillation of DAP RNA is evident.
Only a uniform low level of RNA can be detected in the
endocycling nurse cells. Although relative differences may exist but may be beyond the level of detection, this difference in the expression pattern between DAP RNA and protein suggests that post-transcriptional mechanisms may regulate the levels of Dap protein in the individual nurse cell nuclei (de Nooij, 2000).
The expression pattern of Dap protein is reminiscent of
the expression pattern of Cyclin E in the ovary, i.e. a strong
oscillation in the nurse cell nuclei, and a persistently high
level of protein in the oocyte nucleus. Confocal images of ovaries double labeled with antibodies against both Cyclin E and Dap show that the expression of Cyclin E and Dap are largely overlapping. However, some of the nurse cell nuclei in the individual egg chambers do differ in the relative levels of Cyclin
E and Dap protein, suggesting that the oscillations of Cyclin E and Dap are slightly out of phase. To assess the regulatory relationship between Cyclin E
and Dap in the nurse cells more directly, Dap expression in ovaries was obtained from females homozygous for the cycEfs(2)01672
allele. This allele of cycE specifically
perturbs the oscillation of Cyclin E in the nurse cell nuclei
and hence ovaries from homozygous cycEfs(2)01672
females show a severe reduction in the amplitude of Cyclin E oscillation. Interestingly, these mutant ovaries also show a strong reduction in Dap oscillation, and most nurse cell nuclei show intermediate levels of
Dap expression; nuclei with high or undetectable levels of
Dap were almost never observed in mutant egg chambers.
Thus in the nuclei of the endocycling nurse cells, expression of Cyclin E and Dap appears to be tightly linked. Dap protein levels oscillate strongly and these oscillations are dampened by a reduction in the extent of Cyclin E oscillation. Since DAP RNA levels in the nurse cells seem to remain relatively constant during oogenesis, it appears likely, that in these cells, Cyclin E controls the level of Dap protein predominantly by post-transcriptional mechanisms (de Nooij, 2000).
The regulation of dap at the transcriptional level seems
unlikely to account fully for the dynamic expression pattern
of Dap protein. In the situations where Cyclin E was overexpressed, the increase in Dap protein is much more dramatic than the increase in DAP RNA. Likewise, in the ovary, dramatic oscillations in Dap levels are observed without
appreciable fluctuations in the levels of DAP RNA. Thus,
in these situations, Cyclin E seems to regulate dap at a
post-transcriptional level. This is perhaps reminiscent of the type of regulation observed for mammalian p27. Accumulation of p27 has been shown to depend largely on an increased translation of p27 mRNA. Although mammalian Cyclin E has, so far, not been implicated in the translational regulation of p27 or any of the other CKIs, such a mechanism could potentially operate in mammalian cells. Another mode of post-transcriptional regulation that may involve Cyclin E activity is a regulation at the level of protein stability (de Nooij, 2000).
Cdc2c kinase, homologous to vertebrate cdk2 kinase and distinct from Drosophila cdc2 (the cyclin dependent kinase associated with Cyclin A and Cyclin B), co-immunoprecipitates with Cyclin E (Knoblich, 1994).
In mammalian systems cyclin E targets Retinoblastoma protein, a negative regulator of entry into S phase. RB associates with E2F in humans, thus rendering it inactive. Whereas the ectopic expression of cyclin E activates Drosophila E2F-dependent transcription, cyclin E does not act directly on E2F but targets, by phosphorylation, a negative regulator of E2F activity. Such a regulator might be analogous to the family of RB-related proteins (pRB, p107, and p130) that physically associates with E2F in humans. Drosophila RB family homolog (RBF) combines several of the structural features of pRB, p107, and p130, suggesting that it may have evolved from a common ancestor to the three human genes. RBF associates with Drosophila E2F and DP in vivo and is a stoichiometric component of E2F DNA-binding complexes. RBF specifically repressed E2F-dependent transcription and suppressed the phenotype generated by ectopic expression of dE2F and dDP in the developing Drosophila eye. RBF is phosphorylated by a cyclin E-associated kinase in vitro, and loss-of-function cyclin E mutations enhanced an RBF overexpression phenotype, consistent with the idea that the biological activity of RBF is negatively regulated by endogenous cyclin E. The properties of RBF suggest that it is the intermediary factor that was proposed to allow cyclin E induction of E2F activity. These findings indicate that RBF plays a critical role in the regulation of cell proliferation in Drosophila (Du, 1996).
Drosophila fizzy-related down-regulates mitotic cyclins and is required for cell proliferation arrest and entry into endocycles. In yeast, inactivation of mitotic cyclins results in acquisition of compentence to initiate another round of DNA replication. The subsequent reactivation of B-type cyclin at the G1/S transition triggers initiation of DNA replication in parallel with a reorganization of protein complexes at origins of replication. fizzy-related (fzr), a conserved eukaryotic gene, negatively regulates the levels of cyclins A, B, and B3. These mitotic cyclins that bind and activate cdk1(cdc2) are rapidly degraded during exit from M and during G1. While Drosophila fizzy has previously been shown to be required for cyclin destruction during M phase, fzr is required for cyclin removal during G1 when the embryonic epidermal cell proliferation stops and during G2 preceding salivary gland endoreduplication. Loss of fzr causes progression through an extra division cycle in the epidermis and inhibition of endoreduplication in the salivary gland, in addition to failure of cyclin removal. Conversely, premature fzr overexpression down-regulates mitotic cyclins, inhibits mitosis, and transforms mitotic cycles into endoreduplication cycles. The coincidence of mitotic cyclin disappearance and cyclinE/cdk2 inactivation during G1 arrest raises the possibliity that fzr activity might be inhibited by cyclinE/cdk2. fzr and fizzy encode highly similar proteins with seven WD repeats in the C-terminal region. WD repeats are found in budding yeast Cdc4p, which is required for the ubiquitin-dependent proteolysis of several cell cycle regulators. The closest yeast relative of fzr, however, is not CDC4 but HCT1, which is required for proteolysis of Clb2p, a budding yeast B-type cyclin with a characteristic destruction box. However, Drosophila fzr is unable to provide HCT1 function in yeast. Thus, fzr transcripts accumulate when cells become postmitotic and fzr is required in proliferating cells progressing through cell cycles with G1 phases and in G2 before endoreduplication, but not during mitosis (Sigrist, 1997).
Minichromosome maintenance (MCM) proteins are essential DNA replication factors conserved among eukaryotes. Three Drosophila MCM proteins have been characterized: DmMCM2, DmMCM4, and DmMCM5. MCMs cycle between chromatin bound and dissociated states during each cell cycle. Cyclin:cdks can prevent an assembly of proteins called the "prereplicative complex" on origins of DNA replication. The prereplicative complexes are thought to contain MCMs. Their absence from chromatin is thought to contribute to the inability of the post S phase nucleus to replicate DNA. Passage through mitosis restores the ability of MCMs to bind chromatin and the ability to replicate DNA. In Drosophila early embryonic cell cycles, which lack a G1 phase, MCMs reassociate with condensed chromosomes toward the end of mitosis. To explore the coupling between mitosis and MCM-chromatin interaction, a test was carried out as to whether this reassociation requires mitotic degradation of cyclins. Arrest of mitosis by induced expression of nondegradable forms of cyclins A and/or B shows that reassociation of MCMs to chromatin requires cyclin A destruction but not cyclin B destruction. In contrast to the earlier mitoses, mitosis 16 (M16) is followed by G1, and MCMs do not reassociate with chromatin at the end of M16. Thus MCM-chromosome association is delayed when mitosis is followed by a prolonged G-1 phase. dacapo mutant embryos lack an inhibitor for cyclin E, do not enter G1 quiescence after M16, and show mitotic reassociation of MCM proteins. It is proposed that cyclin E, inhibited by Dacapo in M16, promotes chromosome binding of MCMs. Thus, it is suggested that cyclins have both positive and negative roles in controlling MCM-chromatin association (Su, 1997).
During Drosophila development and mammalian embryogenesis, exit from the cell
cycle is contingent on tightly controlled downregulation of the activity of
Cyclin E-Cdk2 complexes that normally promote the transition from G1 to S phase.
Although protein degradation has a crucial role in downregulating levels of
Cyclin E, many of the proteins that function in degradation of Cyclin E have not
been identified. In a screen for Drosophila mutants that display increased cell
proliferation, archipelago, a gene encoding a protein with an
F-box and seven tandem WD (tryptophan-aspartic acid) repeats, has been identified. archipelago mutant cells have persistently elevated levels of Cyclin E protein without increased levels of cyclin E RNA. They are under-represented in G1
fractions and continue to proliferate when their wild-type neighbors become
quiescent. The Archipelago protein binds directly to Cyclin E and probably
targets it for ubiquitin-mediated degradation. A highly conserved human
homolog is present and is mutated in four cancer cell lines including three of ten derived from ovarian carcinomas. These findings implicate archipelago in developmentally regulated degradation of Cyclin E and potentially in the pathogenesis of human cancers (Moberg, 2002).
To identify genes that restrict cell numbers and tissue growth
during development, a genetic screen was conducted to identify
recessive mutations that gave homozygous mutant cells even a
subtle proliferative advantage over their wild-type neighbors.
Clones of homozygous mutant tissue (marked white) were generated in the eyes of heterozygous flies and their size was compared
with the wild-type twin spots generated from the same recombination events. Flies were retained in which there was more mutant than
wild-type tissue. Of more than 23 loci identified in the screen, multiple alleles were obtained of homologs of several known human
tumor-suppressor genes, including TSC1, TSC2 and PTEN. A
previously unknown locus, represented by a lethal complementation group consisting of three alleles, was named archipelago (Moberg, 2002).
Compared with an unmutagenized control, adult eyes mosaic for
mutations in ago were composed mostly of mutant tissue. Within ago mutant clones, most ommatidial clusters lacked the
wild-type complement of photoreceptor cells, and the distance
between adjacent photoreceptor clusters was increased.
Staining of apical cell profiles during pupal eye development
showed that the enlarged interommatidial spaces in ago mutant
clones contain excess cells. TUNEL revealed no significant decrease in the extent of cell death in ago mutant clones. Moreover, co-expression of the baculovirus p35 protein, which blocks caspase-dependent cell death, resulted in a further increase in the number
of interommatidial cells. These data suggest
that loss of ago leads to increased cell proliferation that is partially offset by apoptosis (Moberg, 2002).
Using meiotic and deletion mapping, ago was localized to position 64B on the left arm of chromosome 3. Sequencing candidate
transcription units has demonstrated that all three ago alleles (ago1, ago3, ago4) have mutations in an open reading frame designated
CG15010. ago encodes a 1,326-amino-acid protein that
contains an F-box and seven WD repeats in its carboxy-terminal
portion. F-boxes and WD motifs are found in proteins that
function as the substrate-recognition component of SCF-type
ubiquitin ligase complexes. Within the C-terminal region, the
Ago protein is most similar to that encoded by an amino-terminally
truncated human expressed sequence tag (EST) previously designated FLJ11071, which has been renamed human Ago. This protein is referred to as hCdc4. A more distant relative is the Caenorhabditis elegans gene
sel-10. Two of the mutations are missense mutations that map to the fourth WD repeat.
