Eukaryotic transcription factors (TFs) form local, high-concentration hubs at specific genomic loci through dynamic, multivalent interactions mediated by their low-complexity domains, a behavior essential for transcriptional activation. Characterizing the dynamics and regulation of TF hubs requires high-resolution imaging of TFs in their native cellular environment; however, such biophysical characterization remains limited. Here, we combine CRISPR/Cas9-mediated genome editing and single-molecule microscopy to investigate the behaviors of endogenous oncogenic fusion TF EWS::FLI1 in Ewing sarcoma cells. EWS::FLI1 forms sub-diffraction-limit hubs with dissolution mechanisms that prevent macroscopic liquid-liquid phase separation. Hub formation is a neomorphic behavior of EWS::FLI1 not conferred by its parental proteins, EWSR1 and FLI1. During mitosis, EWS::FLI1 hubs dissolve, but EWS::FLI1 molecules continue to dynamically associate with mitotic chromosomes, consistent with a mitotic bookmarking role. Nascent RNA destabilizes EWS::FLI1 hubs without affecting their dimensions. Finally, LY2835219 and trabectedin, compounds previously reported to affect EWS::FLI1 function, disrupt and mislocalize endogenous EWS::FLI1 hubs, respectively, highlighting their therapeutic potential for treating Ewing sarcoma. Together, our results reveal new insights into the assembly and regulation of endogenous EWS::FLI1 hubs at an unprecedented resolution and establish a framework for biophysical and functional characterization of other TF hubs in the future.
", "doi": "10.1002/advs.202600048", "pmcid": "PMC13343610", "issn": "2198-3844", "publisher": "Wiley", "publication": "Advanced Science", "publication_date": "2026-07-08", "pages": "e00048" }, { "id": "authors:16dh8-jc232", "collection": "authors", "collection_id": "16dh8-jc232", "cite_using_url": "https://authors.library.caltech.edu/records/16dh8-jc232", "type": "conference_item", "title": "Abstract 623: Targeting PAX3::FOXO1 condensates in fusion-positive alveolar rhabdomyosarcoma via disordered-region mimetic peptides.", "author": [ { "family_name": "Di Martino", "given_name": "Michael" }, { "family_name": "Zhong", "given_name": "Yanghao", "orcid": "0000-0002-0747-3041", "clpid": "Zhong-Yanghao" }, { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" } ], "abstract": "PAX3::FOXO1 (P3F1) is a fusion oncoprotein that drives fusion-positive alveolar rhabdomyosarcoma (aRMS), a highly lethal pediatric cancer with <30% 5-year survival in high-risk patients. Targeted therapy development has been limited by P3F1’s largely disordered structure and poorly understood interaction mechanisms. Recent work has shown that short peptides targeting intrinsically disordered regions (IDRs) can disrupt transcriptional hubs and impair tumor growth, but this strategy requires identifying key interaction partners that often regulate other signaling pathways. We previously discovered that P3F1 demonstrates a neomorphic hub formation propensity that corresponds with its transcriptional activity, suggesting that disrupting these hubs can hinder said activity.
\n\n\n\nTo design short peptides modelled from the P3F1 IDR itself that disrupt P3F1 hubs and suppress P3F1-mediated transcription and aRMS cell proliferation. Experimental Procedures and Results: Using the LacO array assay, an established in-cellulo method that quantifies IDR interaction strength, we discovered that truncating the P3F1 392-510 region significantly decreased IDR interaction strength. Further, the same truncation significantly altered its interaction strength with p300, a key transcriptional coactivator known to promote P3F1’s function. Finally, dual luciferase assay results showed that a 392-510 deletion significantly decreased P3F1’s transactivation ability. Taken together, this suggested that P3F1 392-510 is a key interaction hotspot that influences its transcriptional capability. Sequence analysis revealed that P3F1 392-510 is enriched in cationic and aromatic residues, consistent with cation-π and π-π interactions common in IDRs. FINCHES interaction mapping showed that mutating either all cationic or all aromatic residues in this region reduces P3F1 homotypic interactions, with aromatic-to-alanine mutations producing the stronger effect. Ongoing experiments utilizing the LacO array assay are probing these interaction modes to guide the design of optimized P3F1 392-510 peptide mimics.
\n\n\n\nP3F1 392-510 is critical for P3F1’s IDR-mediated interactions and transcriptional activity. Its cationic and aromatic composition suggests cation-π and/or π-π interactions as key interaction modes. Future work will experimentally dissect these interaction modes and develop peptide inhibitors informed by this mechanistic insight.
", "doi": "10.1158/1538-7445.am2026-623", "issn": "0008-5472", "publisher": "American Association for Cancer Research (AACR)", "publication": "Cancer Research", "publication_date": "2026-04-03", "series_number": "7_Supplement", "volume": "86", "issue": "7_Supplement", "pages": "623-623" }, { "id": "authors:hwz3s-98150", "collection": "authors", "collection_id": "hwz3s-98150", "cite_using_url": "https://authors.library.caltech.edu/records/hwz3s-98150", "type": "article", "title": "Abstract 5950: Dynamic low-complexity domain interactions of PAX3::FOXO1 mediate endogenous pathological transcriptional hub formation in alveolar rhabdomyosarcoma", "author": [ { "family_name": "Zhong", "given_name": "Yanghao", "orcid": "0000-0002-0747-3041", "clpid": "Zhong-Yanghao" }, { "family_name": "Di Martino", "given_name": "Michael", "clpid": "Di-Martino-Michael-T" }, { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" } ], "abstract": "Rhabdomyosarcoma (RMS), a cancer of skeletal muscle tissue, is the most common pediatric soft tissue sarcoma with 5-year survival rate less than 30% in high-risk RMS, despite advances in prognosis and treatment over the past few decades. PAX3::FOXO1 (P3F1) is a fusion oncoprotein found in 60% cases of alveolar RMS (aRMS), the most aggressive subtype of RMS, and has been thought to drive oncogenic transcription in aRMS. However, the molecular mechanisms by which P3F1 regulates transcription remain poorly understood, and the extent to which these transcriptional changes contribute to tumorigenesis is unclear. In the current study, we first demonstrated exogenously expressed P3F1 exhibited neomorphic hub formation propensity which is not observed for its parental proteins. Using CRISPR/Cas9-based gene editing method, we endogenously labeled P3F1 with a fluorogenic HaloTag in patient-derived aRMS cells, allowing us, for the first time, to visualize P3F1 at its native pathological environment. We discovered that endogenous P3F1 forms transcriptional hubs at its target genes, and the average hub size is ∼117 nm characterized by photoactivatable localization microscopy. Single particle tracking experiment showed that P3F1 molecules have longer chromatin bound residence time in the hubs compared to those outside the hubs, suggesting an important role of P3F1 hubs in target gene transcription. We found disrupting the intrinsically disordered low complexity domains (LCDs) of P3F1 impaired its hub formation propensity, target gene transcription and its ability to recruit coactivator p300. Using a phase-separation-induced interactome detection (PhaseID) method, we mapped unique interacting proteins that P3F1 hubs recruit and utilize to influence oncogenic transcription. In summary, our results uncover a neomorphic hub formation behavior of pathological P3F1 and provide a strong rationale of targeting P3F1 hub formation as new therapeutics for aRMS.
