The Futile Cycle

I’ve been thinking a lot lately about picking a lab for my thesis, and part of that has involved thinking about what kind of organism I want to do research on.

Of course, I have a strong interest in medicine, so perhaps my basic science research should be on a human-like system, such as on cultured human cells or human cancer cells, which are pretty common. When I first started learning biology, I thought to myself, “If mice and humans are very different, then what’s the point of even trying to do research on yeast, bacteria, or flies? Who cares? Why not just work on human cells?” After all, that’s the most directly “relevant” research to medicine, and if a lot of research is funded by the NIH for the future benefits to human health, why are they funding research about yeast mating? Human cells don’t even have cell walls, and they don’t bud!

One thing I learned pretty quickly about research, however, is that there’s a lot more to those model organisms than meets the eye.

Of the past 20 or so Nobel prizes in medicine, about 14 were given for research involving model (i.e. non-human) organisms! And 8 of them were given to research on non-mammalian organisms, including yeast, nematodes, bacteria, sea urchins, viruses, and fruit flies. In that time, two Nobel prizes in chemistry were also given for research in model organisms (E. coli and yeast). The most common mammalian organisms, of course, were rat and mouse, but there were also cancer cells in there that count. So clearly, research in model organisms is somehow breaking fundamental grounds, even now!

But why?

It has to do with how easy certain organisms are to handle. Mammals obviously don’t reproduce very fast, stereotypes about rabbits notwithstanding; bacteria, on the other hand, will divide every 30 minutes when they’re happy. That’s partly why they, along with viruses of the bacteria (which grow even faster), were the basis of almost all of the revolutionary molecular biology in the 1940s and 1950s. Genetics was a whole lot easier with them because there were millions and billions of them. Biochemistry was easier, too, because you could grow gallons of them within a day or two. Yeast is easy to handle, too. A decent amount of yeast can grow overnight, and a buckets of it can be grown in a couple of days. Both yeast and bacteria are very hardy, too; you can keep them for basically forever if you stick them in some glycerol and throw them in a freezer. They’re enormously popular, even today, for basic biological research, from bioinformatics to genetics to cell biology. The awesome power of microbial genetics is a wonder to behold.

Even fruit flies, nematodes, zebrafish, and sea urchins are pretty easy to handle, compared to mice and rats. You can grow tons of nematodes and fruit flies in a few days or weeks. Zebrafish and sea urchins take longer, but they produce tons and tons of eggs (and thus, offspring) at a time.

I think in part, these model organisms have the edge because biology tends to be pretty universal, thanks to evolution. A lot of stuff discovered in yeast is relevant to humans, because though our line split off from them a long while back (maybe a billion years or so), we still share a lot of biology, from the shapes and homology of molecules to the ways our cells are organized. Similarly, insect development studies led to major discoveries in our developmental regulation, especially the hox genes. Nematode work led to the discovery of microRNAs as crucial regulators of human development and signaling. Zebrafish gives fundamental insight into vertebrate-specific mechanisms of neural development that wouldn’t be found in flies or worms. Though these animals are different from us in many ways, they have lots of similarities to us that we can find before we’ve seen them all, and we can find them fast because these organisms are easier to work with.

There are certain niches, of course, for human and mammalian research. Immunology, for example, is pretty hard to study in non-mammalian systems, just because it evolved so recently. And cancer is very hard to study in mice and rats, because their lives are so much shorter than us. Still, human cells in culture, even cancer cells, are pretty difficult to use, especially for genetics, because they don’t sexually reproduce, which makes “purebred” lines and new mutations difficult and time-consuming to come by. So work in human cells, while perhaps more “directly relevant” to human biology, is much, much harder.

Ultimately, of course, for research to contribute to understanding human biology, one needs to do experiments in human cells too, but those will get done if and when the need arises, especially by companies interested in making new drugs and cures. In the mean time, there are scientists churning away at bacteria, viruses, yeast, flies, worms, fish, sea urchins, and even sea slugs that are doing breakthrough work from which we’ll benefit years down the line.

