Posts Tagged ‘DNA Damage’
Today I bring you something extra special: A guest post from Lucas Brouwers of the world-famous blog Thoughtomics. He loves genomes, I love plankton, and you get a great story involving spaceships, genomic party crashers, and, of course, a planktonic sea squirt. Enjoy!
Just below the surface of the sea, little animals are floating through a universe where the stars are made of plankton. They travel in what can best be described as gelatinous spaceships, which provide both shelter and food. They are Oikopleura dioica (Oikos is ancient Greek for house or household).
Their spaceships are ingenious constructions, made in such a way that every beat of Oikopleura‘s tail brings in a new flow water. The water and all the plankton it contains, is led through different tubings and chambers of the ship, until it reaches the waiting mouths of Oikopleura. The spaceships are not of a durable design and last for only a couple of hours before they are broken down again. Oikopleura spend the largest part of their short lives (5-10 days) repeating a cycle of building, feeding and destruction.
As small and short-lived as they may be, their genomes are evolving at at an extraordinary speed. A large team of scientists sequenced the full genome of Oikopleura recently, and found that its genome has been reorganized and trimmed down on an unprecedented scale.
One of the first things they noticed was the rapid evolution of introns in the genes of Oikopleura. Introns are the genetic equivalent of party crashers who show up at parties uninvited. They hang around inside genes, without coding for anything. Our cell have to force the introns out of messenger RNAs before they can properly be translated into proteins. Despite their apparent lack of use, many genes of distantly related animals have introns in exactly the same places. These introns have been conserved for millions of years, leading some to believe that they provide an evolutionary advantage somehow.
Oikopleura doesn’t care about these million year old traditions though. A staggering 76,2% (or 4.260) of its introns are unique to Oikopleura. Conversely, 3.917 of introns that are present in other animals (including close relatives) have been lost in Oikopleura. Not only do the newer introns outnumber their older peers, they are also good deal shorter than the few introns that have been retained.
But the extensive remodeling of the genome didn’t stop with introns. Normally, genes tend to hang out in similar neighborhoods in different species. There’s a good chance that two genes that are close to one another in mouse, are also close together in the human genome for example. Just like introns, many of these neighborhood relationships have persisted in species that are as far apart as humans and sponges.
Not so in Oikopleura. Its genes have been shuffled and switched around until the point that conventional gene orders are no longer recognizable. For small sets of genes the gene order in Oikopleura is closer to random than the gene order in other species.
The authors subtly hint at a potential cause for all this genetic upheaval: Oikopleura‘s genome lacks a full set of DNA repair genes. DNA is a robust molecule, but it can still be damaged. One of the nastier things that can happen is that both DNA strands of a single chromosome break. Cells can repair this type of damage in two different ways. The first way of repair works by taking a close look at the sister chromosome, and see how the damaged strands should be repaired, as shown in the video below. Another way is to directly seal the broken ends.
Oikopleura is lacking the genes for this last type of repair work. This means that every double strand break in Oikopleura‘s DNA can only be repaired via a sister chromosome, increasing the recombination rates (the exchange of genetic material between chromosomes) for Oikopleura. The causal link between the missing DNA repair genes and the accelerated evolution of Oikopleura‘s genome remains to be proven, but it isn’t hard to imagine how their absence could have played a large role in the overhaul of genomic architecture.
In addition to having a fast evolving genome, Oikopleura also has one of the smallest animal genomes (70 MB, compared to 3,174 MB for humans), which still contains some 18,000 genes (compared to ~21,000 in humans). Clearly, we don’t need that extra 3,100 MB for only a couple of thousand genes more. Compared to Oikopleura, our genomes are like attics crammed full of boxes and old furniture. Some scientists think that many features of our genomes (such as introns) are quite unnecessary, while others disagree. The debate boils down to this: are we keeping all the stuff because it is worth a lot, or because we never got around to throwing all the junk away?
Since Oikopleura was successful in redecorating and cleaning out its entire genome, our conserved genomic architecture might be nothing more than a product of historical contingency. In the small and slowly reproducing human population, natural selection might not be strong enough to remove all the unnecessary junk.
Despite the large genomic changes, Oikopleura has remained an animal in every way. While it might not be immediately obvious, they are very closely related to us vertebrates (animals that carry a backbone such as fish, reptiles, birds and mammals). They don’t have a spine, but they do have a lining of tough cells (a notochord) and a neural tube running through their entire bodies.
