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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.


Related in: MedlinePlus

TTALE-based imaging indicating physical attrition of NOR-rDNAs in senescent WS-MSCs. (A) SIM images showing mCherry-TTALErDNA-labeled NOR-rDNA in WS-MSCs and WT-MSCs. NLS-EGFP was co-expressed to monitor transfection efficiency. Dashed lines indicate the nuclear boundary. Scale bars, 5 μm. (B) Intensity profiles of TTALErDNA signals across the solid lines (10 μm in length) in A. (C) Histogram showing fluorescence intensity of mCherry-TTALErDNA normalized by that of NLS-GFP in A. n = 50 nuclei; ***P < 0.001. (D) Quantitative FISH (FACS) analysis of NOR-rDNA in WS-MSCs and WT-MSCs. Left panel: primary result of FACS. Right panel: histogram showing lower average intensity of NOR-rDNA FISH signal in WS-MSCs compared with WT-MSCs. Data are presented as mean ± SEM; n = 3; ***P < 0.001. (E) Quantitative PCR analysis showing diminished rDNA copy numbers in WS-MSCs relative to WT-MSCs. Data are presented as mean ± SEM; n = 3; **P < 0.01. (F) qPCR analysis of of telomere length (left) and NOR-rDNA copy number (right) in the genomic DNA of peripheral blood samples collected from young and old donors. n = 8 (young donors) or 9 (old donors); ***P < 0.001; *P < 0.05.
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fig7: TTALE-based imaging indicating physical attrition of NOR-rDNAs in senescent WS-MSCs. (A) SIM images showing mCherry-TTALErDNA-labeled NOR-rDNA in WS-MSCs and WT-MSCs. NLS-EGFP was co-expressed to monitor transfection efficiency. Dashed lines indicate the nuclear boundary. Scale bars, 5 μm. (B) Intensity profiles of TTALErDNA signals across the solid lines (10 μm in length) in A. (C) Histogram showing fluorescence intensity of mCherry-TTALErDNA normalized by that of NLS-GFP in A. n = 50 nuclei; ***P < 0.001. (D) Quantitative FISH (FACS) analysis of NOR-rDNA in WS-MSCs and WT-MSCs. Left panel: primary result of FACS. Right panel: histogram showing lower average intensity of NOR-rDNA FISH signal in WS-MSCs compared with WT-MSCs. Data are presented as mean ± SEM; n = 3; ***P < 0.001. (E) Quantitative PCR analysis showing diminished rDNA copy numbers in WS-MSCs relative to WT-MSCs. Data are presented as mean ± SEM; n = 3; **P < 0.01. (F) qPCR analysis of of telomere length (left) and NOR-rDNA copy number (right) in the genomic DNA of peripheral blood samples collected from young and old donors. n = 8 (young donors) or 9 (old donors); ***P < 0.001; *P < 0.05.

Mentions: We also investigated potential changes of NOR-rDNA loci in aged human stem cells. The fluorescence intensity of mCherry-TTALErDNA was significantly lower in the nuclei of senescent WS-MSCs compared with WT-MSCs (Figure 7A-7C); a co-transfected nuclear-targeted GFP (NLS-GFP) was used as an internal control. Furthermore, flow FISH and PCR indicated that the copy number of NOR-rDNA repeats was significantly decreased in senescent WS-MSCs (Figure 7D and 7E), whereas no significant decrease was observed when the copy number of GAPDH locus was analyzed (Supplementary information, Figure S9A). Similar reductions of NOR-rDNA copy number and mCherry-TTALErDNA fluorescence intensity were also observed in HGPS-MSCs undergoing accelerated senescence and in replicative senescent MSCs (Supplementary information, Figure S9B-S9G). To identify whether the NOR-rDNA attrition observed during premature human MSC senescence can be extended to human physiological aging, peripheral bloods from young (6-10 years old) and old (69-72 years old) donors were obtained to detect the NOR-rDNA copy number, as well as telomere length. As expected, we observed significant shortening of telomeres in blood samples from old individuals (Figure 7F, left panel). Importantly, the copy numbers of NOR-rDNA in peripheral blood of old donors were also diminished relative to those of young donors (Figure 7F, right panel). Together, using TTALE-based imaging, we have not only validated telomere attrition and centromere disorganization in senescent human cells7,40,43, but also provided the strong evidence that human aging is associated with attrition of NOR-rDNA repeats.


