Cell  Cycle  

 
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Be n  An th o ny  A.  Lop e z Cell Cycle Transcription 7

 
TOPIC OUTLINE
I. Cell Cycle
A. Phases of the Cell Cycle
B. Cell Cycle Events Require Temporal Coordination
C. Regulators of Periodic Transcription
D. Regulation by Checkpoints
a. Cell Size Control
b. DNA Damage Responses
c. Monitoring DNA replication
d. S-M Dependency
e. Mitotic Spindle Checkpoint

CELL CYCLE

• Consists of temporally ordered events: DNA replication •

centrosome duplication • mitosis • cytokinesis––that result in
complete duplication of genetic material, followed by equal
segregation into 2 cell bodies (i.e. 2 daughter cells)
• Many of the genes encoding cell-cycle regulators (e.g. cyclins) are Fig. 2 Significance of the periodic transcription program. (a) Genes are expressed
transcribed only in distinct phases of the cell cycle only during the cell-cycle phase needed. Genes required for DNA replication are
expressed during S phase. (b) The temporal order of gene expression may aid in
PHASES OF THE CELL CYCLE the construction of a protein complex only needed once per cycle. (c) While protein
levels of cell-cycle regulators may remain constant, posttranslational modifica-
GAP 1 SYNTHESIS GAP2 MITOSIS tions may alter the activity of the proteins
• “Gap” phases – termed so because they separate the visibly
observable events of S & M phase; no overt cellular changes are § Cells are subject to environmental constrains and will
observable during G1 & G2 characterization ofdivide whengene
periodic conditions permit,
expression will but not when
become more the
• early G1: extracellular signals (e.g. nutrient abundance, mating tractable in othersurrounding is not amenable to cell division.
model systems.
phenomenon) are interpreted by the cell to decide whether to commit § Much of the life of a single cell is spent outside the
Two questions arise from the finding that the transcriptional
to a new cell cycle cell cycle, in quiescence (a state of rest); when a
program is conserved across
signal eukaryotes:
is received (1) what
to initiate cellis division,
the signifithe
cance
cell is
• Upon commitment, cells enter S by activating the expression of genes of cell cycle-regulated
required for duplicating DNA and centrosomes poised to complete cell cycle events with coor-
transcription and (2) what mechanisms the right
dinate this large transcriptional
genes expressed program
at thewith cell time.
proper cycle progression?
• G2: cells interpret intracellular signals from checkpoint pathways that
monitor whether: (2) to build a required structure only once per cell cycle (Fig
2.2 Significance of
(1) duplication events have been completed with fidelity Many hypotheses have beenofposed
2b)—components to explain
a complex the importance
are periodically of
transcribed
(2) the mitotic spindle apparatus is functional Cell Cycle-Regulated cell cycle-regulated
so functiontranscription.
of the complex The explanations
differs can be general-
temporally
o duplicate centrosomes separate to form poles of the Transcription
mitotic (3) tothree
ized into renewcategories.
pools of unmodified
While all proteins that are
are plausible able toforcarry
reasons
spindle (which separates the sister chromatids) regulatingout genecell cycle timing
expression events during
(Fig the2c)––these
cell cycle,proteins
it is not are
• M: sister chromatids separate; cytokinesis follows posttranslationally modified to become inactive
currently possible to discriminate between the multiple hypotheses. or be
. Bristow et al. Moreover,unresponsive
each potential to additional signaling
hypothesis is not mutually exclusive and
may be true for only a subset of cell cycle-regulated genes.
REGULATORS OF PERIODIC TRANSCRIPTION
The first category postulates that cell cycle-regulated transcrip-
• transcription factors – control activation or repression of periodic
tion is a mechanism to expend energy resources efficiently, as tran-
transcription
• scription
these TFs: and translation are energetically expensive. This concept
is often(1)referred to as “just
are themselves in time” transcribed
periodically transcription,
(asin which
they playgene
a role in
products that function
controlling at a specific cell
cell cycle-regulated cycle interval are expressed
transcription)
only when needed TFs2athat
§ In(Fig. ) (reviewed in [10, 32, 33
act in complexes, at]). A variation
least one TF is
on this first explanation hasexpressed
periodically been referred to as the “Sleeping
Beauty” (2) situation,
are affected which takes into activity,
by cyclin/CDK accountinthe fulloflifetime
terms activityof a
(3) bind to the promoters of other TFs shown to regulate periodic
gene expression
Fig. 1 Cell-cycle progression in Saccharomyces cerevisiae. Budding yeast serves § E.g., TFs expressed late in the cell cycle have been
shown to bind to the promoters of TFs responsible for
as an excellent model system to study the cell cycle. Timing and regulation of
early periodic gene expression (suggesting the
events are conserved across species. More importantly, the phase of the cell
transition from one phase
CelltoCycle
the next)
Transcription 13
cycle can be deduced by observing the state and size of the bud, the future
daughter cell