Oikopleura belongs to a group of animals known as tunicates, or sea squirts. Most sea squirts live a sedentary life on the ocean floor where they filter plankton from seawater. They are basically hollow bags with two siphons – one for drawing seawater in, and the other to expel waste and water. But the solitary and free swimming Oikopleura don’t look anything like these stationary creatures, so what are these familial ties based on?
A different life stage holds the answer: every sea squirt starts its life as a larvae, looking very much like Oikopleura. After swimming around for a couple of days the larvae attach themselves to a comfortable looking outcropping and morph into the sedentary sea squirts. By retaining its larval features and delaying its further development, the ancestor of Oikopleura has permanently avoided the fate of settling down (these processes are known as neoteny and progenesis).
The similarity between vertebrates and sea squirt larvae implies that our own vertebrate ancestor once looked very much like larvaceans such as Oikopleura. Like Oikopleura, we are basically sea squirts that have never grown up. But unlike them, we still carry the baggage of 600 million years of animal evolution. I think there is much that we can still learn from the Peter Pans of the oceans.
Denoeud F, Henriet S, Mungpakdee S, Aury JM, Da Silva C, Brinkmann H, Mikhaleva J, Olsen LC, Jubin C, Cañestro C, Bouquet JM, Danks G, Poulain J, Campsteijn C, Adamski M, Cross I, Yadetie F, Muffato M, Louis A, Butcher S, Tsagkogeorga G, Konrad A, Singh S, Jensen MF, Cong EH, Eikeseth-Otteraa H, Noel B, Anthouard V, Porcel BM, Kachouri-Lafond R, Nishino A, Ugolini M, Chourrout P, Nishida H, Aasland R, Huzurbazar S, Westhof E, Delsuc F, Lehrach H, Reinhardt R, Weissenbach J, Roy SW, Artiguenave F, Postlethwait JH, Manak JR, Thompson EM, Jaillon O, Du Pasquier L, Boudinot P, Liberles DA, Volff JN, Philippe H, Lenhard B, Crollius HR, Wincker P, & Chourrout D (2010). Plasticity of Animal Genome Architecture Unmasked by Rapid Evolution of a Pelagic Tunicate. Science (New York, N.Y.) PMID: 21097902
Apoptosis, or programmed cell death, is regulated by the protein RanBPM (Atabakhsh et al. 2009, Molecular Cancer Research)
I have been fearful of molecular biology for most of my life. This is partially because I so clearly defined myself as an ecologist that I partitioned molecules into “little biology” and out of my range. But mostly it was a fear of what I considered unnatural. Scientists who play around with chemicals and little tubes of liquid DNA cannot be real biologists, as they are studying life in a tube and not as it truly exists. These actions seemed so separate from what I considered “real biology,” these little details so far from actual application to learning about life as we know it.
And now look at me. I spend my days playing with chemicals and little tubes of DNA. I HAVE BECOME WHAT I FEAR MOST.
But I’ve been learning that molecular biology isn’t all boring. Molecules can be COOL and some of the little processes really interesting and relevant on a larger scale.
One of my favorite small-scale processes is apoptosis, or programmed cell death. For a long time, I never really thought about what the body does with old or sick cells. When your cells get old, you just die, right? Not exactly. The body can destroy and reabsorb its sick cells — incredible! That the body would kill off parts of itself! Maybe this isn’t so amazing to most, but it was a pretty big revelation to me. Apoptosis is also important in development. We start off as one cell which becomes a ball of cells. In order to create cavities, such as the digestive tract, the cells go through apoptosis and creates functional holes in itself.
Apoptosis is especially relevant to cancer regulation. When a cell is dividing and, inevitably, accidentally mutating its DNA (see my DNA primer for basics), there are processes that essentially spellcheck the DNA to ensure that it is functional. If there is a mutation that the cell cannot fix, it will send out DNA damage signals to neighboring cells, signaling that it’s time for this cell to die, or it could become cancerous.
Additionally, most cancers actually disable apoptosis. That is, they disable the signals so that the body cannot recognize the cell as cancer and force it through apoptosis. As you can imagine, the study of apoptosis has all kinds of implications for cancer treatments. If we could find a way to induce apoptosis in cancerous cells, we could potentially force cancer cells to kill themselves.
