Download The Likelihood of Local Variation in the Gene MCL-1

Raffia Ahmed
Chisom Amaefuna
Molecular Ecology
December 15, 2014
The Likelihood of Local Variation in the
Gene MCL-1 (Induced Myeloid Leukemia)
Abstract
The anti-apoptotic gene MCL-1 was examined for a possible present or future
role in the local adaptation of Amphiprion clarkii populations. Due to its highly
essential nature, MCL-1 is a highly conserved gene, making it unlikely that it is
currently subject to large amounts of local variation. However, observations of the
upregulation of MCL-1 in other fish species exposed to cyanotoxins suggests the
potential for MCL-1 to be upregulated in clownfish in response to such toxins as
well. Depending on the variability of exposure to these toxins across geographically
isolated populations, small amounts of variation - particularly in amounts of MCL-1
expressed in mRNA - could take place over time.
Introduction
Apoptosis is the process of programmed cell death in organisms. It is a
conserved cell death pathway vital in functions for tissue homeostasis, removal of
unwanted and damaged cells, morphogenesis during embryonic development, and the
resolution of inflammation (Thomas, 2010). This carefully regulated type of cell
death is initiated and completed through the activation of a number of genes. The
synthesis of products from many of these genes are required for cell destruction, but
others in the same families enhance cell survival by preventing cell destruction
through anti-apoptosis (Wei et al 2014).
MCL-1 is protein expressed by an anti-apoptosis gene that plays a key role in
mitochondrial and nuclear cell function, helping to protect vertebrate organisms from
pathogens. It is found throughout the body, but particularly in the blood and outer
mitochondrial lining (Feng et al 2009). Both the lack of MCL-1 and the overexpression of MCL-1 cause harmful effects that can be fatal, although temporary
upregulation does occur in response to hazards.
The aim of this paper is to explore the possibility of current and future
intraspecific adaptation of the anti-apoptotic MCL-1 gene in a coral reef fish known
as Amphiprion clarkii. This colorful fish is a widely distributed species that lives
throughout the Indo-West Pacific ocean in areas as disparate as Japan, the
Philippines, and Indonesia. These smaller population groups are likely isolated both
by their distance and by the dispersal of the clownfish’s favored habitat, sea
anemones. Although sea anemones typically feed on nearby fish, clownfish have
certain adaptations that protect them. In many cases, they have also formed a
symbiotic relationship with the anemone and are not subject to this predation.
Apoptosis, Anti-apoptosis, and the MCL-1 Gene
Although apoptosis is a process that is crucial to long-term survival, its
deregulation can result in diseases such as cancer, neurodegeneration, and
autoimmunity. The detrimental effects of deregulation can be seen in the function of
the anti-apoptotic Myeloid Leukemia Cell Differentiation Protein, particularly in the
first of its three isoforms. Isoform 1 is the longest gene product and inhibits
apoptosis, therefore enhancing cell survival. The two shorter gene products, Isoform
2 and 3, enhance apoptosis and are death inducing (Gene Cards). When Myeloid Cell
Leukemia-1, or MCL-1, is upregulated, it can grow out of control and produce large
numbers of white blood cells that cannot mature properly, resulting in myeloid
leukemia. However, because the protein enhances cell survival, it is deeply involved
in normal cell function and its loss leads to poor survivability (Thomas and
Gustaffson 2013).
Encoded by the MCL-1 gene, MCL-1 is a part of the B-cell lymphoma 2 (Bcl2) family of proteins, which partake in regulating mitochondrial integrity and
preventing cell death by inhibiting the permeability of the mitochondria (Thomas,
2010). Unlike other members in the Bcl-2 family, MCL-1 has a larger size of 350
residues because of the presence of two weak and two strong polypeptide enriched in
proline, glutamic acid, serine and threonine. These polypeptides provide
responsibility for MCL-1 stability, localization, dimerization and also function (Fan
et al 2014). MCL-1 also significantly differentiates from its Bcl-2 family members
with its short half-life (Michels et al 2004).
Although MCL-1 and the Bcl-2 family are often coexpressed in the same
tissue, their physiological roles are quite different. MCL-1 deficiency in the heart, of
adult mice, leads to heart failure and early mortality. The lack of MCL-1, although an
anti-apoptotic protein, has little effect on caspase-activation, which plays an essential
role in cell death. However the lack of MCL-1 did result in swollen mitochondria,
and ruptured myocytes which affects the permeability of the mitochondria. MCL-1 is
crucial in the regulation of mitochondrial permeability and necrotic cell death, and
are usually activated when there is an accumulation of deteriorated mitochondria.
