Visualization of aging-associated chromatin alterations with an engineered TALE system

Abstract

Visualization of specific genomic loci in live cells is a prerequisite for the investigation of dynamic changes in chromatin architecture during diverse biological processes, such as cellular aging. However, current precision genomic imaging methods are hampered by the lack of fluorescent probes with high specificity and signal-to-noise contrast. We find that conventional transcription activator-like effectors (TALEs) tend to form protein aggregates, thereby compromising their performance in imaging applications. Through screening, we found that fusing thioredoxin with TALEs prevented aggregate formation, unlocking the full power of TALE-based genomic imaging. Using thioredoxin-fused TALEs (TTALEs), we achieved high-quality imaging at various genomic loci and observed aging-associated (epi) genomic alterations at telomeres and centromeres in human and mouse premature aging models. Importantly, we identified attrition of ribosomal DNA repeats as a molecular marker for human aging. Our study establishes a simple and robust imaging method for precisely monitoring chromatin dynamics in vitro and in vivo.

The Nucleolus: In Genome Maintenance and Repair

“…Cells derived from Bloom syndrome (mutated BLM) or Ataxia Telangiectasia (mutated ATM) patients show high variability of the rDNA copy number due to mitotic recombination in relation to their wild type counterparts [ 137 , 138 ]. Moreover, recent evidence suggests reduction of ribosomal DNA repeats as a trait of human aging [ 139 , 140 ]. Age-related neurological disorders, such as Hodgkin’s disease [ 141 ] and Parkinson’s disease [ 142 ], show deregulated rDNA transcription and nucleolar dysfunction.…”

The past and presence of gene targeting: from chemicals and DNA via proteins to RNA

“…Indeed, TALEs seemed to surpass the use of ZFPs because of their straightforward one-to-one binding approach. However, TALEs are quite big and the high number of repeats can cause assembly problems and aggregation [65], although the latter can be prevented by the fusion of thioredoxin [66].…”

Genome im Fokus: Entwicklung und Anwendungen von CRISPR‐Cas9‐Bildgebungstechnologien

“…Spätere Experimente zeigten, dass Fusionen von ZFs und TALEs mit GFP so programmiert werden konnten, dass sie repetitive DNA‐Elemente wie z. B. Telomere und Centromere abbilden . Unglücklicherweise erwies sich die Bildgebung von nicht‐repetitiven genomischen Regionen mit ZFs und TALEs als weniger handhabbar.…”