", "doi": "10.1158/1538-7445.am2026-5950", "issn": "0008-5472", "publisher": "American Association for Cancer Research (AACR)", "publication": "Cancer Research", "publication_date": "2026-04-03", "series_number": "7_Supplement", "volume": "86", "issue": "7_Supplement", "pages": "5950-5950" }, { "id": "authors:9mj3b-xxr45", "collection": "authors", "collection_id": "9mj3b-xxr45", "cite_using_url": "https://authors.library.caltech.edu/records/9mj3b-xxr45", "type": "article", "title": "Evolving concepts of the protein universe", "author": [ { "family_name": "Kulkarni", "given_name": "Prakash", "orcid": "0000-0001-6736-4333", "clpid": "Kulkarni-Prakash" }, { "family_name": "Porter", "given_name": "Lauren", "orcid": "0000-0003-2031-8326" }, { "family_name": "Chou", "given_name": "Tsui-Fen", "orcid": "0000-0003-2410-2186", "clpid": "Chou-Tsui-Fen" }, { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Chiti", "given_name": "Fabrizio", "orcid": "0000-0002-1330-1289" }, { "family_name": "Schafer", "given_name": "Joseph W." }, { "family_name": "Mohanty", "given_name": "Atish", "orcid": "0000-0003-1464-3165" }, { "family_name": "Ramisetty", "given_name": "Sravani", "orcid": "0000-0001-6462-9658" }, { "family_name": "Onuchic", "given_name": "Jose N.", "orcid": "0000-0002-9448-0388" }, { "family_name": "Tuite", "given_name": "Mick", "orcid": "0000-0002-5214-540X" }, { "family_name": "Uversky", "given_name": "Vladimir N.", "orcid": "0000-0002-4037-5857" }, { "family_name": "Weninger", "given_name": "Keith R." }, { "family_name": "Koonin", "given_name": "Eugene V.", "orcid": "0000-0003-3943-8299" }, { "family_name": "Orban", "given_name": "John", "orcid": "0000-0002-3895-1800" }, { "family_name": "Salgia", "given_name": "Ravi", "orcid": "0000-0001-9643-7626" } ], "abstract": "The protein universe is the collection of all proteins on earth from all organisms both extant and extinct. Classical studies on protein folding suggested that proteins exist as a unique three-dimensional conformation that is dictated by the genetic code and is critical for function. In this perspective, we discuss ideas and developments that emerged over the past three decades regarding the protein structure-function paradigm. It is now clear that ordered (active/functional) and disordered/denatured (and hence inactive/non-functional) represent a continuum of states rather than binary states. Some proteins can switch folds without sequence change. Others exist as conformational ensembles lacking defined structure yet play critical roles in many biological processes, including forming membrane-less organelles driven by liquid-liquid phase separation. Numerous diverse proteins harbor segments with the potential to form amyloid fibrils, many of which are functional, and some possess prion-like properties enabling conformation-based transfer of heritable information. Taken together, these developments reveal the remarkable complexity of the protein universe.
", "doi": "10.1016/j.isci.2025.112012", "pmcid": "PMC11926713", "issn": "2589-0042", "publisher": "Cell Press", "publication": "iScience", "publication_date": "2025-03-21", "series_number": "3", "volume": "28", "issue": "3", "pages": "112012" }, { "id": "authors:bvyhb-v5h44", "collection": "authors", "collection_id": "bvyhb-v5h44", "cite_using_url": "https://authors.library.caltech.edu/records/bvyhb-v5h44", "type": "article", "title": "Roles of intrinsically disordered protein regions in transcriptional regulation and genome organization", "author": [ { "family_name": "Miao", "given_name": "Jiapei", "orcid": "0000-0003-3722-1678", "clpid": "Miao-Jiapei" }, { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" } ], "abstract": "Eukaryotic transcription is a complex process regulated by transcription factors (TFs), coactivators, and RNA polymerase machineries, many of which contain sizable intrinsically disordered regions (IDRs). Many TFs activate transcription through multivalent IDR-IDR interactions. Optimal levels of such multivalent interactions associated with appropriate IDR concentrations, interaction strengths, or interaction valencies are required for effective transcriptional activation. The interaction selectivity of IDRs is crucial for the\u00a0precise regulation of transcription, and this selectivity is dependent on the IDR sequences. Furthermore, IDRs modulate gene expression by bringing chromatin sites together to form transcriptionally active chromatin hubs. Mutations in IDRs may cause dysregulation of their multivalent interactions, contributing to diseases, including cancers and neurodegenerative disorders. Understanding the effects of IDR-related mutations on transcription control and genome organization opens new opportunities for developing targeted therapeutic strategies. In this review, we discuss recent reports documenting important functions of IDRs in transcriptional regulation and their implications for human health and disease.", "doi": "10.1016/j.gde.2024.102285", "issn": "0959-437X", "publisher": "Elsevier", "publication": "Current Opinion in Genetics and Development", "publication_date": "2025-02", "volume": "90", "pages": "102285" }, { "id": "authors:3ww8a-hkc68", "collection": "authors", "collection_id": "3ww8a-hkc68", "cite_using_url": "https://authors.library.caltech.edu/records/3ww8a-hkc68", "type": "article", "title": "How transcription factor binding controls transcriptional bursting dynamics: A single-molecule view", "author": [ { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Yoshida", "given_name": "Shawn", "orcid": "0000-0002-0866-2741", "clpid": "Yoshida-Shawn" } ], "abstract": "In this issue, Pomp et al.1 simultaneously tracked transcriptional bursts of yeast gene GAL10 and transient binding of transcription factor Gal4 at the gene using novel methods. Dynamic exchange and infrequent long binding of Gal4 together enable prolonged transcriptional bursts of GAL10.
\nTranscription factors (TFs) activate enhancers to drive cell-specific gene programs in response to signals, but our understanding of enhancer assembly during signaling events is incomplete. Here, we show that androgen receptor (AR) forms condensates through multivalent interactions mediated by its N-terminal intrinsically disordered region (IDR) to orchestrate enhancer assembly in response to androgen signaling. AR IDR can be substituted by IDRs from selective proteins for AR condensation capacity and its function on enhancers. Expansion of the poly(Q) track within AR IDR results in a higher AR condensation propensity as measured by multiple methods, including live-cell single-molecule microscopy. Either weakening or strengthening AR condensation propensity impairs its heterotypic multivalent interactions with other enhancer components and diminishes its transcriptional activity. Our work reveals the requirement of an optimal level of AR condensation in mediating enhancer assembly and suggests that alteration of the fine-tuned multivalent IDR-IDR interactions might underlie AR-related human pathologies.