Each alters a residue that is conserved among the three proteins. The third mutation would
result in premature termination of translation early in the sixth WD
repeat. As the WD repeats of F-box proteins are thought to be
required for recruitment of substrates to the SCF complex, it is
possible that the alleles recovered in the screen may impair the
interaction of Ago with specific substrates (Moberg, 2002).
Because ago mutant cells proliferate more than wild-type cells, it
seemed likely that ago mutations would result in increased levels of a
positive regulator of the cell cycle. Clones of homozygous mutant tissue were generated in eye imaginal discs and examine them
for changes in cyclin levels. In wild-type third-instar
discs, Cyclin E is expressed at varying levels and is
unpatterned in cells anterior to the morphogenetic furrow, which
is thought to correlate with expression at specific stages of the cell
cycle in cells that are proliferating asynchronously. A strong stripe of expression can be found immediately posterior to the furrow,
which corresponds to cells of the second mitotic wave. Cells
posterior to the second mitotic wave express low levels of Cyclin
E. In clones of ago mutant tissue anterior to the furrow, almost all
cells express high levels of Cyclin E. Clones posterior to
the furrow display mild elevations in Cyclin E levels. In contrast to
the increase in Cyclin E protein, no alteration in the expression
pattern of cyclin E RNA is observed in discs that contain many large
ago mutant clones. In wild-type discs, ago mRNA is also
expressed throughout the disc, but is expressed at particularly high
levels in the morphogenetic furrow. In contrast to the
results obtained with Cyclin E, the levels of Cyclin B, Cyclin A and the SCF substrates Cubitus
interruptus, Armadillo and Tramtrack are not appreciably elevated
in ago mutant clones in the larval imaginal disc, nor are the levels of
the putative substrates Dacapo and the intracellular domain of
Notch (Moberg, 2002).
Because Cyclin E promotes S-phase entry, an increase in the level
of Cyclin E can perturb regulation of the cell cycle. To examine the
proliferative properties of ago mutant cells, ago clones were generated
in the wing disc of third-instar larvae and their DNA
content was compared with that of wild-type cells from the same imaginal discs. In mutant clones, a smaller fraction of cells (21.4%) is
found in G1 when compared with wild-type cells (36.8%). The
proportion of cells found in S phase and in G2/M is increased. These
alterations are extremely similar to those elicited by the overexpression of Cyclin E (Moberg, 2002).
The effect of ago mutations on the patterns of
cell proliferation in vivo was also examined. Cells anterior to the morphogenetic furrow in the larval eye disc proliferate asynchronously. It is therefore difficult to visualize differences in rates of cell proliferation in
mutant clones at this stage of eye development. In contrast, very
few cells proliferate in the wild-type pupal retina. The bristle
precursor cell is the only mitotically active cell type detected
during this stage; it divides twice during the mid-pupal phase to
generate the four cells of the 'bristle complex'. Levels of Cyclin E
rapidly decrease after these divisions. In ago mutant clones, elevated
levels of Cyclin E are detected in the four cells of the bristle complex
well after the levels in the corresponding cells of adjacent wild-type
ommatidia have declined. Some of these ago mutant
cells also continue to incorporate 5-bromodeoxyuridine (BrdU),
suggesting that they continue to cycle after the corresponding cells
in adjacent wild-type tissue have exited from the cell cycle.
Such additional divisions are likely to contribute to the increased
number of interommatidial cells observed in the pupal retina. Thus, the persistence of Cyclin E in ago mutant cells disrupts exit from the
cell cycle in a manner similar to that elicited by Cyclin E
overexpression (Moberg, 2002).
The simplest explanation of the role of ago in cell cycle control is
that Ago binds to Cyclin E and targets it for ubiquitin-mediated
degradation. Genetic and physical interactions between Ago and Cyclin E were therefore sought. A genetic interaction was observed
between ago and the cyclin EJP
allele, which reduces the levels of
cyclin E transcription in the developing eye. The rough-eye
phenotype of cyclin EJP
flies is suppressed in flies that are also
heterozygous for a mutation in ago. In addition, ago
mutations dominantly suppress the small-eye phenotype produced
by eyGAL4-driven overexpression of the cyclin-dependent kinase
inhibitor dacapo, which has been shown to reduce
Cyclin E-Cdk2 activity. Thus flies that are heterozygous for mutant
alleles of ago are likely to have increased levels of Cyclin E (Moberg, 2002).
To test for a direct physical interaction between Archipelago and
Cyclin E, the portion of Archipelago containing the
F-box and WD repeats was expressed as a protein fused to glutathione S-transferase (GST: GST-AgoDeltaN) and its ability to bind Cyclin E
protein was evaluated in lysates of S2 cells transfected with cyclin E and cdk2. Versions of GST-AgoDeltaN were generated that contained the mutations found in the ago1 and ago3
alleles. Binding was readily detected
with the wild-type version of GST-AgoDeltaN and was greatly reduced
with both mutant versions. Thus the ability of Archipelago
to bind Cyclin E in vitro correlates with its ability to downregulate
Cyclin E levels in vivo (Moberg, 2002).
These findings, together with the observation that mutations in the C. elegans genes cul1 and lin-23 (which encode a cullin and an F-box protein respectively) have increased cell divisions, highlight the importance of SCF-mediated degradation in regulating cell proliferation
through Cyclin E. Because ago RNA is expressed in a dynamic
pattern, these results indicate that degradation of Cyclin E is not
constitutive in vivo. Dynamic expression of Ago provides another
mechanism by which cyclin/cdk activity and cell proliferation can
be regulated during development. Finally, impaired proteolysis of Cyclin E is implicated in the pathogenesis of human cancers (Moberg, 2002).
Cullins are the major components of a series of multimeric
ubiquitin ligases that control the degradation of a broad range of
proteins. The ubiquitin-like protein, Nedd8, covalently modifies members of the Cullin family. Nedd8 modifies Cullin 1 (Cul1, also known as Lin-19-like or simply Lin-19) in Drosophila. In mutants of
Drosophila Nedd8 and Cul1, levels of the signal transduction effectors, Cubitus interruptus (Ci) and
Armadillo, and the cell cycle regulator, Cyclin E (CycE), are
unusually high, suggesting that the Cul1-based multimeric SCF ubiquitin ligase complex requires Nedd8 modification for the degradation processes of Ci, Arm, and CycE in vivo. Whether Nedd8 affects the protein level of CycE was examined. Levels of CycE are regulated by the F-box protein, Archipelago. CycE accumulates in Nedd8 mutant cells in the eye disc. These results suggest that Nedd8 might affect the stability of a broad range of proteins through F-box
proteins in flies (Ou, 2002).
Cyclin E-Cdk2 is essential for S phase entry. To identify genes interacting with cyclin E, a genetic screen was carried out using a hypomorphic mutation of Drosophila cyclin E (DmcycEJP), which gives rise to adults with a rough eye phenotype. Among the dominant suppressors of DmcycEJP, brahma (brm) and moira (mor) were identified. These genes encode conserved core components of the Drosophila Brm complex that is highly related to the SWI-SNF ATP-dependent chromatin remodeling complex. Mutations in genes encoding other Brm complex components, including snr1 (BAP45), osa and deficiencies that remove BAP60 and BAP111 can also suppress the DmcycEJP eye phenotype. Brm complex mutants suppress the DmcycEJP phenotype by increasing S phases without affecting DmcycE protein levels. DmcycE physically interacts with Brm and Snr1 in vivo. These data suggest that the Brm complex inhibits S phase entry by acting downstream of DmcycE protein accumulation. The Brm complex also physically interacts weakly with Drosophila retinoblastoma (Rbf1), but no genetic interactions were detected, suggesting that the Brm complex and Rbf1 act largely independently to mediate G1 arrest (Brumby, 2002).
Several recent studies have provided strong connections between metazoan SWI- SNF complexes and regulation of the cell cycle. In yeast, the SWI- SNF complex is not essential for viability, and whole genome analyses of swi/snf mutants have shown roles in activation and repression of transcription. A screen for modifiers of E2F1/DP function in Drosophila identified new alleles of brm and mor as enhancers of the rough eye phenotype associated with ectopic expression of E2F1 and DP in the developing Drosophila eye imaginal disc. In support of this, mammalian homologs of Brm and Mor (hBrm/Brg1 and BAF55, respectively) have been reported to be present in cyclin E complexes and to be phosphorylated by cyclin E- Cdk2. Significantly, human homologs of Brm (hBrm and Brg1) inhibit entry into S phase and achieve this at least in part by cooperation with the tumor suppressor Rb. Furthermore, Rb can bind to Brg1 and hBrm, and the ability of Rb to induce G1 arrest has been shown to depend upon hBrm and Brg1 (Brumby, 2002 and references therein).
The genetic interactions with DmcycE or E2F1/DP and Brm complex genes initially were thought to be due to effects on DmcycE transcription or E2F/DP-dependent transcription, given the role of the Brm complex in transcriptional regulation. Surprisingly, the results of this study suggest that the Brm complex functions downstream of DmcycE transcription and protein accumulation. (1) No significant effect on DmcycE protein levels in DmcycEJP eye discs was observed when the dosage of brm or mor was halved. (2) The rough eye phenotype due to overexpression of DmcycE from the GMR driver is enhanced by halving the dosage of brm and mor, indicating that Brm and Mor act to inhibit S phase entry downstream of DmcycE transcription. (3) DmcycE forms a complex with Brm and Snr1. Taken together, these data provide strong evidence that the Brm complex does not inhibit the G1 to S phase transition by acting to down-regulate DmcycE transcription (Brumby, 2002).
It is also likely that the Brm complex does not act to down-regulate E2F1/DP-dependent gene transcription, since no effect was observed for at least two E2F1/DP targets in brm mutants. Thus, mutations in Brm complex genes suppress the DmcycEJP mutant phenotypes by allowing progression into S phase without increasing either DmcycE protein levels or the expression of E2F1/DP-dependent genes. This suggests that one function of the Drosophila Brm complex is to restrict entry into S phase by inhibiting DmcycE-Cdk2 activity or by acting downstream of DmcycE-Cdk2 function. A function for Brm downstream of DmcycE-Cdk2 is consistent with reports that mammalian cyclin E can bind to and phosphorylate components of the Brm complex and thereby inactivate it. Thus the Brm complex may be acting as a curb to S phase entry that needs to be overcome by phosphorylation and inactivation by cyclin E-Cdk2 (Brumby, 2002).
Consistent with studies in cultured mammalian cells, the Rbf1 protein was found to be present in complexes with Brm or Snr1 in larval and embryonic extracts. However, in embryos, only a small portion of total cellular Rbf1 is present in Snr1 immunoprecipitates, in contrast to a significant fraction of the cellular DmcycE, suggesting that most Brm complexes do not contain Rbf1. The observation that Drosophila Rbf1 and Brm form a complex in vivo is consistent with studies in mammalian cells showing that hBrm and/or Brg1 can bind to and cooperate with Rb in transcriptional repression, and that hBrm and Brg1 are required for Rb-induced G1 arrest. However, in Drosophila, no clear evidence was obtained for cooperation of brm or mor with rbf1 in S phase entry. It is possible that the phenotypes being examining were not sensitive enough for S phase effects to be observed. However, the lack of a strong effect of Brm complex mutants on the rbf1 mutant S phase phenotype, when strong genetic interactions were observed with Brm complex genes and DmcycE, suggests that Rbf1 and Brm primarily function independently in negatively regulating S phase entry. Therefore, the suppression of the S phase defect of DmcycEJP by Brm complex mutants may not involve rbf1. Independent roles for Brm and Rb are also likely in mammalian cells since Rb knockout mice have a different mutant phenotype from that of Brg1 or Brm knockouts (Brumby, 2002).
