Experimental Biology 2016: Early Replication Stress Leads to Abnormal Mitosis and Genome Rearrangement.

Photo by Sean MacEntee

Pressure pushing down on me
Pressing down on you, no man ask for

Under pressure that burns a building down
Splits a family in two
-Under Pressure by Queen and the late and great David Bowie

We all feel the stress sometimes, but how we deal is what is important… and interesting. Dr. Susan Forsburg is interested in understanding how cells respond to replication stress and the survivors. 

Dr. Forsburg studies Schizosaccharomyces pombe, a species of yeast, which might leave you wondering how yeast relates to biomedical research. As it turns out, S. pombe is a good chromosomal model for humans. Also, I’d argue, (to paraphrase Annie Oakley) that whatever yeast can do people can do better—so it’s worth studying how yeast do things.

Her lab uses live cell imaging with the cell membrane and the chromosomes each fluorescently tagged (with different colors). When she looked at her cells, though, some just looked plain odd—the chromosomes were off. This led Dr. Forsburg to start monitoring what was happening to those cells, and her motto has become “the cell will tell you what’s happening if you pay attention to it.”

This story all started with the MCM helicase. It’s the first replicate helicase discovered that’s conserved from archaea to eukaryotes. MCM4 mutations, a subunit within the MCM helicase, are associated with increased chromosomal breaks and micronuclei. Micronuclei are exactly what they sound like—small, separate “nuclei”—and are common in cancer. 

Dr. Forsburg started with a MCM mutation that causes the cell to arrest late in S phase, where DNA Synthesis or replication occurs. (Quick primer on the cell cycle. Cells that aren’t dividing are in the G0 phase.  When they are dividing, they enter the G1 phase, go through S phase, then G2, and finally divide in M phase.)

This mutation caused less DNA replication/synthesis and the replication fork to collapse early. Yet, the cell still divides—albeit without much DNA due to the aforementioned replication and synthesis difficulties. Lack of DNA is a problem, but what if the DNA that’s left is damaged?

The daughter cells still had Replication Protein A (RPA) andRad52, but the phenotype of RPA is different. In the mutated MCM4, the RPAs are clustered as opposed to being spread out like normal. If you do a 3D simulation, you’ll see that RPA and Rad52 are interdigitated. If you keep looking, you’ll see more abnormalities, including an ultrafine anaphase bridge and mitosis with an unreplicated genome, all of which isn’t supposed to happen.

When you analyze these genomes, you’ll see evidence of chromothripsis. The best way I can think of to describe chromothripsis is that it looks like you took a chromosome, fractured it, and glued it back together not in any particular order. Now, what’s odd about these cells displaying this? Well, cancer cells often have chromothripsis. This fractured chromosome was often trapped in micronuclei “where bad things happen to good chromosomes.” Sometimes these micronuclei were absorbed back into the actual nuclei, incorporating the mutation into the cell’s genome.

This means that it might not be S phase stress alone but being unable to stop mitosis that’s the problem. That is, it’s not just the damage to the chromosome that’s is the problem. It’s trying to do something with it that causes issues.

Even more interesting, if you use the imaging technique in Dr. Forsburg’s lab. You can do pedigrees within the yeast cells, where you trace and examine subpopulations of cells with these defects. This is especially relevant to cancer because of the cellular heterogeneity that is commonly observed within just one tumor.

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Sabatinos SA, Ranatunga NS, Yuan JP, Green MD, Forsburg SL. Replication stress in early S phase generates apparent micronuclei and chromosome rearrangement in fission yeast. Mol Biol Cell. 2015 Oct 1;26(19):3439-50.

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Experimental Biology 2016: Interactomic and Enzymatic Analyses of Distinct Affinity Isolated Human Retrotransposon Intermediates.

How does a RNA biologist end up studying the structure-function relationship of proteins? Dr. John LaCava moved from the world of nucleic acid to purifying and studying protein complexes. When he explained his path, it sounded like a natural transition. Dr. LaCava started out studying RNA biology—in a splicing lab. He started to become intrigued about the protein complexes in RNA. From there it was a quick hop, skip, and a jump to studying the structure-function relationship of proteins. However, the real gleam in his eye comes from when he talks about purifying protein complexes—but not just any protein complexes, the physiologically relevant for purifying protein complexes. 

The trick to protein complexes is keeping them, well, in the complex. It’s not uncommon to go through a very complicated protein purification protocol only to find out that a one protein with the complex has “fallen off” and is missing (I’m looking at you, delta subunit of the E. coli F1F0 ATP synthase). He and his lab have developed a method to determine under what conditions you can artificially prolong the life of the protein interaction.