", "doi": "10.1016/j.molcel.2023.08.027", "pmcid": "PMC10592010", "issn": "1097-2765", "publisher": "Cell Press", "publication": "Molecular Cell", "publication_date": "2023-10-05", "series_number": "19", "volume": "83", "issue": "19", "pages": "3438-3456.e12" }, { "id": "authors:ecpdk-79f96", "collection": "authors", "collection_id": "ecpdk-79f96", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20230615-129043000.4", "type": "article", "title": "Visualizing Protein Localizations in Fixed Cells: Caveats and the Underlying Mechanisms", "author": [ { "family_name": "Yoshida", "given_name": "Shawn R.", "orcid": "0000-0002-0866-2741", "clpid": "Yoshida-Shawn-R" }, { "family_name": "Maity", "given_name": "Barun K.", "orcid": "0000-0002-0749-3405", "clpid": "Maity-Barun-K" }, { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" } ], "abstract": "Fluorescence microscopy techniques have been widely adopted in biology for their ability to visualize the structure and dynamics of a wide range of cellular and subcellular processes. The specificity and sensitivity that these techniques afford have made them primary tools in the characterization of protein localizations within cells. Many of the fluorescence microscopy techniques require cells to be fixed via chemical or alternative methods before being imaged. However, some fixation methods have been found to induce the redistribution of particular proteins in the cell, resulting in artifacts in the characterization of protein localizations and functions under physiological conditions. Here, we review the ability of commonly used cell fixation methods to faithfully preserve the localizations of proteins that bind to chromatin, undergo liquid\u2013liquid phase separation (LLPS), and are involved in the formation of various membrane-bound organelles. We also review the mechanisms underlying various fixation artifacts and discuss potential alternative fixation methods to minimize the artifacts while investigating different proteins and cellular structures. Overall, fixed-cell fluorescence microscopy is a very powerful tool in biomedical research; however, each experiment demands the careful selection of an appropriate fixation method to avoid potential artifacts and may benefit from live-cell imaging validation.", "doi": "10.1021/acs.jpcb.3c01658", "pmcid": "PMC10201523", "issn": "1520-6106", "publisher": "American Chemical Society", "publication": "Journal of Physical Chemistry B", "publication_date": "2023-05-18", "series_number": "19", "volume": "127", "issue": "19", "pages": "4165-4173" }, { "id": "authors:08xz6-28191", "collection": "authors", "collection_id": "08xz6-28191", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20230404-290277800.12", "type": "article", "title": "Phase Separation in Biology and Disease; Current Perspectives and Open Questions", "author": [ { "family_name": "Boeynaems", "given_name": "Steven", "orcid": "0000-0002-9705-9871", "clpid": "Boeynaems-Steven" }, { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Gsponer", "given_name": "J\u00f6rg", "orcid": "0000-0003-3672-6594", "clpid": "Gsponer-J\u00f6rg" }, { "family_name": "Holt", "given_name": "Liam", "orcid": "0000-0002-4002-0861", "clpid": "Holt-Liam" }, { "family_name": "Milovanovic", "given_name": "Dragomir", "orcid": "0000-0002-6625-1879", "clpid": "Milovanovic-Dragomir" }, { "family_name": "Mitrea", "given_name": "Diana M.", "orcid": "0000-0003-4501-0846", "clpid": "Mitrea-Diana" }, { "family_name": "Mueller-Cajar", "given_name": "Oliver", "orcid": "0000-0003-2866-5963", "clpid": "Mueller-Cajar-Oliver" }, { "family_name": "Portz", "given_name": "Bede", "orcid": "0000-0002-4698-790X", "clpid": "Portz-Bede" }, { "family_name": "Reilly", "given_name": "John F.", "clpid": "Reilly-John-F" }, { "family_name": "Reinkemeier", "given_name": "Christopher D.", "orcid": "0000-0003-2821-218X", "clpid": "Reinkemeier-Christopher-D" }, { "family_name": "Sabari", "given_name": "Benjamin R.", "orcid": "0000-0002-6411-3907", "clpid": "Sabari-Benjamin-R" }, { "family_name": "Sanulli", "given_name": "Serena", "orcid": "0000-0002-7226-3147", "clpid": "Sanulli-Serena" }, { "family_name": "Shorter", "given_name": "James", "orcid": "0000-0001-5269-8533", "clpid": "Shorter-James" }, { "family_name": "Sontag", "given_name": "Emily", "orcid": "0000-0003-3068-6253", "clpid": "Sontag-Emily" }, { "family_name": "Strader", "given_name": "Lucia", "orcid": "0000-0002-7600-7204", "clpid": "Strader-Lucia" }, { "family_name": "Stachowiak", "given_name": "Jeanne", "orcid": "0000-0003-2501-142X", "clpid": "Stachowiak-Jeanne" }, { "family_name": "Weber", "given_name": "Stephanie C.", "orcid": "0000-0002-7297-2875", "clpid": "Weber-Stephanie-C" }, { "family_name": "White", "given_name": "Michael", "clpid": "White-Michael" }, { "family_name": "Zhang", "given_name": "Huaiying", "orcid": "0000-0002-1784-2664", "clpid": "Zhang-Huaiying" }, { "family_name": "Zweckstetter", "given_name": "Markus", "orcid": "0000-0002-2536-6581", "clpid": "Zweckstetter-Markus" }, { "family_name": "Elbaum-Garfinkle", "given_name": "Shana", "orcid": "0000-0002-3953-7329", "clpid": "Elbaum-Garfinkle-Shana" }, { "family_name": "Kriwacki", "given_name": "Richard", "orcid": "0000-0002-9798-6018", "clpid": "Kriwacki-Richard-W" } ], "abstract": "In the past almost 15 years, we witnessed the birth of a new scientific field focused on the existence, formation, biological functions, and disease associations of membraneless bodies in cells, now referred to as biomolecular condensates. Pioneering studies from several laboratories [reviewed in 1, 2, 3] supported a model wherein biomolecular condensates associated with diverse biological processes form through the process of phase separation. These and other findings that followed have revolutionized our understanding of how biomolecules are organized in space and time within cells to perform myriad biological functions, including cell fate determination, signal transduction, endocytosis, regulation of gene expression and protein translation, and regulation of RNA metabolism. Further, condensates formed through aberrant phase transitions have been associated with numerous human diseases, prominently including neurodegeneration and cancer. While in some cases, rigorous evidence supports links between formation of biomolecular condensates through phase separation and biological functions, in many others such links are less robustly supported, which has led to rightful scrutiny of the generality of the roles of phase separation in biology and disease. During a week-long workshop in March 2022 at the Telluride Science Research Center (TSRC) in Telluride, Colorado, \u223c25 scientists addressed key questions surrounding the biomolecular condensates field. Herein, we present insights gained through these discussions, addressing topics including, roles of condensates in diverse biological processes and systems, and normal and disease cell states, their applications to synthetic biology, and the potential for therapeutically targeting biomolecular condensates.", "doi": "10.1016/j.jmb.2023.167971", "pmcid": "PMC9970028", "issn": "0022-2836", "publisher": "Elsevier", "publication": "Journal of Molecular Biology", "publication_date": "2023-03-01", "series_number": "5", "volume": "435", "issue": "5", "pages": "Art. No. 167971" }, { "id": "authors:zdfhg-1g918", "collection": "authors", "collection_id": "zdfhg-1g918", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20230322-270078000.2", "type": "article", "title": "Mutant