Hypothesis: All-In-G1 assembly

Head Biotech May 2010, Oslo, Norway. Bjørn Sponberg M.Sc.

ABSTRACT

Looking back over 1 billion years in time and peak into the life of unicellular protist cells,
cell division speed was probably under tremendous selection pressure. The protist cells would
constantly seek cell cycle regulatory models that accomplished the division process faster
than competing cells. By designing alternative regulatory models and transcend them into this
ancient world of protist colonies, one can predict the outcome of these evolutionary battles. In
this way, All-In-G1 emerges as a more reliable candidate model compared to the current
view. Three principal arguments support this outcome.
All-In-G1 would give higher rates of progenitor cells via: 1. Faster cell division speed (The
first principal argument) and 2. More evolutionary robust genomes (The second principal
argument). The third principal argument documents that eukaryotic cells have evolved the
molecular mechanisms that underlies the All-In-G1 model. Consequently, All-In-G1 should
have been forced to evolve over the current model, due to its higher fitness. This is the
logic fundament that the three principal arguments provide.
In addition, a blind zone in the work that underlies the current model gives room for All-In-
G1 to exist in vivo, and for the current view to be wrong.
On top of this, All-In-G1 can explain many odd observations made in projects that try to
analyze cell cycle regulation.

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1. A blind zone in the current cell cycle regulatory model

Most protein complexes active during the cell cycle consists of both periodically and
constitutively expressed proteins (1). All-In-G1 argues against the process that brings
about the periodically expressed units. The current model states that periodically
expressed cell cycle genes are transcribed in the same time window in which they are
needed. Most articles today refer to a classic paper on the matter by Simon et al (2), or later
papers that put their trust in the same paper.
The experiments that underlie the current model are not completely waterproof, as I see it
(Figure 1). The usage of Chip-chip to identify and annotate genes that are regulated by
transcription factors (TF’s) can not automatically proof their time of activation. That is, to
use time series gene expression profiles of TF's target genes are no proof that transcriptional
activity takes place in the same time window. Rather, this co-operative linkage is probably
based on the classic transcript experience (Just in time synthesis). This assumption creates a
“blind zone” in the logical link that builds today’s regulatory model. The problem to "see and
believe" within this blind zone is shown in a recent article that focuses on the
problem (3) (Figure 2). Since the Simon et al paper in 2002, a clear trend has emerged.
Identical transcription factors are often shown to be active in several phases via their target
genes, in both yeast and in human cells (5 & 6). Looking through the All-In-G1 model, this is
an expected pattern. Transcription factors work in modules in promoter regions when
transcribing genes (4), they can therefore appear in modules which controls different
transcript routes (Normal, ECIN or assemble and wait) (See figure 4). Consequently, target
genes to a unique transcription factor can be distributed all over the cell cycle, if
necessary. This logic explains why transcription factors target genes so easily
can emerge in multiple cell cycle phases. It is therefore interesting to observe that Wu and Li
in 2008 (6) reports multiple phase activities to the transcription factors that were initially
reported to be phase-specific in the Simon et al paper. E.g. SWI4 which is given a unique role
in G1 phase in 2002 is in 2008 reported by Wu and Li to be active during G1, S, G2/M and
M/G1 phases. This is a new reminder that these kinds of TF data are very sensitive to errors,
and consequently also to "the will to interpret data" towards an already predefined model in a
person’s head. This is a pretty common human error, especially when there is no
obvious alternative model available around to put your strange results into.

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Figure 1. chip-chip experiments in non-synchronized yeast cells (2002). Temporal placement
of TF activation is based on microarray experiments measuring TF’s target genes activity
only. This creates a "blind zone" in which evidence of All-In-G1 can hide (Modified from 2).

