Fanconi
anemia Review article
Abstract
Fanconi anemia is an autosomal recessive disease involving
nine identified proteins that are essential for maintaining
genomic stability [Godthelp B., 2006]. It is a cancer susceptible
disorder characterized by chromosomal instability and hypersensitivity
to DNA cross-linking agents such as mitomycin C. FA also
lead to bone marrow failure by reducing the production of
red blood cells in the body. The report entitled “The
Fanconi anemia core complex associates with chromatin during
S phase” by Mi et al, the authors focused on the FA
core complex and its association with chromatin during the
G1-S border and S phase of the cell division. This study
includes the movement of the complex during the cell cycle,
the complex related DNA damage and the manner in which the
complex localizes to chromatin fibers. There are several
different types of proteins found in the core complex such
as FANCA, FANCC, FANCE, FANCF, FANCG and FANCL. The localization
of these foci was detected by using florescent tagged versions
of FA proteins. The findings showed that FA proteins localized
to nucleus from cytoplasm during G1-S, S phase of the cell
cycle to regulate and maintain genomic stability. This report
did not include the factors that cause Fanconi anemia. It
is only focused on FA pathway, and no implications were
stated by those obtained results.
The
Fanconi anemia core complex associated with chromatin
during S phase
Fanconi anemia (FA) is an autosomal recessive disease
that affects a wide range of people from children to adults.
Both parents must be carriers of the defective gene in
order for a child to inherit this disease. It is a very
deadly disease since it leads to bone marrow failure and
reduces the amount of red blood cells in the body [Mi
J., 2005]. Fanconi anemia is characterized by observing
shorter stature, increased tumors, and bone marrow failure.
Patient’s cells show hypersensitivity to DNA damaging
agents such as mitomycin C. There are many other physical
symptoms that can be characterized such as discolouration
of the skin, learning and mental dilemma, small head,
eyes and other body organs, low birth weight, and abnormalities
in heart, kidney, and skeleton [Titus T., 2006]. There
are nine identified and two unidentified loci have been
found to be essential for maintaining genomic stability
[Titus, T., 2006]. The report entitled “The Fanconi
anemia core complex associates with chromatin during S
phase” by Mi J. et al, and the authors try to prove
the dynamics of FA complex movement between cytoplasm
and nuclear compartments. The study includes the movement
of the complex during the cell cycle, the complex related
DNA damage, and the manner in which the complex localizes
to chromatin fibers.
In this report by Mi J. et al, the authors focused on the
Fanconi anemia core complex and its association with chromatin
during the S phase of the cell division. Chromatin fibers
were stripped away from nucleus and cytoplasmic components
by using situ chromatin preparations. There are several
different types of proteins found in the core complex such
as FANCA, FANCC, FANCE, FANCF, FANCG and FANCL. The purpose
of this study was to decide the movement of these core complex
proteins between cytoplasm and nucleus. This was proved
by using a fluorescent-tagged version of these proteins.
The localization of FA proteins in the cytoplasm, nucleus
and especially chromatin was analyzed.
This study showed that Fanconi anemia proteins control and
maintain the genomic stability by acting on chromosome in
S phase. The treatment with the DNA cross-linker mitomycin
C was done to prove this. It has been shown that FA core
complex exists in chromatin at G1-S border and it starts
to diffuse to the other part of the nuclear compartment,
and at one point it completely expels from chromosome. Immunoblotting
experiment showed that FA anemia protein increases its binding
with chromatin when DNA damage occurs. The localization
of this protein from cytoplasm to nucleus was detected by
this experiment.
The studies have shown that cells derived from patients
with the diseases showed hypersensitivity to DNA cross linking
agents and a decreased in survival [L´eveill´e
F, 2006]. The Functional defects in the Fanconi pathway
can lead to cancer, but it has not been fully studied yet
[Van der Heijden M., 2004]. A G2 phase cycle delay has been
found due to the defect in S or G2 check point. Although
other studies focused on cytokine dysfunction, sensitivity
to oxidative damage and defects in DNA repair, they have
yet to find a defined mechanism for the hypersensitivity
[Mi J, 2005]. According to L´eveill´e, F. et
al, FA protein FANCE is an essential component of the nuclear
FA core complex and it is required for monoubiquitination
of FANCD2, which is a an important step in the FA pathway
of DNA cross linking repair [L´eveill´e F, 2006].