NPM1 Hijacks Transcriptional Hubs to Maintain Pathogenic Gene Programs in Acute Myeloid Leukemia", "author": [ { "family_name": "Wang", "given_name": "Xue Qing David", "orcid": "0000-0003-4951-5788", "clpid": "Wang-Xue-Qing-David" }, { "family_name": "Fan", "given_name": "Dandan", "orcid": "0000-0002-1471-1183", "clpid": "Fan-Dandan" }, { "family_name": "Han", "given_name": "Qinyu", "orcid": "0000-0003-3464-8967", "clpid": "Han-Qinyu" }, { "family_name": "Liu", "given_name": "Yiman", "orcid": "0000-0002-2223-7223", "clpid": "Liu-Yiman" }, { "family_name": "Miao", "given_name": "Hongzhi", "orcid": "0000-0003-4862-9484", "clpid": "Miao-Hongzhi" }, { "family_name": "Wang", "given_name": "Xinyu", "orcid": "0000-0003-0806-4335", "clpid": "Wang-Xinyu" }, { "family_name": "Li", "given_name": "Qinglan", "orcid": "0000-0003-0160-8774", "clpid": "Li-Qinglan" }, { "family_name": "Chen", "given_name": "Dong", "orcid": "0000-0003-2210-1024", "clpid": "Chen-Dong" }, { "family_name": "Gore", "given_name": "Haley", "orcid": "0000-0001-8993-6611", "clpid": "Gore-Haley" }, { "family_name": "Himadewi", "given_name": "Pamela", "orcid": "0000-0001-5721-1974", "clpid": "Himadewi-Pamela" }, { "family_name": "Pfeifer", "given_name": "Gerd P.", "orcid": "0000-0002-5080-9604", "clpid": "Pfeifer-Gerde-P" }, { "family_name": "Cierpicki", "given_name": "Tomasz", "orcid": "0000-0003-2058-0658", "clpid": "Cierpicki-Tomasz" }, { "family_name": "Grembecka", "given_name": "Jolanta", "orcid": "0000-0002-6180-9095", "clpid": "Grembecka-Jolanta" }, { "family_name": "Su", "given_name": "Jianzhong", "orcid": "0000-0003-1054-6042", "clpid": "Su-Jianzhong" }, { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Wan", "given_name": "Liling", "orcid": "0000-0002-5444-0437", "clpid": "Wan-Liling" }, { "family_name": "Zhang", "given_name": "Xiaotian", "orcid": "0000-0002-9533-4761", "clpid": "Zhang-Xiaotian" } ], "abstract": "Nucleophosmin (NPM1) is a ubiquitously expressed nucleolar protein with a wide range of biological functions. In 30% of acute myeloid leukemia (AML), the terminal exon of NPM1 is often found mutated, resulting in the addition of a nuclear export signal and a shift of the protein to the cytoplasm (NPM1c). AMLs carrying this mutation have aberrant expression of the HOXA/B genes, whose overexpression leads to leukemogenic transformation. Here, for the first time, we comprehensively prove that NPM1c binds to a subset of active gene promoters in NPM1c AMLs, including well-known leukemia-driving genes\u2014HOXA/B cluster genes and MEIS1. NPM1c sustains the active transcription of key target genes by orchestrating a transcription hub and maintains the active chromatin landscape by inhibiting the activity of histone deacetylases. Together, these findings reveal the neomorphic function of NPM1c as a transcriptional amplifier for leukemic gene expression and open up new paradigms for therapeutic intervention. Significance: NPM1 mutation is the most common mutation in AML, yet the mechanism of how the mutant protein results in AML remains unclear. Here, for the first time, we prove mutant NPM1 directly binds to active chromatin regions and hijacks the transcription of AML-driving genes.", "doi": "10.1158/2159-8290.cd-22-0424", "pmcid": "PMC9975662", "issn": "2159-8274", "publisher": "American Association for Cancer Research", "publication": "Cancer Discovery", "publication_date": "2023-03", "series_number": "3", "volume": "13", "issue": "3", "pages": "724-745" }, { "id": "authors:hgfgq-w6924", "collection": "authors", "collection_id": "hgfgq-w6924", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20230125-514169000.3", "type": "article", "title": "Fixation can change the appearance of phase separation in living cells", "author": [ { "family_name": "Irgen-Gioro", "given_name": "Shawn", "orcid": "0000-0001-8638-6191", "clpid": "Irgen-Gioro-Shawn" }, { "family_name": "Yoshida", "given_name": "Shawn", "orcid": "0000-0002-0866-2741", "clpid": "Yoshida-Shawn-R" }, { "family_name": "Walling", "given_name": "Victoria", "clpid": "Walling-Victoria" }, { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" } ], "abstract": "Fixing cells with paraformaldehyde (PFA) is an essential step in numerous biological techniques as it is thought to preserve a snapshot of biomolecular transactions in living cells. Fixed-cell imaging techniques such as immunofluorescence have been widely used to detect liquid\u2013liquid phase separation (LLPS) in vivo. Here, we compared images, before and after fixation, of cells expressing intrinsically disordered proteins that are able to undergo LLPS. Surprisingly, we found that PFA fixation can both enhance and diminish putative LLPS behaviors. For specific proteins, fixation can even cause their droplet-like puncta to artificially appear in cells that do not have any detectable puncta in the live condition. Fixing cells in the presence of glycine, a molecule that modulates fixation rates, can reverse the fixation effect from enhancing to diminishing LLPS appearance. We further established a kinetic model of fixation in the context of dynamic protein\u2013protein interactions. Simulations based on the model suggest that protein localization in fixed cells depends on an intricate balance of protein\u2013protein interaction dynamics, the overall rate of fixation, and notably, the difference between fixation rates of different proteins. Consistent with simulations, live-cell single-molecule imaging experiments showed that a fast overall rate of fixation relative to protein\u2013protein interaction dynamics can minimize fixation artifacts. Our work reveals that PFA fixation changes the appearance of LLPS from living cells, presents a caveat in studying LLPS using fixation-based methods, and suggests a mechanism underlying the fixation artifact.", "doi": "10.7554/elife.79903", "pmcid": "PMC9817179", "issn": "2050-084X", "publisher": "eLife Sciences Publications", "publication": "eLife", "publication_date": "2022-11-29", "volume": "11", "pages": "Art. No. e79903" }, { "id": "authors:cfg1v-e0896", "collection": "authors", "collection_id": "cfg1v-e0896", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20230322-101476000.9", "type": "monograph", "title": "Hormone-induced enhancer assembly requires an optimal level of hormone receptor multivalent interactions", "author": [ { "family_name": "Chen", "given_name": "Lizhen", "clpid": "Chen-Lizhen" }, { "family_name": "Zhang", "given_name": "Zhao", "orcid": "0000-0002-3679-8945", "clpid": "Zhang-Zhao" }, { "family_name": "Han", "given_name": "Qinyu", "orcid": "0000-0003-3464-8967", "clpid": "Han-Qinyu" }, { "family_name": "Rodrigues", "given_name": "Leticia", "clpid": "Rodrigues-Leticia" }, { "family_name": "Zboril", "given_name": "Emily", "clpid": "Zboril-Emily" }, { "family_name": "Adhikari", "given_name": "Rashmi", "orcid": "0000-0003-4426-4780", "clpid": "Adhikari-Rashmi" }, { "family_name": "Li", "given_name": "Xin", "clpid": "Li-Xin" }, { "family_name": "Ko", "given_name": "Su-Hyuk", "clpid": "Ko-Su-Hyuk" }, { "family_name": "Xue", "given_name": "Pengya", "orcid": "0000-0002-5887-1760", "clpid": "Xue-Pengya" }, { "family_name": "Smith", "given_name": "Emilie", "clpid": "Smith-Emilie" }, { "family_name": "Xu", "given_name": "Kexin", "clpid": "Xu-Kexin" }, { "family_name": "Wang", "given_name": "Qianben", "orcid": "0000-0003-2636-7145", "clpid": "Wang-Qianben" }, { "family_name": "Huang", "given_name": "Tim