HISTONE TITRATION BY DNA SETS THE THRESHOLD FOR THE MID‐BLASTULAct021wp7863/Amodeo Thesis... ·...
Transcript of HISTONE TITRATION BY DNA SETS THE THRESHOLD FOR THE MID‐BLASTULAct021wp7863/Amodeo Thesis... ·...
-
HISTONE TITRATION BY DNA SETS THE THRESHOLD FOR THE MID‐BLASTULA
TRANSITION IN XENOPUS
A DISSERTATION
SUBMITTED TO THE DEPARTMENT OF BIOLOGY
AND THE COMMITTEE ON GRADUATE STUDIES
OF STANFORD UNIVERSITY
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
Amanda A. Amodeo
February 2013
-
This dissertation is online at: http://purl.stanford.edu/ct021wp7863
© 2014 by Amanda Anne Amodeo. All Rights Reserved.
Re-distributed by Stanford University under license with the author.
ii
http://purl.stanford.edu/ct021wp7863
-
I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.
Jan Skotheim, Primary Adviser
I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.
Michael Simon
I certify that I have read this dissertation and that, in my opinion, it is fully adequatein scope and quality as a dissertation for the degree of Doctor of Philosophy.
Timothy Stearns
Approved for the Stanford University Committee on Graduate Studies.
Patricia J. Gumport, Vice Provost for Graduate Education
This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file inUniversity Archives.
iii
-
iv
Abstract
During early development, animal embryos depend upon maternally deposited
RNA until zygotic genes become transcriptionally active. Prior to this maternal‐
to‐zygotic transition (MZT), many species execute rapid and synchronous cell
divisions without growth phases or cell cycle checkpoints. In Xenopus laevis
embryos, the MZT coincides with cell cycle lengthening, cellular motility onset,
and the addition of cell cycle checkpoints at a stage termed the mid‐blastula
transition (MBT). A long standing model is that the timing of the MBT is
controlled by a maternally loaded inhibitory factor that is titrated against the
exponentially increasing amount of DNA(Newport and Kirschner, 1982b). To
identify the inhibitory factor(s), we developed an assay using Xenopus egg extract
that recapitulates the activation of transcription only above the DNA‐to‐
cytoplasm ratio found in embryos at the MBT. We used this system to
biochemically purify the factor responsible for inhibiting transcription below the
threshold DNA‐to‐cytoplasm ratio. This unbiased approach identified histones H3
and H4 as inhibitory factors. Addition of exogenous H3/H4 tetramers to the
extract increased the threshold concentration of DNA required for transcriptional
activation. Non‐specific removal of endogenous histones reduced the threshold
DNA concentration required for transcription, and the addition of histone H3/H4
tetramers was sufficient to restore the threshold in depleted extracts. Finally,
reduction of H3 protein in embryos induced a premature MBT. Our observations
-
v
support a model for MBT regulation by a maternally loaded titratable factor and
provide an identity for this long‐sought molecule. More broadly, our work
demonstrates how a constant concentration DNA binding molecule can be used
to measure the amount of cytoplasm per genome, a surrogate for cell size.
-
vi
Acknowledgments
This work would not have been possible without the intellectual contributions
and support of several communities of people. Foremost, I must thank my
advisors. As a joint student I have benefited from the mentorship of two very
talented and different faculty members at Stanford. Throughout my graduate
career Jan has supported and encouraged me to explore, even when I didn’t think
things would work. His unyielding optimism has spurred me to take risks and try
experiments that I never would have attempted without his encouragement.
Aaron too, has been a remarkable mentor. He welcomed me into his lab as a
visitor and allowed me to stay on as a true member of his lab. I would have never
predicted that my PhD would involve the biochemical purification of an unknown
factor, and I could never have done it with out them.
Along with two advisors came two wonderful sets of labmates and friends. Their
intellectual contributions and help in troubleshooting my experiments cannot be
overstated. Beyond that, their camaraderie made every day and adventure and
their support helped me though those weeks when nothing worked.
In addition to my labmates, I’ve also benefited from the support of my family and
a number of great friends? I’d particularly like to thank my father and brother
who still understand me better than anyone else even after all these years. Words
cannot express how grateful I am for all that you all have done for me.
-
vii
Contents
Abstract ................................................................................................................................ iv
Acknowledgments ............................................................................................................ vi
Contents .............................................................................................................................. vii
List of Tables ....................................................................................................................... ix
List of Figures ....................................................................................................................... x
Chapter I: Introduction to cell size control ............................................................... 1
1.1 Introduction: Cells maintain a characteristic cell size ............................................ 1
1.2 The relationship between growth and size control ................................................. 4
1.3 Many potential size‐sensing mechanisms ................................................................... 6
1.4 Cell size correlates with DNA content ........................................................................... 8
1.5 Titration of Cyclins in Budding Yeast .......................................................................... 13
1.6 Titration of DNA‐A in E. coli ............................................................................................ 14
1.7 Geometric size sensing in fission yeast ...................................................................... 17
1.8 The ease of generating titration based size sensors .............................................. 18
Chapter II: Titration and the mid‐blastula transition ......................................... 22
2.1 The maternal to zygotic transition (MZT) ................................................................. 22
2.2 The mid‐blastula transition and titration ................................................................. 24
2.3 Potential titrated factors at the MBT ........................................................................... 26
Chapter III: Histone titration times the MBT .......................................................... 29
3.1 Recreating the MBT in vitro ........................................................................................... 29
-
viii
3.2 Unbiased biochemical purification .............................................................................. 33
3.3 Effects of histones in extracts ........................................................................................ 36
3.4 Histone reduction and the MBT in vivo ...................................................................... 38
3.5 Supplemental material .................................................................................................... 40
Chapter IV: Methods ........................................................................................................ 51
Chapter V: Conclusions and future directions ........................................................ 58
5.1 Conclusions .......................................................................................................................... 58
6.2 Future directions ............................................................................................................... 62
References .......................................................................................................................... 68
-
ix
List of Tables
Table of mass spectrometry results ......................................................................... 48
-
x
List of Figures
Figure 1.1: Cell size verses organ and organism size ............................................. 3
Figure 1.2: Mechanisms for cell size measurement ............................................... 8
Figure 1.3: Endocytosis and cell size ........................................................................ 12
Figure 1.4: Titration mechanisms for cell size measurement in bacteria ... 16
Figure 3.1. in vitro transcription is sensitive to the DNA‐to‐cytoplasmic
ratio via a titratable transcriptional inhibitor ...................................................... 32
Figure 3.2. Isolation of histones H3 and H4 as the inhibitory activity .......... 34
Figure 3.3. H3/H4 tetramers set the threshold for transcriptional activation
in extract ............................................................................................................................ 37
Figure 3.4. Reduction of histone H3 causes premature initiation of the MBT
in vivo .................................................................................................................................. 39
Figure S1. DNA‐to‐cytoplasm ratio controls transcription ............................... 40
Figure S2. The major sperm chromatin transcript is a single stranded Pol III
product ............................................................................................................................... 41
Figure S3. Inhibitory activity can be removed from extract‐treated DNA ... 42
Figure S4. Intermediate gels during biochemical purification ........................ 43
-
xi
Figure S5. H3/H4 inhibits transcription ................................................................. 45
Figure S6. H3 morpholinos reduce H3 concentrations at the MBT ................ 46
Figure S7. H3 morphant embryos display early MBT in multiple
independent experiments ............................................................................................ 47
Figure 5.1: Mid‐blastula transition controlled by titration of regulators
against DNA ....................................................................................................................... 61
-
CHAPTER I: INTRODUCTION TO CELL SIZE CONTROL 1
Chapter I: Introduction to cell size control
1.1 Introduction: Cells maintain a characteristic cell size
Cells size varies greatly depending on cell type and species. Among the eukaryotic
cells, 1mm frog oocytes (Wallace et al., 1981), are 1000 times larger than 1
micron diameter phytoplankton, a billion‐fold difference in volume (Palenik et al.,
2007). Bacteria, such as Mycoplasma genitalium, can be as small as 200nm in
diameter (Zhao et al., 2008), while the largest prokaryote, Thiomargarita
namibiensis can be larger than 100 microns (Schulz et al., 1999). Even within an
organism, cells of different type may be of very different sizes. Human blood cells
are tiny (
-
CHAPTER I: INTRODUCTION TO CELL SIZE CONTROL 2
In contrast to unicellular organisms, cells within multicellular organisms
experience much more constant environments. However, the function of these
cells still strongly depends on their size. For example, blood cells must maintain a
sufficiently small size to allow them to pass through capillaries and neurons must
span great distances in order to transduce singles down the lengths of limbs.
