Dataset Information

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.

SUBMITTER: Ren R

PROVIDER: S-EPMC5385610 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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

Ren Ruotong R Deng Liping L Xue Yanhong Y Suzuki Keiichiro K Zhang Weiqi W Yu Yang Y Wu Jun J Sun Liang L Gong Xiaojun X Luan Huiqin H Yang Fan F Ju Zhenyu Z Ren Xiaoqing X Wang Si S Tang Hong H Geng Lingling L Zhang Weizhou W Li Jian J Qiao Jie J Xu Tao T Qu Jing J Liu Guang-Hui GH

Cell research 20170131 4

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 ima ...[more]

PMID: 28139645

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Project description:Alzheimer's disease (AD) is characterized by a progressive cognitive decline and believed to be driven by the self-aggregation of amyloid-? (A?) peptide into oligomers and fibrils that accumulate as senile plaques. It is widely accepted that microglia-mediated inflammation is a significant contributor to disease pathogenesis; however, different microglia phenotypes were identified along AD progression and excessive A? production was shown to dysregulate cell function. As so, the contribution of microglia to AD pathogenesis remains to be elucidated. In this study, we wondered if isolated microglia cultured for 16 days in vitro (DIV) would react differentially from the 2 DIV cells upon treatment with 1000 nM A?1-42 for 24 h. No changes in cell viability were observed and morphometric alterations associated to microglia activation, such as volume increase and process shortening, were obvious in 2 DIV microglia, but less evident in 16 DIV cells. These cells showed lower phagocytic, migration and autophagic properties after A? treatment than the 2 DIV cultured microglia. Reduced phagocytosis may derive from increased CD33 expression, reduced triggering receptor expressed on myeloid cells 2 (TREM2) and milk fat globule-EGF factor 8 protein (MFG-E8) levels, which were mainly observed in 16 DIV cells. Activation of inflammatory mediators, such as high mobility group box 1 (HMGB1) and pro-inflammatory cytokines, as well as increased expression of Toll-like receptor 2 (TLR2), TLR4 and fractalkine/CX3C chemokine receptor 1 (CX3CR1) cell surface receptors were prominent in 2 DIV microglia, while elevation of matrix metalloproteinase 9 (MMP9) was marked in 16 DIV cells. Increased senescence-associated ?-galactosidase (SA-?-gal) and upregulated miR-146a expression that were observed in 16 DIV cells showed to increase by A? in 2 DIV microglia. Additionally, A? downregulated miR-155 and miR-124, and reduced the CD11b+ subpopulation in 2 DIV microglia, while increased the number of CD86+ cells in 16 DIV microglia. Simultaneous M1 and M2 markers were found after A? treatment, but at lower expression in the in vitro aged microglia. Data show key-aging associated responses by microglia when incubated with A?, with a loss of reactivity from the 2 DIV to the 16 DIV cells, which course with a reduced phagocytosis, migration and lower expression of inflammatory miRNAs. These findings help to improve our understanding on the heterogeneous responses that microglia can have along the progression of AD disease and imply that therapeutic approaches may differ from early to late stages.

Dataset Information

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

Publications

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

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