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:



Short News

Simon Elsässer has been appointed to the Global Young Academy

Congratulations to Sigrid Lundin and Rahul Kumar for finishing the Berlin Marathon 2015!

Meet Simon Elsässer at the 65th Lindau Nobel Laureate Meeting, June 28th-July 3rd in Germany

Simon Elsässer will be presenting at the Eukaryotic Synthetic Biology Symposium in Heidelberg, 21-23rd June 2015 in Germany

Zakir Tnimov will give a talk at the International Synthetic and Systems Biology Summer School, 5-9th July in Italy

CONGRATULATIONS to Sigrid Lundin defending her Master thesis!

Good luck to Rahul Kumar running the Stockholm Marathon in torrential rain!

H3.3 silences endogenous retroviral elements

Histone H3.3 is required for endogenous retroviral element silencing in embryonic stem cells.

Elsässer SJ, Noh K-M, Diaz N, Allis CD, Banaszynski LA Nature 2015 In press

The double-stranded DNA molecules that make up the human genome are present inside the nucleus of a cell in a highly condensed state – it requires a ~10000fold compaction to fit its 3 billion base pairs into the tiny available space. So-called histone proteins achieve this task by wrapping the DNA like strings on beads. Through this packaging mechanism, we think that histone proteins are the key to regulating access to the genetic information and provide a molecular basis for indexing or annotating the genome with so-called epigenetic information. Histone proteins carry a large number of distinct chemical modifications or ‘marks’, providing a verbose epigenetic language. As a field, we have only started to appreciate the intricate complexity of this histone code.  Our study investigated the mechanism of silencing transposable elements in a mouse embryonic stem cell system. These parasitic DNA elements can transpose or ‘jump’ and multiply within a host genome and have played an active role in animal evolution, facilitating genetic variation and adaptation. But their activity represents a threat to the host genome and thus they are almost always actively silenced. We have found a new factor that is used to mark specific DNA elements for silencing. It is a variant of one of the core histones, H3, called H3.3, which has been intensely studied in other processes. But no one has looked at transposable elements, possibly because they are often considered to be of no particular function to the cell. Unexpectedly, we found that a large fraction of the histone variant H3.3 occupies transposable elements in mouse embryonic stem cells and our genetic studies show that it is required to efficiently silence the underlying DNA elements. The combination of H3.3 with a known silencing mark, histone H3 lysine 9 trimethylation (which in no other instance are found together) provide an exceptionally strong signal to the cell to ‘not read from this genomic region’. Untitled But why is this mechanism so important? When we deleted all H3.3 genes, we found that the repressive histone modification H3K9me3 is significantly reduced and some previously silenced elements are reexpressed. While we conducted our experiment in mouse cells, the mechanism is very likely conserved to humans. Over the last few years a number of cancer types have been found to harbor frequent and recurrent mutations in the histone variant H3.3 and two other associated genes, DAXX and ATRX, notably pancreatic neuroendocrine tumors and a family of aggressive childhood gliomas. In our study, we found that DAXX and ATRX proteins, like histone H3.3, are required for the silencing mechanism. Thus it is possible that the new molecular detail we describe plays an important role in maintaining genomic stability in the affected human tissues, a hypothesis that can be tested in the future.

A highly efficient system for amber suppression

In Jason Chin’s lab, we have developed an efficient system to incorporate unnatural amino acids in mammalian cells. Read more here: Efficient Multisite Unnatural Amino Acid Incorporation in Mammalian Cells via Optimized Pyrrolysyl tRNA Synthetase/tRNA Expression and Engineered eRF1,

Publications using our system to address novel applications: Genetic Code Expansion Enables Live-Cell and Super-Resolution Imaging of Site-Specifically Labeled Cellular Proteins Selective, rapid and optically switchable regulation of protein function in live mammalian cells