Furthermore, defects in cell size are associated with diseases such as Lhermitte‐
Duclos disease wherein increase in cerebellar granule cell size leads to seizures
and eventual death (Kwon et al., 2001).
In some multicellular organisms, cell size can influence organ and organism size.
This scaling is absolute in C. elegans, where the cell number is fixed and
perturbations to cell size result in proportional changes in organ and organism
size (Cook and Tyers, 2007; Watanabe et al., 2007). However, in a large number
of species, such as the tiger salamander, alterations to cell size can be
compensated for by changes in cell number to preserve organ size (Fankhauser,
1945) (Fig 1.1). In addition, forcing proliferation in the fly wing imaginal discs
alters cell size, but triggers sufficient apoptosis to maintain organ size (Neufeld et
al., 1998).
Due to functional constraints, cells are under pressure to maintain a
characteristic size for their given type. In order to accomplish this size
homeostasis, growth and division must be coupled. For example, yeast grown in
nutrient poor conditions adjust their cell cycle duration to accommodate for
slower growth so that cells divide at roughly similar sizes as in nutrient rich
-
CHAPTER I: INTRODUCTION TO CELL SIZE CONTROL 3
environments (Di Talia et al., 2007; Di Talia et al., 2009). Yet, how size
homeostasis is maintained has remained elusive. Here, we review recent
advances and discuss a conceptual framework that potentially links a large class
of cell size questions.
Figure 1.1: Cell size verses organ and organism size. In multicellular organisms cell
size can interface with organ and organism size in one of two ways. a. If the
number of cells is fixed, changes in cell size will lead to changes in organ and
-
CHAPTER I: INTRODUCTION TO CELL SIZE CONTROL 4
subsequently organism size, as in C. elegans. b. If organ size is regulated
independently of cell size, changes in cell size will be compensated by changes in
cell number resulting in a constant organ and organism size, as happens in
salamanders. A mix of both mechanisms is also possible where cell size affects
organ size, but is partially compensated for by changes in cell number.
1.2 The relationship between growth and size control
At its most fundamental, cell size control within a specified interval ensures that
larger cells grow proportionally less than smaller cells. Importantly, size control
does not have to completely compensate for initial cell size differences. Rather,
size control is a quantitative feature whose amount in an interval can be
measured (Di Talia et al., 2009; Sveiczer et al., 1996). For example, weak size
control might require multiple cell division cycles to return an initially aberrantly
large or small cell to the characteristic size, while strong size control could do this
in one cycle.
For some cell growth dynamics, cell size homeostasis can be maintained without
a cell size sensor. All that is required for size homeostasis is that smaller cells
grow proportionally more than larger cells in a cell cycle so that size variation is
reduced in the next generation. For example, in the case of a constant rate of
linear growth, the cells size , will increase at a constant rate so that .
Then, specifying the duration of the cell cycle determines the amount of growth €
V
!
dVdt
= C
-
CHAPTER I: INTRODUCTION TO CELL SIZE CONTROL 5
so that, in proportion, smaller cells grow more than larger cells and the
population approaches a mean size without any specific size sensing mechanism
(Conlon and Raff, 2003). Thus, some cell growth dynamics do not require a cell
size measurement to maintain size homeostasis.
Other growth dynamics do necessitate a size measurement. One such example is
exponential growth, where the rate of growth is proportional to cell size:
. Here, a timer mechanism will not maintain size homeostasis because
cells born larger would grow proportionally during a specified period.
Fluctuations in division times will then result in a progressively wider size
distribution unless the cell employs a size sensing mechanism to limit cell cycle
duration in larger cells (Tyson and Hannsgen, 1985a; Tyson and Hannsgen,
1985b).
The importance of the dynamics of cell growth for size control has motivated
many studies (Mitchison, 2003). In the case of budding yeast, growth in an
unperturbed cell cycle is likely close to exponential as supported by both some
single cell analysis using fluorescence time‐lapse microscopy and microchannel
resonators, as well as classical bulk experiments using radioactive labeling (Di
Talia et al., 2007; Elliott and McLaughlin, 1978; Godin et al., 2010). However,
other groups have argued that G1 growth is closer to linear based on estimates of
cell volume using time‐lapse phase contrast microscopy (Ferrezuelo et al., 2012)
and that growth rates are altered by cell cycle phase (Goranov et al., 2009). For a
!
dVdt
= "V
-
CHAPTER I: INTRODUCTION TO CELL SIZE CONTROL 6
more complete discussion of yeast size control and growth see Turner ’12.
Fission yeast growth is certainly less likely to be exponential as reviewed in
(Mitchison, 2003), and metazoan cells are almost certainly not (Kafri et al., 2013;
Son et al., 2012; Sung et al., 2013). The clear deviation from exponential growth
in mammalian and other cells does not imply that these cells do not employ a size
sensor. In cases where size sensors are not strictly necessary, they may still be
desirable to keep the cell within an optimal range of sizes for proliferation and
function. Thus, the only way to determine if cell size sensors are operating in a
given cell type is to determine the molecular mechanism.
1.3 Many potential size‐sensing mechanisms
Active regulation of cell cycle progression in response to cell size requires that
cells have a method to accurately measure their size. Cells might accomplish size
measurement though a geometric mechanism if there is little cell‐to‐cell variation
in their geometry. For example, a geometric mechanism seems unlikely in
amoeboid cells or in the microvilli containing cells of the small intestine, but are
much more plausible in rod like bacteria and fission yeast, which grow in a well‐
specified geometry along a single dimension. One potential method to measure
size using geometry would be to measure the ratio of surface area to volume
since for many geometries surface area will increase much less rapidly than
volume. A related possibility is that cells might sense other aspects of their
geometry such as cell length via intracellular gradients of cell cycle regulators
-
CHAPTER I: INTRODUCTION TO CELL SIZE CONTROL 7
(Martin and Berthelot‐Grosjean, 2009; Moseley et al., 2009). In this case,
segregation of critical components to designated sub‐cellular locations would
allow the cell to assess the distance between these compartments as a proxy for
cell size.
In multicellular organisms, external landmarks such as tissue edges or junctions
with target cells could be used to constrain cell growth. For example, a neuron
might grow until it makes contact with its target (Guthrie, 2007). However, this
mechanism requires an external pattern to already be in place and cannot
generate size control de novo so we do not focus our attention on this mechanism
in this chapter.
Finally, cells could measure their volume directly by titration of a constant
concentration sensor against a fixed internal “yardstick”. This mechanism
requires that the production of the volume sensor must be proportional to cell
growth and that there be a fixed “yardstick” to measure, i.e., titrate against. Here,
we explore the relevance of titration mechanisms in several biological contexts
(Fig 1.2).
-
CHAPTER I: INTRODUCTION TO CELL SIZE CONTROL 8
Figure 1.2: Mechanisms for cell size measurement. Cells can potentially sense their
size via a number of different mechanisms including: a. aspects of their cell
geometry such as surface area‐to‐volume or cell length via intracellular
gradients; b. extracellular landmarks; and (C) titration of a constant
concentration regulator, whose amount scales with cell size, against a fixed
“yardstick” to measure cell size.