While you might want to optimize the aforementioned complicated protein purification protocol, you just plain don’t have the time. What you really need is a high throughput way to test out a bunch of different versions of the protocol—but you just don’t have the time. This is exactly what Dr. LaCava figured out and is detailed in his Nature Methods paper last year (Hakhverdyan et al., 2015). I’ll do a quick summary here: You start with a wet pellet of cells that you then extrude into liquid nitrogen. This gives you beads of cells that you can “grind” into a fine powder using stainless steel beads that will get you down to the micron scale. One of the nifty parts of this method is that now the insides of the cells are now on the outside for easy access to protein complexes. Next, you can distribute this powder to different tubes and test out a different protocol in each tube to find the optimal conditions.

So, what does all of this have to do with RNA splicing? Well, RNA splicing is done in a, wait for it, protein complex. That’s where LaCava got his start. He’s now moved on to answering different questions. He got interested, through collaborations with colleagues, in retrotransposons—these so-called “selfish genes.” These are genes that are self-replicating and are present in the brain, in gametes, and in development.

His particular retransposon is in the LINE-1 family. It is bicistronic each with a 5’ UTR, promoter, and 3’ UTR that encodes a poly-A tail. It also displays a cis-preference. That is, its proteins prefer to associate with its own message (there’s that RNA biology again).

His lab purifies it using IDIRT—a spin on a metabolic tag in mass spec. To make a long story short, his lab tags the protein with heavy label and has an untagged version of the protein, which is mixed in a 1:1 ratio. You can read more about the general method in this article (Tackett et al., J Proteome Res, 2005).

The RNAs (either ORF1 or ORF2) that are engaged with tagged protein that are most likely true and stable under experimental conditions. He pulled out ORF2 from the supernatant and bound fraction and measured the average distance of the message. From this, he got a three node cluster (the chances of seeing any three nodes close together). 

For now, this measures the ability of ORF2 to replicate itself. However, it has opened the door for the future. You could use this method to do enzymatic assay on the intermediate structure, or genetic and biochemistry depletion assays, or even electron microscopy to examine the structure-function relationship. And now we’ve come full circle. This is how a RNA biologist ends up studying the structure-function relationship of proteins.

Interactomic and Enzymatic Analyses of Distinct Affinity Isolated Human Retrotransposon Intermediates.

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J. LaCava, K.R. Molloy, D. Fenyö, M.S. Taylor, B.T. Chait, J.D. Boeke, M.P. Rout. The Rockefeller Univ.,NYU Sch. of Med. and Massachusetts Gen. Hosp 

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Experimental Biology 2016: Epigenetics Impacts Copy Number Heterogeneity and Drug Resistant Gene Selection

Photo by Steve Davis

First, a bit of blog business. My travel to San Diego was, well, fraught. I was supposed to arrive on Saturday, but I was delayed until late Sunday night. I've tried to cram all the meeting activities I wanted to do into these two days. So, these posts may be a bit out of order. However, there is a lot of really cool science going on that I want to share.

Now, on to the science.

Before attending the meeting I dutifully went through the EB app (see? At least one person used it) to decide what talks and posters I wanted to see/visit. I was pleased to see that epigenetics would be well represented at the meeting.

Given my interest in epigenetics and frequent discussions I’ve had with people about its importance in cancer, I had to visit the Whetstine Lab Poster on "Epigenetics Impacts Copy Number Heterogeneity and Drug Resistant Gene Selection"  and Dr. Whetstine’s talk. So, how does epigenetics impact copy number heterogeneity and drug resistance?

The simple answer is A Lot.

Let's start with cancer cell heterogeneity. Cancers often have subpopulations of cells within tumors; not every cancer cell is the same. These cells can have distinct genotypes and phenotypes. Copy number variance is just one of these possible genotypes and drug resistance is just one of the phenotypes.

Dr. Whetstine proposes that one of the causes of those differing genotypes is epigenetics and he's recently demonstrated that to be true.

The entry point into his work is really through the KDM4A, a lysine-specific demethylase. Most specifically it demethylates lysine 9 and 36 on histone 3.3. His lab screened over 4,000 cancer types and found that 20% of them had increased KDM4A levels. This also correlated with increased copy gain of genes located in the 1q12-22 locus, which contains a number of pro-survival genes and oncogenes. Dr. Whetstine's lab went on to investigate this link and found that KDM4A brought all the replication machinery to the 1q12-22locus. Moreover, if you chemically modified the histones to interfere with methylation (by substituting the lysine with a methionine), then you see the same copy gain. This demonstrates that the specific epigenetic modifications also matter. Taking the reverse tactic, if you overexpress Suv39h1 (which opposes the action of KDM4A), then you suppress the copy gain.