Hui-Ming", "orcid": "0000-0001-5985-9176", "clpid": "Huang-Tim-Hui-Ming" }, { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Liu", "given_name": "Zhijie", "clpid": "Liu-Zhijie" } ], "abstract": "Transcription factors (TFs) activate enhancers to drive cell-specific gene programs in response to signals, but our understanding of enhancer assembly during signaling events is incomplete. Here, we show that Androgen Receptor (AR), a steroid hormone-regulated transcription factor, forms condensates through multivalent interactions in response to androgen signaling to orchestrate enhancer assembly. We demonstrate that the intrinsically disordered N-terminal domain (NTD) of AR drives 1,6-Hexanediol-sensitive condensate formation and that NTD deletion or aromatic residue mutation reduces AR self-association and abolishes AR transcriptional activity. AR NTD can be substituted by intrinsically disordered regions (IDRs) from selective proteins for AR condensation capacity and transactivation function. Surprisingly, strengthened AR condensation capacity caused by extending the polyQ tract within AR NTD also leads to impaired transcriptional activity without affecting AR binding on enhancers. Furthermore, either NTD deletion or polyQ extension reduces heterotypic multivalent interactions between AR and other enhancer components. These results suggest the importance of an optimal level of AR condensation in mediating AR-AR homotypic and AR-cofactor heterotypic interactions to regulate enhancer assembly in response to signals. Our study supports the notion that alteration of the fine-tuned multivalent IDR-IDR interactions might underlie AR-related human pathologies, thereby providing novel molecular insights for potential therapeutic strategies to treat prostate cancer and other AR-involved diseases by targeting AR multivalent interactions.", "doi": "10.1101/2022.10.28.514297", "publication_date": "2022-10-30" }, { "id": "authors:tyx9y-jnd79", "collection": "authors", "collection_id": "tyx9y-jnd79", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210821-004311309", "type": "article", "title": "Tuning levels of low-complexity domain interactions to modulate endogenous oncogenic transcription", "author": [ { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Graham", "given_name": "Thomas G. W.", "orcid": "0000-0001-5189-4313", "clpid": "Graham-Thomas-G-W" }, { "family_name": "Dugast-Darzacq", "given_name": "Claire", "orcid": "0000-0001-8602-3534", "clpid": "Dugast-Darzacq-Claire" }, { "family_name": "Dailey", "given_name": "Gina M.", "orcid": "0000-0002-8988-963X", "clpid": "Dailey-Gina-M" }, { "family_name": "Darzacq", "given_name": "Xavier", "orcid": "0000-0003-2537-8395", "clpid": "Darzacq-Xavier" }, { "family_name": "Tjian", "given_name": "Robert", "orcid": "0000-0003-0539-8217", "clpid": "Tjian-Robert" } ], "abstract": "Gene activation by mammalian transcription factors (TFs) requires multivalent interactions of their low-complexity domains (LCDs), but how such interactions regulate transcription remains unclear. It has been proposed that extensive LCD-LCD interactions culminating in liquid-liquid phase separation (LLPS) of TFs is the dominant mechanism underlying transactivation. Here, we investigated how tuning the amount and localization of LCD-LCD interactions in vivo affects transcription of endogenous human genes. Quantitative single-cell and single-molecule imaging reveals that the oncogenic TF EWS::FLI1 requires a narrow optimum of LCD-LCD interactions to activate its target genes associated with GGAA microsatellites. Increasing LCD-LCD interactions toward putative LLPS represses transcription of these genes in patient-derived cells. Likewise, ectopically creating LCD-LCD interactions to sequester EWS::FLI1 into a well-documented LLPS compartment, the nucleolus, inhibits EWS::FLI1-driven transcription and oncogenic transformation. Our findings show how altering the balance of LCD-LCD interactions can influence transcriptional regulation and suggest a potential therapeutic strategy for targeting disease-causing TFs.", "doi": "10.1016/j.molcel.2022.04.007", "issn": "1097-2765", "publisher": "Cell Press", "publication": "Molecular Cell", "publication_date": "2022-06-02", "series_number": "11", "volume": "82", "issue": "11", "pages": "2084-2097" }, { "id": "authors:1mtdx-p2151", "collection": "authors", "collection_id": "1mtdx-p2151", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20220509-155322997", "type": "monograph", "title": "Fixation Can Change the Appearance of Phase Separation in Living Cells", "author": [ { "family_name": "Irgen-Gioro", "given_name": "Shawn", "orcid": "0000-0001-8638-6191", "clpid": "Irgen-Gioro-Shawn" }, { "family_name": "Walling", "given_name": "Victoria", "clpid": "Walling-Victoria" }, { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" } ], "abstract": "Fixing cells with paraformaldehyde (PFA) is an essential step in numerous biological techniques as it is thought to preserve a snapshot of biomolecular transactions in living cells. Fixed cell imaging techniques such as immunofluorescence have been widely used to detect liquid-liquid phase separation (LLPS) in vivo. Here, we compared images, before and after fixation, of cells expressing intrinsically disordered proteins that are able to undergo LLPS. Surprisingly, we found that PFA fixation can both enhance and diminish putative LLPS behaviors. For specific proteins, fixation can even cause their droplet-like puncta to artificially appear in cells that do not have any detectable puncta in the live condition. Fixing cells in the presence of glycine, a molecule that modulates fixation rates, can reverse the fixation effect from enhancing to diminishing LLPS appearance. We further established a kinetic model of fixation in the context of dynamic protein-protein interactions. Simulations based on the model suggest that protein localization in fixed cells depends on an intricate balance of protein-protein interaction dynamics, the overall rate of fixation, and notably, the difference between fixation rates of different proteins. Our work reveals that PFA fixation changes the appearance of LLPS from living cells, presents a caveat in studying LLPS using fixation-based methods, and suggests a mechanism underlying the fixation artifact.", "doi": "10.1101/2022.05.06.490956", "publication_date": "2022-05-08" }, { "id": "authors:j246j-11b24", "collection": "authors", "collection_id": "j246j-11b24", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210902-234005496", "type": "article", "title": "TRIM8 modulates the EWS/FLI oncoprotein to promote survival in Ewing sarcoma", "author": [ { "family_name": "Seong", "given_name": "Bo Kyung A.", "orcid": "0000-0002-1398-6230", "clpid": "Seong-Bo-Kyung-A" }, { "family_name": "Dharia", "given_name": "Neekesh V.", "orcid": "0000-0001-5048-067X", "clpid": "Dharia-Neekesh-V" }, { "family_name": "Lin", "given_name": "Shan", "orcid": "0000-0002-9699-0250", "clpid": "Lin-Shan" }, { "family_name": "Donovan", "given_name": "Katherine