Except for the "blind zone" in Simon et als experiment, their experiments are very
elegant and impressive. Especially their follow up experiment on their initial results in which
they are able to establish a network among the active transcription factors, working the cell
cycle. But again, the interpretation of these regulatory networks can suffer from the same
possible "blind zone" as mentioned over. That is, even though their behavior in regulating
each other can be proven, it cannot prove their temporal distribution spanning the cell cycle.
Seen through All-In-G1 glasses, such a regulatory network could perfectly fit between the
control point and S phase (See figure 3 under). Such a transcription factor network would
be necessary to activate the huge family of TF's that are needed just after the G1 control point.
They must fill the many regulatory modules in all cell cycle genes.

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Figure 2. In 2008 Cheng and Li points out that the level of TF expression in yeast cell
cycle are not temporal compatible with their target genes, or it is hard to measure (Modified
from 3). That is why TF’s pattern of activation is used to position their presence, as illustrated
in figure 1.

Figure 3. The TF network established in Simon et al (Left) could in vivo take place in another
time scale (Right) (Modified from 2).

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Figure 4. Illustration of the All-In-G1 assembly regulation model (Modified from my master thesis -
page 104). ECIN is short for "Eukaryotic cell cycle involved Non-permanent microRNA down
regulation”.

2. All-In-G1 gives optimal strategies for genome replication and condensation

Gene transcription can be sectioned into three different regulatory levels. They are annotated
as 1: The DNA level, 2: The chromatin level and 3: The nucleus level (15). All three levels are
involved to bring about correct assembly of transcriptional machineries. The three regulatory
levels must necessarily communicate to coordinate correct transcriptional events. The master
thesis refer to this regulatory process as "complex gene expression regulatory events", since it
is one area in gene expression regulation that is too complex to understand with today’s cell-
investigating tools. The only level we know much about is the DNA level (Level 1), but the
details that underlie how the chromosome coordinates its spatial orientations - are still very

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unclear. It is therefore also difficult to know exactly how sensitive regulatory level two, and
three, are to genome destabilization. All-In-G1 speculates that they must be very sensitive to
the brutal treatment the genome must undergo during cell division. The current
model assumes that transcript regulatory events takes place in combination with genome
replication and condensation. All-In-G1 assumes all transcript regulatory events to take
place at one time point in the cycle, before the genome loses its endogenous state. This time
point must lie between the G1 control point and S phase (Figure 4).

In endogenous states (G1 and G0), the chromosomes are in a non-condensed form but are at
the same time organized into delicate compartments within the cell nucleus called
chromosome territories. Each non-condensed chromosome territory is divided by canals
called interchromosomal domains. These canals are spaces between the chromosome
territories with very little, or no DNA content. Genes in the center of the chromosome
territories must change their 3D position relative to the territories outskirt. This to be exposed
to the Interchromosomal domains that surround each territory (Page 413 ISBN 0-13-196894-
7). Such 3D genome position-sensitive regulation is known as regulatory level three. It is
unlikely that these fine tuned 3D regulatory processes can easily survive the cell division
process. That is, genome replication, condensation and finally separation of the two copies.
Especially when closing in on full condensation in M phase. The same arguments go for
regulatory level two (Chromatin level). The delicate dynamic closing, and opening, of the
chromatin must also become heavily disturbed during cell division - for the same reasons.
In the current model, the cell must probably set up new and unique regulatory networks to
manage the genome throughout the cell cycle. This would probably slow down replication
and condensation for two main reasons. First, if the cell was given a chance to focus
exclusively on how to coordinate its genome for optimal division, it would probably pick a
faster strategy compared to the current model. In contrast, All-In-G1 makes the cell optimize
genome coordination relative to genome replication and condensation speed. Second, the
genome becomes dependent on the conservation of a larger numbers of genes, to keep a more
complex cell alive. Therefore, the current model would become less robust compared to a cell
that has developed All-In-G1. This since All-In-G1 results in a less complex system for the
cell to handle.
Clearing the nucleus system for any need to mix division and gene-regulation, should
therefore give higher fitness compared to the current view. Thus, All-In-G1 would make

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protist cells 1 billion years ago favor its services - before evolving into multicellular life
forms.