1.4 Cell size correlates with DNA content
One of the earliest and most consistent observations about cell size is the
relationship between cell size and ploidy. In the early 1900’s, Theordor Boveri
-
CHAPTER I: INTRODUCTION TO CELL SIZE CONTROL 9
produced sea urchin embryos in a range of ploidies by manipulating the
conditions of fertilization. Haploid embryos divided more than diploid embryos
and thereby maintained a consistent ratio of DNA to cytoplasm. In further
experiments he created triploid and tetraploid embryos through multiple
fertilizations. In these embryos, spindle abnormalities caused an uneven
distribution of chromosomes so that not all cells received integer multiples of the
genome. In all cases, however, Boveri observed that the number of divisions
within a given sector of the embryo compensated for the DNA content that sector
received in the first cleavage. By the pluteus larval stage, cells that had received
more DNA were proportionally larger than those that had received less, which
indicated that the relationship extended beyond a simple size scaling with ploidy.
(Wilson, 1926)
Moreover, the relationship between cell size and ploidy extends well beyond the
embryo. Viable adult salamanders can be created from haploid to pentaploid. In
all cases, the size of the cells within their tissues increased or decreased
correspondingly, though the total organism size remained relatively invariant
due to compensatory alterations to cell number (Fankhauser, 1945). This
relationship is true in unicellular eukaryotes as well. The cell size of budding
yeast correlates with ploidy from haploid to hexaploid (Mayer et al., 1992;
Mortimer, 1958).
Developmentally regulated changes in ploidy correlate with cell size in numerous
large cell types. Often, as in the case of fly salivary gland and the mammalian
-
CHAPTER I: INTRODUCTION TO CELL SIZE CONTROL 10
trophoblast, cells increase their ploidy by undergoing rounds of
endoreduplication. In many cases, such as the wing scale cells in the moth,
Ephestia, the degree of endoreduplication correlates with cell size within a tissue
(Edgar and Orr‐Weaver, 2001) (Fig 1.3b). Similarly, as the land‐slug Limax grows
over its lifetime it increases the size of the neurons within its brain though
endoreduplication (Yamagishi et al., 2011). A very tight relationship has been
demonstrated between DNA content and cell size in the green algae Eudorina
(Tautvydas, 1976). The filamentous yeast, Ashbya, also contains many copies of
its genome within a single cytoplasm. In this case, individual nuclei appear be
carefully spaced within the growing cells in order to retain a relatively constant
ratio of DNA to cytoplasm (Anderson et al., 2013; Nair et al., 2010).
Endoreduplicative cell cycles increase ploidy by entering a relatively normal S‐
phase but then skipping cytokinesis resulting in a doubling of the DNA content
for each iteration. This process can be repeated a large number of times resulting
in very large ploidies (up to 24,000C in the case of some plant endosperms (Traas
et al., 1998)). Endoreduplicating cell cycles are not simply unregulated or
prolonged S‐phases. Instead, these cells go through a fairly normal G1 and S‐
phase in which the DNA is only replicated once. They then either proceed directly
from G2 into another G1‐like state (called an endocycle), or enter into mitosis but
abort before cytokinesis is completed and return to the G1‐like state (called an
endomitosis) (Zielke et al., 2013) (Fig 1.3a). In both cases, the activity of
conventional M‐phase regulators is absent or prematurely lost. However, G1/S
-
CHAPTER I: INTRODUCTION TO CELL SIZE CONTROL 11
regulation in endoreduplicating cells appears to be broadly similar to what is
found in conventional mitotic cell cycles (Edgar and Orr‐Weaver, 2001). This
implies that if a mechanism to measure cell size relative to DNA content were
operable at the G1/S transition, it might also explain the ploidy‐size relationship
observed for endoreduplicated cell types.
We note that the relationship between DNA content and cell size is not only true
within single species, but seems to be constant across most eukaryotes with only
small variations between clades across 6 orders of magnitude. A similar
relationship between cell size and DNA content has also been observed in
prokaryotes, although the slope is not the same (Cavalier‐Smith, 2001; Gregory,
2000). However, this does not imply that the mechanism of size measurement is
identical in all clades, but rather that there exist selective pressures that maintain
a roughly linear relationship between DNA content and cell size across
eukaryotes.
-
CHAPTER I: INTRODUCTION TO CELL SIZE CONTROL 12
Figure 1.3: Endocytosis and cell size. DNA content correlates with cell size in a
variety of organisms and contexts. a. Endoreduplicative cell cycles are similar to
mitotic cycles without cytokinesis. Endocycling cells proceed directly from a S/G2
like state to another G1, while endomitotic cells undergo a partial mitosis but
abort before cytokinesis. b. The wing scale cells of the moth Ephestia vary in
-
CHAPTER I: INTRODUCTION TO CELL SIZE CONTROL 13
ploidy from 8 to 32N with higher ploidy cells being larger than lower ploidy cells
(modified from (Edgar and Orr‐Weaver, 2001)).
1.5 Titration of Cyclins in Budding Yeast
Cell size control can be most directly studied in unicellular rather than multi‐
cellular organisms because cell size is more easily measured and the number of
cell types limited. For this reason, the yeasts S. cerevisiae and S. Pombe have been
favorite models for the study of cell size regulation.
Budding yeast divide asymmetrically, and size control occurs in the smaller
daughter cells primarily in G1 (Di Talia et al., 2009). Smaller cells spend more
time in G1, but this size control is imperfect since variations in size are not
eliminated in a single cell cycle (Di Talia et al., 2007; Johnston, 1977). Progression
through G1 is initiated by the cyclin Cln3, which binds and activates CDK1 to
partially inactivate the transcriptional inhibitor Whi5 (Bertoli et al., 2013).
Inactivation of Whi5 relieves the inhibition the transcription factor SBF whose
target genes include the additional cyclins, Cln1 and Cln2. These two cyclins
complete inactivation of Whi5 via a positive feedback loop that drives further cell
cycle progression (Eser et al., 2011). Activation of this feedback loop ensures
irreversible commitment to cell cycle progression (Charvin et al., 2010; Doncic
and Skotheim, 2013; Skotheim et al., 2008). Interestingly, the levels of the
upstream rate‐limiting cyclin Cln3 oscillate only weakly within the cell cycle and
-
CHAPTER I: INTRODUCTION TO CELL SIZE CONTROL 14
may be at constant concentration during early G1 (Tyers et al., 1993). Therefore,
Cln3 might be used to sense cell size via a titration mechanism.
Cln3 may be titrated against the genome itself, or specific binding sites.
Consistent with the later, cell size was shifted by a high‐copy plasmid containing
multiple SBF binding sites in a Cln3‐Whi5‐dependent manner (Wang et al., 2009).
In either case, Cln3‐Cdk complex, whose number is likely proportional to cell size,
would be titrated against a constant DNA yardstick. However, implications of
these Cln3‐titration conjectures remain largely untested.
1.6 Titration of DNA‐A in E. coli
Titration of regulatory molecules against a cellular component whose size is
invariant with growth has been proposed to regulate specific points of the
bacteria cell cycle (Chien et al., 2012). While any point in the cell cycle can
potentially be regulated via a size checkpoint, two specific points of control have
been identified through studies of E. coli and B. subtilis. Bacteria size control has
been reported to impact the onset of DNA replication and cell division. In both
cases, a titration mechanism has been suggested for the origin of size‐dependent
cell cycle progression signals (Chien et al., 2012; Donachie, 1968; Teather et al.,
1974).
To control replication, Donachie (Donachie, 1968) proposed that a constant
concentration activator would accumulate at the origins of replication. In this
-
CHAPTER I: INTRODUCTION TO CELL SIZE CONTROL 15
model, a regulator whose number increases with cell size would titrate against
the fixed number of origins to yield a size‐dependent activating signal. Consistent
with this model, cell size at the onset of replication in E. coli was similar
regardless of birth size and growth rate. Even in the case of the fastest growing E.
coli, where mass doubling outpaces the duration of replication so that large cells
contain multiple copies of partial genomes, replication is triggered at a constant
size‐to‐origin ratio (Donachie, 1968).
