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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. References Coelho, S., Simon, N., Ahmed, S., Cock, J., & Partensky, F. (2012). Ecological and evolutionary genomics of marine photosynthetic organisms. Molecular Ecology, 22, 867-907. Fan, F., Tonon, G., Bashari, M., Vallet, S., Antonini, E., Goldschmidt, H., ... Podar, K. (2013). Targeting Mcl-1 for multiple myeloma (MM) therapy: Drug-induced generation of Mcl-1 fragment Mcl-1128–350 triggers MM cell death via c-Jun upregulation. Cancer Letters, 343, 286-294. Feng, C., & Rise, M. (2009). 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