The Futile Cycle

In an earlier post a two days ago, I mentioned that recently people have been speculating about the existence of “transcriptional factories”, because they saw that some genes might move to different parts of the nucleus when they’re turned on. They propose theories like this:

Scientists have also seen pictures like this

(which I showed last time, from this review article). Stuff like this drives them wild with speculation. Why do proteins form these shapes? What does it mean?

My answer: maybe nothing?

A new paper in Molecular Cell attempts to find evidence that genes don’t need to relocate in order to be activated; that is, it tries to disprove the notion of the “transcriptional factory.”

The problem with looking at how DNA is organized in the nucleus is that DNA is very small and hard to see, especially in a cell that’s alive (as opposed to killed, fixed, and stained with various dyes). DNA is invisible under a normal light microscope except when the cell is going into mitosis, when the DNA gets wrapped into chromosomes that get separated. Normally, though, the DNA is in this diffuse mass throughout the nucleus, and thus completely invisible.

The authors here get around this problem at first by using a trick called “polytene cells”; in the salivary glands of fruit flies, certain cells will accumulate a huge number of copies of DNA, leading to these really fat bundles of DNA called “polytene chromosomes,” which are actually visible under a microscope! The cool thing is that these chromosomes have different densities along the DNA, causing it to have distinctive stripes. Biologists can use these stripes to figure out where genes are on chromosomes, and then follow those genes by sight!

Here’s a picture of polytene chromosomes from that paper:

The little green bands are genes that are being transcribed (”read”) by RNA Polymerase II, which the authors have stuck to a green fluorescent protein (GFP) in order to make visible.

Another neat thing is that when genes turn on in polytene chromosomes, the bundle of DNA opens up to let proteins bind and start reading the gene sequence, causing the chromosome to “decompress” or “puff” (and thus become invisible). The authors here track the puffs with the labeled RNA polymerase II in order to see where heat shock genes go in the nucleus when they are turned on.

In polytene cells, the authors couldn’t find any sign that the heat shock genes moved around a lot, and the genes that did respond to heat shock did not move to the same place. But these are polytene chromosomes; maybe in normal diploid cells, that doesn’t happen?

So the authors also use “FISH” (fluorescent in situ hybridization) in order to see where specific genes are located in normal, non-polytene cells. Basically, one takes a piece of fluorescent DNA and sticks it into a fixed cell, hoping that the piece of DNA will base pair with its complementary partner DNA inside the nucleus. Using this, the authors saw very little change in the location of most genes when they turned on, although one gene did move a little.

Thus, the authors don’t find, at least for heat shock genes, that it’s necessary for them to move around the nucleus in order to get transcribed. In the end, perhaps the aggregates of proteins and such in the nucleus are just that: aggregates.

The simplest explanation may sometimes be the best one, even if it’s a little boring. I’m excited to see how the research in this field develops, and whether this paper turns out to be horribly wrong (it can happen to the best of papers), or whether the idea of transcriptional factories becomes outdated.

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Yao, J., Ardehall, M.B., Fecko, C.J., Webb, W.W., Lis, J.T. (2007). Intranuclear Distribution and Local Dynamics of RNA Polymerase II during Transcription Activation. Molecular Cell, 28(6), 978-990.

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.

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):

Via Ars Technica, there’s apparently a new database online called Deja Vu, which catalogues duplicate (or near duplicate) entries in MEDLINE, using an algorithm very similar to BLAST.

The majority of the entries retained in the database seem to be cases of outright plagiarism. Going through a few of the entries is like dredging the bottoms of the academic pool; most of the journals are pretty obscure or quite poor in quality, and I bet most of the authors are pretty unknown, too. It’s a strange world down in the pits…

As I was studying like mad for my prokaryotic molecular genetics final exam, I found an (alas) engrossing anecdotal essay from Seymour Benzer, called Adventures in the rII Region, which contains the following rather amusing story.

Seymour Benzer, who died just one and a half months ago, was one of the giants of early molecular biology. He was one of the first geneticists to show that genes weren’t indivisible subunits on DNA (like beads on a string), but rather linear “regions” of DNA, which he called cistrons. Many consider his mapping of mutations within individual genes to be one of the most elegant and amazing achievements of early molecular biology.