Visualization of aging-associated chromatin alterations with an engineered TALE system
TTALE-based imaging indicating physical attrition of NOR-rDNAs in senescent WS-MSCs. (A) SIM images showing mCherry-TTALErDNA-labeled NOR-rDNA in WS-MSCs and WT-MSCs. NLS-EGFP was co-expressed to monitor transfection efficiency. Dashed lines indicate the nuclear boundary. Scale bars, 5 μm. (B) Intensity profiles of TTALErDNA signals across the solid lines (10 μm in length) in A. (C) Histogram showing fluorescence intensity of mCherry-TTALErDNA normalized by that of NLS-GFP in A. n = 50 nuclei; ***P < 0.001. (D) Quantitative FISH (FACS) analysis of NOR-rDNA in WS-MSCs and WT-MSCs. Left panel: primary result of FACS. Right panel: histogram showing lower average intensity of NOR-rDNA FISH signal in WS-MSCs compared with WT-MSCs. Data are presented as mean ± SEM; n = 3; ***P < 0.001. (E) Quantitative PCR analysis showing diminished rDNA copy numbers in WS-MSCs relative to WT-MSCs. Data are presented as mean ± SEM; n = 3; **P < 0.01. (F) qPCR analysis of of telomere length (left) and NOR-rDNA copy number (right) in the genomic DNA of peripheral blood samples collected from young and old donors. n = 8 (young donors) or 9 (old donors); ***P < 0.001; *P < 0.05.
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fig7: TTALE-based imaging indicating physical attrition of NOR-rDNAs in senescent WS-MSCs. (A) SIM images showing mCherry-TTALErDNA-labeled NOR-rDNA in WS-MSCs and WT-MSCs. NLS-EGFP was co-expressed to monitor transfection efficiency. Dashed lines indicate the nuclear boundary. Scale bars, 5 μm. (B) Intensity profiles of TTALErDNA signals across the solid lines (10 μm in length) in A. (C) Histogram showing fluorescence intensity of mCherry-TTALErDNA normalized by that of NLS-GFP in A. n = 50 nuclei; ***P < 0.001. (D) Quantitative FISH (FACS) analysis of NOR-rDNA in WS-MSCs and WT-MSCs. Left panel: primary result of FACS. Right panel: histogram showing lower average intensity of NOR-rDNA FISH signal in WS-MSCs compared with WT-MSCs. Data are presented as mean ± SEM; n = 3; ***P < 0.001. (E) Quantitative PCR analysis showing diminished rDNA copy numbers in WS-MSCs relative to WT-MSCs. Data are presented as mean ± SEM; n = 3; **P < 0.01. (F) qPCR analysis of of telomere length (left) and NOR-rDNA copy number (right) in the genomic DNA of peripheral blood samples collected from young and old donors. n = 8 (young donors) or 9 (old donors); ***P < 0.001; *P < 0.05.
Mentions: We also investigated potential changes of NOR-rDNA loci in aged human stem cells. The fluorescence intensity of mCherry-TTALErDNA was significantly lower in the nuclei of senescent WS-MSCs compared with WT-MSCs (Figure 7A-7C); a co-transfected nuclear-targeted GFP (NLS-GFP) was used as an internal control. Furthermore, flow FISH and PCR indicated that the copy number of NOR-rDNA repeats was significantly decreased in senescent WS-MSCs (Figure 7D and 7E), whereas no significant decrease was observed when the copy number of GAPDH locus was analyzed (Supplementary information, Figure S9A). Similar reductions of NOR-rDNA copy number and mCherry-TTALErDNA fluorescence intensity were also observed in HGPS-MSCs undergoing accelerated senescence and in replicative senescent MSCs (Supplementary information, Figure S9B-S9G). To identify whether the NOR-rDNA attrition observed during premature human MSC senescence can be extended to human physiological aging, peripheral bloods from young (6-10 years old) and old (69-72 years old) donors were obtained to detect the NOR-rDNA copy number, as well as telomere length. As expected, we observed significant shortening of telomeres in blood samples from old individuals (Figure 7F, left panel). Importantly, the copy numbers of NOR-rDNA in peripheral blood of old donors were also diminished relative to those of young donors (Figure 7F, right panel). Together, using TTALE-based imaging, we have not only validated telomere attrition and centromere disorganization in senescent human cells7,40,43, but also provided the strong evidence that human aging is associated with attrition of NOR-rDNA repeats.

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