Cardiac myocytes, which contain vast extensions of mitochondria, often clear out the
dysfunctional mitochondria in order to maintain the production of ATP (Thomas and
Gustaffson 2013).
MCL-1 deficient hearts are incapable of creating activating autophagy,
however this is observed before dysfunctional mitochondria and heart failure,
inferring that MCL-1 does not have direct control over the production of the
autophagy. MCL-1 is present in both the outer membrane and the matrix of
mitochondria with different functions. The function of the outer membrane is to
control apoptosis, whereas the inner matrix is involved in regulation of mitochondrial
functioning. Studies have been unable to pinpoint which form partakes in the
production of autophagy and mitophagy. MCL-1 is crucial in the functioning of both
the heart as well as, in mitochondria (Thomas and Gustaffson 2013). The importance
of MCL-1 in the heart has made it difficult for scientists to create successful forms of
chemotherapy that won’t inhibit the function of MCL-1 in the heart causing
cardiotoxicity, a condition where there is damage to the heart muscle, making it
difficult for the heart to properly pump blood through the body. In order to properly
use chemotherapy mechanisms in the heart, MCL-1 needs to be taken into account
and unaffected by the methods in order to maintain the proper functioning of the
heart while still attacking the harmful cells (Thomas and Gustaffson 2013).
Yellowtail Clownfish
Also known as Clark’s anemonefish or yellowtail clownfish, A. clarkii is a
species of clownfish widely distributed through sea anemones throughout shallow
coral reefs in the Indo-West Pacific region. They are known to naturally be able to
inhabit all ten species of sea anemones, but are commonly found around specific sea
anemones. The tropical fish ranges in color, but typically has a base body color and
orange areas of varying size on the head and fins. Amphiprion clarkii have roughly
ten dorsal spines, fifteen to sixteen soft dorsal rays, and teeth that are close set which
act as a defense mechanism (Steer 2012).
Clownfish larvae are protandrous hermaphrodites, meaning they all develop
as males and retain the capability to change into females later in life. They live
together in groups of one adult female, one adult male, and several juveniles and are
often hostile to new arrivals. Female fish usually dominate with their larger size,
preventing the male from getting any larger. Male anemonefish in turn dominate the
juvenile fish in order to prevent competition. The juveniles retain that life stage until
either the dominant male or female dies; then the largest male fish transitions to
being the dominant female fish, and one of the juveniles takes the place of the adult
male. Because there is only one adult male and female pair at a time, the species
breeds in monogamous pairs. Upon spawning, the male fish take on the primary
duties of caring for the eggs until they hatch into independent larvae (Steer 2012).
An aggressive fish living in an environment hostile to other fish, clownfish
are well adapted to their situation. Sea anemone have stinging venom filled tentacles
that they use to prey on fish that swim nearby. Most are also covered with a toxincontaining mucus. At the same time, the clownfish are also covered with a mucosal
layer that protects them from their living habitat’s venom and toxins. The layer
develops through acclimatization - the young fish swims over the anemone and rubs
its belly and ventral fins over the tentacle tips until the fish begins producing its
protective coating. This mucus layer protects the clownfish during direct contact with
the anemone. When it is removed through abrasion, the fish typically dies (Mebs
1994).
Anemones that host anemonefish are generally healthier, so it is
not surprising that a variety of sea anemone have created symbiotic relationships
with clownfish. The clownfish protect the sea anemone from their prey and also
receive shelter from the sea anemone. Since they are hostile to intruders, clownfish
often defend their host and themselves from predatory fish with their sharp teeth.
Unfortunately, any damage to the fish’s epidermis could remove its protective
mucosal layer, putting it in danger if it then makes physical contact with its host.
Clownfish occasionally eat parasites harming their host anemones, but their primary
food source is zooplankton, copepods, and algae within close proximity of their host.
Larger fish are willing to venture farther for food, but doing so puts them at risk of
predation (Steer 2012).