L´eveill´e, F. et al also have investigated
the nuclear localization, especially FANCE FA protein and
they have found that FANCE has a strong tendency to localize
in nucleus and recruit the binding of FANCD2 to the core
complex [L´eveill´e F, 2006].
In the study by Mai et al the results were collected by
performing various experiments and other factors that can
affect the results were also analyzed. First of all, by
transfecting mutant cells with florescent (fluorescent tags
such as EGFP-FANCG, EYFP-FANCC and ECFP-FANCG with FA cDNAs)
and nonfluorecent tagged protein, it has been proven that
fluorescent tagged did not interfere with the normal function
of FA proteins. Localization of tagged FANCA, FANCC, and
FANCG into nucleus and chromatin were found by doubly transfected
HeLa cells with either EGFP-FANCA and ECFP-FANCG or ECFP-FANCG
and EYFP-FANCC. When double thymidine blocked HeLa cells
were released into regular media, the visibility of the
foci appeared to diffuse and move to the outside of nucleus
as it progresses to G2. This suggests that FA proteins form
foci at G1-S, S phase and diffuse as it goes to mitosis.
In order to find the FA core complex localization in chromatin
fibers, nuclei were extracted out by vertical immersion.
The co-localizing FANCA and FANCG were seen irregularly
spaced foci along the chromatin fibers. This was seen predominantly
in entry of G1-S and S phase and spread apart when the cycle
goes to G2 phase. This result was consistent with the previous
finding where they used fluorescent tagged protein model.
The findings show that there is an increase of FA core complex
localization after the treatment with mitomycin C (MMC),
and this complex can also be localized to chromatin in primary
cells. The results were proven by the examination of chromatin
and the whole cells at various times of MMC treatment, and
by fluorescent tagged transfected primary mutant cells.
Synchronization and chromatin fiber experiments compare
FA protein during S phase and after MMC treatment, and they
suggest that functional engagement of S-phase check point
may have been the reason for the DNA damage and FA protein
localization. This was also suggested by another study done
by Akkari et al [Mi J, 2005].
In order to evaluate the degree of MMC effect on chromatin
localization of FA protein, researchers synchronized HeLa
cells to the G1-S border and asynchronized HeLa cells with
MMC. After performing FANCA immunoblotting peak levels,
FANCA in chromatin were observed during S-phase and this
is greater than asynchronous cells with MMC. FACS data showed
that MMC induce a marked extent of S and G2- M phase after
12 and 24 hours, but did not alter the DNA histogram. This
result was consistent with the data from Akkari et al.
Finally, all of the findings were combined and authors concluded
that Fanconi anemia proteins localize to chromatin predominately
after MMC treatment and during S phase. In discussion they
also included various researches and future studies. Authors
also reconfirmed their research results as that the FA core
complex forms chromatin foci at the G1-S and S phase, the
diffusion of foci is due to damage of DNA during S phase
and protein exits the nucleus when mitosis starts.
Future studies include both biochemical functions for the
FA pathway and the link between replication and repair of
DNA damage. They also suggested that by finding the functional
pathways of biochemical substances the process of DNA repair,
DNA replication and other genomic activities can be found.
No treatment for this disease was suggested by authors,
but other studies refer that Hematopoietic stem cell transplantation
is the only way of curing this disease [Van der Heijden
M., 2004].
The central theme of this experiment is based on aspects
such as the movement of FA protein complex during the cell
cycle, how the complex responds to DNA damage and the localization
of chromatin fibers. The purpose of this article is clearly
stated. All results are proved clearly and factors that
can affect the results are also examined and proved to have
no effect on the results. Some of the examples include the
usage of fluorescent tagged FA protein and FA complex localization
in primary cells. They have performed florescent tagged
experiment to make sure that these tags did not interfere
with normal FA function. They have validated every single
result by analyzing further outcome.
When FA proteins form foci at G1-S and during S phase, HeLa
transfected cells were placed under time lapse photography,
and the film showed that FANCA and FANCG are cytoplasmic.
However, after 6 hours of observation, these proteins were
found mostly at the periphery of nucleus and not within
the nucleus. Time-lapse photography taken of asynchronous
cells shows a similar trend, specifically, FA proteins initially
in the cytoplasm and as time goes it moves to nucleus. They
did not prove particularly in the nucleus, but just in the
periphery of the nucleus. This shows the over interpretation
of the data.