When he first showed Delbruck (his mentor and another giant back in the day) his manuscript, apparently Delbruck commented, “Delusions of grandeur.” Benzer follows:

Delbruck knocked the paper so badly that not until a visit to Caltech the following spring did I dare to approach him with another version. He submitted it to the Proceedings of the National Academy of Sciences, and it contained an appropriate acknowledgement of his “moderating influence.”

Tough love, indeed!

There are all sorts of anecdotes in that essay that a pleasure to read, about the entire molecular biology revolution of the mid-20th century, and the close relationships of the biggest players of the time. Some of those scientists were probably more than a little crazy! Check it out!

There’s an interesting article in Genetics on the luckiness of biology’s initial choice of bacteria to study. One of the most common bacterial types that molecular biologists studied back in the mid-20th century was E. coli K12. K12 was used a lot because it harbored a dormant λ virus (allowing the discovery of one of the most elegant genetic switches ever found), an F⁺ plasmid, and many suppressors of amber mutants (which I mentioned before).

All three of these features of K12 were extraordinarily important to the development of modern molecular biology, including the nature of the genetic code, tools for understanding gene regulation, fundamental mechanisms of transcription and translation, the circular nature of the bacterial chromosome, the mechanisms of transposition, the nature of recombination, and so on.

The paper talks in particular about amber mutation suppressors. Apparently strain K12, back when Edward Tatum first established it, had somehow acquired an amber nonsense mutation in rpoS, which codes for the RNA polymerase σ^S sigma factor needed for survival in stationary phase (i.e. semi-starvation and crowding phase). Lab strains often hit stationary phase when grown on plates, which would lead to a positive selection for amber suppressors. Thus, K12 acquired a large number of amber suppressors, leading to the discovery and utilization of a large number of conditional and conditionally lethal mutations!

Amazing how much biology has been discovered due to sheer chance…

I’ll be writing on microRNAs soon, but here’s yet another example of the extraordinary speed at which some hot fields (like microRNAs) move.

There’s a review paper on microRNAs that was published in advance (i.e. online before its print publication) in Nature Reviews Genetics this past Wednesday. It summarizes the latest and cutting edge of our current understanding of microRNAs, how they’re made, and how they work.

And it might already be (slightly) obsolete, even before it is published!

The review paper almost didn’t get to mention the new upregulatory mechanism of microRNAs paper that I wrote about one month ago, but they managed to squeeze it in at the last moment (there’s an addendum at the end of the paper mentioning this new result). The review certainly didn’t mention last month’s Cell paper on Ago2’s possible role in microRNA maturation and the PNAS paper on the current understanding of the RISC-loading complex composition that was published just over a week ago.

Certainly, the review paper isn’t useless; far from it, as it summarizes much research that is still relevant to our understanding of microRNA mechanisms, and it’s a useful resource for reference and for those who want to learn about the field for the first time. But it is still the slightest bit obsolete even before the ink has hit the page.

Alas, the curse and thrill to live in exciting times.

I’ve always had a strong dislike about certain biology naming conventions, but one thing that I always found rather strange was some of the whimsical and utterly meaningless names given to some genes and mutants. Take hedgehog, for example, or Bicoid. Meaningless names, really; they vaguely correspond to some sort of phenotype, but other than that, have very little to do with the actual biological function.

A paper I’m reading for a class, however, gleefully points out that meaningless names have their purpose: they don’t go obsolete with new information:

Epstein was struck by the similarity of the amber mutants and the so-called hd, or host-defective mutants….Who first had the idea that amber mutations are a general class of “suppressor-sensitive” mutations, I don’t recall….That year, Campbell did further experiments to show that the hd mutants (then renamed sus, for suppressor-sensitive) were in fact responding to a bacterial suppressor gene…

(It is amusing to note that Campbell found it necessary to rename his mutants after learning more about them, whereas the name amber is just as meaningless, and thus just as useful, now, as when the mutants were first discovered and named for Mrs. Bernstein.)

Edgar, R.S. 1966. In: Phage and the Origins of Molecular Biology, Cold Spring Harbor Laboratory, NY. p. 166-170.

hd and amber mutants are the same type of mutant, and “amber” is the English version of the German name “Bernstein.”

So, perhaps those Drosophila geneticists are doing a service to the rest of us after all.