The most likely candidate for Donachie’s titrated replication activator is DnaA,
which binds cooperatively to origins to promote loading of the replication
machinery (Kaguni, 2006). Consistent with the DnaA‐titration model, the size‐to‐
origin ratio at replication initiation is sensitive to DnaA expression levels
(Lobner‐Olesen et al., 1989). Furthermore, mutations affecting cell size at
division, but not cell growth rate, do not change DnaA concentration or the size at
which cells initiate replication. Mutations making cells twice as large lead to cells
containing twice the amount of DNA (Hill et al., 2012). Taken together, this
evidence suggests molecular titration as the most plausible mechanism for
coupling cell size to the onset of DNA replication in E. coli (Fig 1.4a).
In addition to coordinating S‐phase with cell size, E. coli may also coordinate cell
division with cell size (Chien et al., 2012). The accumulation of the constant
concentration regulator FtsZ at the cytokinetic ring could function as a titration
base cell size sensor (Chien et al., 2012; Teather et al., 1974). Since cell growth of
rod shaped bacteria maintains a constant width, the circumference at the midcell
-
CHAPTER I: INTRODUCTION TO CELL SIZE CONTROL 16
is constant. Thus, titration of FtsZ against the midcell perimeter could yield a
size‐dependent signal to initiate cytokinesis (Fig 1.4b).
Figure 1.4: Titration mechanisms for cell size measurement in bacteria. a. ATPase
DnaA accumulates on origins of replication in a size dependent manner until a
critical threshold triggers replication. b. FtsZ accumulates at the site of
cytokinesis as the cell grows until a threshold triggering cytokinesis
-
CHAPTER I: INTRODUCTION TO CELL SIZE CONTROL 17
1.7 Geometric size sensing in fission yeast
Similar to E. coli, fission yeast are rod‐shaped and grow by lengthening at a
constant width so that volume is proportional to length (Mitchison, 2003). It has
been proposed that fission yeast employ a spatial gradient of the mitotic inhibitor
Pom1 emanating from its poles to measure cell size geometrically via length
(Martin and Berthelot‐Grosjean, 2009; Moseley et al., 2009). In this model,
division ensues when the concentration of Pom1 dips below a critical
concentration at the midcell, which occurs at a specific cell length.
Pom1 regulates the mitotic trigger network centered on the competing kinase
and phosphatase pair Wee1 and Cdc25 that regulates Cdk activity. Pom1 inhibits
the Wee1‐inhibitory kinases Cdr1 and Cdr2, which localize to specific nodes at
the midcell (Deng and Moseley, 2013). As a cell grows, the gradient in Pom1 from
the poles ensures that its concentration at the midcell decreases, which could
allow Cdr1 and Cdr2 to inactivate Wee1 and thereby promote mitotic entry.
Perturbations of the Pom1 gradient result in both changes to the Cdr2
distribution and cell size (Bhatia et al., 2013; Hachet et al., 2011; Martin and
Berthelot‐Grosjean, 2009; Moseley et al., 2009).
However, several recent results have begun to cast doubt on the gradient model.
First, the length scale of the exponential Pom1 gradient (~1.5µm) appears to be
too short to accurately measure the length of a >10µm cell (Saunders et al.,
2012). Second, the gradient has a difficult time explaining why the DNA‐to‐
cytoplasm ratio is maintained in fission yeast cells of different ploidy and in
-
CHAPTER I: INTRODUCTION TO CELL SIZE CONTROL 18
temperature sensitive cytokinesis mutants (Neumann and Nurse, 2007). Third, in
fission yeast mutants of different width, volume at mitosis was more variable
than area, suggesting a surface area‐based model (Pan and Chang in publication).
Finally, cell size control occurs, albeit a bit less effectively, in cells lacking
regulation of Cdk1 by Wee1 and Cdc25, indicating that the Pom1 pathway is not
the only size control mechanism (Coudreuse and Nurse, 2010; Wood and Nurse,
2013).
1.8 The ease of generating titration based size sensors
To link growth and proliferation, size sensing mechanisms regulate cell cycle
transitions, which are typically switch‐like and associated with threshold
ultrasensitive responses. Switch‐like transitions are used to ensure coordination
of downstream responses. For example, switch‐like increases in Cdk activity
ensure the coherent transcription of G1/S phase regulators, the all‐or‐none
replication of the genome during S phase, and the robust phosphorylation of
mitotic targets (Ferrell et al., 2009; Georgi et al., 2002; Koivomagi et al., 2011;
Skotheim et al., 2008; Yang et al., 2013b).
Under some conditions titration mechanisms can directly generate ultrasensitive
responses (Buchler and Louis, 2008). For example, titration with a high affinity
stoichiometric inhibitor can generate an ultrasensitive response curve. Consider
two proteins A and B that bind to form an inactive complex AB. The response can
be characterized as the amount of active A as a function of the total amount of A
given a fixed amount of B. For high affinity interactions with B, low amounts of A
-
CHAPTER I: INTRODUCTION TO CELL SIZE CONTROL 19
will be fully inactivated by B. However, for increasing amounts of A, the inhibitor
B is eventually titrated out so that above a threshold the amount of active A
increases rapidly. In the case of DNA‐binding proteins, such as DnaA high affinity
decoy sites (Kitagawa et al., 1996) could serve the purpose of stoichiometric
inhibitors as these sites will be occupied before the relevant target site and
therefore titrate out the regulator to create an ultrasensitive response.
Although titration mechanisms can produce ultrasensitivity, they need not
generate the switch themselves. Graded upstream signals can be converted
downstream in order to produce a switch‐like response. For example, positive
feedback loops at the G1/S and G2/M transitions convert small changes in the
upstream kinase input signal to dramatic increases in downstream kinase activity
associated with the next cell cycle phase (Pomerening et al., 2003; Skotheim et al.,
2008). In case of budding yeast, a graded Cln3 signal is coupled via Whi5 to a
Cln1/2 positive feedback response in order to generate a sharp transition in Cdk
activity. Thus, downstream switches can generate threshold responses thereby
alleviating any such requirement on the upstream size‐dependent signal.
Whether ultrasensitive or not, titration‐based size sensors are relatively easy to
generate. All they require is one component that is produced proportionally to
cell size and another that is constant. Many proteins fit the profile for the titrated
molecule because the majority of proteins and RNAs are found at roughly
constant concentration during the cell division cycle. This is supported by the fact
that
-
CHAPTER I: INTRODUCTION TO CELL SIZE CONTROL 20
et al., 1998; Whitfield et al., 2002). Furthermore, a genome wide analysis of GFP
tagged ORFs in budding yeast concluded that most of the variability in protein
abundance arose from variations in cell size (Newman et al., 2006). This is a
natural consequence of larger cells having more protein and that the proportions
of most protein species remain relatively constant during cell growth. Any one of
these “cell cycle independent” proteins could potentially be titrated against a
constant “yardstick” to yield a size sensor.
Constant “yardsticks” are much less common within growing cells because, much
like proteins, the majority of organelles scale with cell size. For example, nuclear
size is always 8‐10% of cell size in yeast mutants (Neumann and Nurse, 2007;
Zhao et al., 2008). In contrast, DNA is a promising candidate because its amount
is fixed for all segments of the cell cycle except S‐phase. Moreover, size scales
with DNA content in many experimental and endogenous contexts as discussed
above. Thus, most DNA binding proteins could potentially be employed as a size
sensor.
Cell size is one of the most basic and defining features of a given cell type, yet the
molecular mechanisms underlying its control have remained elusive. We are
excited by recent work revisiting this long‐standing problem. Here, this chapter
reviewed current understandings of size sensing mechanisms and highlighted the
potential of titration‐based mechanisms. However, because of the ease with
which size‐dependent signals might be generated by any constant concentration
DNA binding protein, we do not expect conservation of the specific regulatory
-
CHAPTER I: INTRODUCTION TO CELL SIZE CONTROL 21
molecules. One important context in which titration‐based cell size sensors are
likely employed is a developmental transition known as the mid‐blastula
transition (MBT).