The purpose of adding fluorescent tag to FA protein is to
visualize the movement of protein from cytoplasm to nucleus,
but fluorescent tag can also have an effect on the localization
of FA protein. This was experimented and proved to have
no effect on the normal function of the FA protein. Transfection
efficiency was found to be 50% in all cases and this was
determined by counting fluorescent cells prior to extraction.
In order to see if the mutation of the FA proteins could
disrupt proper chromatin localization, they prepared the
tagged version of FA proteins and this experiment proved
that all mutants are cytoplasmic and did not alter the visualization
of chromatin. In the article they have mention that the
tagged construct related to the complex localization also
validated by patient-derived point mutations, but they did
not clearly explain how it related to the tagged fluorescent
method.
In order to correct discrepancy of obtained results, researchers
did various checks and concluded that fluorescent tag did
not disrupt the normal function of the FA proteins. The
patient-derived point mutations also revealed the nuclear
or chromatin localization. The chromatin fiber prep model
on mitotic cells showed the complete absence of FA protein.
In order to show the chromatin movement after the treatment
with MMC, the whole cell and chromatin were examined at
various time points and no obvious difference were found.
It took 12 hours to visualize the chromatin. To validate
this result, researchers made a similar slide, and the experimental
results illustrated the consistency with the situ appearance
in figure 5B and immunoblotting data. All these points are
considered and analyzed to validate the experiment.
Many of the experiments are performed using HeLa cells due
its easily synchronizable characteristics, where effects
on primary cells can differ from these results. In order
to solve that, primary mutant cells (FA) were transfected
with EGFP-FANCA and ECFPP- FANCG, and results showed the
co-localization of FANCA and FANCG to foci in the nucleus.
This confirms the correct outcome of the results that have
been obtained. For example, in order to compare the extent
of MMC effect on the localization, HeLa cells were synchronized
and results were found. It suggests that the DNA damage
may be a function of engagement of an S-phase checkpoint.
This result is consistent with another study by Akkari et
al.
In this article, authors did not include any information
on FA disease such as the causes of this disease, the normal
function of FA protein, how it relates to Fanconi anemia,
presences of FA protein in normal people, the current treatments
and etc. The report was only focused on FA pathway and although
the results are precise and clear they did not relate those
findings with the actual disease. They did not mention that
the defect in this FA pathway can lead to Fanconi anemia,
or how does that relate to cancer and what is the implication
of the hypersensitivity of these proteins to MMC. People
who read this study will not be able to get the general
idea of what FA protein means; is it a defected protein?;
can it be found in normal people?; how does these findings
relate to the disease?; therefore only after reading other
sources will help to understand and identify the causes
of this disease. By not having a prior knowledge of Fanconi
anemia, relating these findings to the actual disease is
impossible.
All the hypotheses are logical and well written in terms
of the style and the structure and main points are well
described and supported by experimental results. All the
experimental outcomes that can affect the results are considered
and further analyzed. Conclusions are made by validating
the experiments with other studies. Mainly this study clearly
answered the Fanconi anemia core complex association with
chromatin during the G1-S and S- phase of the cell cycle.
Authors also included previous findings in order to explain
complex phenomena. All the actual experiments are performed
in way to support the hypothesis. Experiments included correct
controls in order to confirm the results. This is a well
designed study and there is no other method that can be
constructed in order to verify the hypotheses. In an attempt
to find the movement of FA core complex from one part of
the cell to another, fluorescent tagged model is very useful
and no other methods can be used to verify the results.
This method is very effective and easy to detect and it
gives very efficient results.
Results are adequately interpreted and the diagrams are
also well explained. Discrepancies were taken into consideration
and supported by performing control experiments, but in
some cases authors were over interpreted few information.
For example, when considering the location of FA proteins
during the S phase, results actually showed periphery of
the nucleus as the location of FA proteins. However, in
the study they have stated that it occurs in the nucleus
and as the cell cycle progresses complex diffuse to the
periphery of the nucleus and then starts to disappear.
Authors in this research identified various discrepancies
as stated above and verified their results according to
the different outcomes. Finally they included other studies
as well in order to support their findings and values which
were also consistent with the theoretical value.
Reference:
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Titus, T., Postlethwait, M., Hoatlin, M., Joenje, H., &
de Winter, J. (2006) ‘The nuclear accumulation of
the Fanconi anemia protein FANCE depends on FANCC’,
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