CELL CYCLE EVENTS REQUIRE TEMPORAL COORDINATION

• chromatids (Fig. event––e.g.
Each cell cycle 1). Sister chromatid segregation
DNA replication, is initiated
centrosome during
duplication,
M phase, or mitosis
chromosome (Fig. 1).
segregation––is so complex that it requires the
G1 and G2
coordination were termed
of many different“gap” phases,
proteins as together
acting they separate the
to complete
visibly
the taskobservable
at hand events of S and M phase. Although no overt
• cellular changes
Cell cycle or events
events are observed
are triggered andduring G1 and primarily
coordinated G2, cells areby 2
interpreting
proteins: (a) signals
cyclinsfrom & (b)their extracellular and
cyclin-dependent intracellular
kinases (CDKs)envi-
o Cyclins to
ronments activate
ensure CDKsthat conditions are appropriate for cellular
o CDKsevents.
division activateInand inhibit
early G1,different events at
cells interpret the proper signals
extracellular time & in
the proper order
(e.g., nutrient abundance, mating pheromone) to decide whether
• Up to a fifth of the S. cerevisiae genome, including cyclins, is Fig. 3  
4 Model of cell-cycle regulation. A transcription factor network is responsible
to commit to a new cell cycle. Following this point of commitment for regulating the timing of the periodic transcriptional program, including cyclins.
transcribed once per cell cycle; this is referred to as the cell cycle-
(called START
regulated in Saccharomyces
transcriptional programcerevisiae), cells prepare for entry Cyclins, in complex with CDKs, then act as effectors to trigger events at the
• While cyclin-CDK activity is the
into S phase by activating expression
implicated of control
in the genes ofrequired for
coordination, proper time after periodic synthesis
duplicating
recent studiesDNA andfound
have centrosomes
out that (Fig. 1). In G2, cells transcription
“cell cycle-regulated interpret
intracellular
has the capacitysignals fromlargely
to occur checkpoint pathways that monitor
without CDKs” • A TF network acts as an oscillator that drives the timing of periodic
whether
o This duplication
suggests that eventsanother
have been completed
regulatory with fidelity,
mechanism and be
may transcription, including transcription of cyclin genes
network
• Cyclin may function
(in complex as an feedbacks
with CDKs) underlyingonto
cell the
cycleTFoscillator
network that
via
whether the mitotic
responsible spindle apparatus
for controlling periodic istranscription
functional. and coordinating
cell cycle controls the periodic
phosphorylation, thereby transcriptional
enhancing orprogram 5], transactivation
reducing([the reviewed in [33of ]).
Each cell events (although it could
cycle event—such as DNA be that an alternative
replication, process
centrosome
compensates the TF’sIntarget
yeast genes
and somatic cells, several experiments have shown that
duplication, and for the lack of CDKs––idea