-
CHAPTER II: TITRATION AND THE MID‐BLASTULA TRANSITION 22
Chapter II: Titration and the mid‐blastula
transition
2.1 The maternal to zygotic transition (MZT) To successfully develop from a single fertilized cell into a functioning adult, cells
within a dividing embryo must shift from utilizing maternally contributed mRNAs
and proteins to expressing zygotically encoded genes, which is known as the
Maternal to Zygotic Transition (MZT). In most species, the MZT switch is abrupt
and involves large‐scale degradation of maternal RNAs in addition to activation
of zygotic transcription (Tadros and Lipshitz, 2009). The MZT occurs at different
stages of development in different species. In mammals, the transition occurs as
early as the two‐cell stage (Piko and Clegg, 1982). Drosophila undergo 14
syncytial nuclear divisions before the zygotic genome becomes active. Zebrafish
divide 10 times, while Xenopus divide 12 times prior to the MZT (Kane and
Kimmel, 1993; Newport and Kirschner, 1982a).
The MZT is the period when the embryo becomes dependent on zygotic genes.
Although several genes critical for later development have been shown to escape
global transcriptional repression prior to the MZT (Merrill et al., 1988; Skirkanich
et al., 2011; Yang et al., 2002), most are not (Heyn et al., 2014; Lecuyer et al.,
-
CHAPTER II: TITRATION AND THE MID‐BLASTULA TRANSITION 23
2007; Tan et al., 2013; Yang et al., 2013a). Moreover, many maternal transcripts
are degraded at the MZT and must be replaced by their zygotic counterparts. For
example, around 35% of all maternal transcripts are destabilized at the MZT in
Drosophila (De Renzis et al., 2007). Although maternal RNAs are deadenylated
throughout early development their destruction does not occur until the onset of
zygotic transcription in many organisms including Xenopus (Audic et al., 1997;
Bashirullah et al., 1999; Duval et al., 1990). In fish, the MZT‐controlled microRNA
mir‐430 marks its targets via their three prime UTRs for destruction (Giraldez et
al., 2006), while in flies, the MZT‐controlled protein Smaug destabilizes RNAs by
binding their three prime UTRs (Tadros et al.).
Several transcription factors are critical for zygotic genome activation. In
Drosophila, a class of pre‐ and early post‐MZT genes has been identified whose
promoters contain heptad repeats which bind the transcriptional regulators
Zelda, and Grainyhead (De Renzis et al., 2007; Harrison et al., 2010; Liang et al.,
2008). Zelda competes with Grainyhead to bind a conserved CAGGTAG cis‐
regulatory element in the promoters of numerous developmental genes.
Depletion of Zelda and overexpression of Grainyhead results in major
developmental defects due to a failure to express Zelda targets at the MZT
(Harrison et al., 2010; Liang et al., 2008). Zelda appears to work as a general
transcriptional regulator in combination with other transcription factors that
determine spatial and temporal expression (Kanodia et al., 2012; Pearson et al.,
2012 2012). Interestingly, Zelda binds its target enhancers several cycles before
-
CHAPTER II: TITRATION AND THE MID‐BLASTULA TRANSITION 24
transcription is initiated and its abundance is predictive of expression levels
during ZGA. This led to the suggestion that Zelda is a “pioneer transcription
factor” that marks early genes for activation (Harrison et al., 2011; Satija and
Bradley, 2012). The transcription factors Nanog, Pou5f1 (Oct4), and SoxB1 are
present on early genes in zebrafish and have been suggested to play a similar role
in vertebrates (Lee et al.; Leichsenring et al.).
However, while necessary, pioneer transcription factors cannot explain
important aspects of transcriptional activation such as the dependence of many
transcripts on the ratio of DNA to cytoplasm. Interestingly, in Drosophila two
classes of genes are activated at the MBT. One class is likely controlled by the
time after fertilization, while others are controlled by the DNA‐to‐cytoplasmic
ratio (Lu et al., 2009). These results indicate that multiple mechanisms and
pathways could contribute to the MZT in Drosophila.
2.2 The mid‐blastula transition and titration
In a large number of organisms, including flies, fish, and frogs, the MZT coincides
with a more general developmental transition known as the Mid‐Blastula
Transition (MBT) (Tadros and Lipshitz, 2009). The MBT is found in a number of
organisms with large, externally developing eggs. The MBT is characterized by a
transition from rapid synchronous cell divisions without transcription to
asynchronous cell cycles containing growth phases and zygotic transcription.
-
CHAPTER II: TITRATION AND THE MID‐BLASTULA TRANSITION 25
It is not currently known whether these processes are interdependent or
whether they are separately controlled. It has been suggested that the rapid cell
cycles of the early embryo prevent transcription because there is insufficient
time to initiate and elongate transcripts between each cell cycle without growth
phases (Howe et al., 1995; Kimelman et al., 1987; Newport et al., 1985).
Consistent with this hypothesis, substantial inhibition of transcription in Xenopus
embryos had no effect on cell cycle elongation at the MBT (Newport and
Kirschner, 1982a). However, early transcription at the MBT has been shown to be
important for G1 acquisition in Zebrafish embryos (Zamir et al., 1997). In
Drosophila, the number of syncytial divisions appears to be controlled by zygotic
transcripts including the Cdc25 regulator tribbles (Di Talia et al., 2013; Farrell
and O'Farrell, 2013; Farrell et al.; Sung et al., 2013). Thus, understanding how
changes in cell cycle timing, transcriptional activation and the acquisition of
motility are coordinately controlled at the MBT is an important unresolved
problem.
Several compelling experiments suggest that the ratio of DNA to cytoplasm
controls the MBT. Each round of DNA replication prior to the MBT doubles the
DNA concentration while the cytoplasmic volume remains constant due to the
lack of cell growth in the early embryo. Artificially altering the DNA content by
creating polyspermic embryos, injecting exogenous DNA, or initiating
development in haploid cells shifts the timing of the MBT as predicted by the
-
CHAPTER II: TITRATION AND THE MID‐BLASTULA TRANSITION 26
DNA‐to‐cytoplasm ratio (Newport and Kirschner, 1982a; Newport and Kirschner,
1982b). Perhaps the most convincing demonstration of the importance of the
DNA‐to‐cytoplasm ratio was performed by Newport and Kirschner (Newport and
Kirschner, 1982a). In this experiment, they constricted a newly fertilized embryo
to restrict the nucleus to one half of the embryo for 2 cell divisions and then
allowed a single nucleus from the nuclear side of the embryo to reenter the
anucleate side. The nucleated side of the embryo had undergone both nuclear
and cell divisions and thus contained cells with a much higher nuclear to
cytoplasmic ratio than the originally anucleate side. Significantly, the cells with
higher nuclear to cytoplasmic ratio underwent the MBT approximately 2 cycles
earlier than cells in the other half of the embryo, providing strong evidence that
the DNA‐to‐cytoplasm ratio is crucial to MBT timing. These experiments also
show that pre‐MBT embryos are capable of transcription but that transcription is
repressed. These observations have lead to the hypothesis that a repressive
factor present in the early embryo is titrated out by the DNA. However, a
definitive test of this model through the identification of the factors controlling
transcription has remained an outstanding question in developmental biology.
2.3 Potential titrated factors at the MBT
A molecular understanding of how transcriptional activation is governed by the
DNA‐to‐cytoplasmic ratio is essential for understanding the coordination
between cell cycle changes and transcription at the MBT. Several candidate
-
CHAPTER II: TITRATION AND THE MID‐BLASTULA TRANSITION 27
factors have been implicated as part of the transcriptional repressor activity in
the embryo. Chromatin components were first proposed as the potential
titratable factor(s) due to the observation that exogenous DNA is rapidly
incorporated into chromatin (Almouzni et al., 1990; Almouzni et al., 1991;
Newport and Kirschner, 1982b; Prioleau et al., 1994). Coinjection of histones
with exogenous DNA reduces the ability of that DNA to be transiently transcribed
in Xenopus embryos, but these experiments cannot differentiate whether histones
themselves are inhibiting transcription rather than simply facilitating the binding
of another factor (Almouzni and Wolffe, 1995). Another chromatin factor, the
DNA methyl transferase, xDnmt1 has also been proposed to regulate the MBT.