Cyanotoxins
Found almost everywhere, cyanobacteria are a type of of photosynthetic
bacteria that were in the past known as blue-green algae. Although it is widely
acknowledged now that cyanobacteria are not algae, the misnomer remains. The
oldest photosynthesizing organisms on Earth, they have surprising little diversity in
the ocean, belonging to only four different species (Coelho et al 2013). In aquatic
environments, cyanobacteria populations benefit from warmer temperatures and can
explode exponentially into large, dense blooms under certain conditions. An
example, called eutrophication, results from the oversupply of essential nutrients
such as nitrogen and phosphates and can result in hypoxic environments that support
little diversity. Such blooms normally appear in coastal environments and have been
spotted near Japan (Coelho et al 2013).
There is another danger associated with cyanobacteria - they produce a
number of toxins known as cyanotoxins. These can be harmful to a wide variety of
species, including mammals and fish. One such cyanotoxin group, microcystins, can
cause significant damage. Microcystins are cyclic peptides, which, due to their shape,
are extremely stable and resistant to digestion. This stability results in
bioaccumulation, particularly in the liver, and can be fatal. In the past, microcystin
exposure was mostly considered a problem in freshwater environments; however, it
has been shown that outflows of microcystin-contaminated freshwater can result in
impacts on marine organisms such as copepods, corals, and fish that may be
exacerbated by a warming climate and increased oceanic pollution (Miller et al
2010).
Discussion
It is unlikely that the MCL-1 gene’s function shows much local adaptation in
clownfish, or any other species. The gene is so important in cell and mitochondrial
function that it is observed over multiple vertebrate classes, indicating that there is
high selection pressure favoring its retention. Its presence in mitochondria also shows
that it its presence is by common linear descent. Therefore, MCL-1 has been part of
organismal function for an extremely long time, and yet retains the same function
over its various taxa. Its importance in mammal species such as mice and humans
also indicates MCL-1’s regulation and expression is under stabilizing selection.
When MCL-1 is not expressed, such as in Thomas et al’s (2013) experiment where
the MCL-1 deficient mice died, it generally results in death. Meanwhile, uncontrolled
upregulation leads to leukemia, particularly in children.
However, that does not mean that there is no variation in the MCL-1 gene. For
instance, duplications of MCL-1 can occur, possibly because of teleost-specific
genome duplication, and this may play a role in preventing necrotic cell death (Feng
et al 2009). Since clownfish are teleost fish, it is possible that such duplication could
have appeared in their lineage as well, but would have likely occurred in a common
ancestor to the sub-populations of A. clarkii since it did not result in further
speciation.
Another interesting possible variation is Feng et al’s (2009) study finding that
the Atlantic cod MCL-1 protein contains a putative monopartite nuclear localization
signal not identified in their other MCL-1 putative orthologues with the exception of
zebrafish. Two other fish, Atlantic salmon and green pufferfish, were among the
orthologues, suggesting that this change is not spread throughout all fish species,
making it difficult to determine whether this adaptation exists in clownfish species.
Moreover, they argue that investigations at the protein level are needed to confirm
expression. With more confidence, they also note that all the intron/exon boundaries
were conserved between their all various orthologues, including humans, concluding
that these boundaries are likely conserved across all vertebrates (Feng et al 2009).
Feng et al (2009) were also able to demonstrate the upregulation of MCL-1
transcripts in Atlantic cod in response to viral mimics, confirming MCL-1’s role in
protecting organisms from pathogenic attacks. This type of upregulation is of
particular interest in local adaptation because of the diversity of pathogens; the
community in one location could be quite different from another. Any successful
species would need to adapt to the local community, and the level of MCL-1
upregulation needed to fight off a pathogen could vary. This level of upregulation
would also need to be adaptable to changes in the local community over time, as any
type of disruption can introduce new species. An example of this disruption is the
potential spread of large cyanobacteria blooms across environments where they were
not previously common.
Due to a number of factors, it is likely that cyanobacteria could have a
deleterious impact on clownfish if they were in the same environment. Cyanotoxins
would affect clownfish’s coral habitat as well a copepods, a common food source for
clownfish. Clownfish could also mistake cyanobacteria for their typical algal food
source, increasing the potential for bioaccumulation of toxins like microcystin.