From this, All-In-G1's existence should therefore depend on its realism in vivo (If All-In-G1
is possible in vivo). The third principal argument provides multiple examples proving that the
molecular events needed to fulfill All-In-G1, are already available to the cell (Page 108 in the
master thesis).

3. An Ishida et al experiment gave All-In-G1 its first push

After establishing the three principal arguments, my attention was drawn to one
particular issue that emerged in many cell cycle papers. That is, many observations identified
a functional connection between G1/S with late phases (like G2, M and M/G1). A paper
by Ishida et al illustrated such a problem clearly.
The Ishida group artificially expressed E2F isoforms 1, 2 and 3 in cells resting in G0 phase
(8). As a result the Ishida group identified expressed genes known to work in G2, as a
response to transcription factors that were previously known to regulate transcription in G1/S.
This was seen as odd since it was believed that no E2F activities took place in G2, only in G1.
Under are two citations from the discussion in the Ishida paper.

“..the finding that a number of genes induced by E2F proteins are normally regulated at G2 is
surprising in light of the fact that these E2F activities are essentially undetectable at this time
of the cell cycle.”

“..The fact that cells expressing E2F1, E2F2, or E2F3 do not complete S phase or enter
mitosis (data not shown) argues that the induction of these G2-specific genes is not the simple
consequence of induced cell cycle progression.”

In Ishidas case described over, they could have come across ECIN-genes destined for p-
bodies - genes that were not meant to function until many hours later.
The ECIN model speaks for Ishidas G2 transcripts to be expressed in G1/S and thereby

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detectable before the cell reaches G2. In principle it is not odd at all to detect G2 transcripts
after experimentally isolating the three E2F isoforms activity in G1 - as seen from All-In-G1.
The Ishida paper only speculates on the reasons behind their strange observation. One
suggestion is that not yet activated repression mechanisms are unable to hinder their release.
However, they cannot find hard proof to back up any reasonable explanation.
The intensiveness in cell cycle investigation over the years, coupled with the
experienced Ishida group's lack of finding a good probable cause, should strengthen the All-
In-G1 hypothesis candidature.

4. Meaningful distribution of genes to late phases strengthens ECIN

The next argument I bring up in my master thesis gives another dimension to why All-In-G1
actually can take place in vivo. Several experiments in Yeast cells have identified genes in M
phase, that are known to function in S phase ( 9 and 10). The question is; how can early
phase genes merge so late in the cell cycle? One must expect that the regulation of their
transcription takes place earlier than the time of their detection in late M phases (Figure 5).
Using the current model, the same genes must thus have two time points in the cycle in which
they are transcribed. This since a yeast cell in its resting state (G0) cannot get its G1/S genes
from M phase. To enter cyclic state, from G0, the cell needs to transcribe G1/S genes in G1.
In contrast, All-In-G1 only needs to achieve the transcriptional events once, and still be able
to supply identical transcripts in both G1/S and late M phase.
That is, a mechanism which can serve to take the Yeast cell from a resting G0 to a cyclic
state, and at the same time provide the identical genes in M phase in the cyclic state. All this
without engaging two separate transcriptional events, one in G1-S and a second in M phase.
This observation also indicates that All-In-G1 gives eukaryotic cells the possibility to
distribute specific genes throughout the cycle, to respond to different cellular contexts. In
multicellular organisms such a context could be development, wound healing or in diseases -
such as cancer.
This gene-swooping feature that seems to yield for Yeast cells is interesting to All-In-G1.
Already available key-genes in S phase could make the G1-S transition go faster. If such a
mechanism takes place in vivo, it would make All-In-G1 even more energetic and timely
favorable - compared to the current view. The cell can have mRNA's pre-stored in p-bodies,
and release them at the perfect moment, without interrupting the cycle momentum with

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transcriptional activity. However, it yields probably for a minority of key-genes that stalls the
cycle momentum more than others, in the G1-S phase.

Figure 5: Two examples from the literature that locates G1-S genes in M phase in Yeast
cells (Modified from 9 (A) and 10 (B)).

