Chromatin perturbances

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 (Fig. 1). 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.

 

Figure 1. Blue Light-Induced Chromatin Recruitment (BLinCR). Transcription activation kinetics for the VP16 activator domain at the reporter array are shown in dependence of histone hyperacetylation.

 

The introduction of fluorescent labels or targeting of effector domains can also established by using the catalytically-dead Cas9 (dCas9) together with appropriate guide RNAs. We utilize this system to reveal structure-function relationships between the organization of chromatin domains and their activity, e.g., pericentric heteochromatin or telomores and their transcriptional activity (Fig. 2).

Figure 2. Live cell microscopic imaging of a fluorescently tagged dCas9 protein targeted to the telomeric repeats in human osteosarcoma cells. 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.

Key references

Trojanowski J, Frank L, Rademacher A, Mücke N, Grigaitis P, Rippe K (2022) Transcription activation is enhanced by multivalent interactions independent of phase separation. Mol Cell 82, 1878-1893. doi: 10.1016/j.molcel.2022.04.017 | Abstract | Reprint | Article metrics

Frank L, Weinmann R, Erdel F, Trojanowski J, Rippe K (2021) Transcriptional activation of heterochromatin by recruitment of dCas9 activators. Meth Mol Biol 2351, 307-320. doi: 10.1007/978-1-0716-1597-3_17 | Abstract | Reprint | Article metrics

Rademacher A, Erdel F, Trojanowski J, Schumacher S & Rippe K (2017). Real-time observation of light-controlled transcription in living cells. J Cell Sci 130, 4213-4224. doi: 10.1242/jcs.205534 | Abstract | Reprint (12.9 MB) | JCS First person | Article metrics