A.", "orcid": "0000-0002-8539-5106", "clpid": "Donovan-Katherine-A" }, { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Robichaud", "given_name": "Amanda", "clpid": "Robichaud-Amanda" }, { "family_name": "Conway", "given_name": "Amy", "clpid": "Conway-Amy" }, { "family_name": "Hamze", "given_name": "Amanda", "clpid": "Hamze-Amanda" }, { "family_name": "Ross", "given_name": "Linda", "clpid": "Ross-Linda" }, { "family_name": "Alexe", "given_name": "Gabriela", "orcid": "0000-0002-5668-6297", "clpid": "Alexe-Gabriela" }, { "family_name": "Adane", "given_name": "Biniam", "clpid": "Adane-Biniam" }, { "family_name": "Nabet", "given_name": "Behnam", "orcid": "0000-0002-6738-4200", "clpid": "Nabet-Behnam" }, { "family_name": "Ferguson", "given_name": "Fleur M.", "orcid": "0000-0003-4091-7617", "clpid": "Ferguson-Fleur-M" }, { "family_name": "Stolte", "given_name": "Bj\u00f6rn", "clpid": "Stolte-Bj\u00f6rn" }, { "family_name": "Wang", "given_name": "Emily Jue", "clpid": "Wang-Emily-Jue" }, { "family_name": "Sun", "given_name": "Jialin", "clpid": "Sun-Jialin" }, { "family_name": "Darzacq", "given_name": "Xavier", "orcid": "0000-0003-2537-8395", "clpid": "Darzacq-Xavier" }, { "family_name": "Piccioni", "given_name": "Federica", "orcid": "0000-0003-1210-3210", "clpid": "Piccioni-Federica" }, { "family_name": "Gray", "given_name": "Nathanael S.", "orcid": "0000-0001-5354-7403", "clpid": "Gray-Nathanael-S" }, { "family_name": "Fischer", "given_name": "Eric S.", "orcid": "0000-0001-7337-6306", "clpid": "Fischer-Eric-S" }, { "family_name": "Stegmaier", "given_name": "Kimberly", "orcid": "0000-0003-0218-7895", "clpid": "Stegmaier-Kimberly" } ], "abstract": "Fusion-transcription factors (fusion-TFs) represent a class of driver oncoproteins that are difficult to therapeutically target. Recently, protein degradation has emerged as a strategy to target these challenging oncoproteins. The mechanisms that regulate fusion-TF stability, however, are generally unknown. Using CRISPR-Cas9 screening, we discovered tripartite motif-containing 8 (TRIM8) as an E3 ubiquitin ligase that ubiquitinates and degrades EWS/FLI, a driver fusion-TF in Ewing sarcoma. Moreover, we identified TRIM8 as a selective dependency in Ewing sarcoma compared with >700 other cancer cell lines. Mechanistically, TRIM8 knockout led to an increase in EWS/FLI protein levels that was not tolerated. EWS/FLI acts as a neomorphic substrate for TRIM8, defining the selective nature of the dependency. Our results demonstrate that fusion-TF protein stability is tightly regulated and highlight fusion oncoprotein-specific regulators as selective therapeutic targets. This study provides a tractable strategy to therapeutically exploit oncogene overdose in Ewing sarcoma and potentially other fusion-TF-driven cancers.", "doi": "10.1016/j.ccell.2021.07.003", "issn": "1535-6108", "publisher": "Cell Press", "publication": "Cancer Cell", "publication_date": "2021-09-13", "series_number": "9", "volume": "39", "issue": "9", "pages": "1262-1278" }, { "id": "authors:68y2p-bav50", "collection": "authors", "collection_id": "68y2p-bav50", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210902-234004411", "type": "article", "title": "Towards Decoding the Sequence-Based Grammar Governing the Functions of Intrinsically Disordered Protein Regions", "author": [ { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Mir", "given_name": "Mustafa", "orcid": "0000-0001-8280-2821", "clpid": "Mir-Mustafa" } ], "abstract": "A substantial portion of the proteome consists of intrinsically disordered regions (IDRs) that do not fold into well-defined 3D structures yet perform numerous biological functions and are associated with a broad range of diseases. It has been a long-standing enigma how different IDRs successfully execute their specific functions. Further putting a spotlight on IDRs are recent discoveries of functionally relevant biomolecular assemblies, which in some cases form through liquid-liquid phase separation. At the molecular level, the formation of biomolecular assemblies is largely driven by weak, multivalent, but selective IDR-IDR interactions. Emerging experimental and computational studies suggest that the primary amino acid sequences of IDRs encode a variety of their interaction behaviors. In this review, we focus on findings and insights that connect sequence-derived features of IDRs to their conformations, propensities to form biomolecular assemblies, selectivity of interaction partners, functions in the context of physiology and disease, and regulation of function. We also discuss directions of future research to facilitate establishing a comprehensive sequence-function paradigm that will eventually allow prediction of selective interactions and specificity of function mediated by IDRs.", "doi": "10.1016/j.jmb.2020.11.023", "issn": "0022-2836", "publisher": "Elsevier", "publication": "Journal of Molecular Biology", "publication_date": "2021-06-11", "series_number": "12", "volume": "433", "issue": "12", "pages": "Art. No. 166724" }, { "id": "authors:hzqf2-3ap31", "collection": "authors", "collection_id": "hzqf2-3ap31", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210902-234003301", "type": "article", "title": "Overcoming the bottleneck to widespread testing: a rapid review of nucleic acid testing approaches for COVID-19 detection", "author": [ { "family_name": "Esbin", "given_name": "Meagan N.", "orcid": "0000-0002-2157-3831", "clpid": "Esbin-Meagan-N" }, { "family_name": "Whitney", "given_name": "Oscar N.", "orcid": "0000-0002-4858-2615", "clpid": "Whitney-Oscar-N" }, { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Maurer", "given_name": "Anna", "orcid": "0000-0003-2768-7757", "clpid": "Maurer-Anna" }, { "family_name": "Darzacq", "given_name": "Xavier", "orcid": "0000-0003-2537-8395", "clpid": "Darzacq-Xavier" }, { "family_name": "Tjian", "given_name": "Robert", "orcid": "0000-0003-0539-8217", "clpid": "Tjian-Robert" } ], "abstract": "The current COVID-19 pandemic presents a serious public health crisis, and a better understanding of the scope and spread of the virus would be aided by more widespread testing. Nucleic-acid-based tests currently offer the most sensitive and early detection of COVID-19. However, the \"gold standard\" test pioneered by the U.S. Centers for Disease Control and Prevention takes several hours to complete and requires extensive human labor, materials such as RNA extraction kits that could become in short supply, and relatively scarce qPCR machines. It is clear that a huge effort needs to be made to scale up current COVID-19 testing by orders of magnitude. There is thus a pressing need to evaluate alternative protocols, reagents, and approaches to allow nucleic-acid testing to continue in the face of these potential shortages. There has been a tremendous explosion in the number of papers written within the first weeks of the pandemic evaluating potential advances, comparable reagents, and alternatives to the \"gold-standard\" CDC RT-PCR test. Here we present a collection of these recent advances in COVID-19 nucleic acid testing, including both peer-reviewed and preprint articles. Due to the rapid developments during this crisis, we have included as many publications as possible, but many of the cited sources have not yet been peer-reviewed, so we urge researchers to further validate results in their own laboratories. We hope that this review can urgently consolidate and disseminate information to aid researchers in designing and implementing optimized COVID-19 testing protocols to increase the availability, accuracy, and speed of widespread COVID-19 testing.", "doi": "10.1261/rna.076232.120", "pmcid": "PMC7297120", "issn": "1355-8382", "publisher": "RNA