Interestingly, you only get this copy gain if the cells are in S phase. This is one mechanism by which you can have the cellular heterogeneity. If one cancer cell is in S phase and the other isn’t, then one cell will have the copy gain and the other won’t.

Another mechanism for this heterogeneity can be the oxygenation of the tumor. Hypoxia results in this transient site specific gains as well. If you return the cells to normoxic conditions, then you reverse the copy gain. So, why is this important? Well, you could imagine that the interior of the tumor would have different oxygenation than the exterior—more room to breathe, if you will. Moreover, hypoxia is related to drug resistance. That is, some cancer drugs are less effective under hypoxic conditions—not necessarily due to the chemistry of the drug, but because the cells themselves are resistant.

Now, miRNAs. What do you think? Well, miRNAs appear to regulate KDM4A. If you transfect cells with hsa-mir-23a-3p, hsa-mir-23b-3p, and hsa-mir-137, then you decrease KDM4A expression and thereby copy gain. However, if you transfect the cells with anti-mirs (which block miRNAs), then you reverse the effect of miRNAs.

That’s all well and good, but is there a therapeutic response? Why, yes, there is. miRNAs give a reduced response to cisplatin.

Now, I’ve told you that KDM4A and specific epigenetic markers cause copy gain of a particular region, but what does this mean in terms of cancer development? Well, it turns out that the cancer treats this as a sort of “DNA suitcase.” That is the mechanism of gene over expression is the same—through these copy gains—but different cancer types overexpress different genes. For example, in breast cancer you see CKS1B overexpression, in myeloma there’s both CKS1B and BCL9, but in epithelial cancers you see neither.

Through “typing” the cells based on KDM4A expression, you could block drug resistance and sensitize cells to various cancer treatments. For example, you could decrease KDM4A expression through the use of miRNAs and, therefore, render certain cancer treatments more effective.

Introduction

A T-Rex Name Sue. Photo by Me

“Tell them what you’re going to tell them.” That was my grad school advisor’s motto for introductions.  So, here it is: I’m going to tell you stories. Throughout my time working in science (first as an undergraduate research assistant, then grad school, and now at a postdoc), when I mentioned at parties what I did, the most common reply was, “I could never do that! Science is so hard.” I always disagreed. Learning science, doing science isn’t that difficult. I would posit that it’s not finding the right angle to make it easy to understand. I think you do that by telling stories.

Science is really cool. Look at your smart phone, the internet, disease rates, the average lifespan—that’s all science! Science isn’t scary. Science is cool and helpful. Also, science isn’t done by robots. People do science. People who have families, friends… and stories. Therefore, (by the transitive property) science is full of stories. Everyone loves stories.

Within academia (and I’d imagine science-at-large), we often compliment a seminar speaker by saying that s/he told a good story or had a good story. We don’t mean in the fictional sense or in the lyrical, though. We mean that s/he put the big problem in context, broke down their methods, led us nicely through the results, and clearly drew conclusions.

These stories, though, follow a fairly set format. You have the introduction, which sets up the problem. This is usually followed by the methods section that tells you how the authors solved the problem. The authors then reveal the outcomes of the experiments in the results section. Finally, the researchers tell you what those results mean both in terms of the piece of the problem they solved and in the big picture in the discussion section.

Within each section, there is a format. In the case of the introduction, the authors first set up the big problem. They then narrow down that big problem to the smaller problem that the paper will address. Then, finally, they tell you how they plan to address it. The final paragraph generally begins “In this paper, the authors show….”

In this blog, I will write stories about science. All angles of science. In particular, the next couple of posts will be those stories about research presented during Experimental Biology. For those of you who don’t know, Experimental Biology is the big conference of several different scientific societies—I’m blogging for the American Society of Biochemistry and Molecular Biology. Indeed, these stories will be the first science stories of this blog.

Abstract

When you read scientific papers, you start with the abstract. This is the first paragraph that you encounter in an article. This paragraph gives you the run down of what the paper will be about. The format is generally the same: a brief background, a bit about the methods, key results, and conclusions. That's what I want to start with here.

I started Scientific Dispatches to write about things that interest me (and hopefully you) about science. The entire time that I've been in science, I've had people tell me how complicated it must be. Science doesn't have to be. All of science is just taking something that seems complicated and breaking it down one variable at a time. That's what I plan to do here. Take complicated, but cool, things and break it down. This way science will be accessible to everyone. Scientific literacy is becoming more and more important. This blog serves two purposes (1) to contribute to increasing scientific literacy and (2) to give me an excuse to read more science.

Photo by: Alex