Limits...
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.


Related in: MedlinePlus

Conventional TALEs were insufficient to precisely track telomeres in four human tumor cell lines tested. (A-B) Co-localization analysis of telomeric FISH (red) and EGFP-TALEtelo (green) signals in the indicated human cell lines (U2OS, HeLa, HepG2, and MCF7). TALEtelo was designed using a 19-bp (A) or a 15-bp (B) telomeric repetitive sequence. Dashed lines indicate the nuclear boundaries; arrowheads indicate overlapping signals. Scale bars, 5 μm. (C) Histograms showing numbers of EGFP-TALEtelo(15 bp)-, EGFP-TALEtelo(19 bp)-, and telomeric FISH-positive nuclear dots in each cell line. n = 50 nuclei per cell line.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5385610&req=5

fig1: Conventional TALEs were insufficient to precisely track telomeres in four human tumor cell lines tested. (A-B) Co-localization analysis of telomeric FISH (red) and EGFP-TALEtelo (green) signals in the indicated human cell lines (U2OS, HeLa, HepG2, and MCF7). TALEtelo was designed using a 19-bp (A) or a 15-bp (B) telomeric repetitive sequence. Dashed lines indicate the nuclear boundaries; arrowheads indicate overlapping signals. Scale bars, 5 μm. (C) Histograms showing numbers of EGFP-TALEtelo(15 bp)-, EGFP-TALEtelo(19 bp)-, and telomeric FISH-positive nuclear dots in each cell line. n = 50 nuclei per cell line.

Mentions: To adapt TALEs for visualizing specific genomic locus, we generated an EGFP-tagged TALE construct targeting a 19-bp telomeric DNA repeat (5′-TAACCCTAACCCTAACCCT-3′ referred to as TALEtelo). This construct was transiently introduced into four human tumor cell lines (U2OS, HeLa, HepG2, and MCF7). To examine the efficiency of TALE-based fluorescence imaging of telomeric sequences, 3D-FISH with a telomeric peptide nucleic acid (PNA) probe was used to visualize the 3D distribution of telomeres inside the nucleus7. Approximately 20% of FISH-positive foci overlapped with EGFP-TALEtelo in U2OS cells. In other cell types examined, EGFP-TALEtelo appeared mostly as large bright foci (referred to hereafter as “aggregates”) distinct from telomeric foci, with only a small percentage (< 5%) of foci co-localized with the FISH signals (Figure 1A and 1C). In addition, an EGFP-tagged TALE against a 19-bp centromeric satellite DNA repeat (5′-TCCATTCCATTCCATTCCA-3′), referred to as TALEcentro, also failed to faithfully identify centromeres in the same cell types, as determined by 3D-FISH with a centromeric PNA probe (Supplementary information, Figure S1A and S1B). Similar results were also observed using previously reported EGFP-TALEs against a 15-bp telomeric repeat (5′-TAACCCTAACCCTAA-3′ Figure 1B and 1C) or a 20-bp centromeric repeat (5′-TAGACAGAAGCATTCTCAGA-3′ data not shown)20. We further verified that these aggregates were not localized in nucleoli (Supplementary information, Figure S1C-S1D). These data indicate that conventional TALEs are inefficient for tracking human telomeric and centromeric DNAs.


Visualization of aging-associated chromatin alterations with an engineered TALE system
Conventional TALEs were insufficient to precisely track telomeres in four human tumor cell lines tested. (A-B) Co-localization analysis of telomeric FISH (red) and EGFP-TALEtelo (green) signals in the indicated human cell lines (U2OS, HeLa, HepG2, and MCF7). TALEtelo was designed using a 19-bp (A) or a 15-bp (B) telomeric repetitive sequence. Dashed lines indicate the nuclear boundaries; arrowheads indicate overlapping signals. Scale bars, 5 μm. (C) Histograms showing numbers of EGFP-TALEtelo(15 bp)-, EGFP-TALEtelo(19 bp)-, and telomeric FISH-positive nuclear dots in each cell line. n = 50 nuclei per cell line.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5385610&req=5

fig1: Conventional TALEs were insufficient to precisely track telomeres in four human tumor cell lines tested. (A-B) Co-localization analysis of telomeric FISH (red) and EGFP-TALEtelo (green) signals in the indicated human cell lines (U2OS, HeLa, HepG2, and MCF7). TALEtelo was designed using a 19-bp (A) or a 15-bp (B) telomeric repetitive sequence. Dashed lines indicate the nuclear boundaries; arrowheads indicate overlapping signals. Scale bars, 5 μm. (C) Histograms showing numbers of EGFP-TALEtelo(15 bp)-, EGFP-TALEtelo(19 bp)-, and telomeric FISH-positive nuclear dots in each cell line. n = 50 nuclei per cell line.
Mentions: To adapt TALEs for visualizing specific genomic locus, we generated an EGFP-tagged TALE construct targeting a 19-bp telomeric DNA repeat (5′-TAACCCTAACCCTAACCCT-3′ referred to as TALEtelo). This construct was transiently introduced into four human tumor cell lines (U2OS, HeLa, HepG2, and MCF7). To examine the efficiency of TALE-based fluorescence imaging of telomeric sequences, 3D-FISH with a telomeric peptide nucleic acid (PNA) probe was used to visualize the 3D distribution of telomeres inside the nucleus7. Approximately 20% of FISH-positive foci overlapped with EGFP-TALEtelo in U2OS cells. In other cell types examined, EGFP-TALEtelo appeared mostly as large bright foci (referred to hereafter as “aggregates”) distinct from telomeric foci, with only a small percentage (< 5%) of foci co-localized with the FISH signals (Figure 1A and 1C). In addition, an EGFP-tagged TALE against a 19-bp centromeric satellite DNA repeat (5′-TCCATTCCATTCCATTCCA-3′), referred to as TALEcentro, also failed to faithfully identify centromeres in the same cell types, as determined by 3D-FISH with a centromeric PNA probe (Supplementary information, Figure S1A and S1B). Similar results were also observed using previously reported EGFP-TALEs against a 15-bp telomeric repeat (5′-TAACCCTAACCCTAA-3′ Figure 1B and 1C) or a 20-bp centromeric repeat (5′-TAGACAGAAGCATTCTCAGA-3′ data not shown)20. We further verified that these aggregates were not localized in nucleoli (Supplementary information, Figure S1C-S1D). These data indicate that conventional TALEs are inefficient for tracking human telomeric and centromeric DNAs.

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.


Related in: MedlinePlus