In order to test mechanistic models by which chromatin networks and epigenetic signaling operate, we use a number of methods to modify the chromatin state. These are referred to here as "chromatin editing" and involve the targeting of protein or RNA to specific loci as well as the microinjection of chromatin modifiers.
- Lac Operator (lacO)/ Lac Repressor (LacI) System
- CRISPR/Cas9-based epigenetic editing
Optogenetics in the nucleus
We have develpoed and optogenetics approach to trace transcription activation in living cells in real-time (Rademacher et al. 2017). The BLinCR (Blue Light-Induced Chromatin Recruitment) approach is very versatile and works not only for tethering a transcription factor to chromatin. It can be used just as well for recruiting a protein of choice to the nuclear lamina, the telomeres, nucleoli, PML nuclear bodies etc. So check out our BLinCR toolbox with all constructs being available from Addgene for more exciting applications of the technique.
In order to visualize chromatin in living cells, several groups employ the lac operator (lacO)/lac repressor (LacI) system. Naturally, the lacO sequence is part of the E. coli lac operon regulating the bacterial lactose transport and metabolism. However, it can be amplified and stably integrated in multiple copies into eukaryotic genomes resulting in a chromosomal binding site array for LacI. Upon transfection of LacI fused to a fluorescence protein like the green fluorescence protein (GFP), the integration can be detected in a conventional light microscope (Figure). Using this technique a specific genomic locus can be observed in fixed as well as in living cells, thus allowing to address the dynamics of chromatin. In our lab, several clones with stable lacO integrations derived from the osteosarcoma cell line U2OS have been established. Most of the lacO arrays are integrated at telomeric regions, which enabled us to use these cells for studies on the dynamics of telomeres (Jegou et al. 2009, Chung et al. 2011)
|Left: Schematic representation of the lacO/LacI system for labeling chromatin in living cells. Arrays of the lacO repeats are integrated into the genome of the cell. The lac repressor LacI binds with high affinity to these arrays. Thus, transfection of LacI fused to a fluorescence protein like GFP visualizes these genomic loci. Right: Schematic representation of the fluorescence three-hybrid system. The GFP-binding protein (GBP) is bound to the chromosomal lacO array by its fusion to the LacI repressor. This construct can additionally be tagged with an RFP-domain. A co-transfected GFP fusion protein is recruited via the GBP-GFP interaction. Thereby induced accumulations of endogenous proteins can subsequently be detected by immunofluorescence. Modified from .|
The RNA-guided Cas9 nuclease from the bacterial CRISPR (Clustered Regularly Interspaced Short Palindomic Repeats) system can be repurposed as a vector for targeting effector molecules to the chromatin of endogenous gene loci. This is achieved by introducing mutations into the Cas9 protein that attenuate its nuclease activity. The resulting catalytically-dead Cas9 (dCas9) can be fused to effector proteins that can be designed to e.g. specifically modify chromatin or DNA. The flexibility of dCas9 complex design also enables site-specific recruitment of RNA molecules to chromatin and live cell microscopic imaging of single genomic loci. We utilize this system to dissect the causality between chromatin states (e.g. histone modification status, binding of RNA to chromatin) and regulation of gene expression in mammalian cells. This technology represents a powerful approach to differentiate between causes and consequences of epigenome de-regulation in human cancers.
Live cell microscopic imaging of a fluorescently tagged dCas9 protein targeted to the telomeric repeats in human osteosarcoma cells (Figure). The visualization of targeted dCas9-effectors enables real-time observation of nuclear organization changes, for example after specific histone modifications or RNA molecules have been deposited via dCas9 complexes.
Microinjection allows injecting small volumes of various molecules directly into specific parts of living cells via a glass capillary. We are using a computer assisted microinjection system as depicted below. It is easy to handle, highly accurate and fast. Using this system, we microinjected various enzymes directly into cell nuclei of mammalian cells and observed their respective effects on genome organization as depicted in the figure and described in Caudron-Herger et al 2011 and 2015.
People working in this research field
Caudron-Herger, M., Müller-Ott, K., Mallm, J.-P., Marth, C., Schmidt, U., Fejes-Tóth, K. & Rippe, K. (2011). Coding RNAs with a non-coding function: maintenance of open chromatin structure, Nucleus 2, 410-424. doi: 10.4161/nucl.2.5.17736 | Abstract | Reprint (4.4 MB)
Caudron-Herger M, Pankert T, Seiler J, Nemeth A, Voit R, Grummt I & Rippe K (2015). Alu element-containing RNAs maintain nucleolar structure and function. EMBO J 34, 2758-2771. doi: 10.15252/embj.201591458 | Abstract | Reprint (4.6 MB)
Chung I, Leonhardt H & Rippe K (2011). De novo assembly of a PML nuclear subcompartment occurs through multiple pathways and induces telomere elongation. J. Cell Sci. 124, 3603-3618. doi: 10.1242/jcs.084681 | Abstract | Reprint (9.4 MB)
Jegou, T., Chung, I., Heuvelmann, G., Wachsmuth, M., Görisch, S. M., Greulich-Bode, K., Boukamp, P., Lichter, P. & Rippe, K. (2009). Dynamics of telomeres and promyelocytic leukemia nuclear bodies in a telomerase negative human cell line. Mol Biol Cell 20, 2070-2082. Abstract | Reprint (3.3 MB pdf file)
Rademacher A, Erdel F, Trojanowski J, Schumacher S & Rippe K (2017). Real-time observation of light-controlled transcription in living cells. J Cell Sci published online 9 November 2017. doi: 10.1242/jcs.205534 | Abstract | Reprint (12.3 MB) | Article metrics