In mammalian cells, Rb can form a complex containing both Brg1 and Hdac1, which is required to repress DmcycE transcription and may also have a role at replication origins. However, reducing the dose of the Drosophila Hdac gene, rpd3, did not suppress the DmcycEJP rough eye phenotype. It is possible that no interaction was observed for rpd3 and DmcycE, because there are a least three other Hdacs in flies that may perform overlapping functions with rpd3. However, mutations in sin3a, which encodes a Hdac-interacting protein, enhance the DmcycEJP rough eye phenotype, suggesting that Sin3a functions in opposition to Brm in regulating DmcycE or S phase entry. Further studies using specific mutations in other Drosophila Hdacs, and Hdac-interacting proteins are required to analyze further their role in the G1 to S phase transition (Brumby, 2002).
How does the Brm complex mediate negative regulation of the G1 to S phase transition? The results suggest that the Brm complex is playing a role independent of DmcycE transcription and E2F/DP-dependent transcription in negatively regulating the G1 to S phase transition. One way in which this may occur is by transcriptional regulation of other critical G1/S phase genes. For example, there is evidence that in Drosophila, the Brm complex is important in negatively regulating Armadillo-dTCF target genes in the Wingless signaling pathway. Although as yet there have been no studies showing directly that G1/S phase-inducing genes are targets of the Wingless signaling pathway in Drosophila, this is possible based on studies in mammalian cells. Furthermore, the Wingless pathway clearly has a role in cell proliferation in some Drosophila tissues. Whether this is the mechanism by which the Brm complex mediates negative regulation of cell cycle entry requires further investigation (Brumby, 2002).
Another way in which the Brm complex may function is by restricting or regulating access to chromosomal origins of replication. Several studies have shown that ATP-dependent chromatin remodeling is important for modulating the initiation of chromosomal DNA replication. The data are consistent with the view that the Brm complex may play a role in this process, possibly functioning to restrict entry into S phase by acting directly to remodel nucleosomes at replication origins. In this scenario, DmcycE-Cdk2 may then act to phosphorylate and inactivate the Brm complex, allowing assembly or function of the pre-replication complex and replication origin firing. Indeed, cyclin E-Cdk2 has been shown to be recruited by the Cdc6 pre-replication complex protein to replication origins at the G1 to S phase transition (Brumby, 2002).
Intriguingly, recent studies have shown that the E2F/DP complex also acts directly at replication origins. In the amplification of the chorion gene clusters during the ovarian follicle cell endoreplicative cycles, it has been shown that E2F1/DP is important in localizing the origin of replication complex specifically to the chorion gene origins and activating replication, and that Rbf1 is important in limiting DNA replication. This mechanism is not limited to these specialized cycles, since transcription-independent roles for E2F1 in inducing S phase have also been documented in the eye imaginal disc. Taken together, these studies suggest that the E2F1/DP-Rbf1 complex plays a non-transcriptional role in S phase by acting directly at DNA replication origins. In mammalian cells, a similar non-transcriptional role for Rb in DNA replication inhibition has been demonstrated, possibly through its functional association with the pre-replication complex protein Mcm7 and its localization to replication foci (Brumby, 2002).
Given the data for a role for Rb-E2F/DP directly at replication origins and the evidence that chromatin remodeling is important in replication initiation, it is possible that Brm and Rbf1 may both have a role at replication origins to prevent premature origin firing in G1. However, the failure to detect a genetic interaction between brm complex genes and rbf1 suggests that they also have other important roles, independent of each other, in the G1 to S phase transition (Brumby, 2002).
In summary, these results have shown that mutations in genes encoding components of the Brm chromatin remodeling complex can dominantly suppress a DmcycE hypomorphic allele by increasing the number of S phase cells without affecting cyclin E protein levels. Consistent with this view, DmcycE physically interacts with Brm and Snr1. Although a complex was also observed between the Brm complex and Rbf1, no genetic interactions have been detected between Brm complex genes and rbf1, suggesting that Rbf1 and Brm function largely independently in negatively regulating the G1 to S phase transition. Taken together, these data suggest that the Brm complex negatively regulates entry into S phase, possibly in partial collaboration with Rbf1, and that this negative regulation can be abrogated by the action of cyclin E at the G1 to S phase transition (Brumby, 2002).
The Notch signaling pathway controls the follicle cell mitotic-to-endocycle transition in Drosophila oogenesis by stopping the mitotic cycle and promoting the endocycle. To understand how the Notch pathway coordinates this process, a functional analysis was performed of genes whose transcription is responsive to the Notch pathway at this transition. These genes include String, the G2/M regulator Cdc25 phosphatase; Hec/CdhFzr, a regulator of the APC ubiquitination complex and Dacapo, an inhibitor of the
CyclinE/CDK complex. Notch activity leads to downregulation of String and Dacapo, and activation of Fzr. All three genes are independently responsive to Notch. In addition, CdhFzr, an essential gene for
endocycles, is sufficient to stop mitotic cycle and promote precocious endocycles when expressed prematurely during mitotic stages. In contrast, overexpression of the growth controller Myc does not induce premature endocycles but accelerates the kinetics of normal endocycles. F-box/WD40-domain protein Ago/hCdc4 (Archipelago), a SCF-regulator is dispensable for mitosis, but
crucial for endocycle progression in follicle epithelium.
CycE oscillation remains critical for endocycling; continuous high level of CycE expression blocks the cell cycle in G2. The regulation of CycE levels is achieved by the function of Ago that presumably binds to auto-phosphorylated CycE and directs it to SCF-complex degradation: high levels of CycE and no endocycling is observed in ago-clones.
The results support a model in which Notch activity executes the mitotic-to-endocycle switch by regulating all three major cell cycle transitions. Repression of String blocks
the M-phase, activation of Fzr allows G1 progression, and repression of Dacapo assures entry into the S-phase. This study provides a comprehensive picture of the logic that external signaling pathways may use to control cell cycle
transitions by the coordinated regulation of the cell cycle (Shcherbata, 2004).
The exit from mitosis and/or progression through G1 requires the inactivation of cyclin-dependent kinases, mediated by the APC/C-dependent destruction of cyclins. APC/C is regulated by multiple mechanisms, such as phosphorylation and by spindle checkpoints. Key factors for APC/C function and regulation are the WD proteins Cdc20 and Hec1/Cdh. These proteins seem to bind directly to substrates and recruit them to the APC/C core complex. Importantly, Cdc20 and Hec1/Cdh bind and activate APC/C in a sequential manner during mitosis. APC/C-Cdc20 is activated at the metaphase/anaphase transition, and gets replaced by APC/C-Hec1/Cdh in telophase. This second complex remains active in the subsequent G1 phase. In Drosophila the homolog of Hec1/Cdh, Fzr, also induces the APC/C-complex-dependent proteolysis of CycA and B and is required for the G1-phase progression. Fzr is required for cyclin removal during G1 when the embryonic epidermal cell or follicle epithelial proliferation stops and the cells enter endocycles. Premature Hec1/CdhFzr transcription in follicle cells is sufficient to block mitosis and initiate precocious endocycling. This suggests that Fzr is a powerful player in the mitotic-to-endocycle switch, yet regulation of other components is also required for the efficiency of this process. Regulators of G1-S transition, such as Dacapo/CIP/KIP, which also turns out to be a Notch-regulated component, possibly abort premature attempts by follicle cells to enter the endocycle (Shcherbata, 2004).
In addition to Myc and Cyclin D, Cyclin E also plays an important role in the regulation of the G1/S-transition. Cyclin E binds to and activates the cyclin-dependent kinase Cdk2, and thereby promotes the transition from G1 to S. Oscillation of Cyclin E activity is a mechanism responsible for the timely inactivation of this G1 cyclin/Cdk complex and an arrest in cell proliferation. The oscillation of Cyclin E level is controlled partly by a SCF-ubiquitin-dependent proteolysis. Fluctuations of Cyclin E are critical for multiple rounds of endocycles. Cyclin E is critical for endocycles in follicle cells as well, and this analysis shows that the CycE level is controlled by an SCF-regulator, F-box protein, Ago/hCdc4/Fbw7. Fbw7 (Ago) associates specifically with phosphorylated Cyclin E, and catalyzes Cyclin E ubiquitination in vitro. Depletion of Ago leads to accumulation and stabilization of Cyclin E in vivo in human and D. melanogaster. This leads to increased mitosis in certain mammalian and Drosophila cell types. In addition, ago loss-of-function clones in the germ line will cause extra mitotic divisions or, in contrast, cell cycle arrest and polyploidy. However, increased Cyclin E levels observed in ago loss-of-function mutant clones do not affect the mitotic cycles in follicle cells but do halt the transition to endocycles that normally occurs at stage 6 (Shcherbata, 2004).
The CUL4 (cullin 4) proteins are the core components of a new class of ubiquitin E3 ligases that regulate replication and transcription. To examine the roles of CUL4 in cell cycle regulation, the effect of inactivation of CUL4 in both Drosophila and human cells was analyzed. Loss of CUL4 in Drosophila cells causes G(1) cell cycle arrest and an increased protein level of the CDK inhibitor Dacapo. Coelimination of Dacapo with CUL4 abolishes the G(1) cell cycle arrest. In human cells, inactivation of CUL4A induces CDK inhibitor p27(Kip1) stabilization and G(1) cell cycle arrest which is dependent on the presence of p27, suggesting that this regulatory pathway is evolutionarily conserved. In addition, the Drosophila CUL4 also regulates the protein level of cyclin E independent of Dacapo. Evidence is provided that human CUL4B, a paralogue of human CUL4A, is involved in cyclin E regulation. Loss of CUL4B causes the accumulation of cyclin E without a concomitant increase of p27. The human CUL4B and cyclin E proteins also interact with each other and the CUL4B complexes can polyubiquitinate the CUL4B-associated cyclin E. These studies suggest that the CUL4-containing ubiquitin E3 ligases play a critical role in regulating G(1) cell cycle progression in both Drosophila and human cells (Higa, 2006).
The COP9 signalosome (CSN) is an eight-subunit complex that regulates multiple signaling and cell cycle pathways. The CSN has been linked to the degradation of Cyclin E, which promotes the G1-S transition in the cell cycle and then is rapidly degraded by the ubiquitin-proteasome pathway. Using CSN4 and CSN5/Jab1 mutants, it has been shown that the CSN acts during Drosophila oogenesis to remove Nedd8 from Cullin1, a subunit of the SCF ubiquitin ligase. Overexpression of Cyclin E causes defects similar to those caused by mutations in CSN or SCFAgo subunits -- extra divisions or, in contrast, cell cycle arrest and polyploidy. Because the phenotypes are so similar and because CSN and Cyclin E mutations reciprocally suppress each other, Cyclin E appears to be the major target of the CSN during early oogenesis. Genetic interactions among CSN, SCF, and proteasome subunits further confirm CSN involvement in ubiquitin-mediated Cyclin E degradation (Doronkin, 2003).