chromosome segregation—is mine)a complex process • This
cyclin/CDKs have
model (Fig 3) the in
differs capacity to alter
the older TFinactivity.
models What
that a TF is the
network
that requires the coordination of many different proteins acting (instead
effect of thecyclin/CDK
that cyclin/CDK activity)
feedbackacts as the
acting onoscillator that has
TF activity keepson
together to complete the task at hand. In turn, each of these com- thetranscriptional
timing and ordering of cell Comparing
cycle progression, and functions to
• Genes involved in cell cycle-regulated transcription are expressed in oscillations? transcriptional dynamics
plex
an events must manner
oscillating be coordinately controlled
periodically duringwiththethecell
othercycle
events.(e.g. control the temporal program of transcription.
reveals that the overall amplitude of periodic gene expression
What, then, are the
histones)—i.e., mechanisms
gene expression that orchestrate
correlated withthea complex setevent
cell cycle of
dynamics decreases as cyclin/CDK activity is removed [12].
events
knownrequired
to occurfor cellular
only division?
once per cycle Over the past three decades, an REGULATION BY CHECKPOINTS
Additionally, the period of transcriptional oscillations also increase
• overwhelming
Hypotheses that number of studies
explain have identifiof
the importance ed and
cell characterized
cycle-regulated
with decreasing cyclin/CDK activity [12]. These observations
two proteins that act in a complex to trigger cell cycle events:
transcription: • CDKs – serine/threonine
suggest that cyclin/CDKprotein
activitykinases
plays anthat phosphorylate
important key
role in regu-
cyclins(1)and
to expend energy resources
cyclin-dependent kinases efficiently,
(CDKs). Bothgenesbiochemical
are expressed substrates to promote DNA
only when their products lating the amplitude andsynthesis
period of and mitotic progression
transcriptional oscillations. In
and genetic approaches have shownare needed
that, (Fig 2a)—“just
throughout in time”
the cell cycle,
addition to cyclin/CDK feedback on the TF network, cyclins
CDKs aretranscription
activated by different cyclins, whose role is to activate
themselves are periodically transcribed (reviewed in [9–11, 33]).
and inhibit different events at the proper time and in the proper
Yet in the absence of CDK activity, cell cycle progression is halted.
order (reviewed in [1–3]). More recently, it has been shown that up Page  1  of  2  
A study showed that in the absence of all S-phase and mitotic
to a fifth of the S. cerevisiae genome, including cyclins themselves,
cyclins, except for a single S-phase cyclin, periodic cycles of DNA
replication occur together with transcriptional activation of the
S-phase cyclin [12]. This observation implies that cyclin/CDK
activity also acts as an effector of the TF network oscillator.
Cell  Cycle  
 