Knockdown of xDmnt1 has a modest effect on transcription onset and is only
expressed in one half of the embryo, indicating that it cannot be the sole factor
responsible for pre‐MZT transcriptional repression (Dunican et al., 2008;
Stancheva and Meehan, 2000).
More recently, two sets of factors have been implicated in S‐phase lengthening at
the Xenopus MBT. The first is the protein phosphatase 2 (PP2A) regulatory
subunit, B55α. PP2A is an important suppressor of ATR, which signals to inhibit
origin firing in response to replication stress. ATR slows S‐phase at high DNA
concentrations in vitro. This appears to be due to limiting quantities of PP2A at
high DNA concentrations as exogenous PP2A restores rapid S‐phases in vitro
(Murphy and Michael, 2013). However, it is unclear if PP2A is, in fact limiting in
vivo. Furthermore, the function of S‐phase checkpoints does not appear to
-
CHAPTER II: TITRATION AND THE MID‐BLASTULA TRANSITION 28
depend on transcription (Dasso and Newport, 1990) and there the regulation of
their acquisition and the other aspects of the MZT such as transcription may be
independent.
The other set of proposed S‐phase regulators are the replication factors Cut5,
RecQ4, Treslin, and Drf1. These factors become unstable at the MBT, and appear
to be limiting well below the DNA concentrations present at the MBT in vitro.
Greater than five fold overexpression of these four factors in combination is able
to induce one additional fast S‐phase in vivo (Collart et al., 2013). The fact that
these factors become unstable at the MBT suggests that they are likely
downstream of the titrated factor, though this remains to be tested.
Nearly three decades after Newport and Kirshner first proposed the titratable
factor hypothesis, its molecular underpinnings are still unclear. Although the
factors discussed above all have their merits, each was identified through a
specific candidate approach, and none has been conclusively shown to be titrated
at the MBT.
-
CHAPTER III: HISTONE TITRATION TIMES THE MBT 29
Chapter III: Histone titration times the
MBT
3.1 Recreating the MBT in vitro
To identify the mechanism by which embryos sense the DNA‐to‐cytoplasm ratio,
we developed a cell free system for Xenopus ZGA where we could directly
manipulate the ratio of DNA to cytoplasm. We added varying concentrations of
purified sperm chromatin into a constant volume of cytosolic extracts from
unfertilized Xenopus eggs and used 32P‐α‐UTP to measure RNA synthesis (Fig.
3.1b). The addition of sperm chromatin to the extract induced transcription
dependent on DNA concentration, as predicted by the titration model. Moreover,
the threshold DNA concentration required for transcriptional activation in
extract (~50 ng/µl) (Fig. 3.1c,d) was strikingly similar to the DNA concentration
present in embryos at ZGA (~48 ng/µl). We controlled for the effects of
increasing the number of sperm transcriptional template in the extract by
keeping the sperm number constant and varying the extract volume (Fig. S1). We
measured a similar threshold DNA‐to‐cytoplasmic ratio indicating that the
observed transcripts are responsive to this ratio and not among the small
number of genes known to escape repression prior to ZGA(Skirkanich et al.,
2011; Tan et al., 2013; Yang et al., 2002) The most abundant transcript we
-
CHAPTER III: HISTONE TITRATION TIMES THE MBT 30
observed off of the sperm chromatin (Fig. 3.1c) is approximately 180‐220
nucleotides, predominantly single stranded based on its ribonuclease sensitivity,
and likely a Pol III transcript based on its sensitivity to transcriptional inhibitors
(Fig. S2). This transcript is likely Sat I/OAX, a 181 nucleotide Pol III‐mediated
transcript activated at the MBT, and previously observed to be highly expressed
from sperm chromatin in Xenopus egg extract (Kimelman et al.; Wolffe, 1989).
Preliminary qPCR results suggest that this transcript increases more than a
thousand fold in transcribing compared to transcriptionally inhibited extracts
(data not shown).*
In order to determine if nonspecific DNA could induce transcription, as has been
observed in Xenopus embryos(Newport et al.), we added λ‐phage DNA to our cell
free system in the presence of low numbers of reporter sperm nuclei. We found
that λ‐DNA was sufficient to induce transcription from the sperm nuclei in a
dose‐dependent manner with a threshold similar to the sperm chromatin alone
(Fig. 3.1e). λ‐DNA alone was not transcribed likely due to a lack of suitable
Xenopus promoters (data not shown). These experiments indicate that titration of
the transcriptional inhibitory activity is not DNA sequence specific and does not
require the titrating DNA to be a template for transcription.
Because DNA binding proteins are likely candidates for a DNA titratable inhibitor
of transcription, we tested whether we could deplete the inhibitory activity from
extracts using DNA as an affinity reagent. To do this, we coupled non‐specific
* Sat I qPCR experiments preformed by David Jukam
-
CHAPTER III: HISTONE TITRATION TIMES THE MBT 31
plasmid DNA to magnetic beads and added these DNA‐beads to an extract so that
there was a 5‐fold excess of DNA above the threshold required for transcriptional
activation. We then removed the DNA‐beads and any associated factors and
measured the transcriptional response of sperm chromatin titrated into the
extract. We found that extracts depleted with DNA‐beads activated transcription
at lower concentrations of sperm DNA than mock depleted controls (Fig. 3.1f,g).
This is consistent with the removal of an inhibitory factor by the DNA‐beads.
To determine if factor removed from extract retained its activity, we moved DNA‐
beads from one extract to another and assayed for transcription (Fig. 3.1h). If
transfer occurred, DNA‐beads saturated with inhibitory activity from a first
extract would be unable to titrate out additional inhibitor in a second extract.
Since the bead‐associated DNA is not transcribed in extracts, we added small
quantities of sperm chromatin as a reporter for transcription. DNA‐beads
pretreated with extract were unable to induce transcription, consistent with a
transcriptional inhibitor binding to the DNA‐beads (Fig. 3.1i). Loss of titration
ability was not due to DNA degradation because DNA‐beads boiled after
incubation in the first extract regained their ability to titrate out the inhibitory
activity from the second extract (Fig. S3).
-
CHAPTER III: HISTONE TITRATION TIMES THE MBT 32
Figure 3.1. in vitro transcription is sensitive to the DNA‐to‐cytoplasmic ratio via a
titratable transcriptional inhibitor. a. A schematic of the titratable factor
hypothesis(Newport and Kirschner, 1982b). A maternally loaded inhibitor is
titrated against an exponentially increasing amount of DNA until a threshold is
-
CHAPTER III: HISTONE TITRATION TIMES THE MBT 33
reached to initiate zygotic transcription. b. Experimental design for (c), (d), and
(f): a constant volume of extract is titrated against variable quantities of sperm
chromatin. c. A representative autoradiograph of new transcription above a
threshold DNA concentration. Transcription in d, e, f and h was quantified using
the indicated boxed band. d. The mean transcriptional response to increasing
amounts of sperm chromatin. Transcription initiates above 50ng sperm DNA/µl
of extract. e. Titration of lambda‐DNA into egg extract activates transcription
from reporter sperm chromatin at a similar DNA concentration as in (d). f. and h.
Schematics of experiments corresponding to panels (g) and (i) respectively. DNA‐
beads are used to bind, deplete and transfer the inhibitory activity in egg extracts.
g. Extracts depleted of a transcriptional inhibitor with DNA coated beads (purple
curve) activate transcription at a lower sperm concentration than extracts
exposed to beads not coupled to DNA (green curve). i. DNA coated beads pre‐
incubated in an extract are unable to induce transcription in a naïve extract
(orange curve) compared to DNA‐beads pre‐incubated with buffer (green curve).