It’s very tempting to join their ranks. It’s something I’ll be pondering for the next few months.

A lot of what we know about transcription and gene regulation in eukaryotes has been from experiments in test tubes. The problem of course, is that test tubes aren’t the same as our cells. We have things like nuclei that aren’t homogeneous; there’s all sorts of stuff in there! Lamins, other nuclear cytoskeletal elements, histones, large loops of chromatin (DNA and the proteins stuck to it), and so on. Clearly, nuclei aren’t just bags of DNA and protein.

A lot of research has gone into trying to figure out how DNA is organized in the nucleus, and what that has to do with gene expression. Some people like to call this field “cytogenetics.” We now know a lot about how histones move and shift along DNA when genes turn on and off, but a lot less is known about higher order chromatin structures. One idea about gene expression is that of “chromatin territories” and “transcriptional factories.” A good review paper on the concept is appeared in Nature Reviews Genetics in 2001;¹ if that’s too long for you, here’s bullet point summary of the review (kind of a sad concept, if you ask me).

The basic idea is that chromosomes occupy particular mutually exclusive spatial “territories” of the nucleus. An idea that kind of got melded with that theory (but which is actually still a separate idea) is the “transcriptional factory” or “interchromatin compartment.” When genes turned on, some scientists have thought that the DNA of that gene moves to special parts of the nucleus enriched in the proteins needed to transcribe, splice, and explore the genes.

This is a really cool picture from that review paper showing why so many biologists think that chromatin territories might be the real thing, even if the evidence for them is still a little vague. It shows a chicken cell nucleus, with each chromosome “painted” a different color by combinatorial immunofluorescence:

One problem with the theory of chromosomal territories and with this sort of visualization is that it’s unclear whether the nucleus just happens to form like this spontaneously, or whether it’s actually regulated to be this way. I’d argue for the former; bacteria, for example, may spontaneously segregate their chromosomes simply due to their polymer physics. Here’s a paper (open access!) summarizing the results of that research.²

In this figure from that paper, the bacterial chromosomes start to segregate spontaneously as the DNA replicates. There’s no specific transport or regulatory mechanism necessary other than rigid confinement of the DNA polymers. It looks too similar to the situation above in the chicken nucleus to dismiss it outright as an explanation, I think.

The other related theory is the “transcriptional territory”, which is the idea that genes that are turned on migrate to certain areas of the nucleus that are enriched for transcription factors, RNA polymerase II, splicing proteins, and export factors. A lot of the evidence for this comes from microscopy, such as this figure from the review paper:

The red staining is a splicing factor, SC-35, and the green is the chromatin labeled with GFP-tagged H2B. It’s clear that there’s certainly some sort of protein aggregate in the nucleus, but whether it’s functional is certain up for definitive proof. Another good review paper for transcriptional factories is Chakalova et al., again in Nature Reviews Genetics.³

Next time, I’ll dig into a new paper from Molecular Cell that seems to strike a blow against the whole “transcriptional factory” idea.

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1. Cremer, T. and C. Cremer. (2001) Nat Rev Gen 2, 292-201.
2. Jun, S. and B. Mulder. (2006) PNAS 103, 12388-12393.
3. Chakalova et al. (2005) Nat Rev Gen 6, 669-677.

In confirmation of my suspicions, transcription initiation seems to be more randomized and probabilistic than people had previously thought.

One theory that had been proposed for a while was the idea of “transcriptional oscillation”, where transcription factors would hop onto a gene, transcribe for a little while (20-30 minutes or so), hop off, then start the cycle over again.