To investigate cyst formation and differentiation in CSN5 germaria, wild-type and CSN5 ovaries were stained with anti-Hts antibody to highlight the fusome that connects all the cells of a cyst through the ring canals. Fusome development is essential for germline cyst formation. In CSN5 mutant germaria the fusome was often less branched, and sometimes there were more individual fusomes than in wild-type germaria. Furthermore, spherical spectrosomes (fusome precursors) are frequently found in more posterior regions of the germaria, probably indicating retarded fusome development. CSN5 null mutant clones eventually cease mitotic divisions and often become enormously polyploid. Along with the increase in DNA, these cells often contain oversized spectrosomes or structures similar to a fragmented fusome, indicating dramatic changes in fusome development. Some mutant clones lacked spectrosomes/fusomes. Usually, these clones were found a significant time after heat shock and were localized in ovarioles with no subsequent germline development. CSN4N mutant clones show similar undifferentiated cysts with enlarged cell nuclei and defective fusome development. These data suggest that the intact CSN complex is required for proper cyst divisions and fusome development.
The polyploid, nondividing germ cells may be the germline stem cells. More than three of these large polyploid cells are never seen in a particular germarium, and they retain contact with somatic cells that probably correspond to the basal and terminal filament cells of normal germaria (Doronkin, 2003).
The Drosophila F box protein Archipelago (Ago) has been proposed to target Cyclin E for ubiquitin-mediated degradation in imaginal discs. The hypomorphic alleles ago1, ago3, and ago4 were used to test for a similar role in Cyclin E degradation during oogenesis. Immunostaining shows that ago mutant clones marked by lack of GFP persistently accumulate Cyclin E at high levels. With one addition, these clones showed a similar range of phenotypes as those seen in CSN5 or CSN4 mutants or after overexpression of Cyclin E. Some mutant cysts had extra nurse cells and some had fewer than normal, and many were degenerating. Some cysts had been arrested after the stem cell division and some of these single-cell cysts were polyploid. Prominent in the ago clones was a phenotype that had not been previously noticed. Cyclin E accumulation in ago clones correlates with significantly DAPI-bright regions in nurse cell nuclei. Because these regions are likely to include heterochromatic sequences that are usually underreplicated during endoreplication, their enlargement may indicate a more complete replication of both heterochromatic and euchromatic sequences in ago clones. Although enlarged heterochromatin-rich regions are occasionally seen in CSN5 mutants and after Cyclin E overexpression, this phenotype is stronger in ago mutants, possibly suggesting a more specific role for ago in regulation of late replication (Doronkin, 2003).
In addition to the similar phenotypes between ago mutants and CSN, Nedd8, or cullin1 mutants, dominant interactions were found between ago and CSN mutants. CSN5/ago and CSN4/ago double heterozygotes show familiar ovarian defects: extra cystocyte divisions, fewer divisions but higher ploidy, and apoptotic egg chambers. These defects are very similar to the CSN5 mutant phenotype and to defects in oogenesis induced by Cyclin E overexpression. In addition, these double heterozygotes have enlarged heterochromatic regions in nurse cell nuclei, suggesting mutual CSN-ago control of late replication (Doronkin, 2003).
The regulatory lid of the proteasome is an eight-subunit complex that is closely related to the CSN. It appears to be necessary for the removal of ubiquitin side chains from the target protein as it is fed into the barrel of the proteasome for proteolysis. A mutation in the RPN6 subunit of the regulatory lid was tested for genetic interactions in oogenesis with CSN4 and CSN5 mutations. Both double heterozygotes show a strong interaction and the full range of CSN5-like ovarian defects, including apoptosis, incorrect number of mitotic divisions, and fusions of neighboring egg chambers (Doronkin, 2003).
The effect of the CSN on the activity of the SCF complex has been controversial. Although Nedd8 modification of Cullin1 stimulates SCF activity, the opposite process, deneddylation, has also been shown to be important for SCF function and cell cycle progression. For example, point mutations in the JAMM domain of the S. cerevisiae CSN5 homolog Rri abolish its deneddylation activity and enhance the growth defect shown by ts alleles of SCF genes. These results have led to the proposal that repeated cycles of neddylation and deneddylation are required for the sustained activity of the SCF. However, a recent gain-of-function analysis suggests that deneddylation by the CSN inhibits degradation of the SCF target p27kip1 (Doronkin, 2003).
The results of this study strongly support the idea that deneddylation of Cullin1 by the CSN is necessary for activity of the SCF complex. CSN mutations have the same, not opposite, effects on oogenesis as do Nedd8, cullin1, or ago mutations. CSN5 and CSN4 mutations also interact dominantly with cullin1 and ago mutations, further suggesting that the CSN works along with the SCF to promote Cyclin E degradation. These requirements for the CSN appear to demand its deneddylase activity, because the CSN5quo2 mutation, with a single amino acid substitution in the metalloprotease domain, behaves similarly to a CSN5 null (Doronkin, 2003).
Cycles of neddylation and deneddylation might control the association of an F box protein with an E3 ubiquitin ligase core complex or the association of a ubiquitin-loaded E2-conjugating enzyme with the E3 complex. Neddylation might also affect Cullin1 stability as suggested by the Cullin1 accumulation that is seen in CSN5 mutants and its reduction in Nedd8 mutants (Doronkin, 2003).
Conjugation of Nedd8 to cullins may regulate not only their activity, but also their subcellular distribution. Shuttling between the nucleus and cytoplasm has been proposed as a regulatory mechanism for E3 ubiquitin ligases when the target protein is ubiquitinated in the nucleus. The results showing that in CSN mutants, Nedd8-modified Cullin1 accumulates in the cytoplasm suggest that neddylation may be one way to regulate shuttling. Neddylation might favor nuclear export of Cullin1, and nuclear CSN would be required to remove Nedd8 and prevent export. Alternatively, neddylation might prevent Cullin1 nuclear import, and recycling of SCF into the nucleus would require cytoplasmic CSN. On either model, the CSN would be an important regulator of SCF activity. For example, modulation of SCF nuclear shuttling might affect the timing of Cyclin E degradation and entry into S phase of the cell cycle (Doronkin, 2003).
One of the important results of the current work is the demonstration that the CSN regulates the cell cycle in ovaries primarily through the turnover of Cyclin E. The apparent perdurance and gradual dilution of wild-type CSN5 protein in genetically null germline clones shows that reduced Cyclin E degradation affects both cell division and DNA replication. Slight reductions cause an extra division of the cystocytes. In contrast, continuous or strong accumulation of Cyclin E in null CSN5 mutants is able to reduce or stop cell divisions though often allowing endoreplication to continue. This switch from overproliferation to inhibition of cell divisions is sometimes visible in a single CSN5 mutant ovariole as the wild-type CSN5 protein is diluted by stem cell divisions. These observations support the view that different Cyclin E levels can lead to distinct and sometimes opposite effects (Doronkin, 2003).
Mutations in Drosophila ago, the C. elegans gene cul1, or the F box-encoding lin23 have been shown to cause increased cell proliferation, suggesting a critical role for SCF in regulating cell divisions. Extra cell division is found to be a frequent phenotype produced by mutations in CSN5, CSN4, cullin1, ago, or by overexpression of Cyclin E. However, SCF and CSN mutations have also been shown to cause the opposite effect on the cell cycle. In null mutant clones of cullin1 or Nedd8, cell proliferation in Drosophila eye discs is arrested. Similarly, loss of CSN5, CSN4, Cullin1, or ago inhibits and finally stops cell proliferation and often leads to enlarged nuclei. The abundance of Cyclin E and giant polyploid nuclei are also present in mice that are mutant for cul1 (Doronkin, 2003).
Elevated levels of Cyclin E that may give cells a proliferative advantage are found in many human tumors. In many of these tumors the Cyclin E gene itself is amplified. However, among breast and ovarian cancer cell lines that overexpress Cyclin E protein without amplification, several lines have mutations in hCDC4, the human homolog of archipelago, suggesting that SCF[hcdc4] acts to suppress tumor formation. The results suggest that the CSN might have a similar effect (Doronkin, 2003).
In summary, these genetic and functional relationships between the CSN, the SCF, and the proteasome link these complexes in the regulation of Cyclin E degradation during normal development. When either the CSN or SCF are disrupted, the periodic degradation of Cyclin E is prevented, and cell cycle deregulation ensues (Doronkin, 2003).
Skp1 proteins function in protein degradation as a component of the SCF (SKP1, cullin/CDC53, F-box protein) complex to link the substrate-recognition subunit (F-box protein) to a cullin (see Drosophila Cullin1) that in turn binds the ubiquitin-conjugating enzyme. Centrosome duplication must be coupled to the main cell cycle to ensure
that each cell has precisely two centrosomes at the onset of mitosis.
Supernumerary centrosomes are commonly observed in cancer cells, and may
contribute to tumorigenesis. Drosophila SkpA, the Skp1 component of Drosophila SCF ubiquitin ligases, regulates the link between the cell and centrosome cycles.
Lethal skpA null mutants exhibit dramatic centrosome overduplication
and additional defects in chromatin condensation, cell cycle progression and
endoreduplication. Surprisingly, many mutant cells are able to organize
pseudo-bipolar spindles and execute a normal anaphase in the presence of extra
functional centrosomes. SkpA mutant cells accumulate higher levels of
cyclin E than wildtype cells during S and G2, suggesting that elevated
cdk2/cyclin E activity may account for the supernumerary centrosomes in
skpA- cells. However, centrosome overduplication
still occurs in skpA-;
cycE- mutant animals, demonstrating that high cyclin E
levels are not necessary for centrosome overduplication. These data suggest
that additional SCF targets regulate the centrosome duplication pathway and that Drosophila SkpA regulates centrosome duplication independently of cyclin E accumulation (Murphy, 2003).
This study has directly tested the role of cyclin E in centrosome overduplication by genetically manipulating cyclin E levels in wildtype and
skpA- cells. Strikingly, drastically reducing cyclin
E levels with a near-null allele does not suppress centrosome overduplication
in cycling skpA- cells. One possibility is that
cyclin E is not required for centrosome duplication in Drosophila.
This seems unlikely, because Drosophila cdk2 does not associate with
cyclin A and lacks in vitro kinase activity when immunoprecipitated from
cyclin E-deficient embryos, and other functions of cdk2 are conserved between
Drosophila and vertebrates. In any
case, centrosome overduplication occurs independently of SCF control of cyclin
E accumulation (Murphy, 2003).
How do SCF components regulate centrosome duplication? One possibility is
that simply lengthening the cell cycle introduces enough time for multiple
cycles of centrosome duplication to occur. Although this model cannot be ruled
out, it seems unlikely given that a centrosome must duplicate in as little as
55 minutes in a cycling neuroblast but does not reduplicate in the 12-hour
cycle of an imaginal wing disc cell.
Furthermore, slowing the cell cycle in abdominal histoblasts by overexpressing
the Drosophila retinoblastoma-family protein RBF is not sufficient to
induce centrosome overduplication (Murphy, 2003).