Clownfish exposure to cyanobacteria has not been recorded in the literature as of yet,
but the possibility should not be ignored. Blooms have already been observed in the
Indian Ocean and off the coast of Japan, both of which are quite close to the A.
clarkii’s natural environment (Miller et al 2010). Since a warmer ocean will
encourage the growth of cyanobacteria, climate change could have an exacerbating
effect on the growth of these blooms, as well as putting the clownfish’s coral habitat
at risk of death due to higher temperatures.
To predict how clownfish might respond to this exposure, it is necessary to
look to other fish. The effects of one of the most common members of the
microcystin family, microcystin-LR has been observed in zebrafish, where it was
found that MCL-1 provided a protective effect. In the study, the change in 16
different apoptosis-related genes in response to microcystin-LR was observed.
Although it was not the most upregulated, the MCL-1 gene sequence experienced a
2.5-fold mRNA increase, suggesting that its anti-apoptosis effects on cell function
are desirable in this scenario. However, this upregulation was only measurable within
six hours of exposure to the toxin, matching what is known about the MCL-1
protein’s short half-life (Wei et al 2014).
Although zebrafish are a freshwater fish, it is possible that the same effect
could be seen in clownfish. Over time, consistent exposure to microcystin-LR could
even lead to a fitness benefit for the permanent upregulation of MCL-1 when
compared to populations that are not located in cyanotoxin-exposed locations. It is
important to note that these assertions are purely speculative. Because the spread of
marine algal blooms has only been observed recently and not directly in clownfish
habitats, it is extremely unlikely that such genomic differences are currently
noticeable in clownfish. However, it remains an interesting area for possible future
study, especially as a case study of potential human-triggered microevolution. If
blooms become commonplace in coral habitats, sampling and testing for upregulation
of apoptosis-related genes and then comparing between different populations would
not be an impossible project.
Of course, before testing for change, it is important that a baseline be
established. MCL-1 proteins have been observed in many different areas of an
organism that are essential to organismal function, so samples could be taken from
many different areas. In Wei et al’s (2014) zebrafish study, samples were taken from
the liver at different timepoints, so the fish survived the sampling process. It would
be difficult to ensure that the same clown fish were caught for multiple samples in a
wild population, but individual samples could still be taken where the fish were
returned to the water mostly unharmed.
If a related but clownfish-specific experiment was conducted, the effect of
anemone toxins on the apoptosis-related genes could be observed. Although MCL-1
prevents cell death, it is unlikely that it is involved in the composition of the mucus
that protects clownfish from anemone toxins. Because MCL-1 has a short half-life of
only six hours, the protein would have to be constantly generated at a large fitness
cost to the fish. However, because the mucus layer develops after exposure to the tips
of the anemone’s tentacles, it is possible that MCL-1 is upregulated during this stage
to keep the fish alive and healthy until the mucosal coating forms. Different anemone
species produce differing levels of toxins (Mebs et al 1994). As a result, MCL-1
levels may differ in this stage for Amphiprion species depending on the toxicity of
their preferred host species. Measuring such changes would be a difficult undertaking
because of the specific development stage needed and the fact that clownfish of that
size would be unlikely to leave the safety of close proximity to their host. It could,
however, result in some interesting findings.
The material given on MCL-1 for this review samples taken from the heart,
where MCL-1 is known to occur. It showed that for this locus, there were three
SNPs, located close to each other at 805, 806, and 808. The species predicted was
Oreochromis niloticus, a species of tilapia native to Africa. This species was
predicted at multiple loci, indicating that it may actually be surprisingly closely
related to A. clarkii despite its spatial difference. However, it could also provide
further evidence of the highly conserved nature of MCL-1 over taxa and time. It is
possible that the three SNPs have been retained not because they provide some type
of selective advantage, but because they do not result in a non-synonymous mutation,
but finding which amino acids are produced at those codons is required to either
confirm or deny this assertion.
It is most likely that if MCL-1 with A. clarkii is subject to local variation, it is
in the magnitude of its expression in response to pathogens and toxins in the local
environment. Such effects are likely to be exacerbated in the future due to increasing
levels of pollutants and climate effects that encourage the growth of toxic
cyanobacteria. At the present time, however, such effects likely play a negligible to
small role in local adaptation because of MCL-1’s importance in essential functions
means there is significant selection pressure against large changes in the gene.
Changes at only three SNPs are unlikely to result in a large adaptive effect in such a
highly conserved gene, so it is probable that the site changes observed are due to the
small sample size or representative of non-synonymous mutations.
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