Society", "publication": "RNA", "publication_date": "2020-07", "series_number": "7", "volume": "26", "issue": "7", "pages": "771-783" }, { "id": "authors:vs74q-0ah73", "collection": "authors", "collection_id": "vs74q-0ah73", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210902-234002184", "type": "article", "title": "Impaired cell fate through gain-of-function mutations in a chromatin reader", "author": [ { "family_name": "Wan", "given_name": "Liling", "clpid": "Wan-Liling" }, { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Xuan", "given_name": "Fan", "orcid": "0000-0002-8070-3309", "clpid": "Xuan-Fan" }, { "family_name": "Liang", "given_name": "Angela", "clpid": "Liang-Angela" }, { "family_name": "Cui", "given_name": "Xiaodong", "clpid": "Cui-Xiaodong" }, { "family_name": "Gates", "given_name": "Leah", "clpid": "Gates-Leah" }, { "family_name": "Carroll", "given_name": "Thomas S.", "orcid": "0000-0001-9753-2385", "clpid": "Carroll-Thomas-S" }, { "family_name": "Li", "given_name": "Yuanyuan", "orcid": "0000-0002-0897-5270", "clpid": "Li-Yuanyuan" }, { "family_name": "Feng", "given_name": "Lijuan", "clpid": "Feng-Lijuan" }, { "family_name": "Chen", "given_name": "Guochao", "orcid": "0000-0003-4888-1786", "clpid": "Chen-Guochao" }, { "family_name": "Wang", "given_name": "Shu-Ping", "clpid": "Wang-Shu-Ping" }, { "family_name": "Ortiz", "given_name": "Michael V.", "orcid": "0000-0001-9379-314X", "clpid": "Ortiz-Michael-V" }, { "family_name": "Daley", "given_name": "Sara K.", "clpid": "Daley-Sara-K" }, { "family_name": "Wang", "given_name": "Xiaolu", "clpid": "Wang-Xiaolu" }, { "family_name": "Xuan", "given_name": "Hongwen", "clpid": "Xuan-Hongwen" }, { "family_name": "Kentsis", "given_name": "Alex", "orcid": "0000-0002-8063-9191", "clpid": "Kentsis-Alex" }, { "family_name": "Muir", "given_name": "Tom W.", "orcid": "0000-0001-9635-0344", "clpid": "Muir-Thomas-W" }, { "family_name": "Roeder", "given_name": "Robert G.", "orcid": "0000-0003-3865-8572", "clpid": "Roeder-Robert-G" }, { "family_name": "Li", "given_name": "Haitao", "orcid": "0000-0001-6741-293X", "clpid": "Li-Haitao" }, { "family_name": "Li", "given_name": "Wei", "clpid": "Li-Wei" }, { "family_name": "Tjian", "given_name": "Robert", "orcid": "0000-0003-0539-8217", "clpid": "Tjian-Robert" }, { "family_name": "Wen", "given_name": "Hong", "orcid": "0000-0001-8739-4572", "clpid": "Wen-Hong" }, { "family_name": "Allis", "given_name": "C. David", "orcid": "0000-0001-7589-713X", "clpid": "Allis-Charles-David" } ], "abstract": "Modifications of histone proteins have essential roles in normal development and human disease. Recognition of modified histones by 'reader' proteins is a key mechanism that mediates the function of histone modifications, but how the dysregulation of these readers might contribute to disease remains poorly understood. We previously identified the ENL protein as a reader of histone acetylation via its YEATS domain, linking it to the expression of cancer-driving genes in acute leukaemia. Recurrent hotspot mutations have been found in the ENL YEATS domain in Wilms tumour, the most common type of paediatric kidney cancer. Here we show, using human and mouse cells, that these mutations impair cell-fate regulation by conferring gain-of-function in chromatin recruitment and transcriptional control. ENL mutants induce gene-expression changes that favour a premalignant cell fate, and, in an assay for nephrogenesis using murine cells, result in undifferentiated structures resembling those observed in human Wilms tumour. Mechanistically, although bound to largely similar genomic loci as the wild-type protein, ENL mutants exhibit increased occupancy at a subset of targets, leading to a marked increase in the recruitment and activity of transcription elongation machinery that enforces active transcription from target loci. Furthermore, ectopically expressed ENL mutants exhibit greater self-association and form discrete and dynamic nuclear puncta that are characteristic of biomolecular hubs consisting of local high concentrations of regulatory factors. Such mutation-driven ENL self-association is functionally linked to enhanced chromatin occupancy and gene activation. Collectively, our findings show that hotspot mutations in a chromatin-reader domain drive self-reinforced recruitment, derailing normal cell-fate control during development and leading to an oncogenic outcome.", "doi": "10.1038/s41586-019-1842-7", "pmcid": "PMC7061414", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "2020-01-02", "series_number": "7788", "volume": "577", "issue": "7788", "pages": "121-126" }, { "id": "authors:qt1vb-8d218", "collection": "authors", "collection_id": "qt1vb-8d218", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210902-234001057", "type": "article", "title": "Imaging dynamic and selective low-complexity domain interactions that control gene transcription", "author": [ { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Dugast-Darzacq", "given_name": "Claire", "orcid": "0000-0001-8602-3534", "clpid": "Dugast-Darzacq-Claire" }, { "family_name": "Liu", "given_name": "Zhe", "orcid": "0000-0002-3592-3150", "clpid": "Liu-Zhe" }, { "family_name": "Dong", "given_name": "Peng", "orcid": "0000-0003-4946-1409", "clpid": "Dong-Peng" }, { "family_name": "Dailey", "given_name": "Gina M.", "clpid": "Dailey-Gina-M" }, { "family_name": "Cattoglio", "given_name": "Claudia", "orcid": "0000-0001-6100-0491", "clpid": "Cattoglio-Claudia" }, { "family_name": "Heckert", "given_name": "Alec", "orcid": "0000-0001-8748-6645", "clpid": "Hekert-Alec" }, { "family_name": "Banala", "given_name": "Sambashiva", "orcid": "0000-0002-9463-7664", "clpid": "Banala-Sambashiva" }, { "family_name": "Lavis", "given_name": "Luke", "orcid": "0000-0002-0789-6343", "clpid": "Lavis-Luke-D" }, { "family_name": "Darzacq", "given_name": "Xavier", "orcid": "0000-0003-2537-8395", "clpid": "Darzacq-Xavier" }, { "family_name": "Tjian", "given_name": "Robert", "orcid": "0000-0003-0539-8217", "clpid": "Tjian-Robert" } ], "abstract": "INTRODUCTION. DNA binding transcription factors (TFs) are quintessential regulators of eukaryotic gene expression. Early studies of TFs revealed their well-structured DNA binding domains (DBDs) and identified functionally critical activation domains (ADs) required for transcription. It later became evident that many ADs contain intrinsically disordered low-complexity sequence domains (LCDs), but how LCDs activate transcription has remained unclear. Although it is known that transcriptional activation by LCDs requires selective interaction with binding partners, it has been challenging to directly measure selective LCD-LCD recognition in vivo and unravel its mechanism of action. \n\nRATIONALE. Traditional biochemical reconstitution and genetics studies have identified most of the molecular players central to transcription regulation. However, the mechanism by which weak, dynamic protein-protein interactions drive gene activation in living cells has remained unknown. Advances in live-cell single-molecule imaging have opened a new frontier for studying transcription in vivo. In this study, we used synthetic LacO (Lac operator) arrays as well as endogenous GGAA microsatellite loci to study LCD-LCD interactions of TFs such as EWS/FLI1, TAF15, and Sp1 in live cells. To probe the dynamic behavior of TF LCDs at target genomic loci, we have combined CRISPR-Cas9 genome editing, mutagenesis, gene activation, cell transformation