Instead, the idea is favored that a target of SCF-mediated degradation acts as
a Centrosome Licensing Factor (CLiF) that limits centrosome duplication to
once per cell cycle. CLiF would be expressed early in the cell cycle, loaded
onto centrosomes, and excess CLiF would be targeted to the proteasome by an
SCF complex. One cycle of centrosome duplication could then be triggered by
Cdk2-E activity, but the daughter centrosomes would not be relicensed until
the next cell cycle. SCF mutants would fail to degrade excess CLiF, allowing
duplicated centrosomes to relicense and reduplicate in the course of a single
cell cycle. One candidate CLiF is nucleophosmin/B23, which is phosphorylated
by Cdk2-E and associates specifically with unduplicated centrosomes
(Okuda, 2000). Future
experiments will need to determine if nucleophosmin/B23 or other candidate
CLiFs are targeted for degradation by an SCF complex (Murphy, 2003).
In Drosophila, egg development starts at the anterior tip of the ovary, in the germarium, where the germline stem cells divide to produce a cystoblast and a self-renewing stem cell. Each cystoblast undergoes four mitotic divisions with incomplete cytokinesis. The resulting 16 cells of each egg chamber are connected by intercellular bridges called ring canals. Exit from the cell cycle at the end of these four mitotic divisions requires the downregulation of Cyclin/Cdk activity. In the ovary of Drosophila, Encore activity is necessary in the germline to exit this division program (Ohlmeyer, 2003).
In encore mutant germaria, Cyclin A persists longer than in wild type. In addition, Cyclin E expression is not downregulated after the fourth mitosis and accumulates in a polyubiquitinated form. Mutations in genes coding for components of the ubiquitin-protease pathway such as cul1, UbcD2 and effete enhance the extra division phenotype of encore. Encore physically interacts with the proteasome, Cul1 and Cyclin E. The association of three factors, Cul1, phosphorylated Cyclin E, and the proteasome 19S-RP subunit S1, with the fusome is affected in encore mutant germaria. It is proposed that in encore mutant germaria the proteolysis machinery is less efficient and, in addition, reduced association of Cul1 and S1 with the fusome may compromise Cyclin E destruction and consequently promote an extra round of mitosis (Ohlmeyer, 2003).
Overexpression or loss-of-function mutations in a third group of genes such as Cyclin A, Cyclin B, Cyclin E and mutations in the gene encoding the E2 Ubiquitin conjugating enzyme UbcD1 lead to the production of cysts with 32 or 8 cells. These genes do not affect fusome integrity and thus timing and spatial characteristics of cell division appear to be intact. The encore gene belongs to this group of genes: its product is necessary for exit from mitosis. Loss of Encore activity results in egg chambers containing 32 rather than 16 cells. Mutations in the encore gene produce additional phenotypes, which show differential temperature sensitivity. encore mutant females raised at 18°C produce egg chambers with 16 cells, but they give rise to ventralized eggs. The extra cell division phenotype is only observed when encore mutant females are raised at high temperatures (25°-29°C). The encore gene encodes a 200 kDa protein with no homolog of a defined biochemical function. The mechanism by which Encore promotes exit from the cell cycle after four germline mitoses has been investigated (Ohlmeyer, 2003).
Cell cycle progression is controlled by a series of cell cycle dependent kinases (Cdk). Cdk activity is carefully regulated by the levels of the Cyclin subunits, by Cdk inhibitors (CKI) and by post-translational modification of the Cdk subunit through both activating and inactivating phosphorylation. Transition from G1 to S phase depends on Cdk2/Cyclin E activity, and on the timely destruction of the Cdk2/Cyclin E inhibitor p27. The Drosophila p27 homologue, Dacapo, is required for exit from the cell cycle in the embryo and eye imaginal disc. In addition, exit from the cell cycle requires destruction of the cyclins by the ubiquitin-proteasome system (UPS). The addition of ubiquitin requires three different activities; the ubiquitin activating enzyme (E1), the ubiquitin conjugating enzyme (E2) and the ubiquitin ligase enzyme (E3). The ubiquitinated protein bound to E3 is presented to the proteasome, isopeptidase activities in the 19S-recognition particle (RP) of the proteasome cleave the ubiquitin tail, the protein is unfolded and finally destroyed by the proteasome 20S-core particle (CP) (Ohlmeyer, 2003).
There are two E3 enzyme complexes that regulate the cell cycle progression. The first, the APC/cyclosome, regulates progression from G2 to M phase transition. The second, the SCF complex regulates the G1 to S phase transition. The SCF complex is composed of Skp/Cullin/Rbx1 and F-box proteins and controls substrate ubiquitination via an interaction between the F-box component and the phosphorylated target protein. In Drosophila and mammalian systems, mutations in the Cul3 and Ago genes have been shown to cause the accumulation of Cyclin E, entry to S-phase and doubling of cell number. Thus, proper regulation of the destruction machinery is important for maintaining normal levels of Cyclin E and assuring proper cell cycle progression (Ohlmeyer, 2003).
The work presented in this study demonstrates that the encore gene product associates with the SCF-ubiquitin-proteasome system and is required for proper exit from germline mitosis. The failure to downregulate Cyclin E after four cell divisions in conjunction with an accumulation of Cyclin A protein provide the conditions to promote an extra cell division. Encore can bind to Cul1, Cyclin E-Ub(n) and the proteasome. Cul1 and the proteasome 19S-RP subunit S1 are associated with the fusome and these associations are very much attenuated in encore mutant ovaries. It is proposed that as a direct consequence, Cyclin E is not degraded properly, its activity is misregulated and the cyst undergoes one extra cell division (Ohlmeyer, 2003).
This study shows that the fusome is a regulator of cell division during early oogenesis. Some of the functions ascribed to the fusome are to synchronize cyst
mitosis and to provide the scaffold for the transport system necessary for
oocyte determination. Limiting the number of cell divisions in the germarium
could be achieved by regulating the association of proteins such as the
cyclins and/or other cell cycle regulators with the fusome. The expression
pattern of Cyclin A, Cul1, P-Cyclin E and 19S-S1 proteins in the germarium
supports the idea that the fusome plays an important role in the regulation of
mitosis. Indeed, Cyclin A association with the fusome is transient and occurs
only during cyst division. In encore mutant germaria, Cyclin A remains associated with the fusome after cell division has stopped. Cul1 localization to the fusome suggests that the rest of the SCF complex also associates with the fusome and that substrate ubiquitination may happen at the fusome. The SCF component Cul1 is mainly associated with the fusome in the wild-type germaria. In encore mutant germaria, Cul1 localization to the fusome is very poor, leading to a proposal that this may be one reason why Cyclin E is not degraded properly. This also suggests that the degradation of Cyclin E and perhaps of other proteins degraded by the SCF-UPS may occur at the fusome. The association of P-Cyclin E supports this idea. The localization of P-Cyclin
E in the wild type seems to be dynamic, consistent with the idea that the
phosphorylated substrate is localized to the fusome, and then rapidly degraded via the SCF-UPS. In encore mutant germaria, the poor localization of Cul1 may result in an inefficient assembly of SCF complexes at the fusome. P-Cyclin E is localized to the fusome, but its degradation is compromised and as a result a consistent expression of P-Cyclin E is observed at the fusome. The partial association of the proteasome 19S-RP subunit S1 to the fusome supports the idea that proteolysis may occur at the fusome. The proteasome 19S-RP would recognize the polyubiquitinated substrate and recruit the rest of
the proteasome to the fusome (Ohlmeyer, 2003).
The results suggest that Encore can associate with the SCF ubiquitin-proteasome system machinery and
assists with the degradation of Cyclin E and perhaps other SCF substrates. Since the mutant Encore protein can still interact with SCF-UPS components, the mutant protein may form complexes but these might be inactive and/or the mutant protein poisons the degradation machinery. Consistent with such a
hypothesis, the encore extra cell division phenotype is milder in
hemizygous versus homozygous females at 25°C
(Hawkins, 1996). Encore
is required for the proper localization of Cul1, P-Cyclin E, S1 and presumably
the rest of the proteolysis complex to the fusome. This localization may be
more crucial at 29°C, whereas at lower temperatures a less efficient
degradation system may have enough time for normal cell cycle regulation.
encore mutations do not affect the 20S-Core Particle activity as
measured by the rate of degradation of a fluorogenic peptide. It is not known
whether Encore retains Cul1 at the fusome or whether Encore directly or
indirectly modifies Cul1 in order to promote its localization at the fusome. Cul1 is known to be modified by the addition of Nedd8;
however, Cul1 seems to be equally neddylated in encore and wild-type ovary extracts (Ohlmeyer, 2003).
In summary, the results suggest that the Encore protein assists with proper
cell cycle progression in the Drosophila germarium by ensuring that
Cul1 and the proteolysis machinery is localized at the mitosis coordination
center, the fusome (Ohlmeyer, 2003).
It is important that chromosomes are duplicated only once per cell cycle. Over-replication is prevented by multiple mechanisms that block the reformation of a pre-replicative complex (pre-RC) onto origins in S and G2 phase. The developmental regulation of Double-parked (Dup) protein, the Drosophila ortholog of Cdt1, a conserved and essential pre-RC component found in human and other organisms, has been studied. Phosphorylation and degradation of Dup protein at G1/S requires cyclin E/CDK2. The N terminus of Dup, which contains ten potential CDK phosphorylation sites, is necessary and sufficient for Dup degradation during S phase of mitotic cycles and endocycles. Mutation of these ten phosphorylation sites, however, only partially stabilizes the protein, suggesting that multiple mechanisms ensure Dup degradation. This regulation is important because increased Dup protein is sufficient to induce profound rereplication and death of developing cells. Mis-expression has different effects on genomic replication than on developmental amplification from chorion origins. The C terminus alone has no effect on genomic replication, but it is better than full-length protein at stimulating amplification. Mutation of the Dup CDK sites increases genomic re-replication, but is dominant negative for amplification. These two results suggest that phosphorylation regulates Dup activity differently during these developmentally specific types of DNA replication. Moreover, the ability of the CDK site mutant to rapidly inhibit BrdU incorporation suggests that Dup is required for fork elongation during amplification. In the context of findings from human and other cells, these results indicate that stringent regulation of Dup protein is critical to protect genome integrity (Thomer, 2004).
To determine whether oscillation of Dup protein levels during cell cycles is due to Dup protein degradation at G1/S, Dup expression within the synchronized cell cycles of the larval eye primordium was examined. Late in third instar, a wave of differentiation sweeps across the eye imaginal disc, which is visible as a morphogenetic furrow (MF). Cells are synchronized in G1 upon entering the furrow. Specific cells posterior to the furrow then enter a synchronous S phase, which is visible as a stripe of BrdU labeling. Labeling with affinity-purified rabbit polyclonal Dup antibody indicates that the protein is abundant in nuclei of late G1 cells, but is undetectable in S phase cells incorporating BrdU. Labeling with a guinea pig anti-Dup antibody gave identical results suggesting that immunolabeling reflects Dup protein in vivo. Double labeling for Dup and cyclin E indicates that both are abundant in nuclei of cells in late G1, but then Dup rapidly declines while cyclin E persists into S phase. Labeling for the G2 and M phase marker cyclin B also indicates that Dup levels decline significantly before cells enter G2. Similar results were obtained for the non-synchronized cell cycles in the eye and other imaginal discs. This rapid decline in protein is primarily due to post-transcriptional regulation because in situ hybridization indicates that dup mRNA persists after G1. Moreover, expression of a dup transgene from the strong hsp70 promoter does not result in detectable Dup protein during S phase. The data suggest that, similar to Cdt1 in humans and other organisms, Dup protein is abundant in G1 when origins are licensed, but is then rapidly degraded when cyclin E appears at G1/S (Thomer, 2004).
