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Visualization of Aging-Associated Chromatin Alterations with an Engineered TALE System

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Visualization of Aging-Associated Chromatin Alterations with an Engineered TALE System Cell Research (2017) 27:483-504. ORIGINAL ARTICLE www.nature.com/cr Visualization of aging-associated chromatin alterations with an engineered TALE system Ruotong Ren1, 2, *, Liping Deng1, 2, 3, *, Yanhong Xue1, 2, *, Keiichiro Suzuki4, *, Weiqi Zhang1, 2, Yang Yu5, Jun Wu4, Liang Sun6, Xiaojun Gong7, Huiqin Luan1, Fan Yang8, Zhenyu Ju9, Xiaoqing Ren1, Si Wang1, Hong Tang7, Lingling Geng1, Weizhou Zhang10, Jian Li6, Jie Qiao5, Tao Xu1, 2, Jing Qu2, 3, Guang-Hui Liu1, 2, 8, 11 1National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; 2University of Chinese Academy of Sciences, Beijing 100049, China; 3State Key Lab- oratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; 4Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA; 5De- partment of Gynecology and Obstetrics, Peking University Third Hospital, Beijing 100191, China; 6The MOH Key Laboratory of Geriatrics, Beijing Hospital, National Center of Gerontology, Beijing 100730, China; 7Department of Pediatrics, Beijing Shijitan Hospital Capital Medical University, Peking University Ninth School of Clinical Medicine, Beijing 100038, China; 8Key Laborato- ry of Regenerative Medicine of Ministry of Education, Institute of Aging and Regenerative Medicine, Jinan University, Guangzhou, Guangdong 510632, China; 9Institute of Aging Research, Hangzhou Normal University School of Medicine, Hangzhou, Zhejiang 311121, China; 10Department of Pathology, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA; 11Beijing Institute for Brain Disorders, Beijing 100069, China Visualization of specific genomic loci in live cells is a prerequisite for the investigation of dynamic changes in chro- matin 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 thioredox- in-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. Keywords: thioredoxin-fused TALE; aging; ribosomal DNA repeat; telomere; centromere Cell Research (2017) 27:483-504. doi:10.1038/cr.2017.18; published online 31 January 2017 Introduction varying size. Although we can obtain sequence informa- tion for any genomic locus with ease, we are still in the In the human nuclear genome, ~3.2 billion base pairs early stages of unraveling the organization of the human of DNA are tightly packed into 23 chromosome pairs of genome and the spatiotemporal relationships between genomic loci in three-dimensional (3D), which will con- sequently improve our understanding of genetic and epi- genetic regulation during cell differentiation, aging, and *These four authors contributed equally to this work. pathophysiological processes [1-7]. The importance of a b c Correspondence: Guang-Hui Liu , Jing Qu , Tao Xu maintaining a proper 3D genome topology is underscored aE-mail: [email protected] bE-mail: [email protected] by recent discoveries that aging and several severe hu- cE-mail: [email protected] man diseases can be attributed to genomic disorganiza- Received 11 October 2016; revised 6 December 2016; accepted 28 Decem- tion [7-9]. Therefore, exploring the spatial organization ber 2016; published online 31 January 2017 An engineered TALE system for live imaging 484 of human chromatin and its dynamic interactions with Here we report that conventional TALEs frequently protein and RNA regulators emerges as an important part form large aggregates in human cells, thereby compro- of understanding the cellular processes underlying aging mising their imaging efficiency in various cell types and human diseases [3, 10, 11]. examined. To overcome this barrier, we developed a Despite recent advances, there is a lack of imaging novel thioredoxin-fused TALE (TTALE) imaging sys- tools suitable for accurate and long-term tracking of to- tem that can effectively eliminate aggregates and enable pological changes at specific genomic loci. A series of high-contrast visualization of the 3D dynamics of spe- tools have been reported for visualization of repetitive cific genomic structures under diverse physiological and DNA sequences or specific gene loci in fixed or live hu- pathological contexts (e.g., aging) across a wide range of man cells, but they suffer from several limitations. (1) cell