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Be n  An th o ny  A.  Lop e z
 
  o In molar excess, but lack activity until bound by their cognate cyclin • The mitotic checkpoint is unique in that it functions to maintain CDK
subunits, which are tightly regulated at (a) synthesis and (b) activity, while those functioning in interphase aim to maintain CDK
ubiquitin-dependent proteolysis inactivity
o Can be negatively regulated by the binding of small inhibitory
proteins, CKIs, or by inhibitory tyrosine phosphorylation which
blocks phosphate transfer to substrates

CELL CYCLE CHECKPOINTS

(1) Cell Size Control
o In G1 and G2
o Unclear, but proposed mechanisms include: monitoring of protein
translation, monitoring of cell geometry (cells grow lengthwise prior
to division)
(2) DNA Damage Responses
o In Interphase, DNA damage elicits a cell cycle arrest that allows
time for repair pathways to operate prior to commitment to
subsequent phases of the cell cycle
o Controlled by either: (a) the tumor suppressor and transcription
factor p53, or (b) the checkpoint kinase Chk1
§ p53 is regulated by posttranslational modifications [e.g. N-
terminal phosphorylation on serine-15, which is catalyzed by
ATR (Ataxia Telangiectasia and Rad3-Related), ATM (Ataxia
Telangiectasia Mutated), DNA PKcs (DNA-dependent protein
kinase, catalytic subunit)—which all target double-strand
DNA breaks by interacting proteins]; activated p53 is
stabilized through protection from its E3 ubiquitin ligase
Mdm2; as a tetramer transactivates the expression of
multiple genes, including the cyclin-dependent kinase
inhibitor (CKI) p21
§ Chk1 is activated by all known forms of DNA damage,
though is more efficient in S- and G2-phase than in G1, and
restricted to post-replicative lesions; Replication Protein A
(RPA)-coated single-strand DNA lesions are where
complexes of checkpoing proteins assemble: ATR (Ataxia
Telangiectasia and Rad3-Related), the phosphorylation of
which recruits and activates Chk1; Chk1 is released to
maintain the mitotic CDK Cdc2 in its Y15 phosphorylated,
inactive state (by phosphorylating both the kinase Wee1 and
phosphatase Cdc25, which both regulate the Y15
phosphorylation); Chk1 is subject to dephosphorylation by
type 1 phosphatases, upon which cells resume cycling into
mitosis
(3) Monitoring DNA replication
o DNA replication is initiated at replication origins, which are
epigenetically defined by a number of proteins that ensure they fire
(start replicating) once and only once per cell cycle
o Replication origin firing is controlled by the phosphorylation of Cdt1
and Cdc6, which is catalyzed by both CDKs and the Dbf4-
dependent protein kinase (DDK) Cdc7; such phosphorylation not
only initiates replication but also leads to degradation of these
proteins, so the origin cannot refire
o When the replisome encounters a blockade to progression (e.g. in
the form of modified dNTPs, abasic sites, protein-DNA complexes,
or result from the depletion of dNTPs), the replisome remains
stably associated with the replicating chromatid for resumption to
occur once blockade is removed––intra-S-phase checkpoint
o The effector kinase of the intra-S-phase checkpoint is known as
Cds1 in fission yeast or Chk2 in humans; activated Cds1/Chk2
stabilizes the stalled replisome by phosphorylation of several
subunits, notably the MCM helicase
(4) S-M Dependency
o Only until replication is complete should mitosis start
o Y15 phosphorylation of Cdc2 blocks mitotic entry
(5) The Mitotic Spindle Checkpoint
o Segregation of sister chromatids at anaphase is under the
mechanical control of the mitotic spindle (comprised of
microtubules and several motor proteins at both centrosomal and
kinetochore ends)
o Sister chromatids should be under tension at metaphase, through
both poles of the spindle
o Once kinetochores are attached and aligned at the metaphase
plate, anaphase proceeds through the activity of a large E3
ubiquitin ligase known as the Anaphase-Promoting Complex or
Cyclosome (APC/C), which targets: (a) mitotic cyclins (abolishes
CDK activity) and (b) securin (the degradation of which allows
separase to be released and cleaves cohesin complexes at the
kinetochores)
o APC activity is controlled by: (1) Cdc20, which functions up to the
metaphase-anaphase; and (2) Cdh1, which facilitates APC-
mediated ubiquitination (once cyclin and separase degradation
have begun)
Cdc20
o The spindle checkpoint functions to prevent activation of APC
under conditions where kinetochores aren’t occupied by spindle
microtobules, or are attached but not under tension
o Spindle checkpoint protein Mad2 (Mitotic Arrest Deficient) inhibits
Cdc20 activity both at unattached kinetochores (where a mitotic
checkpoint complex is formed) and at APC-bound molecules
o Cdc20 is regulated by Bub1 (yeast) and Bub1R (mammals)
o When Cdc20 is inactive, so is APC, so cells do not enter anaphase
o The formation of the spindle and the detection and correction of
spindle defects are under the control of the Polo, Aurora and NIMA-
related (Nek) kinases

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