Beginning in late mitosis, origins of replication are prepared for replication by binding of a pre-replicative complex (pre-RC), which is subsequently activated to initiate replication at the onset of S phase. The building of the pre-RC onto origins in late mitosis/early G1 is a stepwise process. The origin recognition complex (ORC) serves as a scaffold for subsequent association of Cdc6 and Cdt1, both of which are required to load the Minichromosome Maintenance (MCM) complex, the replicative helicase. Once MCMs are loaded, the origin is considered to be licensed for subsequent replication. Cdc7 kinase, with its activating subunit Dbf4, and CDK2 kinase, activated by cyclin E or cyclin A, are then required for initiation of replication. Initiation is associated with departure of Cdc6, Cdt1, MCMs, and, in multicellular eukaryotes, certain ORC subunits from the origin. Continued CDK activity in S, G2, and early M phases inhibits reassembly of the pre-RC to block origin refiring. Unique to multicellular eukaryotes is another inhibitor of pre-RC assembly, Geminin, which binds Cdt1 and renders it incapable of loading the MCM complex. It is only after Geminin and cyclins are degraded at the subsequent metaphase that the pre-RC can reform, thereby restricting origin licensing, and DNA replication, to once per segregation of chromosomes (Thomer, 2004 and references therein).
It is likely that part of the cyclin E/CDK2 dependent regulation is direct because Dup associates with CDK2 protein and activity in embryos. The results of the mutagenesis show that the N terminus of Dup is necessary and sufficient for degradation at G1/S. Mutation of the CDK sites in the N terminus, however, only partially stabilize the protein, suggesting the existence of other CDK2-dependent mechanisms for degradation. It is crucial to tightly regulate the abundance of Dup protein because its over-expression is sufficient to induce a full genome reduplication and cell death in the ovary and imaginal discs. The different effects on amplification and genomic replication suggest that phosphorylation of the N terminus of Dup protein may be required for replication fork elongation during amplification and provides insight into the mechanism of this developmentally specific replication program (Thomer, 2004).
The results suggest that cyclin E/CDK2 phosphorylates the Dup N terminus contributing to its instability at G1/S. Dup was degraded during periodic endocycle S phases that are solely regulated by oscillating cyclin E/CDK2, further supporting a link between this kinase and Dup degradation. Although the N terminus was necessary and sufficient for degradation, mutation of the ten N-terminal CDK sites within Dup 10(A) only partially stabilized the protein. This suggests that there are other cyclin E/CDK2-dependent mechanisms that trigger Dup degradation independent of these ten sites during S phase. It has been noted that the C terminus of Dup contains a PEST sequence, and there are several serines and threonines in the C terminus that are potential targets of phosphorylation. Although the requirement for these sites has not been directly tested, the stability of C-Dup indicates that they are not sufficient for degradation at G1/S. To explain these results, a bi-phasic degradation model is suggested where cyclin E/CDK2 phosphorylation promotes Dup degradation in late G1, whereas other fail-safe mechanisms become operative only during S phase. This would explain why inhibiting CDK2 and S phase entry with GMRp21 completely blocked Dup degradation (Thomer, 2004).
A number of recent publications describe results for Cdt1 in human cells that are similar to those in flies. These results suggest that cyclin A/CDK2 phophorylates the human Cdt1 N terminus, which enhances its binding to the Skp2 subunit of the SCF ubiquitin ligase. Like Dup, non-phosphorylatable Cdt1 mutants are only partially stabilized, but simultaneously inhibiting CDK2 and S phase entry with p21 completely blocks degradation. Previous evidence in C. elegans, human and Drosophila cells have suggested that destruction of Cdt1 may be mediated by two ubiquitin ligases, an SCF complex containing Skp2, and an SCF-like complex based on Cul4. For many substrates of the SCF, prior phosphorylation is required for their subsequent recognition and ubiquitinylation, including substrates phosphorylated by CDK2 at G1/S. It is not known whether prior phosphorylation is required for substrate recognition by Cul4-based ubiquitin ligases. It is tempting to speculate, therefore, that the bi-phasic degradation of Cdt1 that may reflect its modification by two distinct ubiquitin ligases: a phosphorylation-dependent ubiquitinylation by the SCF complex, and a phosphorylation-independent ubiquitinylation by a Cul4-based complex. Clearly, more experiments are needed to sort out the complexity of this regulation. Nonetheless, the similar results from flies and humans suggest that tight regulation of Cdt1 abundance is a generally conserved and important mechanism to protect genome integrity in eukaryotes (Thomer, 2004).
Cyclin A expression is only required for particular cell divisions
during Drosophila embryogenesis. In the epidermis, Cyclin A
is strictly required for progression through mitosis 16 in cells that become
post-mitotic after this division. By contrast, Cyclin A is not
absolutely required in epidermal cells that are developmentally programmed for
continuation of cell cycle progression after mitosis 16. These analyses suggest
the following explanation for the special Cyclin A requirement during
terminal division cycles. Cyclin E is known to be downregulated
during terminal division cycles to allow a timely cell cycle exit after the
final mitosis. Cyclin E is therefore no longer available before terminal
mitoses to prevent premature Fizzy-related/Cdh1 activation. As a consequence,
Cyclin A, which can also function as a negative regulator of
Fizzy-related/Cdh1, becomes essential to provide this inhibition before
terminal mitoses. In the absence of Cyclin A, premature Fizzy-related/Cdh1
activity results in the premature degradation of the Cdk1 activators Cyclin B
and Cyclin B3, and apparently of String/Cdc25 phosphatase as well. Without
these activators, entry into terminal mitoses is not possible. However, entry
into terminal mitoses can be restored by the simultaneous expression of
versions of Cyclin B and Cyclin B3 without destruction boxes, along with a
Cdk1 mutant that escapes inhibitory phosphorylation on T14 and Y15. Moreover,
terminal mitoses are also restored in Cyclin A mutants by either the
elimination of Fizzy-related/Cdh1 function or Cyclin E
overexpression (Reber, 2006).
Mitotic cyclins accumulate during the S and G2 phases of the cell cycle.
Their C-terminal cyclin boxes mediate binding to cyclin-dependent kinase 1
(Cdk1). Their rapid degradation during late M and G1 phase depends on the D-
and KEN-boxes in their N-terminal domains. These degradation signals are
recognized by Fizzy/Cdc20 (Fzy) and Fizzy-related/Cdh1 (Fzr), which recruit
the mitotic cyclins to the anaphase-promoting complex/cyclosome (APC/C) during
M and G1, respectively. The ubiquitin ligase activity of the APC/C allows
cyclin poly-ubiquitination and consequential proteolysis (Reber, 2006).
Metazoan species express three different types of mitotic cyclins: A, B and
B3. The specific functions of these different cyclins are not understood in
detail. The presence of single genes coding for either Cyclin A (CycA), Cyclin
B (CycB) or Cyclin B3 (CycB3) has facilitated a genetic dissection of their
functional specificity in Drosophila melanogaster. In this organism,
development to the adult stage requires the zygotic function of CycA,
but not of CycB or CycB3.
Initial analysis of the embryonic cell proliferation program in CycA
mutants revealed that epidermal cells fail to progress through the sixteenth
round of mitosis. Cyclin A is also required for mitosis 16 in the epidermis
of dup/Cdt1 mutant embryos, in which mitosis 16 is no longer
dependent upon completion of the preceding S phase. The
failure of mitosis 16 in CycA mutants therefore does not simply
result from the activation of a DNA replication or damage checkpoint -- a
possibility suggested by evidence obtained in vertebrate cells in which Cyclin
A binds not only to Cdk1 but also to Cdk2, and provides crucial functions
during S phase (Reber, 2006 and references therein).
The accumulation of Cyclin B and Cyclin B3 during cycle 16, which also
occurs in CycA mutants, complicates the explanation of
why mitosis 16 in the epidermis requires Cyclin A. In Xenopus egg
extracts, Cyclin B can trigger entry into mitosis in the absence of Cyclin A.
Conversely, mitosis is clearly inhibited in cultured human cells after the
microinjection of antibodies against cyclin A. Cyclin
A-Cdk1 complexes are thought to have special properties, important for
starting up a positive-feedback loop that confers a switch-like behavior on
the Cdk1 activation process. In this feedback loop, Cdk1 activity results in
phosphorylation and suppression of the inhibitory Wee1 kinase, as well as in
phosphorylation and activation of the String/Cdc25 phosphatase, which removes
the inhibitory phosphate modifications from Cdk1. However, the analyses described in this study indicate that the Cyclin A requirement in Drosophila is not linked to this positive-feedback loop. Rather, it is linked to the fact that the
sixteenth round of mitosis during embryogenesis is the last cell division for
the great majority of the epidermal cells (Reber, 2006).
After mitosis 16, most epidermal cells enter a G1 phase and become
mitotically quiescent. By contrast, all the previous embryonic divisions (mitoses
1-15) are followed by an immediate onset of S phase. The G1 phase after
mitosis 16 is therefore the first G1 phase during development. Entry into this
G1 phase is dependent upon a complete, developmentally controlled inactivation
of Cyclin E-Cdk2 and Cyclin A-Cdk1, because both complexes can trigger entry
into S phase. Cyclin E-Cdk2 inactivation results from transcriptional
CycE downregulation and concomitant upregulation of dacapo,
which encodes the single Drosophila CIP/KIP-type inhibitor specific
for Cyclin E-Cdk2. Cyclin A-Cdk1 inactivation is dependent on Fzr, which is also transcriptionally upregulated. Moreover, Fzr is activated as a consequence of Cyclin E-Cdk2 inactivation. Importantly, this cell cycle exit program is initiated already during G2 of the final division cycle (Reber, 2006).
Although cycle 16 is the final division cycle for most epidermal cells,
some defined regions do not activate the cell cycle exit program during cycle
16. Instead, they maintain CycE expression, enter S phase immediately
after mitosis 16 and complete an additional division cycle 17. In these
regions, mitosis 16 is not fully inhibited in CycA mutants. Cyclin A
is therefore especially important for terminal mitoses preceding G1 and cell
cycle exit. This study shows that the downregulation of Cyclin E-Cdk2 before terminal divisions, in preparation for the imminent cell cycle exit, converts Cyclin A from a redundant into an indispensable, negative regulator of
Fizzy-related/Cdh1, preventing premature degradation of the mitotic inducers
String/Cdc25 and the mitotic cyclins. The significance of the basic cell cycle
regulator Cyclin A therefore depends on the developmental context (Reber, 2006).
The phenotypical characterization of mutations in the Drosophila genes encoding the A-, B- and B3-type cyclins have indicated that Cyclin A is the most crucial of these co-expressed mitotic cyclins. Although zygotic CycB or CycB3 function is not essential for cell proliferation and development to the adult stage, null mutations in CycA result in embryonic lethality.