Since both cells (Daughter and mother cell) needs the pre-stored G1-S genes, there must be a
mechanism that distribute the transcripts to both cells. Such a distribution should not be very
controversial but its detailed mechanism is difficult to predict. It depends on the timing of
transcript distribution relative to the division process itself.

The current model would force the cell to transcribe the same set of genes in both phases
(G1/S and M). Thus, signs that speak for eukaryotic cells to implement such a multi-usage
strategy would at the same time strengthen All-In-G1's candidature. Said in another way, the
principal arguments in All-In-G1 would become even stronger than before. All-In-G1 could
take care of the Yeast cells transcript needs in both the cycling state, and the resting
state (G0), without duplicating the transcriptional event itself. Rather, regulate the distribution
of specific genes to distinct transcript routes (Normal or ECIN).
Dynamically moving genes between cell cycle phases, depending on the cells context, would
perfectly fit into the All-In-G1 model. If All-In-G1 won the first battle against the current
view in early evolution, it would certainly be capable of evolving a dynamic usage of the
system. Therefore, any signs of locating G1-S genes in M phase in the literature, and in my
results, were embraced.

On that note, the discovery of EVI-1’s involvement in both cell lines (HeLa and HaCaT) S
phases, made me look deeper into EVI-1's output data from the detection process. I made a

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graph of the average detection of EVI-1's binding motifs in each phase, in both cell lines
and in all genes. That is, EVI-1's average number of registered footprint (Binding motifs) was
counted. Such manual counting of EVI-1 binding sites in all phases created a pattern that is
shown in figure 6 (You might get small deviations in the average count numbers, but the
overall pattern is unmistakable). EVI-1’s activity shoots up in early S phase, and reemerges in
late M phase. Since the same pattern emerges in both cell lines, this pattern is probably no
coincidence. Further, EVI-1's involvement in the cell cycle has been very well documented
(11 and 12). Thus, once again we can see the G1/S - M phase pattern involving a transcription
factor known to work in cell cycle. However, this time the result comes from our own data.

The possible realism in All-In-G1 made me doubt the statistical overrepresentation strategy
used to identify phase specific TF (This conserns details described in the method section in
the thesis). A unique combination of the same set of transcription factors in modules could
very well be as important for phase-specific control. In addition, when it comes to ECIN
regulation, microRNA binding sites and microRNA availability can also play a role in the
discrimination process. However, these detailed questions are not very important for now.
The important thing is that All-In-G1 could serve as an example to why analysis of gene
regulation during cell cycle becomes problematic.

Figure 6. Measurement of EVI-1 binding sites was discovered in genes by the search
algorithm (Motif Scanner) in the different phases during the cell cycle (Taken from page 92 in
the thesis). Again, we can see that S phase activation is linked to M phase activation, this time
in our own data.

As also argued in the master thesis, the function of p-bodies has shown signs to affect both
cancer disease (13) and developmental processes (14). This could be another sign that
ECIN is involved in both processes. This, since both processes is dependent on p-bodies as a

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gene-storage chamber. What I try to indicate is that the strategic placement of G1/S genes
is dependent on p-bodies. That is, strategic gene placements that affect cell cycle speed, which
All-In-G1 suggests to take place in both cancer and developmental processes. For example,
under developmental processes, extra fast cell cycle speed would be an advantage for rapid
gain of strength for a new born infant. One solution committed by cells in the developing
tissue can be to move some of the G1/S critical genes to M phase, to speed up the M-G1-S
phase transition. After differentiation, the cells could go back to a "normal" usage of the
ECIN route. If the differentiated tissue is damaged later in life, the cell can go back to using a
"fast cell cycle state" it used during development. Such a shift of gears to control cell cycle
speed could therefore by very useful for multicellular organisms in nature.

