Cracking the histone code

Our paper in Nature Methods (“Genetic code expansion in stable cell lines enables encoded chromatin modification“, DOI:10.1038/NMETH.3701) is the first one to generate and characterize stable amber suppression cell lines for unnatural amino acid mutagenesis . The principle of genetic code expansion via amber suppression is shown below

Amber Suppression

We then apply the system to generate genetically encoded synthetic histone acetylation marks to directly test the function of this posttranslational modification in chromatin, one position at a time. This approach highlights the potential of the methodology to perform experiments with biochemical precision in living cells that could otherwise only be achieved in vitroIn vitro experiments can provide a clear link between molecular cause and effect, but are abstracted from the appreciable complexity of the cellular environment. In contrast, in vivo experiments typically provide a wealth of correlative information about changes of chromatin state in a native context, but it is commonly impossible to infer direct causation from these experiments. For example, all histone acetyl transferases (HATs) are known to acetylate a range of sites and substrates, including non-histone proteins, thus genetic knockout or enzymatic inhibition of HATs does not directly and exclusively test the function of histone acetylation. Employing a synthetic route to modulate  cognate posttranslational modifications has the power to show direct causality between the modifications and their downstream effects, abstracted from the  complexity of enzymes that set and erase the modifications. We believe that in the future such approaches to synthetic epigenetics will be very powerful for defining the function of posttranslational modifications, in particular the complex modification code present on histones.

Screen Shot 2016-01-02 at 8.13.12 PM

In a second publication, we have employed stable amber suppression in  HEK293 cell lines to synchronously activate a mutant Isocitrate-dehydrogenase enzyme (IDH2) in the entire populaiton of cells by light and followed changes in metabolic and epigenetic products:

http://pubs.acs.org/doi/abs/10.1021/jacs.5b07627

 

 

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