This study has clarified the molecular basis of the distinct importance of
Cyclin A. The results indicate that the crucial role of Cyclin A is linked to
its ability to inhibit Fzr-APC/C-mediated degradation. Moreover,
this Cyclin A-dependent negative regulation of the Fzr-APC/C-degradation
pathway is of particular importance for progression through the very last
mitotic division preceding cell cycle exit and the proliferative quiescence of
epidermal cells during embryogenesis. This particular Cyclin A requirement
during terminal divisions is caused by a cell cycle exit program that is
initiated already before the terminal mitosis. The cell cycle exit program
includes downregulation of Cyclin E-Cdk2, which has a comparable ability to
inhibit the Fzr-APC/C-degradation pathway to Cyclin A. The downregulation of
Cyclin E-Cdk2 by the cell cycle exit program turns Cyclin A into an
indispensable inhibitor of the premature degradation of mitotic cyclins and
String/Cdc25 via Fzr-APC/C before the terminal mitosis. Accordingly, the terminal mitosis in the epidermis of CycA mutants can be restored by overexpression of Cyclin E, by genetic elimination of Fzr, or by simultaneous expression of the String/Cdc25-independent Cdk1AF mutant and B-type cyclin versions that are no longer Fzr-APC/C substrates (Reber, 2006).
The fact that Cyclin A is also a substrate of Fzr-APC/C-mediated
degradation complicates the interpretation of the results. Two
findings, however, strongly suggest that Cyclin A functions not just
downstream of Fzr, but also upstream as a negative regulator. The observed
premature loss of B-type cyclins in CycA mutants is readily explained
by a negative effect of Cyclin A on Fzr-APC/C activity and is difficult to
explain if Cyclin A was only a Fzr-APC/C substrate. Moreover, the suppression
of the UAS-fzr overexpression phenotype by co-expression of
UAS-CycA, which is described here, includes the re-accumulation of
B-type cyclins and not just the restoration of terminal mitosis 16 (Reber, 2006).
Work in mammalian cells has clearly established that Cyclin A functions as
a negative regulator of Fzr/Cdh1. Human Cyclin A can bind directly to Cdh1.
Moreover, Cyclin A-dependent Cdk activity phosphorylates Cdh1, resulting in
the dissociation of Cdh1 from APC/C. Conversely, mutations in Cdk consensus phosphorylation sites of human CDH1 were reported to abolish inhibition by Cyclin A. The current findings point to alternative modes of Fzr-APC/C-inhibition by Cyclin A. Fzrpsm variant no longer contains canonical Cdk consensus
phosphorylation sites (S/T P) and yet its activity is still suppressed by
CycA overexpression. Fzr inhibition by CyclinA-dependent
phosphorylation of non-consensus sites remains a possibility in
Drosophila. However, it is pointed out that, apart from a potential
control by Cdk phosphorylation, Fzr is inhibited by the Emi1-like
Drosophila protein Rca1. Rca1 overexpression has been shown to prevent
premature Cyclin B degradation and restore mitosis 16 in the epidermis of
CycA mutant embryos. Based on these observations, the failure of
mitosis 16 in CycA mutants was proposed to reflect premature Fzr
activation, a suggestion fully confirmed by the current work. It is
conceivable, therefore, that the Cyclin A-mediated suppression of
Fzrpsm activity involves Rca1 or other unknown targets. The fact
that not only Cyclin A, but also Cyclin E, effectively suppresses
Drosophila Fzr and Fzrpsm provides further support of
additional regulatory complexity. In vertebrate systems, only Cyclin A and not
Cyclin E was shown to bind and inhibit Cdh1 (Reber, 2006).
The current findings demonstrate that the Cyclin A requirement in epidermal cells is maximal for progression through the last mitosis of Drosophila
embryogenesis, which precedes cell cycle exit and proliferative quiescence. A
prominent Cyclin A requirement for terminal mitoses appears to exist in
neuroblast lineages during development of the embryonic CNS, although
definitive proof will require further work. On the basis of this analysis in
epidermal cells, a high Cyclin A requirement for entry into mitosis is
expected whenever Fzr levels are high and Cyclin E levels low. During the
comparatively slow postembryonic cell cycles of imaginal cells, the
periodicity of Cyclin E expression is presumably far more pronounced than
during the rapid embryonic cycles in which the persistent presence of
maternally contributed Cyclin E eliminates G1 phases. In imaginal cell cycles,
which have a G1 phase, Cyclin E expression might therefore be low before each
mitosis, and not just before terminal divisions. In combination with Fzr
expression, every imaginal mitosis might therefore be strongly dependent upon
Cyclin A. By contrast, in the absence of Fzr, progression through mitosis
appears to be almost completely independent of Cyclin A, as is evidenced by
the observation that the epidermal cells in fzr CycA double mutant
embryos not only progress successfully through mitosis 16, but also complete
an extra division cycle 17. Nevertheless, 10% of the late mitosis 17 figures
in these double mutants displayed lagging chromosomes, indicating that cell
cycle progression is not entirely normal in the absence of Cyclin A (Reber, 2006).
The cell cycle exit program, which is activated during the final division
cycle in the embryonic epidermis, includes the strong transcriptional
upregulation of the CIP/KIP-type Cyclin E-Cdk2 inhibitor Dacapo, apart from
the downregulation of Cyclin E and the upregulation of Fzr.
Accordingly, genetic elimination of dacapo function should also
restore progression through terminal mitosis 16 in CycA mutants.
However, mitosis 16 was not observed in the epidermis of dacapo CycA
double mutants. The contribution of Dacapo to Cyclin E-Cdk2 inhibition appears
to be insignificant before mitosis 16. After the stage of mitosis 16, however,
the epidermal cells in these double mutants entered an endoreduplication
cycle, a behavior that is also displayed by some cells in the prospective
anterior spiracle region of CycA single mutants. This
region does not downregulate Cyclin E during cycle 16 in the wild type, it
does not upregulate Dacapo, and it progresses through an additional cycle 17
instead of becoming postmitotic after mitosis 16, in contrast to the great
majority of the other epidermal cells. The premature activation of Fzr in
CycA mutants, therefore, appears to result in DNA replication origin
re-licensing, perhaps as a result of B-type cyclin and geminin degradation.
Cyclin E-Cdk2 activity might subsequently trigger endoreduplication in cells
in which it is not effectively eliminated by both Cyclin E downregulation and
Dacapo upregulation. Importantly, not all cells in the anterior spiracle
region of CycA mutants endoreduplicate, some of the cells still
manage to divide. This variability could reflect minor differences in the
onset and strength of the zygotic Cyclin E expression. The outcome of
insufficient Cyclin A levels appears to be highly dependent on the levels of
Cyclin E and Fzr, which, in turn, are subject to developmental regulation, in
particular during cell cycle exit. The significance of basic cell cycle
regulators in vivo is therefore different in various tissues and developmental
stages, and most likely in various cultured mammalian cell types as well (Reber, 2006).
The CUL4 (cullin 4) proteins are the core components of a new class of ubiquitin E3 ligases that regulate replication and transcription. To examine the roles of CUL4 in cell cycle regulation, the effect of inactivation of CUL4 was examined in both Drosophila and human cells. Loss of CUL4 in Drosophila cells causes G1 cell cycle arrest and an increased protein level of the CDK inhibitor Dacapo. Coelimination of Dacapo
with CUL4 abolishes the G1 cell cycle arrest. In human cells, inactivation of CUL4A
induces CDK inhibitor p27Kip1 stabilization and G1 cell cycle arrest which is dependent on the presence of p27, suggesting that this regulatory pathway is evolutionarily conserved.
In addition, it was found that the Drosophila CUL4 also regulates the protein level of cyclin E independent of Dacapo. Evidence is provided that human CUL4B, a paralogue of
human CUL4A, is involved in cyclin E regulation. Loss of CUL4B causes the accumulation of cyclin E without a concomitant increase of p27. The human CUL4B and cyclin E proteins also interact with each other and the CUL4B complexes can polyubiquitinate the CUL4B-associated cyclin E. These studies suggest that the CUL4-containing ubiquitin E3 ligases play a critical role in regulating G1 cell cycle progression in both Drosophila and human cells (Higa, 2006).
The CUL1 (cullin 1; see Drosophila Cul1) containing SCF (SKP1, CUL1/CDC53, F-box proteins) ubiquitin
E3 ligases are key regulators of cell cycle progression from yeast to human. The
SCF E3 ligases use different F-box proteins to bind and target various cell cycle regulators for ubiquitin-dependent proteolysis. In mammalian cells, it has been shown that SKP2, an F-box protein, primarily binds and targets phosphorylated CDK inhibitors p27Kip1 and p21Cip1 for ubiquitin-dependent proteolysis, while another F-box protein, human CDC4/AGO/FBXW7 regulates the proteolysis of phosphorylated cyclin E protein. In mammalian cells, the G1 cell cycle is regulated by the relative abundance of G1 cyclin/CDKs and CDK inhibitors such as p27 and p21. Similarly, the Drosophila G1 cell cycle is regulated by the balance between the CDK inhibitor Dacapo, which shares substantial homology to p27, and cyclin E. While cyclin E is regulated by the conserved Drosophila SCFAgo E3 ligase, it is not clear how the level of Dacapo is regulated in the cell cycle (Higa, 2006).
Like other cullin family members, CUL1 is regulated by the covalent linkage of an ubiquitin like protein, NEDD8, through the neddylation activating enzyme E1 (APPBP1 and UBA3) and the E2 enzyme, UBC12. Neddylation of CUL1 dissociates CAND1,
an inhibitor of SCF, from CUL1, and consequently promotes the binding of SKP1 and
F-box proteins such as SKP2 to CUL1 and the assembly of the SCF E3 ligase complex.
The neddylation of CUL1 is removed (deneddylated) by the peptidase activity of the
COP9-signalosome complex (CSN; see Drosophila COP9 complex homolog subunit 5). Many lines of evidence suggest that the activity
of cullins is regulated by the elegant balance between the neddylation and deneddylation
activities (Higa, 2006).
Cullin 4 (CUL4) is a conserved core component of a new class of ubiquitin E3 ligase
that also contains the UV-damaged DNA-binding protein 1 (DDB1) and Ring finger
protein ROC1 (also called RBX1 or HRT1). Unlike Drosophila or other metazoans,
mammals encode two paralogues of CUL4, CUL4A and CUL4B. CUL4A and CUL4B are coexpressed in many cells but the functional differences between them remain
unclear. Like other cullin E3 ligases, the CUL4 proteins also bind to CAND1 and CSN, and are regulated by neddylation and deneddylation processes. Previous studies suggest that CUL4-containing E3 ligase complexes and CSN regulate
the proteolysis of replication licensing protein CDT1 (see Drosophila Cdt/Double parked) in
response to UV or gamma-irradiation. Additional studies suggest
that DDB1, a potential SKP1-like adaptor for CUL4 E3 ligase is
also involved in UV-induced CDT1 proteolysis. The CUL4ADDB1
complex also regulates the proteolysis of c-Jun and DDB2.
However, the roles of CUL4-containing ubiquitin E3 ligases in cell
cycle regulation remain uncharacterized. This study has investigated the regulation of cell cycle regulators by neddylation and CAND1 and reports the unexpected finding that CUL4 E3 ligase plays a critical role in regulating G1 cell cycle progression (Higa, 2006).
