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Single-chromosome transcriptional profiling reveals chromosomal gene expression regulation.
Feb 27, 2013   Nature methods
Levesque MJ, Raj A


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The power of n=1

Jul 02, 2013
Marshall and I recently published a paper on our findings resulting from measuring chromosome structure and transcription simultaneously in single cells, and I think it's a good example of the usefulness of thinking laterally in research. Lately, there's been a lot of activity in the field of chromosome structure (primarily measured by 3C and its variants), and we were thinking that it would be nice to approach the same problems from an imaging perspective, so we developed a method that let us look at the position and transcriptional activity of 20 genes along a single chromosome simultaneously in single cells. It was awesome! I actually came up with the idea for the method right around when I started at Penn, and it took a year or so of painstaking problem solving and iteration, but we finally got the whole thing working and it was just oh so cool when it did! Hats off to Marshall for all his efforts in getting the whole thing to hold together–it's definitely the most technically challenging piece of work I've been involved in.

So, like I said, our initial goal was really to look at the conformation of chromosomes (in this case, chromosome 19) and see how it affects transcription. We figured we should start collecting tons of data using primary foreskin fibroblasts [insert foreskin joke here], since they have nice normal chromosomes, etc. But in the course of testing out the method, we looked in HeLa cells, mostly because we just had them around. Marshall analyzed one of the cells and showed it to me, primarily just to show me that the method was working. In particular, we knew that HeLa cells have a lot of chromosomal translocations, and indeed, we could see an extra piece of chromosome 19. But when I looked at it, I saw something I found very curious: it looked like there was somehow way more transcription than normal on the translocated piece! This was crazy, because the translocation is so big that this would point to some sort of chromosome-wide transcriptional change. Sure, it was just one cell, could have been a fluke, could have been any number of technical artifacts. Whatever. I knew we were on to something!

And so began the journey. It took a bit of convincing to divert attention away from then "main" goal of the project, but Marshall did all the experiments and controls, etc., to show that it was indeed the case that there was chromosome-wide transcriptional differences on the translocated chromosome 19. What a cool result! At least I think so. And that's how a diversion based on n=1 cell turned into one of the most important results from our manuscript.

So what happened to the initial idea with the chromosome structure and transcription, etc.? Turns out to be a pretty big negative result. Hidden in the morass of 20+ supplementary figures is the finding that the shape of a chromosome per se doesn't really have much to do with transcriptional regulation, at least for the genes we looked at. There is so much hoopla about chromosome structure these days, and many people have a vested interest in there being a relationship, so I'm always very circumspect about making too much of this negative result in public. But I think it's an important point. It's also the sort of thing that makes publishing a hassle. We ran into one such reviewer with such a vested interest at Science, who raised all sorts of irrelevant and frankly bizarre "concerns" that ultimately scuppered our chances there after 6 annoying months of "review". Sigh. I've got more to say about that, but I don't want to get into trouble. What I can say is that we were in the end very happy with Nature Methods and their editors, who believed in our paper and got our paper out the door relatively quickly.

But this publication process did leave some serious scars. I was SO excited about this paper because I felt like it really used a pretty cool method and used it to find some interesting new biology. It was also the first paper from the lab, and so I was really emotionally invested in it, perhaps overly so. I have to say that getting rejected at Science was pretty crushing, and I'm also sad to say that this was the first time I've had a paper not eventually get published at a journal after it went to review (not that we haven't had to fight in virtually all cases, but still). I know, "Boo-hoo, your paper got rejected at Science. Wah-wah, little science baby." But it's not the fact that it got rejected, it's the way it went down. It really made me doubt myself as a scientist for many months, and it definitely somewhat tempered my personal excitement once we finally published it, along with the fact that many important results ended up in the supplement with only the scarcest of mentions after the paper got demoted to a "Brief Communication". What's even worse about it is that I think the process really turned Marshall off of academic science. Perhaps he should have a thicker skin about it, and perhaps it's a lesson about how the world is not always a fair place. Fine, but I think we should strive to make things better in the scientific process rather than just shrug and tell people to "suck it up".

Whatever, enough whining. The main thing is that we finished up a piece of work that I truly think is one of the more important things we've done, and we're very interested in following up on the translocation stuff. I sometimes wonder what would have happened if we hadn't taken a close look at that one single cell, now a couple of years ago...
Copyright: © 2013 Arjun Raj. The above content is licensed under the Creative Commons Attribution License (CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.  
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