There was interesting data showing all this, which was nicely summarized by Métivier et al.¹ in the following graph:

The close details of the plot aren’t that important; the main point is that a lot of the machinery for transcriptional initiation seemed to oscillate with a cycle on the order of 30 mins. The plot took a heck of a lot of work to generate; each data point on a curve is one chromatin immunoprecipitation (ChIP), and in one of the group’s papers, they took a time point every five minutes. It must have been a nightmare of a project to make work, considering everything that could go wrong.

Now, the data is interesting, but their interpretation of it goes wrong. Their model supposes that this sort of mechanism is very regulated, that the cycles occur because factors hop on, do their thing, and get knocked off in a very timed, controlled manner.

Deterministic models like this make me sad. Sure, videos like “The Inner Life of a Cell” make biology look pretty, showing a smoothly operating machine in which motors step along nicely, molecules steadily march along tracks in the right direction, and everything swooshes together into assemblies that make things happen. But molecules just don’t work like this! Take a look at the elongation of RNA polymerase in the plots below from Stephen Block’s lab:

This is probably the smoothest anything ever gets in the cell. RNA transcription is fast, the elongation complex is super-stable (with a half-life of weeks), and not much is going to stop the polymerase. And yet, there’s still so much noise!

At long last, a new paper in Science² shows that the deterministic model (surprise!) might not be accurate! These authors use FRAP (fluorescence recovery after photobleaching) in order to show that the occupancy of promoters (at least in yeast) is very transient (on the order of 30 seconds).

They also show that this is how transcription initiation happens by using a clever experiment. If a few promoters were stable (in the sea of noisy binding) and responsible for the majority of transcription, then with ten copies of a gene, one particular copy would be transcribed only in about 1/10th of the cell. What the authors show is that any one particular copy gets transcribed in basically all of the cells, which means that the noisy bouncing on-and-off of transcription factors is how genes work, and that it isn’t something that’s obscuring a more stable, deterministic mechanism underneath.

On the other hand, they too find the slow-cycle oscillations when they do ChIP and RT-PCR analysis of the promoter, so clearly, there must be some sort of longer time-length phenomenon. Since this reflects an average phenomenon of all the noise, it must mean that some sort of global energetics is changing, something that would be at a slower time scale than just the binding of some other protein (which would seem to have the same noisiness and time scale as a transcription factor); the authors hypothesize that something like chromatin remodeling is happening, and I’m inclined to agree with them.

There’s more on nuclear dynamics within cells in the next blog post, on a paper that goes after the theory of chromosome territories!

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1. Métivier, R., Reid, G., Gannon, F. (2006). Transcription in four dimensions: nuclear receptor-directed initiation of gene expression. EMBO reports, 7(2), 161-167. DOI: 10.1038/sj.embor.7400626
2. Karpova, T.S., Kim, M.J., Spriet, C., Nalley, K., Stasevich, T.J., Kherrouche, Z., Heliot, L., McNally, J.G. (2008). Concurrent Fast and Slow Cycling of a Transcriptional Activator at an Endogenous Promoter. Science, 319(5862), 466-469. DOI: 10.1126/science.1150559

I finally got around to reading in depth the Science paper¹ on how serum-starvation may cause microRNAs to increase the expression of their cognate genes. I’ve got to say that, at the very least, the paper got me thinking a lot about the mechanisms of microRNAs, since if this paper is right about upregulating genes with miRNAs, then the picture that has been built over the past few years may change dramatically.

First, a little scene-setting.

Lately, it seems like everyone wants to tie their research to microRNAs (a.k.a. miRNAs); they’re hot, they’re interesting, and it’s up-for-grabs really on how they work. Over the past few years, a lot of really vague and contradictory data has started to emerge about how microRNAs actually inhibit the expression of genes. Along with that, of course, are a whole swathe of theories, ranging from the idea that they work at the level of mRNA stability or translation to ideas that miRNAs recruit proteases that chew up proteins as they get made from the RNA.

