Another possible paradigm shift (if replicated)

        I’m a sucker for papers that turn our previously held ideas upside down.  The more spectacular they are, the more replication they need. Such papers happen all the time in molecular biology, but fairly rarely in chemistry.   Here’s a previous one — — the author was going to write with more data, but so far nothing has been forthcoming.  Then way back at the beginning of this blog, there was a paper implying that DNA had to be oxidatively damaged for transcription to occur. Unfortunately I can’t find the link (in my own blog yet !).  

       Here’s yet another intellectual cherry bomb.   To get your molecular biology up to speed, click on the category “Molecular Biology Survival Guide” on the left side of the blog.    

      [ Proc. Natl. Acad. Sci. vol. 108 pp. 11918 – 11923 ’11 ] An incredible paper.   The RNA containing a polyAdenine 3′ tail (polyA tail) from cardiomyocytes (heart muscle cells) was put into fibroblasts (connective tissue cells) in culture using 3 cationic lipids.  Note: this really isn’t the whole transcriptome, which includes a lot of pseudogenes, and lots of RNA without a polyA tail.   But this collection of RNAs does contain all the mRNA coding for proteins.

       This changed the fibroblasts into cells which looked like heart muscle cells which they call transcriptome-effected cardiomyocytes (tCardiomyocytes).  The treated cells show structure, immunocytochemical properties, protein expression profiles of postnatal cardiomyocytes.  They are elongated and have a similar length to width ratio as adult ventricular myocytes (which are much longer than fibroblasts.  The tCardiomycyites are electrically excitable as well (something fibroblasts are not).  So the (mostly protein) gene expression profile maintains (and controls) cellular phenotype (the way cells look and act).   It took two weeks in culture for the change to occur, but it was stable for 2 months (the period of culture).  The authors have also been able to produce cardiomyocytes from astrocytes (but not from neurons) by the technique.

         Why is this paper (if it holds up) such a paradigm shifter?  We know that all cells (except some immune cells) contain exact;u the same collection of genes (e.g. the genome).  Yet a liver cell looks nothing like any other cell.  Each cell type is thought to differ from others, by the types and amounts of proteins they express.  This has been attributed to two processes.  The first is the formation of heterochromatin (densely compacted DNA) around certain stretches of DNA, effectively shutting it down so that proteins coded by it aren’t made.  The second process is that of epigenetics, in which the DNA, or the histone proteins are chemically modified (again determining which proteins are made or not made).

I would have thought that epigenetics and heterochromatin would make  the transformation of one cell type into another by the above technique impossible.  Maybe they are less important that we’ve thought (or maybe no one will be able to replicate the paper).  Stay tuned.

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  • Handles  On July 31, 2011 at 10:39 pm

    The DNA oxidative damage paper was this one I think:

  • Bryan  On August 1, 2011 at 11:30 am

    The idea that you can change the fates of these cells by injecting mRNAs fits in nicely with the work that stem cell biologists have done with induced pleuripotent stem cells (iPSCs). Back in 2006, Shinya Yamanaka showed that mouse fibroblasts could be converted into cells that act very much like embryonic stem cells by injecting just four transcription factors into the cell (Takahashi and Yamanaka 2006 Cell 126 (4):663). So, injecting the entire set of cardiomyocyte mRNAs into the cells is likely overkill; I’d bet the researchers could find a small subset of these mRNAs that are sufficient for reprogramming, just as Yamanaka could find a subset of four factors required for reprogramming into stem cells.

    These results seem to imply that cell fate is maintained by positive feedback loop whereby the genes that specify cell fate maintain their own expression as well as the required chromatin state of the cell. Given a strong enough perturbation, however, one can disrupt these feedback loops enough to knock the cells out of their stable minimum in “phase space” and knock them into a different local minimum.

  • luysii  On August 1, 2011 at 11:56 am

    Bryan — thanks for the comment.
    “These results seem to imply that cell fate is maintained by positive feedback loop whereby the genes that specify cell fate maintain their own expression as well as the required chromatin state of the cell.”

    That’s just what I find remarkable. It seems to throw epigenetics and heterochromatin to the winds. This is essentially transdifferentiation, without going through the middleman of the embryonic pluripotent state. A few examples are around — fibroblasts to neurons with 5 factors (Brn2, Mut1l, Diz1, Olig2, Ascl1) Nature 463 pp. 1031 – 1032 ’10, pancreatic exocrine cells to ‘beta-like’ cells using a viral vector and 3 factors (nanog3, pdx1, mafa) Nature vol. 455 pp.604 – 605, 627 – 632 ’08.

  • luysii  On August 1, 2011 at 12:16 pm

    Bryan — Also the IPSC work and the stuff I just cited seems terribly UNphysiologic — blasting away with high concentrations of a few genes (or mRNAs). Back in med school, there was a pharmacology prof named Schmidt, who told us what’s I’ve since called Schmidt’s law — enough of anything will do anything. The work here just used the mix of polyA+ RNAs present and marching around in the cardiomyocytes and presented it to the fibroblasts. Of course we don’t know just which ones got inside, and it may be as you say, just a few were crucial.

  • luysii  On August 2, 2011 at 2:53 pm

    Bryan — not as current on my reading as I’d like, but Nature vol. 475 pp. 390 – 393 ’11 (21 July ’11) transformed mouse fibroblasts to hepatocyte-like (liver-like) cells using 3 genes (Gata4, Hnf1alpha, Foxa3) and inactivating a fourth (p19^Arf). They used lentiviral transfection to get the genes into the fibroblasts — again this may be due to a huge overexpression of what was transfected, rather than the day to day population of polyA+ RNAs used in the paper in the post.

  • Bryan  On August 4, 2011 at 1:26 pm

    I’m not so sure that transfecting cells with the pool of mRNAs from a different cell type is any less unphysiological than the transfection used to generate iPSCs; in fact, since fibroblasts won’t normally turn into cardiomyocytes, isn’t this mRNA transfection approach by definition unphysiological?

    As for epigenetics and heterochromatin, perhaps I see these forms of regulation as more dynamic than you do. Certainly epigenetic modifications are dynamic. The fact that the DNA and histones are covalently modified does not necessarily imply that these modifications are harder to change — for example, phosphorylation is a covalent modification that is very dynamic and able to change relatively quickly inside the cell. We know that various deacetylases and demethylases exist in the cell, so it is reasonable to think that these could reverse epigenetic changes once the machinery maintaining these modifications go away.

    Similarly, the packaging of chromatin is dynamic as well. We know that various chromatin remodeling proteins exist in the cell that can relatively quickly change the packing of chromatin. A good example of this is during mitosis. When the cell needs to divide, essentially all of the DNA in the cell becomes tightly packed in a heterochromatin-like state but this rapidly goes away afterward.

    Yes, epigenetic modifications and heterochromatin seem to be inherited by daughter cells after division, but again, this may be due to the presence of the regulatory factors that maintain the fate of the cell. Once these factors go away, the cells seem to be more amenable to reprogramming.

    Although I do seem to remember some labs doing detailed analysis of iPSCs (looking specifically at epigenetic marks) and seeing some differences between iPSCs and normal embryonic stem cells. Maybe I should look up that paper again.

  • luysii  On August 4, 2011 at 1:56 pm

    I guess it all depends on what strikes one as surprising. Have a look at Nature vol. 467 pp. 280 – 281, 285 – 290 ’10 for the epigenetics of iPSCs and also ESCs produced by somatic nuclear transfer. Both retained some of the epigenetic marks of the tissue of origin, and the IPSCs retained more of them.

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