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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.


TTALE-mediated imaging of telomeres and centromeres in mitotic HeLa cells, hESCs, and the isogenic cell derivatives of hESCs. (A) Dynamic distribution of telomeres and centromeres at different stages of mitosis. Live HeLa cells co-expressing EGFP-TTALEcentro and mCherry-TTALEtelo were imaged at different mitotic phases. Scale bars, 5 μm. (B) Schematic diagram showing derivation of isogenic cell types from hESCs. hMSCs, hNSCs, and hVSMCs were differentiated from hESCs, and hNSCs were further differentiated into postmitotic neurons or reprogrammed into iPSCs. (C-D) Structured illumination microscopy (SIM) images showing co-localization of centromeric FISH (red) and Flag-TTALEcentro (green) (C) or telomeric FISH (red) and Flag-TTALEtelo (green) (D) signals in the indicated cell types. The number of each scatter plot represents the Pearson r value to show the linear correlation between FISH and TTALEtelo or TTALEcentro signals. Dashed lines indicate the nuclear boundary. Scale bars, 5 μm.
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fig4: TTALE-mediated imaging of telomeres and centromeres in mitotic HeLa cells, hESCs, and the isogenic cell derivatives of hESCs. (A) Dynamic distribution of telomeres and centromeres at different stages of mitosis. Live HeLa cells co-expressing EGFP-TTALEcentro and mCherry-TTALEtelo were imaged at different mitotic phases. Scale bars, 5 μm. (B) Schematic diagram showing derivation of isogenic cell types from hESCs. hMSCs, hNSCs, and hVSMCs were differentiated from hESCs, and hNSCs were further differentiated into postmitotic neurons or reprogrammed into iPSCs. (C-D) Structured illumination microscopy (SIM) images showing co-localization of centromeric FISH (red) and Flag-TTALEcentro (green) (C) or telomeric FISH (red) and Flag-TTALEtelo (green) (D) signals in the indicated cell types. The number of each scatter plot represents the Pearson r value to show the linear correlation between FISH and TTALEtelo or TTALEcentro signals. Dashed lines indicate the nuclear boundary. Scale bars, 5 μm.

Mentions: To visualize the dynamic distribution of telomeres and centromeres, we imaged HeLa cells at different mitotic phases using TTALEtelo and TTALEcentro probes. As in interphase cells, we observed nearly perfect overlap between TTALEtelo and telomeric FISH signals and between TTALEcentro and centromeric FISH signals during prophase, metaphase, anaphase, and telophase (Supplementary information, Figure S4A and S4B). In addition, telomeres and centromeres could be simultaneously visualized at all stages of mitosis by co-expression of mCherry-TTALEtelo and EGFP-TTALEcentro(Figure 4A and Supplementary information, Movie S1). These data indicate that binding of TTALEtelo and TTALEcentro probes to telomeric and centromeric DNAs, respectively, is stable and specific throughout mitosis, and further suggest that the expression of TTALEs has minimal effect on mitosis in HeLa cells.


Visualization of aging-associated chromatin alterations with an engineered TALE system
TTALE-mediated imaging of telomeres and centromeres in mitotic HeLa cells, hESCs, and the isogenic cell derivatives of hESCs. (A) Dynamic distribution of telomeres and centromeres at different stages of mitosis. Live HeLa cells co-expressing EGFP-TTALEcentro and mCherry-TTALEtelo were imaged at different mitotic phases. Scale bars, 5 μm. (B) Schematic diagram showing derivation of isogenic cell types from hESCs. hMSCs, hNSCs, and hVSMCs were differentiated from hESCs, and hNSCs were further differentiated into postmitotic neurons or reprogrammed into iPSCs. (C-D) Structured illumination microscopy (SIM) images showing co-localization of centromeric FISH (red) and Flag-TTALEcentro (green) (C) or telomeric FISH (red) and Flag-TTALEtelo (green) (D) signals in the indicated cell types. The number of each scatter plot represents the Pearson r value to show the linear correlation between FISH and TTALEtelo or TTALEcentro signals. Dashed lines indicate the nuclear boundary. Scale bars, 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: TTALE-mediated imaging of telomeres and centromeres in mitotic HeLa cells, hESCs, and the isogenic cell derivatives of hESCs. (A) Dynamic distribution of telomeres and centromeres at different stages of mitosis. Live HeLa cells co-expressing EGFP-TTALEcentro and mCherry-TTALEtelo were imaged at different mitotic phases. Scale bars, 5 μm. (B) Schematic diagram showing derivation of isogenic cell types from hESCs. hMSCs, hNSCs, and hVSMCs were differentiated from hESCs, and hNSCs were further differentiated into postmitotic neurons or reprogrammed into iPSCs. (C-D) Structured illumination microscopy (SIM) images showing co-localization of centromeric FISH (red) and Flag-TTALEcentro (green) (C) or telomeric FISH (red) and Flag-TTALEtelo (green) (D) signals in the indicated cell types. The number of each scatter plot represents the Pearson r value to show the linear correlation between FISH and TTALEtelo or TTALEcentro signals. Dashed lines indicate the nuclear boundary. Scale bars, 5 μm.
Mentions: To visualize the dynamic distribution of telomeres and centromeres, we imaged HeLa cells at different mitotic phases using TTALEtelo and TTALEcentro probes. As in interphase cells, we observed nearly perfect overlap between TTALEtelo and telomeric FISH signals and between TTALEcentro and centromeric FISH signals during prophase, metaphase, anaphase, and telophase (Supplementary information, Figure S4A and S4B). In addition, telomeres and centromeres could be simultaneously visualized at all stages of mitosis by co-expression of mCherry-TTALEtelo and EGFP-TTALEcentro(Figure 4A and Supplementary information, Movie S1). These data indicate that binding of TTALEtelo and TTALEcentro probes to telomeric and centromeric DNAs, respectively, is stable and specific throughout mitosis, and further suggest that the expression of TTALEs has minimal effect on mitosis in HeLa cells.

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.