A new review paper in Nature Reviews Genetics² is out arguing for the idea that microRNAs stop translation initiation; other observations about microRNAs are either secondary consequences of this mechanism, or are minor, supplementary mechanisms for repression. It’s a very nice review, and it builds a very convincing case for their idea that the putative cap-binding activity of Argonaute 2 is one of the primary mechanisms for translational repression.

It seems to me like competition for 5′ cap binding could occur right after mRNA export and the putative pioneer translation round,³ which is thought to occur before the nuclear cap binding complex (CBC) gets replaced by the cytoplasmic eIF4F complex. If Ago2 manages to get its claws on the 5′ cap then, it’s certainly possible that eIF4F would never bind the cap, repressing translation, circularization, and even stabilization of the poly(A) tail.

(One of the problems with this theory, of course, is that non-capped RNAs, such as those with internal ribosome binding sites, can still sometimes be repressed by miRNAs (it seems). I’m not really sure how that fits into the larger picture; it’s entirely possible that these mRNAs are repressed by secondary mechanisms from miRNAs.)

Now, back to the paper I just read.

The cap-competition theory of miRNAs might explain the result of that paper in Science. The cool thing is that there is a paper from 2005 in Nucleic Acids Research⁴ showing that the cap binding of mRNAs by Poly(A)-specific ribonuclease (PARN) is at least partially serum-starvation dependent. So clearly, serum-dependent post-transcriptional regulation by cap-binding proteins has been seen before.

Now, this new Science paper purports that when cells are serum starved, miRNAs direct Ago2 to upregulate mRNA translation rather than depress it, like with others. It would be really interesting if these results pan out, because then they might unlock a cool and interesting piece of the miRNA puzzle, and it would definitely have vast implications for developmental biology, wound-healing, and overall gene regulation in the body.

The paper has some interesting experimental results, but there are a few fishy things about the paper that make its conclusions a bit forced. For one thing, the authors don’t really show miRNA-mediated repression of translation unless they synchronize their cells in their cell cycle. You can see this if you compare the expression levels of CX and CX with its cognate miRNA in the figure below (figure 2A from the paper):

As you can see, in the +Serum (grey) bars, CX and CX+miRcxcr4 have the same translation efficiency; the authors don’t see repression of CX when they transfected in miRcxcr4 unless they synchronize their cells (+Snc). (CX is an artificial sequence that they constructed with an artificial target site. miRcxcr4 is the artificial miRNA that targets CX.)

The problem here, of course, is that many other labs have seen repression of translation by miRNAs even when they don’t synchronize their cells. Thus, either the tons of other papers seeing this effect are wrong, or this one paper has some strange experimental quirks that may make the system very different from those of other labs (I’m inclined to think the latter). The authors argue (in the supplementary material) that perhaps other labs’ results are due to toxic effects of transfection reagents, or due to differences in cell culture protocols. It’s strange that so many other labs have seen such down-regulation without synchronization. Can every single one of those effects be an artifact? It seems unlikely.

So in the end, I’d like to see some sort of independent confirmation of the results of this paper before I’m likely to accept that somehow, almost the entire scientific community had screwed up their miRNA protocols and missed this crucial and drastically different role for miRNAs in post-transcriptional regulation.

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1. Vasudevan, S., Tong, Y., Steitz, J.A. (2007). Switching from Repression to Activation: MicroRNAs Can Up-Regulate Translation. Science, 318(5858), 1931-1934. DOI: 10.1126/science.1149460
2. Filipowicz, W., Bhattacharyya, S.N., Sonenberg, N. (2008). Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight?. Nature Reviews Genetics, 2008(2), 102-114. DOI: 10.1038/nrg2290
3. Chiu et al. (2004) Genes Dev 18, 745-754.
4. Seal et al. (2005) Nuc Acids Res 33, 376-387.

At the risk of alienating part of my family and a bunch of my friends from college, hell yes, the Giants win a huge upset victory! And Eli’s absolutely amazing pass under pressure to Tyree was too appropriate a way to set up the final, last minute touchdown. Congrats to the Giants! Eli no longer needs to live under his brother’s shadow.