5. Proteome investigation in Yeast cells strengthens the principal arguments

From the first principal argument, cell division speed is the process that has been most
affected by selection pressure, during early evolution. The protist cell lived over 1 billion
years ago (16). The protist cell is seen in science as the higher eukaryotic organisms (Fungi,
plants and vertebrates) earliest ancestor. Due to their non-mobile phenotypes they were
probably dependent on division speed to compete for available nutrients. Their mobility was
probably very limited. Thus, it should not be very risky to compare the contextual behavior of
protist cell types - to the present day’s unicellular Yeast cells. It is therefore interesting to
look closer at the protein content in Yeast cells, since a Yeast cells proteome can project non-
mobile unicellular lifestyles general needs - for survival.
Comparison of protein contents in yeast (Unicellular) and C. elegans (Which is a
multicellular organism) have shown that Yeast cells lack the regulatory cell cycle proteins
which are found in multicellular lifeforms, such as Retinoblastoma (RB) and p53 (17). Since
Yeast cells do probably share the protists non-mobile phenotype, it is tempting to assume that
protist neither used cell cycle regulatory proteins. In other words, this observation supports
the assumption in the first principal argument that early non-mobile unicellular cells
also consisted of a proteome that was designed to compete by cell division speed. That is, one
forward cell cycle speed-modus: ON - with no breaks. Whenever nutrients became available
to the cells, they replicated until the source was empty. The evolutionary argument in the All-
In-G1 hypothesis thus states that the fastest strategy possible to divide has been forced to
evolve in protist cells.

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6. Assemble and wait increases cell division speed

I want to clarify some aspects on what takes place in the border going from normal
expression in G1, to assemble and wait in S phase (See overview in figure 4).
The reason for the cell to change expression strategy from normal, to assemble and wait, and
not directly to ECIN would be to avoid a contradiction in the evolutionary argument (First
principal argument). Since, much time is wasted by the cell if it went directly to ECIN, from
normal transcription, in the G1-S transition. If a constant flow of gene transcripts are needed
in the G1-S transition, a swop from normal to ECIN would make the cell cycle process stall.

Figure 7. Change from normal gene expression to "assemble and wait" in the G1-S phase
border. It could have changed directly to the ECIN model, but would contradict the
evolutionary argument (See page 91 in the master thesis).

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This swop would take place when normal transcription no longer can be achieved due to
genome destabilization. However, assemble and wait can replace normal transcription and
thereby keep a continuous flow of mRNA supply. This, since the transcriptional machineries
already is assembled, and no longer needs genome stability to transcribe its downstream
genes.
At the same time ECIN genes can slowly make their way to p-bodies for storage. The
time difference created in the two alternatives in the G1-S transition is illustrated in
figure 7 over.

Conclusion

By transcending All-In-G1 over 1 billion years back in time into the pools of struggling
unicellular colonies fighting over nutrients, All-In-G1 would be more fit, compared to the
regulatory mechanism that underlies the current regulatory model. This, since the All-In-G1
model would give a higher frequency of progenitor cells via; 1) Faster cell division speed
(First principal argument) and 2) More evolutionary robust genomes (Second principal
argument) which give higher survivor rate per progenitor cell. The third principal argument
documents that eukaryotic cells have evolved the molecular mechanisms that underlies All-
In-G1. Consequently, All-In-G1 should have evolved to be implemented in cell cycle
regulation as well, if it was not in the cell cycle itself the mechanism originally evolved. This
is the logic fundament that the three principal arguments provide. In addition to this
fundament, the model can solve many of the strange observations that cause problems in
today’s cell cycle regulation analysis. All-In-G1 should still be at work in human cells to this
day, for example fast cell division speed would be critical to multicellular organisms during
development.
A blind zone in the work that underlies the current cell cycle regulatory model can give room
for All-In-G1 to exist in vivo, and for the current model to be wrong.

Figure 8 and 9 (Next page). Illustration of the ECIN model. White lines illustrate microRNA-
mRNA complexes. These complexes are transported to p-bodies for storage. When the time is
rigth they are released to precede with traditional activities (Modified from 13).

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References

See http://blogs.myspace.com/index.cfm?fuseaction=blog.ListAll “Hypothesis: All-In-G1

assembly” (www.myspace.com/headbiotech). Click the direct linked references (Blue) in the
text.

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