Now commonly accepted as the major risk factor for a wide range of chronic diseases, aging is accompanied by correlative and/or causative changes in the genome and epigenome1. While genome sequencing and whole genome monitoring of epigenetic changes are possible, these approaches are not viable in vivo or in live cells in culture. Accurate tools in live single cells to track chromosome dynamics in three dimensions (3D) could dramatically enhance fundamental knowledge of the aging process. Here, Ren et al.2 develop a promising new technique to monitor the genome in live cells and exploit it to understand aging-related changes at locations previously implicated in modulation of lifespan.

Fluorescent in situ hybridization (FISH), fluorescent Lac or Tet operator and repressor systems, engineered zinc finger proteins and the CRISPR/Cas9 system have each been used to visualize genome organization3,4,5,6. However, each of these are hampered by restrictive labeling to certain sequences, low signal-to-noise ratio, poor transfection efficiency, and/or formation of non-specific protein aggregates. These factors also limit the utility of current editing techniques to visualize changes in genome topology.

Transcriptional activator-like effectors (TALEs) are proteins secreted by Xanthomonas that have 33-35 amino acid repeats, each of which specifically recognizes and binds to a DNA base7. They function as transcriptional activators in plant species that are targets of infection by the bacteria. Importantly for this study, TALEs can be engineered to bind to specific DNA sequences and are functionally validated in mammalian cells7,8. Compared to CRISPR/Cas9, TALEs overcome technical difficulties, such as low transfection efficiency due to the high molecular weight of Cas9 and low signal-to-noise ratio. Fluorescent TALEs have recently been used for live imaging of centromeres and telomeres to visualize chromatin dynamics in vitro and in vivo9,10. However, TALEs tend to aggregate, resulting in their inaccurate localization. They are thus not optimal as a live-cell imaging tool to visualize human telomeric and centromeric DNAs, or other sequences in the genome.

Ren and colleagues2 have now taken a creative approach to improve upon conventional fluorescent TALEs by fusing the proteins to thioredoxin. Thioredoxin catalyzes steps in oxidative protein folding, facilitating the reduction of disulfide linkages in other proteins11. Thioredoxin is often fused to other proteins during recombinant expression as a means to assist their protein folding and prevent aggregation, which it can achieve independently of its latter activity in some cases. It is this redox activity that may be most beneficial in the case of TALEs. By fusing fluorescent TALEs to thioredoxin (TTALEs), Ren et al.2 found a dramatic improvement of signal-to-noise ratio in imaging studies.

TTALEs were assessed in a variety of contexts, including not only human cancer cell lines, but also human embryonic stem cells, iPSCs, differentiated cells, and mouse cells both in vitro and in vivo. In all cases, TTALE-based imaging was found to be comparable to 3D-FISH and better than the CRISPR/Cas9 system in imaging quality and transfection efficiency.

Theoretically, TTALEs can be used to track any region of the genome, but Ren et al. focused on three repeated regions all linked to aging in this initial study: telomeres, centromeres and ribosomal DNA (rDNA), as well as a specific gene locus, MUC4. Their success with this latter locus emphasizes the potential utility of the TTALE system for a wide range of experimental questions.

Several genomic loci have been linked to aging, including telomeres, centromeres and rDNA. Consistent with numerous reports of telomere shortening with aging, Ren et al. found that telomere TTALE signals were reduced in mesenchymal stem cells (MSCs) from three human cellular models of aging. Using one of these models, MSCs derived from Werner helicase (WRN)-deficient embryonic stem cells, the telomere TTALE signals were found to co-localize with γ-H2AX foci marking DNA damage, indicative of telomere-associated genome stress. Finally, telomere attrition was tracked successfully in multiple tissues from telomerase-deficient mice, showing that the TTALE system can be used successfully in vivo. WRN-deficient MSCs also show indications of age-related changes at centromeres, with more dispersed TTALE signals correlated with aberrant transcriptional activation.

Whereas TTALE signals at telomeres and centromeres generally corroborate prior findings, the studies examining rDNA repeats and their residence, the nucleolar organizer region (NOR), break new ground, in part because this region of the genome had previously not been amenable to live-cell imaging. Interestingly in a variety of contexts, rDNA repeat signals diminished, including models of aged MSCs and peripheral blood cells isolated from aging humans. These findings indicate a high rate of genome instability at the rDNA with aging, and may mirror that reported in replicatively aging yeast cells12. Further study needs to be conducted in both yeast and mammals, but these initial findings open the possibility that the rDNA repeats may be a common source of genome instability and a driver of aging across diverse eukaryotes.

These findings emphasize the utility of TTALEs to understand genomic events with aging, but the possibilities extend far beyond. For instance, TTALEs may provide a means to study pathologies with single nucleotide polymorphisms, such as autism-related disorders. Moreover, since TTALEs label gene loci in spite of dynamic chromatin changes during mitosis, they could also be invaluable in examining chromosomal and cell cycle aberrations in cancer.

TTALEs promise to provide a robust platform for imaging chromatin dynamics under physiological conditions, as well as a potential tool for genome editing in therapeutic approaches to various pathologies. While it remains to be determined whether TTALEs influence chromatin dynamics in real time or alter transcription states of target loci, the future seems bright for these new players in genome imaging.