To coincide with that amusing pseudoscience advertisement, I just came across a new paper in Cell explaining how cyclosporine A can lead to excessive hair growth in patients. Apparently, by modulating calcium signaling in hair follicle stem cells, cyclosporine A can tip stem cells out of their quiescent state earlier than normal, leading to more cell division and thus more hair. Elaine Fuchs has been studying hair follicle stem cells as a model system for differentiation and development, and this is a really neat paper that ties those topics together with cell-cycle regulation via an interesting signaling system.

One amusing assay that they conduct on mice is to shave them or dye their hair to look at the rates of hair growth. It seems to be a pretty common assay in the hair-growth and baldness fields of research, but I haven’t seen it before, and the pictures are funny to look at (my apologies to those who are touchy about mouse humiliation):

Today I was emailing data to myself with Gmail from the lab computer when I found this amusing advertisement:

Yes, we’re all looking for that option that won’t get away! Or maybe it means, the first unambiguous option! I also like how it doesn’t even say what the “option” is for, or what “activating” stem cells really means.

Today is apparently Blogroll Amnesty Day, in which small blogs link to smaller blogs, and so on. Alas, my blog is one of the smaller ones around, but since I tend to read across genres pretty broadly, perhaps I can help some people find some new blogs that they’d not seen before.

New to the blogroll, which has been un-changed for a very long time:

Computational Biology News

This blog is mostly links to interesting articles rather than a bunch of articles themselves, but Animesh Sharma nicely curates and finds stories on the net that I wouldn’t find otherwise.

DrugMonkey

DrugMonkey, which was recently assimilated into the SciBorg collective, is a great blog on health regulation in America.

Ars Technica: Nobel Intent

I’ve read Ars Technica for almost as long as it’s been around, starting from way back in my days in which I was really into computer science. Basically, I thought of Ars Technica as the thinking man’s Slashdot: less frenetic, more in-depth, and not pandering to the masses. Lately, Ars has gotten more pop-news-ish, but for the most part, it still seems to retain its journalistic integrity. Nobel Intent is Ars’ science journal, and they’ve got some great writers there. Apparently, a lot of science blogs have never heard of Ars Technica, so check it out if you haven’t read it before.

UPDATE:Forgot to add Evolving Thoughts, which covers biology, the philosophy thereof, history, politics, creationism, and really a whole swath of things that I find interesting.

In the first major casualty of the etBLAST algorithm and Deja Vu database has been found at Harvard Medical School, where Prof. Lee Simon’s review paper has been found to have large sections copied from another professor’s paper.

I had hoped that Deja Vu would consist of articles from random foreign countries and small, obscure universities, but alas, I was perhaps a little naïve. Perhaps the good part about this will be that it encourages authors to be much more reluctant to plagiarize.

On the other hand, it depends on whether the journals care. Elsevier, in this case, did the right thing and acted upon the evidence to retract the paper, but other journals don’t have such “enlightened” policies. I heard once about a professor that was reviewing a manuscript for a journal when he found that the other author had plagiarized sections from one of the professor’s own papers! When the professor notified the journal editor, they informed him that this was commonplace, and that he should just review the article anyway.

What floats to the top in science is often beautiful, but there’s a lot of crap that sinks to the bottom.

Yesterday I watched online the Republican debate, which happened at the Reagan Library, and I heard Mike Huckabee and John McCain both mention leaving Iraq “with honor.” Apparently, Dick Cheney has used that term, too.

Now, I’m not going to comment on the US operations in Iraq, but seriously, what politician in their right mind would invoke Nixon’s statements on Vietnam in order to describe the policy of staying in Iraq? “Leave with honor” sounds very much like Nixon’s “Peace with honor.” It would seem to me like political suicide to utter those words; but perhaps Vietnam is fading from the memories of the public?