Epigenetic regulation and genomic engineering
Previous and Current Research
Our work focuses on two complementary aspects of genomics, (i) mechanisms of epigenetic regulation in eukaryotic chromatin and (ii) technologies of genetic engineering.
Epigenetic regulation in chromatin
Although the complete DNA sequence of an organism encodes the primary information, additional information is added by epigenetic regulation. In eukaryotic chromatin, epigenetic regulation is conveyed by covalent modifications of DNA (DNA methylation) and histone tails (acetylation, phosphorylation, methylation, ubiquitinylation). Much attention worldwide is now focused on the histone tails and the proposition that patterns of covalent modifications serve as an epigenetic code. Our approach to unravelling epigenetic mechanisms and hierarchies is based on complementary uses of the yeast, S. cerevisiae and the mouse as experimental systems. We apply advanced reverse genetic strategies, some of which were developed by us, to analyze select classes of epigenetic regulators in both organisms. In yeast, we are using protein-tagging and mass spectrometry to characterize complexes containing epigenetic regulators. Amongst other complexes that we have identified in the proteomic environment of chromatin, we have recently identified a new histone methyltransferase activity for lysine 4 of histone 3.
In mice, we are studying two candidate histone methyltransferases by knock-out and conditional strategies using Cre/lox, as well applying proteomic approaches to characterize the complexes. A future aspect of our mouse work is directed towards use of ES cell differentiation in culture as a model for epigenetic decisions and stem cell manipulations.
We have developed several aspects of genetic engineering technology using site specific and homologous recombination. We aim at more fluent manipulation of mammalian cells, particularly ES cells and in mice. Most recent work involves exploration and implementation of a novel homologous recombination system that we discovered in E.coli phages. This permits fluent engineering of BACs in E.coli, and may offer new routes for directly engineering eukaryotic cells.
Future Projects and Goals
Further work on epigenetic regulators in eukaryotes will be accompanied by advanced engineering strategies to examine roles of epigenetic regulation in mammalian development, stem cells, ageing and disease.
Methodological and Technical Expertise
- Recombineering and other DNA modification methods for BAC tagging, targeting constructs, point mutations; Site-specific recombination
- Chromatin IP
- Expression Profiling
- Genome Engineering (Gene Targeting, BAC transgenesis, Transposons, TALENS, etc.)
- Mouse model studies (developmental studies, engineering)
Rostovskaya, M., Fu, J., Obst, M., Baer, I., Weidlich, S., Wang, H., Smith, A., Anastassiadis, K. and Stewart, A. F.
Transposon Mediated BAC Transgenesis in Human ES Cells.
Nucleic Acids Res (2012) [Epub ahead of print]
Bird, A. W., Erler, A., Fu, J., Hériché, J. K., Maresca, M., Zhang, Y., Hyman, A. A. and Stewart, A. F.
High efficiency counterselection recombineering for site-directed mutagenesis in bacterial artificial chromosomes.
Nat Methods 9:103–109 (2011)
Hofemeister, H., Ciotta, G., Fu, J., Seibert, P. M., Schulz, A., Maresca, M., Sarov, M., Anastassiadis, K. and Stewart, A. F.
Recombineering, transfection, Western, IP and ChIP methods for protein tagging via gene targeting or BAC transgenesis.
Kranz, A., Fu, J., Duerschke, K., Weidlich, S., Naumann, R., Stewart, A. F. and Anastassiadis, K.
An improved Flp deleter mouse in C57Bl/6 based on Flpo recombinase.
Maresca, M., Erler, A., Fu, J., Friedrich, A., Zhang, Y. and Stewart, A. F.
Single-stranded heteroduplex intermediates in lambda Red homologous recombination.
BMC Mol Biol 11: 54 (2010)
Glaser, S., Lubitz, S., Loveland, K. L., Ohbo, K., Robb, L., Schwenk, F., Seibler, J., Roellig, D., Kranz, A., Anastassiadis, K. and Stewart, A. F.
The histone 3 lysine 4 methyltransferase, Mll2, is only required briefly in development and spermatogenesis.
Epigenetics Chromatin 2: 5 (2009)