Scientists from the University of Western Ontario just published a paper online on December 8th in Molecular Cancer Research identifying a protein that activates apoptotic pathways in DNA damaged cells (doi:10.1158/1541-7786.MCR-09-0098). RanBPM, or Ran-binding protein M, had a previously unknown function. They discovered it’s potential for apoptosis regulation in a large screen looking for proteins that bind with Oct-1, a transcription factor implicated in cell survival after DNA damage, and decided to look further.
One problem in studying molecular biology is that living tissue doesn’t take well to being cultured in the lab, so biologists often have to use certain cells types that will grow under laboratory conditions. Where I work, we use yeast, which is beneficial because it is easy to take care of and grow, cheap, and easy to manipulate because it is a simple, single-celled fungus; the downside is that it is not human. The authors here used Hela cells, which are derived from human cervical cancer, and are essentially immortal. These have the benefit of being actual human cells — but they have been described jokingly as their own species because of their immortality, ability to thrive under laboratory conditions, and their abnormal chromosome number (caused by contamination from an HPV virus). However, they are still a good starting point for studying human biology because they are human derived, if a little mutated.
The first two steps in molecular biology are usuallyto overexpress the molecule, and underexpress the molecule. The researchers inserted an extra copy of the RanBPM gene into the cells, causing them to make twice as much RanBPM protein. These cells did not survive well compared to normal cells, indicating a potential link with apoptosis. Now they had to double check that this was actually due to a cellular pathway, and not because too much RanBPM poisons cells and kills them, known as necrosis. Caspase 3 is an enzyme commonly associated with apoptosis; the authors then looked at caspase 3 levels in RanBPM overexpressed cells compared to normal cells, and saw a dramatic increase in caspase 3 activation, showing that it is related to apoptosis and not necrosis.
So now we know what happens when we have too much RanBPM in a cell. What happens when we have too little? To downregulate RanBPM, the authors used siRNA, or small interfering RNAs. (Read my Protein Synthesis Basics primer to learn about RNA.) These siRNAs have a complementary sequence to the RNA used to produce RanBPM protein. When they are released into the cell, these siRNAs bind with the RanBPM RNA, making it double-stranded and unable to be made into protein, blocking its production. The authors did several experiments where they damaged cell DNA on purpose by radiating it and observed how the cells survived. In the image to the right, you can see that cell survival in downregulated RanBPMcells is greater than controls (injected with a random shRNA, a kind of siRNA, to ensure that the shRNA process doesn’t affect the cells’ survival) even with high radiation levels. This demonstrates that a loss of RanBPM protein affects the ability of a cell to undergo apoptosis when it is affected by DNA damage, further implicating this molecule in being important to this pathway.
The next step, after over- and underexpressing a gene, is to work out it’s molecular function. The authors looked at the expression of two proteins known to be involved in apoptosis, Bax and Bcl2. When there is a DNA damage signal present, Bax relocates to the mitochondria to do its part in apoptosis. The authors found that in the shRNA RanBPM cells, with reduced apoptosis, Bax never relocated to the mitochondria after radiation, showing that RanBPM affects the Bax pathway. They then looked at another protein which regulates Bax, Bcl2, and also found increased expression in shRNA RanBPM cells. Previous studies have shown that Blc2 overexpression blocks the apoptotic pathway, and can even lead to cancer. When they reintroduced RanBPM to nearly wild-type levels, Bcl2 expression decreased. These studies show that RanBPM is in fact affecting the expression of other proteins involved in apoptosis, and a known relevant apoptotic pathway at that.
This study, and many molecular biology studies, rely heavily on correlation. That is, looking at changed levels without showing causation. We know now that RanBPM is part of this pathway and affects other molecules, but it still isn’t clear how it affects these other molecules and how it functions in a wild-type cell. If only we could simply use the methods the authors used to overexpress or underexpress these proteins in cancer cells, but regular human cells don’t accept these kinds of treatments. While doing this sort of research is important to pinpoint particular molecules that regulate cell survival, until we have developed treatments to actually utilize proteins in human therapy, we won’t be able to cure cancer.
Atabakhsh, E., Bryce, D., Lefebvre, K., & Schild-Poulter, C. (2009). RanBPM Has Proapoptotic Activities That Regulate Cell Death Pathways in Response to DNA Damage Molecular Cancer Research DOI: 10.1158/1541-7786.MCR-09-0098