Transcription is measured from reporter sperm chromatin. All error bars
represent SEM.
3.2 Unbiased biochemical purification
To identify the transcriptional inhibitor, we biochemically fractionated the egg
extract and tested the activity of each fraction by adding DNA‐beads to each
fraction and assaying the DNA’s ability to induce transcription in naïve extract
-
CHAPTER III: HISTONE TITRATION TIMES THE MBT 34
(Fig. 3.2a, Methods, Fig. S4). Two fractions from the purification’s final
chromatographic step exhibited the bulk of the inhibitory activity and contained
4 protein bands as observed by silver staining. These bands were identified by
mass spectrometry as histones H3 and H4 (Fig. 3.2b; Supplemental table) with
the slower mobility bands containing ubiquitinated forms of H4.
The titration hypothesis predicts that the titratable factor should be present in
the embryo at a relatively constant concentration, but should be depleted from
the cytoplasm over the course of the cleavage divisions. To test this, we examined
the concentrations of H3 and H4 in whole embryos by western blot at time points
starting immediately after fertilization until approximately 4 hours post‐MBT.
The total embryo concentrations for both H3 and H4 do not increase over this
time period while the DNA concentration increases exponentially, suggesting that
cytoplasmic histone stores are likely being depleted during the MBT in vivo (Fig.
3.2c,d).
-
CHAPTER III: HISTONE TITRATION TIMES THE MBT 35
Figure 3.2. Isolation of histones H3 and H4 as the inhibitory activity. a. Top:
autoradiograph of the transcriptional inhibition activity of fractions from the
final size exclusion step of the purification. Fractions 8 and 9 show inhibitory
activity. Bottom: Silver‐stain gel of the proteins bound to the beads in each
fraction. b. and c. Histone H3 (c) and H4 (d) protein levels normalized to tubulin
levels throughout early embryonic development. For each time point, n=3; error
bars represent SEM.
-
CHAPTER III: HISTONE TITRATION TIMES THE MBT 36
3.3 Effects of histones in extracts
If histones H3/H4 are the inhibitory factors titrated during division, then
increasing histone concentration in the extract should increase the threshold
amount of DNA required for transcription. Adding bacterially produced H3/H4
increased the amount of DNA required to activate transcription in a dosage‐
dependent manner (Fig. 3.3a). We observed a linear trend over a 4‐fold
concentration range in the relationship between the amount of histone H3 in the
extract and the amount of DNA required to activate transcription (Fig. 3.3b).
H3/H4 addition also inhibited transcription from plasmid DNA (Fig. S5a) and
although H2A/H2B dimers could inhibit transcription they did so less efficiently
than H3/H4 (Fig. S5b). The bacterially purified histones we use are not post‐
translationally modified indicating that any histone modifying enzymes that
contribute to transcriptional inhibition in this assay must be present in
concentrations that are not rate limiting.
We tested whether depleting histones from the extract would reduce the amount
of DNA required for transcriptional activation. We removed histones using DNA‐
beads (histone immunodepletion is inefficient from egg extracts) and found that
transcription initiated at lower DNA concentrations (Fig. 3.1f; 3.3c). The specific
addition of purified H3/H4 tetramers to these depleted extracts restored the DNA
threshold to mock‐depleted levels (Fig. 3.3c, d), strongly supporting histones as
the titrated inhibitory molecule.
-
CHAPTER III: HISTONE TITRATION TIMES THE MBT 37
Figure 3.3. H3/H4 tetramers set the threshold for transcriptional activation in
extract. a. Increasing the levels of histone H3 and H4 in egg extracts increases the
concentration of sperm chromatin required to induce transcription. b. The
concentration of DNA required for transcription activation scales roughly linearly
with histone concentration for both sperm chromatin (blue circles) and λ‐phage
DNA (red diamonds). Each point represents a titration as depicted in (a). Points
above dashed line had thresholds beyond the detection limit. c. Depletion of
histone H3/H4 with DNA‐beads reduces the amount of DNA required to activate
-
CHAPTER III: HISTONE TITRATION TIMES THE MBT 38
transcription (purple curve) compared to the control (green curve), and adding
back H3 and H4 restores the transcriptional response (blue curve). d.
Relationship between histone concentration and amount of DNA required for
transcriptional activation as described in (b) for add‐back experiments depicted
in (c). Histone H3 concentrations were quantified by Western blot.
3.4 Histone reduction and the MBT in vivo
We tested whether reducing histone H3 levels in embryos altered the timing of
the MBT in vivo using morpholinos(Szenker et al.). Histone H3 reduction by 40‐
56% at 7.5 hours post fertilization (Fig. S6) caused premature initiation of the
MBT. Both cell cycle period lengthening and cell cycle asynchrony begin earlier,
resulting in larger cells (Fig. 3.4a,c; Fig. S7)†. Despite initiating one cell cycle
earlier, H3 morphant embryos displayed otherwise normal cell cycle lengthening
across the MBT (Fig 3.4d and S7). Ultimately, most H3 morphants died during
gastrulation as previously reported(Szenker et al.) (89/92 morphant vs 3/31
control embryos; Fig. 3.4b).
† Data quantified by David Jukam
-
CHAPTER III: HISTONE TITRATION TIMES THE MBT 39
Figure 3.4. Reduction of histone H3 causes premature initiation of the MBT in vivo.
a. H3 morphant embryos appear normal until the MBT when they display
increased cell cycle lengthening and larger cells compared to controls. The
location of the zoomed panels is marked by the black box. b. H3 morphant
embryos die at gastrulation. c. The cumulative effect of longer cell cycles results
in larger cells and therefore lower cell density in the H3 morphant embryos (H3
MO) compared to control injected embryos (Control). Data shown for 215
minutes after the 9th division (n=3 embryos) d. H3 morphant embryos display
-
CHAPTER III: HISTONE TITRATION TIMES THE MBT 40
early cell cycle lengthening compared to control embryos. The mean and cell
cycle duration increases one cell cycle early in H3 morphant embryos compared
to control morpholino. Circles represent embryos that did not reach a 15th
division before the movie limit. All data shown for >15 cells per embryo, with 3
control embryos and 4 H3 morphant embryos from the same clutch. The box
height represents 25th to 75th percentile, and the centerline represents the
median. The whiskers extend to the furthest data point that is not an outlier.
Asterisks (*) represent p‐values less than 10‐4 Outliers are plotted as diamonds.
Results from two similar experiments are shown in Figure S7.
3.5 Supplemental material
Figure S1. DNA‐to‐cytoplasm ratio controls transcription. Extract volume was
varied (as labeled on top x‐axis) while the quantity of sperm chromatin was kept
-
CHAPTER III: HISTONE TITRATION TIMES THE MBT 41
constant (1µg per sample), resulting in indicated ratios of DNA to cytoplasm
without varying the quantity of template. Reactions containing less extract
resulted in greater total transcription.
Figure S2. The major sperm chromatin transcript is a single stranded Pol III product.
Concentrations of α‐amanitin or actinomycin D as indicated were added during
the transcription reaction. The low sensitivity of our product to both inhibitors
indicates that it is likely a Pol III transcript. The purified RNA was incubated with
DNase, inactive RNase H‐D10A, RNase H, RNase III, or RNase A and showed the
greatest sensitivity to RNase A indicating that the product is likely single
stranded, and comparisons with non‐radiolabeled RNA ladder suggest it is a
transcript between 180‐220 nt long.
-
CHAPTER III: HISTONE TITRATION TIMES THE MBT 42
Figure S3. Inhibitory activity can be removed from extract‐treated DNA. DNA‐beads
were exposed to crude extract and then boiled or left on ice. DNA‐beads that had
been boiled regained their ability to titrate out factor in a fresh extract indicating
that boiling removed the inhibitory factor.
