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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Precise labeling of telomeres with thioredoxin-fused TALEs (TTALEs). (A) Schematic illustration of TALEs fused with various solubility-enhancing peptides (X) to label telomeres. (B) Co-localization analysis of telomeric FISH (red) and Flag-TALEtelo (green) fused with the indicated peptides in HeLa cells. Engineered TALEtelo was visualized by immunostaining with anti-Flag antibody. Representative images using various fusion partners show that precise co-localization with telomeric FISH signals was obtained only using the thioredoxin-fused TALEtelo (TTALEtelo). Dashed lines indicate the nuclear boundary. Scale bars, 5 μm. (C) Histograms showing numbers of telomeric FISH- and peptide-fused TALEtelo(19 bp)-positive dots in nuclei of HeLa cells. n = 50 nuclei per group.
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fig2: Precise labeling of telomeres with thioredoxin-fused TALEs (TTALEs). (A) Schematic illustration of TALEs fused with various solubility-enhancing peptides (X) to label telomeres. (B) Co-localization analysis of telomeric FISH (red) and Flag-TALEtelo (green) fused with the indicated peptides in HeLa cells. Engineered TALEtelo was visualized by immunostaining with anti-Flag antibody. Representative images using various fusion partners show that precise co-localization with telomeric FISH signals was obtained only using the thioredoxin-fused TALEtelo (TTALEtelo). Dashed lines indicate the nuclear boundary. Scale bars, 5 μm. (C) Histograms showing numbers of telomeric FISH- and peptide-fused TALEtelo(19 bp)-positive dots in nuclei of HeLa cells. n = 50 nuclei per group.

Mentions: TALEs are composed of multiple highly repetitive modules, a feature that likely predisposes them to self-assemble into bulky protein aggregates especially when being simultaneously tethered to multiple copies of genomic repetitive DNA sequences, preventing their binding to cognate DNA sequences. We thus screened a panel of peptides known to facilitate expression of insoluble proteins in Escherichia coli30, and fused them with TALEs (Figure 2A). We used the following fusion partners for initial screening: ubiquitin (UB), small ubiquitin-related modifier (SUMO), glutathione S-transferase (GST), maltose-binding protein (MBP), and thioredoxin (TRX) (Figure 2B and 2C). Among all peptides tested in telomeres, TRX was the best candidate, as the use of TRX-fused TALEtelo (TTALEtelo) led to nearly perfect co-localization of telomeric FISH and TTALEtelo signals (Figure 2B and 2C). Likewise, the TRX-fused TALEcentro (TTALEcentro) also yielded specific signals precisely marking centromeres and overlapping with centromeric FISH signals (Figure 3A-3C) as well as the centromeric protein CENPA (Supplementary information, Figure S2A).


Visualization of aging-associated chromatin alterations with an engineered TALE system
Precise labeling of telomeres with thioredoxin-fused TALEs (TTALEs). (A) Schematic illustration of TALEs fused with various solubility-enhancing peptides (X) to label telomeres. (B) Co-localization analysis of telomeric FISH (red) and Flag-TALEtelo (green) fused with the indicated peptides in HeLa cells. Engineered TALEtelo was visualized by immunostaining with anti-Flag antibody. Representative images using various fusion partners show that precise co-localization with telomeric FISH signals was obtained only using the thioredoxin-fused TALEtelo (TTALEtelo). Dashed lines indicate the nuclear boundary. Scale bars, 5 μm. (C) Histograms showing numbers of telomeric FISH- and peptide-fused TALEtelo(19 bp)-positive dots in nuclei of HeLa cells. n = 50 nuclei per group.
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Related In: Results  -  Collection

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fig2: Precise labeling of telomeres with thioredoxin-fused TALEs (TTALEs). (A) Schematic illustration of TALEs fused with various solubility-enhancing peptides (X) to label telomeres. (B) Co-localization analysis of telomeric FISH (red) and Flag-TALEtelo (green) fused with the indicated peptides in HeLa cells. Engineered TALEtelo was visualized by immunostaining with anti-Flag antibody. Representative images using various fusion partners show that precise co-localization with telomeric FISH signals was obtained only using the thioredoxin-fused TALEtelo (TTALEtelo). Dashed lines indicate the nuclear boundary. Scale bars, 5 μm. (C) Histograms showing numbers of telomeric FISH- and peptide-fused TALEtelo(19 bp)-positive dots in nuclei of HeLa cells. n = 50 nuclei per group.
Mentions: TALEs are composed of multiple highly repetitive modules, a feature that likely predisposes them to self-assemble into bulky protein aggregates especially when being simultaneously tethered to multiple copies of genomic repetitive DNA sequences, preventing their binding to cognate DNA sequences. We thus screened a panel of peptides known to facilitate expression of insoluble proteins in Escherichia coli30, and fused them with TALEs (Figure 2A). We used the following fusion partners for initial screening: ubiquitin (UB), small ubiquitin-related modifier (SUMO), glutathione S-transferase (GST), maltose-binding protein (MBP), and thioredoxin (TRX) (Figure 2B and 2C). Among all peptides tested in telomeres, TRX was the best candidate, as the use of TRX-fused TALEtelo (TTALEtelo) led to nearly perfect co-localization of telomeric FISH and TTALEtelo signals (Figure 2B and 2C). Likewise, the TRX-fused TALEcentro (TTALEcentro) also yielded specific signals precisely marking centromeres and overlapping with centromeric FISH signals (Figure 3A-3C) as well as the centromeric protein CENPA (Supplementary information, Figure S2A).

View Article: PubMed Central - PubMed

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.

No MeSH data available.