{"lab": {"correspondence": [{"contact_email": "aHVhaXlpbnpAYW5kcmV3LmNtdS5lZHU=", "@id": "/users/c55c809d-8ffc-42f9-a528-12db012f846f/", "display_title": "Huaiying Zhang"}], "@id": "/labs/huaiying-zhang-lab/", "display_title": "Huaiying Zhang, CMU", "status": "current", "@type": ["Lab", "Item"], "uuid": "3525602a-81b2-4db9-92bd-858ef82e1b53", "title": "Huaiying Zhang, CMU", "pi": {"error": "no view permissions"}, "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin", "role.lab_submitter", "submits_for.3525602a-81b2-4db9-92bd-858ef82e1b53"]}}, "award": {"center_title": "Zhang", "description": "NI-OFHHD: All cancer cells need to maintain telomere length for immortality. While most cancer cells reactivate telomerase, a reverse transcriptase, to elongates telomere from an RNA template, about 10-15% of cancer cells are telomerase-negative and adopt a homologous-recombination based alternative lengthening of telomeres (ALT) pathway. ALT cells exhibit many abnormalities in nuclear organization, including the formation of nuclear bodies called APBs for ALT telomere-associated promyelocytic leukemia nuclear bodies, clustering of telomeres within APBs, and the formation of RNA foci on telomeres with a long non-coding RNA called telomere repeat-containing RNA (TERRA). These unique features are used as biomarkers for ALT diagnosis and can be attractive therapeutic targets because of reduced side effects on healthy cells that do not share these features. However, how these features contribute to telomere maintenance and ALT cancer cell growth remain elusive, due to the lack of conceptual model as well as experimental tools to monitor and control their assembly and function in live cells. Based on our observation that APBs exhibit liquid behavior and long non- coding RNAs can phase separate with RNA-binding proteins, we propose a liquid-liquid phase separation model for the assmembly and function of these ALT specific features. We hypothesize TERRA phase separates with its interacting proteins to nucleate APB liquid droplets. The liquid nature of APBs droplets (also called condensates) would promote coalescence of APBs to drive telomere clustering. Meanwhile, condensation of APB droplets can concentrate DNA repair factors, providing opportunities for telomeres to use one another as repair templates to elongate within APBs. To test our hypothesis, we developed a state-of-the- art optogenetic approach to control APB assembly. We demonstrate that liquid phase separation underlies APB assembly and coalescence of APB droplets indeed drives telomere clustering. Building on our ability to control telomere clustering and APB assembly and by collaborating with experts in super resolution microscopy, nuclear mechanics, chromosome organization and ALT cancer, we will investigate how DNA repair factors are recruited to and organized in APB condensates for ALT telomere DNA synthesis (Aim 1) and how telomere clustering leads to unique genome organization and gene expression in ALT cells (Aim 2). We will then extend our optogenetic tools to control RNA and dissect TERRA contributions in ALT (Aim 3). Results obtained by manipulating cultured ALT cells will be confirmed by characterizing ALT tissue or creating de novo ALT phonotypes in primary human cells. Our results will provide mechanistic understanding on how protein and/or RNA phase separation contributes to ALT cancer, which will offer the potential to develop strategies specifically targeting these unique phase separation processes, rather than the existing molecules that shared by heathy cells, for ALT cancer treatment. 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(2021)", "uuid": "0007f833-05a1-40c8-b1a5-996dec2ad130", "@type": ["Publication", "Item"], "status": "current", "authors": ["Xu M", "Chigumira T", "Chen Z", "Tones J", "Zhao R", "Dahl KN", "Chenoweth DM", "Zhang H"], "display_title": "Xu M et al. (2021) PMID:35174207", "@id": "/publications/0007f833-05a1-40c8-b1a5-996dec2ad130/", "journal": "Frontiers in molecular biosciences", "abstract": "TERRA, TElomeric Repeat-containing RNA, is a long non-coding RNA transcribed from telomeres. Emerging evidence indicates that TERRA regulates telomere maintenance  and chromosome end protection in normal and cancerous cells. However, the mechanism of how TERRA contributes to telomere functions is still unclear, partially owing to the shortage of approaches to track and manipulate endogenous  TERRA molecules in live cells. Here, we developed a method to visualize TERRA in  live cells via a combination of CRISPR Cas13 RNA labeling and SunTag technology.  Single-particle tracking reveals that TERRA foci undergo anomalous diffusion in a manner that depends on the timescale and telomeric localization. Furthermore, we  used a chemically-induced protein dimerization system to manipulate TERRA subcellular localization in live cells. Overall, our approaches to monitor and control TERRA locations in live cells provide powerful tools to better understand its roles in telomere maintenance and genomic integrity.", "date_published": "2021", "ID": "PMID:35174207", "principals_allowed": {"view": ["system.Everyone"], "edit": ["group.admin"]}}, "publications_of_exp": [{"ID": "PMID:35174207", "title": "CRISPR Cas13-Based Tools to Track and Manipulate Endogenous Telomeric Repeat-Containing RNAs in Live Cells.", "uuid": "0007f833-05a1-40c8-b1a5-996dec2ad130", "short_attribution": "Xu M et al. 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Overall, our approaches to monitor and control TERRA locations in live cells provide powerful tools to better understand its roles in telomere maintenance and genomic integrity.", "date_published": "2021", "authors": ["Xu M", "Chigumira T", "Chen Z", "Tones J", "Zhao R", "Dahl KN", "Chenoweth DM", "Zhang H"], "@type": ["Publication", "Item"], "journal": "Frontiers in molecular biosciences", "display_title": "Xu M et al. 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