-
CHAPTER III: HISTONE TITRATION TIMES THE MBT 43
Figure S4. Intermediate gels during biochemical purification. a. Titration of DNA
beads treated with buffer, crude extract, or high speed extract shows that both
-
CHAPTER III: HISTONE TITRATION TIMES THE MBT 44
crude and high‐speed extract are able to inhibit the ability of beads to induce
transcription in a fresh extract. b. Flow through and elute of dilute high speed
extract from Q‐ and S‐columns bound in 20mM KCl and eluted with 1M KCl
indicates that under these conditions the inhibitory factor binds the S‐column but
not the Q‐column. c. Flow through from Q‐column in 20mM KCl directly bound by
S‐column. Fractions result from an 8‐fraction linear gradient from 20mM to 1M
KCl elution and the same Q‐column eluted with 1M KCl. These results indicate
that the inhibitory factor does not come off the S‐column until high KCl
concentrations and does not bind the Q‐column. d. Similar to (c), flow through
from Q‐column in 20mM KCl directly bound by S‐column and was then washed
with 400mM KCl. Fractions result from an 8‐fraction linear gradient from 400mM
to 1M KCl elution. The fractions marked with an asterisk were collected, pooled,
concentrated, and run over a gel filtration column as in (f). e. Schematic of the
final resulting biochemical purification used to isolate the inhibitory factor. f. The
final output of the total purification protocol resulting from running fractions
prepared as in (d) over a gel filtration column.
-
CHAPTER III: HISTONE TITRATION TIMES THE MBT 45
Figure S5. H3/H4 inhibits transcription. a. H3/H4 tetramers are able to inhibit
transcription from Xenopus U6 plasmid DNA indicating that the observed effect is
-
CHAPTER III: HISTONE TITRATION TIMES THE MBT 46
not specific to sperm DNA. b. H2A/H2B dimers were also able to inhibit
transcription, but not as efficiently as H3/H4 tetramers.
Figure S6. H3 Morpholinos reduce H3 concentrations at the MBT. Dilution series of
control injected and H3 morphant embryos for the three experiments shown in
Fig. S7. Embryos produced 56, 41, and 40% reduction in H3 normalized to
tubulin loading controls compared to control injected embryos respectively.
-
CHAPTER III: HISTONE TITRATION TIMES THE MBT 47
-
CHAPTER III: HISTONE TITRATION TIMES THE MBT 48
Figure S7. H3 Morphant embryos display early MBT in multiple independent
experiments. Three independent clutches with 2‐3 control embryos and 3‐4 H3
morphant embryos. A minimum of 15 cells were counted from each embryo. All
experiments show an early cell cycle lengthening in the morphant embryos as
compared to controls from the same clutch. Experiment 1 is also displayed in Fig.
3.4.
-
CHAPTER III: HISTONE TITRATION TIMES THE MBT 49
Band Protein ID Type Number of hits
1 H4_XENLA H4 14
1 H32_XENLA H3 6
1 H2B11_XENLA H2B 3
1 H33_XENLA H3 3
1 H2AV_XENLA H2A 2
1 A2BDA0_XENLA H4 2
1 H2AZL_XENLA H2A 1
1 H2A1_XENLA H2A 1
2 H32_XENLA H3 16
2 H4_XENLA H4 12
2 H2B11_XENLA H2B 7
2 CENPA_XENLA H3 5
2 Q6PH89_XENLA H2A 3
2 H33_XENLA H3 3
2 UBIQP_XENLA Ubiquitin 3
2 Q6DKE3_XENLA H2A 2
2 H2A1_XENLA H2A 2
2 A2BDA0_XENLA H4 1
2 Q3KQC9_XENLA Ribosomal 1
3 H4_XENLA H4 11
3 1433T_XENLA 12‐3‐3 theta 4
3 Q7T0R9_XENLA Ribonuclear 4
-
CHAPTER III: HISTONE TITRATION TIMES THE MBT 50
3 UBIQP_XENLA Ubiquitin 4
3 H32_XENLA H3 3
3 RAN_XENLA Ran GTPase 3
3 RS8_XENLA Ribosomal 3
3 H33_XENLA H3 2
3 RL10A_XENLA Ribosomal 2
3 A2BDA0_XENLA H4 1
3 PSA7A_XENLA Proteasome subunit 1
3 Q6AZR7_XENLA Proteasome subunit 1
3 Q8AVT2_XENLA Ribonuclear 1
4 H4_XENLA H4 10
4 Q7T0R9_XENLA Ribonuclear 5
4 H32_XENLA H3 3
4 UBIQP_XENLA Ubiquitin 3
4 EF1B_XENLA Elongation fact (translation) 1
4 A2BDA0_XENLA H4 1
4 Q68A88_XENLA Proteasome subunit 1
Table of mass spectrometry results: Total results from mass spectrometry of
bands shown in Fig. 3b, including the number of unique reads identified for each
protein.
-
CHAPTER IV: METHODS 51
Chapter IV: Methods
Transcription assay: 32P‐α‐UTP was added to interphase extracts or without
DNA and bacterially purified histones as indicated. RNAs were extracted using
either Qiagen RNAeasy kits or Tripure (Roche). RNA samples were run on a 5%
acrylamide, 8M urea gel and exposed to a phosphor screen overnight. Pixel
intensity within the band denoted in Fig. 3.1c was quantified after background
subtraction using ImageJ (NIH). Background subtraction was preformed using an
identical region below the end of the same lane. All titration series were
normalized to a reaction lacking DNA. The threshold for transcriptional
activation was defined as the first point 1.5 fold above the no DNA sample.
H3 Morpholinos: H3 (which target both H3.2 and H3.3) and control morpholinos
(previously described(Szenker et al.)) were injected four times distributed
equally around the animal cap with ~5nl of 1mM morpholino per injection.
Measurement of histone levels: Equal protein concentrations were separated
by SDS‐PAGE and transferred to polyvinylidenefluoride membrane in CAPS
transfer buffer. Membranes were incubated in rabbit anti‐H3 (1:1000; Millipore:
04‐928), 2 μg/ml rabbit anti‐H4 (Abcam: ab7311), or 0.25 μg/ml mouse anti‐
tubulin (Sigma‐Aldrich: T9026), washed and then incubated for a minimum of
one hour in Alexa Fluor 488– or Alexa Fluor 647–conjugated goat anti–rabbit or
-
CHAPTER IV: METHODS 52
anti–mouse antibodies (1:2,500; Invitrogen). Fluorescence was quantified in
Image J. Histone concentrations were normalized to tubulin loading controls.
Xenopus egg extract and sperm chromatin: Preparation of extracts was
performed as previously described(Desai et al., 1999). 750µM CaCl2 was added
to release the extracts into interphase. To monitor new transcription, 50nl/µl of
10mCi/ml or 12.5nl/µl of 40mCi/ml 32P‐α‐UTP (Perkin Elmer BLU507H or
BLU507Z) was added to each extract. Clarified extracts were prepared as in
ref.(Desai et al., 1997). Briefly, crude extracts not containing 32P‐α‐UTP or CaCl2
were spun for a second time at 50,000RPM in a Beckman SW55Ti rotor for 2
hours at 4°C. The clear middle layer was extracted using a 22g needle.
DNA immobilization: pBluescript DNA was cut using EcoRI and BamHI (New
England Biolabs) and end filled using klenow (NEB) and biotin‐dATP
(Invitrogen). DNA was coupled to streptavadin dynabeads (Invitrogen: Cat#
65305) following the protocol outlined in (Hannak and Heald, 2006). For factor
and histone depletion assays, ~250ng/µl DNA on beads was incubated in
interphase extract at 4°C for 1‐2 hours and then removed. In assays that transfer
the factor(s) from one extract or fraction to another, DNA‐beads were first
incubated in an excess of extract or fraction for 1‐2 hours, and then added to a
fresh extract at ~50ng/µl.
Transcription assay: Interphase extracts containing 32P‐α‐UTP were prepared
as described above and distributed into 20µl aliquots with or without DNA and
-
CHAPTER IV: METHODS 53
bacterially purified histones as indicated. Histones were purified as previously
described (Guse et al., 2012; Luger et al., 1997). In cases where sperm chrom