Loss of CUL4 E3 ligases causes a G1 cell cycle arrest that is dependent on CDK inhibitors
Dacapo in Drosophila and p27 in human cells. The regulation of
Dacapo and p27 by CUL4 E3 ligases occurs at the post-transcriptional
levels of protein stability. Although it has not been
demonstrated that p27 can be directly polyubiquitinated by the
CUL4 E3 ligase complexes in vitro due to technical difficulties, this
study raises the possibility that CUL4 E3 ligases may regulate
Dacapo or p27 by directly targeting them for ubiquitin-dependent
proteolysis. Several lines of evidence support this hypothesis. Dacapo protein is regulated by CUL4 but not by CUL1 in Drosophila cells. Although in human cells, SCFSKP2
regulates p27, there is no structural and functional evidence that
SKP2 is conserved in Drosophila cells. In addition, although
Dacapo shares substantial homology to p27 or p21 in the
core region that mediates cyclin or CDK binding, it
diverges greatly at the carboxy terminal end with p27 in
which the critical threonine 187 is located for the SCFSKP2-
dependent proteolysis of p27 (this threonine is absent
in Dacapo). Furthermore, it was found that there are no
significant differences in the SCF-dependent p27 degradation between extracts derived from the control and DDB1 or CUL4A siRNA treated cells, suggesting that reduced
levels of DDB1 and CUL4A proteins does not significantly
affect SCFSKP2 activity. However, these experiments do not
completely rule out the possibility that CUL4A/DDB1 are
catalytically involved in SCFSKP2-mediated p27 degradation
since small amounts of DDB1 and CUL4A proteins remain in
the siRNA treated cells. Moreover, although SKP2 represents
a major proteolysis pathway for regulating p27 degradation
in S phase of human cells, substantial evidence suggests
there are additional pathways that regulate the stability of
CDK inhibitors. For example, it was found that the Xenopus p27 homologue p27Xic1 is polyubiquitinated on chromatin only when DNA replication starts in the Xenopus egg extracts. Replication licensing protein CDT1 is proteolyzed by CUL4/ROC1 E3 ligase in response
to UV or gamma-irradiation. CDT1 is also degraded in
S phase in mammalian cells and such an event can be reproduced
in Xenopus egg extracts in which CDT1 was found to undergo ubiquitin-dependent proteolysis once DNA replication starts. In C. elegans, loss of CUL4 stabilizes
CDT1 in S phase and causes the accumulation of polyploid
nuclei containing 100C DNA content. It is possible that
CUL4 may also regulate the proteolysis of Dacapo or p27 in
similar processes in Drosophila or human cells (Higa, 2006).
Cyclin E protein accumulates in CUL4 silenced Drosophila and human cells often in the absence of CDK inhibitors Dacapo or p27. Although this effect is more pronounced in
Drosophila cells, the CUL4 E3 ligase may represent one of
several pathways that regulate cyclin E in response to
certain signals in mammalian cells. It has been
shown that CUL1- and CUL3-containing E3 ligases
regulate cyclin E stability in mammalian cells. Cyclin E
expression and its protein stability are also regulated by an
E2F/DP-1 dependent process. This study found that cyclin E
directly interacts with Drosophila CUL4 and human CUL4B and
the isolated CUL4A or CUL4B immunocomplexes can polyubiquitinate
the associated cyclin E in vitro. These observations raise the possibility that cyclin E may also be a direct ubiquitination target of CUL4 E3 ligases in vivo.
These studies indicated that loss of CAND1, APPBP1, or CSN has
differential effects on Armadillo/β-catenin and cyclin E.
This effect could be partly explained by the observation that while
Armadillo is regulated by CUL1-containing SCF ligase, cyclin E is
controlled by both CUL1 and CUL4 E3 ligases. Evidence is also provided
that the effects of CAND1, APPBP1 or CSN deficiency on the substrates of various cullin E3 ligases may be different. Further analysis is required to investigate the
mechanisms for these observations (Higa, 2006).
These data reveal that CUL4 E3 ligase represents a novel and
conserved pathway from Drosophila to human cells in regulating
CDK inhibitors and cyclin E. In the G1 cell cycle, the CDK inhibitors Dacapo and p27 appear to be the primary targets of CUL4 E3 ligases, since loss of CUL4 in Drosophila or CUL4A in
human leads to the G1 cell cycle arrest rather than enhanced S phase
entry. Since the gene encoding CUL4A is amplified in many breast
cancers and hepatocellular carcinomas and since low or absent
expression of p27 is often associated with malignant cancers, these
studies also highlight how altered regulation of CUL4 E3 ligase may
contribute to the genesis and progression of human cancers (Higa, 2006).
Terminal differentiation is often coupled with permanent exit from the cell cycle, yet it is unclear how cell proliferation is blocked in differentiated tissues. The process of cell cycle exit was examined in Drosophila wings and eyes; cell cycle exit can be prevented or even reversed in terminally differentiating cells by the simultaneous activation of E2F1 and either Cyclin E/Cdk2 or Cyclin D/Cdk4. Enforcing both E2F and Cyclin/Cdk activities is required to bypass exit because feedback between E2F and Cyclin E/Cdk2 is inhibited after cells differentiate, ensuring that cell cycle exit is robust. In some differentiating cell types (e.g., neurons), known inhibitors including the retinoblastoma homolog Rbf and the p27 homolog Dacapo contribute to parallel repression of E2F and Cyclin E/Cdk2. In other cell types, however (e.g., wing epithelial cells), unknown mechanisms inhibit E2F and Cyclin/Cdk activity in parallel to enforce permanent cell cycle exit upon terminal differentiation (Buttitta, 2007).
Current models for cell cycle exit invoke repression of Cyclin/Cdk activity by CKIs or repression of E2F-mediated transcription by RBs as the proximal mechanisms by which cell cycle progression is arrested. Since these models include the potential for positive feedback between E2F and CycE/Cdk2, they predict that the induction of either E2F or a G1 Cyclin/Cdk complex should be sufficient to maintain the activity of the other and thereby sustain the proliferative state. However, in differentiating Drosophila tissues, both E2F and G1 Cyclin/Cdk activities had to be simultaneously upregulated to bypass or reverse cell cycle exit. An explanation for this resides in two observations. First, the ability of Cyclin/Cdk activity to promote E2F-dependent transcription is lost or reduced in the wing and eye after terminal differentiation. Second, increased E2F cannot sustain functional levels of CycE/Cdk2 activity after terminal differentiation, despite an increase in cycE and cdk2 mRNA to levels higher than those observed in proliferative-stage wings. Thus, crosstalk between E2F and Cyclin/Cdk activity appears to be limited, in both directions, as a consequence of differentiation (Buttitta, 2007).
How are these two regulatory interactions altered? One possibility is that Rbf2- or E2F2-dependent repression prevents ectopic Cyclin/Cdk activity from promoting E2F-dependent transcription after prolonged exit. While mRNA expression data and the existing genetic data on E2F2 and Rbf2 do not support this possibility, the roles of Rbf2 or E2F2 have not been tested in the presence of continued Cyclin/Cdk activity. Therefore, transcriptional repression of E2F targets by Rbf2 or E2F2 remains an important issue to address in future experiments (Buttitta, 2007).
More enigmatic is the inability of the ectopic CycE/Cdk2 provided by overexpressed E2F to promote cell cycle progression. One plausible explanation for this is that novel inhibitors of CycE are expressed with the onset of differentiation, and that these raise the threshold of Cyclin/Cdk activity required to promote cell cycle progression. Such inhibitors might make the critical substrates of CycE/Cdk2, which reside on chromatin in DNA-replication and -transcription initiation complexes, less accessible or otherwise recalcitrant to activation. The notion of an increased Cdk threshold is consistent with the observation that the >10-fold increase in CycE/Cdk2 provided by direct overexpression of the kinase bypassed cell cycle exit in conjunction with E2F, while the ~4-fold increase provided indirectly by ectopic E2F is insufficient to drive the cell cycle. Although a >10-fold increase in Cdk activity as applied in these experiments is far above the normal physiological range, such dramatic deregulation of cell cycle genes may be physiologically relevant to cancers, in which gene expression can be greatly amplified (Buttitta, 2007).
Recent studies of cycle exit in larval Drosophila eyes have concluded that Rbf1 and Dap are required to inhibit E2F and CycE/Cdk2 in differentiating photoreceptors. Other studies document the roles of Ago/Fbw7 and components of the Hippo/Warts-signaling pathway in downregulating CycE for cell cycle exit in nonneural cells in the eye. Although the data are consistent with these studies in the eye, Ago and the Hippo/Warts pathway are dispensable for cell cycle exit in the wing. Furthermore, deletion of Rbf1 did not prevent cell cycle exit in the epithelial wing, even when high levels of CycE/Cdk2 were provided. Conversely, deletion of Dap was not sufficient to keep wing cells cycling, even when excessive E2F activity was provided. These observations suggest that unknown inhibitors of E2F and Cyclin/Cdk activity mediate cell cycle exit in specific contexts, such as the wing (Buttitta, 2007).
In attempts to identify upstream factors regulating cell cycle exit, a variety of growth and patterning signals were manipulated in the pupal wing and eye, and their effects on cell cycle exit were examined. Surprisingly, signals that act as potent inducers of proliferation in wings and eyes at earlier stages did not prevent or even delay cell cycle exit upon terminal differentiation. Thus, an important focus for future studies will be the nature of the signals upstream of E2F and CycE that mediate cell cycle exit. These could be novel signals, or combinations of known signals delivered in unappreciated ways (Buttitta, 2007).
How general is double assurance? Studies of cell cycle exit in mammals do not offer a consistent answer to this question. S phase re-entry can be achieved in differentiated cells by activating E2F, CycE/Cdk2, or CycD/Cdk4 alone, but this does not lead to cell division or continued proliferation. Several studies with mammalian cells in vivo have shown that neither increased E2F nor Cyclin/Cdk activity alone is sufficient to fully reverse differentiation-associated quiescence, consistent with the double-assurance model propose in this study. Also consistent with this model is the ability of proteins from DNA tumor viruses, such as adenovirus E1A, SV40 LargeT, and HPV E6 and E7, to fully reverse differentiation-associated cell cycle exit in many cell types. These viral onco-proteins stimulate cell cycle progression by targeting multiple cell cycle factors, which ultimately increase both E2F and G1 Cyclin/Cdk activities simultaneously. For example, LargeT and E1A inhibit both RBs and CKIs, such as p21Cip1 and p27Kip1 (Buttitta, 2007).
There are some instances, however, in which differentiation-associated cell cycle exit has been bypassed, not just delayed, by the deletion of CKIs or RBs. In one such case, p19Ink4d and p27Kip1 were knocked out in the mouse brain, and ectopic mitoses were documented in neuronal cells weeks after they normally become quiescent. Similar results have been obtained with hair and support cells in the mouse inner ear, where deletion of p19Ink4d, p27Kip1, or pRB can bypass developmentally programmed cell cycle exit. In light of these findings, it is interesting to speculate that certain differentiated tissues may retain some ability to repair or regenerate by maintaining the capacity for positive feedback between E2F and CycE/Cdk2 activity. Inner-ear hair cells may be such an example, since in many vertebrates they are capable of regeneration, although this ability has been lost in mammals. Although the mammalian brain has a very limited capacity for regeneration, the cell cycle can be reactivated in the brains of other vertebrates, such as fish, in response to injury. Thus, the retention of crosstalk between E2F and Cyclin/Cdk activities in the evolutionary descendents of regeneration-competent cells might explain some of the tissue-specific sensitivities to loss of CKIs or RBs observed in mammals (Buttitta, 2007).
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