types in vitro and in vivo. Fluorescent Lac or Tet operator and repressor systems have been used to visualize target genomic sequences in Results live cells [12, 13], but the integration of large artificial sequences (~10 kb) into the genomic region of interest Precise visualization of telomeres and centromeres using suffers from low efficiency and a risk of disturbing ge- TTALEs nomic structures. (2) Fluorescent in situ hybridization To adapt TALEs for visualizing specific genomic (FISH) has been widely used to study nuclear localiza- locus, we generated an EGFP-tagged TALE construct tion of specific sequences and genomic aberrations [14], targeting a 19-bp telomeric DNA repeat (5′-TAAC- but can only be performed on fixed cells after DNA de- CCTAACCCTAACCCT-3′; referred to as TALEtelo). This naturation. (3) Recently, the CRISPR/dCas9 system has construct was transiently introduced into four human been adapted for visualization of specific genomic loci tumor cell lines (U2OS, HeLa, HepG2, and MCF7). To (e.g., protein-coding mucin genes such as MUC4) and examine the efficiency of TALE-based fluorescence im- repetitive telomere sequences in live cells [15-17]. How- aging of telomeric sequences, 3D-FISH with a telomeric ever, low signal-to-noise ratios, inefficient delivery of the peptide nucleic acid (PNA) probe was used to visualize bulky Cas9 and single-guide RNA (sgRNA) constructs, the 3D distribution of telomeres inside the nucleus [7]. and potential cellular stresses caused by overloading Approximately 20% of FISH-positive foci overlapped sgRNA have limited their utility [18, 19]. (4) Fluores- with EGFP-TALEtelo in U2OS cells. In other cell types cent protein-labeled transcription activator-like effectors examined, EGFP-TALEtelo appeared mostly as large (TALEs) have been used for live imaging of genomic bright foci (referred to hereafter as “aggregates”) distinct repetitive sequences [17, 20, 21]. TALEs are proteins se- from telomeric foci, with only a small percentage (< 5%) creted by Xanthomonas bacteria that infect various plant of foci co-localized with the FISH signals (Figure 1A and species. TALEs are DNA-binding proteins that contain 1C). In addition, an EGFP-tagged TALE against a 19-bp tandem 33- to 35-amino acid (aa) repeats, each of which centromeric satellite DNA repeat (5′-TCCATTCCATTC- specifically recognizes and binds to a single target DNA CATTCCA-3′), referred to as TALEcentro, also failed to base [22, 23]. The smaller size of TALEs and simple faithfully identify centromeres in the same cell types, as correlation between TALEs and target DNA bases makes determined by 3D-FISH with a centromeric PNA probe them extremely useful for designing artificial constructs (Supplementary information, Figure S1A and S1B). capable of recognizing genomic sequences in diverse Similar results were also observed using previously re- experimental systems. Indeed, engineered TALEs have ported EGFP-TALEs against a 15-bp telomeric repeat been harnessed for a variety of applications, including (5′-TAACCCTAACCCTAA-3′; Figure 1B and 1C) or a genome editing (when fused to the cleavage domain of 20-bp centromeric repeat (5′-TAGACAGAAGCATTCT- FokI nuclease or to meganucleases) [24] and design of CAGA-3′; data not shown) [20]. We further verified that customized transcriptional modulators [25, 26] and re- these aggregates were not localized in nucleoli (Supple- combinases [27]. Due to their relatively small size, fluo- mentary information, Figure S1C-S1D). These data indi- rescently tagged TALEs have been used as small protein cate that conventional TALEs are inefficient for tracking probes to track specific genomic DNA sequences, espe- human telomeric and centromeric DNAs. cially within telomeres and centromeres, in live cells [20, TALEs are composed of multiple highly repeti- 21, 28, 29]. Despite these advances, a careful validation tive modules, a feature that likely predisposes them to of TALE-based imaging in different cellular systems is self-assemble into bulky protein aggregates especially still needed. Importantly, TALE- and Cas9/sgRNA-based when being simultaneously tethered to multiple copies imaging systems have seldom been tested in physiologi- of genomic repetitive DNA sequences, preventing their cal and pathological contexts such as human aging. binding to cognate DNA
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