Live visualization of chromatin dynamics with fluorescent TALEs

Abstract

The spatiotemporal organization of genomes in the nucleus is an emerging key player to regulate genome function. Live imaging of nuclear organization dynamics would be a breakthrough toward uncovering the functional relevance and mechanisms regulating genome architecture. Here, we used transcription activator-like effector (TALE) technology to visualize endogenous repetitive genomic sequences. We established TALE-mediated genome visualization (TGV) to label genomic sequences and follow nuclear positioning and chromatin dynamics in cultured mouse cells and in the living organism. TGV is highly specific, thus allowing differential labeling of parental chromosomes by distinguishing between single-nucleotide polymorphisms (SNPs). Our findings provide a framework to address the function of genome architecture through visualization of nuclear dynamics in vivo.

Visualization of repetitive DNA sequences in human chromosomes with transcription activator-like effectors

“…For live imaging, cells were grown on Lab-Tek two-well coverglasses in Hepes-buffered DMEM containing 10% (vol/vol) FBS, penicillin (100 units/mL), and streptomycin (100 μg/mL) and then overlaid with mineral oil. A total of 50 ng of TALEColor plasmids were transfected using Lipofectamine 2000 (Life Technologies) and the cells were incubated for another 24 h. The microscope stage incubation chamber was maintained at 37°C as described previously (29). Phase-contrast and fluorescence microscopy were performed with a Leica DM-IRB inverted microscope equipped with a mercury arc lamp, a 10-position filter wheel (Sutter Instrument), CFP/YFP/HcRed filter set, GFP/DsRed filter set (Semrock), a CCD camera (Photometrics), and MetaMorph acquisition software (Molecular Devices).…”

Nanoscale histone localization in live cells reveals reduced chromatin mobility in response to DNA damage

“…Particle-tracking toolkits are well suited to measure the dynamics of macromolecules with slow diffusion in a confined medium such as chromatin (Marshall et al, 1997;Heun et al, 2001;Vazquez et al, 2001;Neumann et al, 2012;Miyanari et al, 2013). With single-particle tracking (SPT) approaches, time-lapse images are registered to compensate for cellular movements, often using the center of the nucleus, the nuclear envelope or nuclear bodies as offsets.…”

A CRISPR view of development

“…By attaching a fluorescent protein to dCas9, it is possible to follow the position of repetitive telomeric sequences, satellite repeats, and individual genes within nuclei of fixed and living cells Anton et al 2014). This method is analogous to the zinc finger-and TALE-based approaches in which a fluorescent protein is joined to a programmable DNA-binding domain (Lindhout et al 2007;Miyanari et al 2013;Thanisch et al 2014). By following fluorescently tagged dCas9 (or TALE or zinc finger) in living cells, it is possible to explore how genomic architecture changes as cells divide and differentiate.…”