assays, and various high-resolution imaging approaches including fluorescence correlation spectroscopy, fluorescence recovery after photobleaching, lattice light-sheet microscopy, three-dimensional DNA fluorescence in situ hybridization, and live-cell single-particle tracking. \n\nRESULTS. Live-cell single-molecule imaging revealed that TF LCDs interact to form local high-concentration hubs at both synthetic DNA arrays and endogenous genomic loci. TF LCD hubs stabilize DNA binding, recruit RNA polymerase II (RNA Pol II), and activate transcription. LCD-LCD interactions within hubs are highly dynamic (seconds to minutes), selective for binding partners, and differentially sensitive to disruption by hexanediols. These findings suggest that under physiological conditions, rapid, reversible, and selective multivalent LCD-LCD interactions occur between TFs and the RNA Pol II machinery to activate transcription. We observed formation of functional TF LCD hubs at a wide range of intranuclear TF concentrations. Although we detected apparent liquid-liquid phase separation with gross overexpression of LCDs, transcriptionally competent TF LCD hubs were observed at physiological TF levels at endogenous chromosomal loci in the absence of detectable phase separation. In addition, mutagenesis, gene expression, and cell transformation assays in Ewing's sarcoma cells revealed a functional link between LCD-LCD interactions, transactivation capacity, and oncogenic potential. \n\nCONCLUSION. The use of various imaging methods in live cells powerfully complements in vitro studies and provides new insights into the nature of LCD interactions and their role in gene regulation. We propose that transactivation domains function by forming local high-concentration hubs of TFs via dynamic, multivalent, and specific LCD-LCD interactions. It also seems likely that weak, dynamic, and transient contacts between TFs play a role in disease-causing dysregulation of gene expression (i.e., EWS/FLI1 in Ewing's sarcoma), suggesting that LCD-LCD interactions may represent a new class of viable drug targets. Although we examined a small subset of TF LCDs, the principles uncovered regarding the dynamics and mechanisms driving LCD-LCD interactions may be applicable to other classes of proteins and biomolecular interactions occurring in many cell types.", "doi": "10.1126/science.aar2555", "pmcid": "PMC6961784", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "2018-07-27", "series_number": "6400", "volume": "361", "issue": "6400", "pages": "Art. No. eaar2555" }, { "id": "authors:58jk3-a5p46", "collection": "authors", "collection_id": "58jk3-a5p46", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210902-233959965", "type": "article", "title": "Mechanism of Transcriptional Bursting in Bacteria", "author": [ { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Chen", "given_name": "Chongyi", "orcid": "0000-0002-0504-5369", "clpid": "Chen-Chongyi" }, { "family_name": "Ge", "given_name": "Hao", "orcid": "0000-0003-0404-5268", "clpid": "Ge-Hao" }, { "family_name": "Xie", "given_name": "X.\u00a0Sunney", "clpid": "Xie-X-Sunney" } ], "abstract": "Transcription of highly expressed genes has been shown to occur in stochastic bursts. But the origin of such ubiquitous phenomenon has not been understood. Here, we present the mechanism in bacteria. We developed a high-throughput, in vitro, single-molecule assay to follow transcription on individual DNA templates in real time. We showed that positive supercoiling buildup on a DNA segment by transcription slows down transcription elongation and eventually stops transcription initiation. Transcription can be resumed upon gyrase binding to the DNA segment. Furthermore, using single-cell mRNA counting fluorescence in situ hybridization (FISH), we found that duty cycles of transcriptional bursting depend on the intracellular gyrase concentration. Together, these findings prove that transcriptional bursting of highly expressed genes in bacteria is primarily caused by reversible gyrase dissociation from and rebinding to a DNA segment, changing the supercoiling level of the segment.", "doi": "10.1016/j.cell.2014.05.038", "pmcid": "PMC4105854", "issn": "0092-8674", "publisher": "Cell Press", "publication": "Cell", "publication_date": "2014-07-17", "series_number": "2", "volume": "158", "issue": "2", "pages": "314-326" }, { "id": "authors:ar12p-fym48", "collection": "authors", "collection_id": "ar12p-fym48", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210902-233958840", "type": "article", "title": "Probing Allostery Through DNA", "author": [ { "family_name": "Kim", "given_name": "Sangjin", "clpid": "Kim-Sangjin" }, { "family_name": "Brostr\u00f6mer", "given_name": "Erik", "clpid": "Brostr\u00f6mer-Erik" }, { "family_name": "Xing", "given_name": "Dong", "clpid": "Xing-Dong" }, { "family_name": "Jin", "given_name": "Jianshi", "clpid": "Jin-Jianshi" }, { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Ge", "given_name": "Hao", "orcid": "0000-0003-0404-5268", "clpid": "Ge-Hao" }, { "family_name": "Wang", "given_name": "Siyuan", "orcid": "0000-0002-8110-5714", "clpid": "Wang-Siyuan" }, { "family_name": "Gu", "given_name": "Chan", "clpid": "Gu-Chan" }, { "family_name": "Yang", "given_name": "Lijiang", "clpid": "Yang-Lijiang" }, { "family_name": "Gao", "given_name": "Yi Qin", "clpid": "Gao-Yi-Qin" }, { "family_name": "Su", "given_name": "Xiao-dong", "clpid": "Su-Xiao-Dong" }, { "family_name": "Sun", "given_name": "Yujie", "clpid": "Sun-Yujie" }, { "family_name": "Xie", "given_name": "X. Sunney", "clpid": "Xie-X-Sunney" } ], "abstract": "Allostery is well documented for proteins but less recognized for DNA-protein interactions. Here, we report that specific binding of a protein on DNA is substantially stabilized or destabilized by another protein bound nearby. The ternary complex's free energy oscillates as a function of the separation between the two proteins with a periodicity of ~10 base pairs, the helical pitch of B-form DNA, and a decay length of ~15 base pairs. The binding affinity of a protein near a DNA hairpin is similarly dependent on their separation, which\u2014together with molecular dynamics simulations\u2014suggests that deformation of the double-helical structure is the origin of DNA allostery. The physiological relevance of this phenomenon is illustrated by its effect on gene expression in live bacteria and on a transcription factor's affinity near nucleosomes.", "doi": "10.1126/science.1229223", "pmcid": "PMC3586787", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "2013-02-15", "series_number": "6121", "volume": "339", "issue": "6121", "pages": "816-819" }, { "id": "authors:8hh97-tgg88", "collection": "authors", "collection_id": "8hh97-tgg88", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210902-233957747", "type": "article", "title": "Ground-State Depletion Microscopy: Detection Sensitivity of Single-Molecule Optical Absorption at Room Temperature", "author": [ { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Min", "given_name": "Wei", "orcid": "0000-0003-2570-3557", "clpid": "Min-Wei" }, { "family_name": "Xie", "given_name": "X. Sunney", "clpid": "Xie-X-Sunney" } ], "abstract": "Optical studies of single molecules in ambient environments, which have led to broad applications, are primarily based on fluorescence detection. Direct detection of optical absorption with single-molecule sensitivity at room temperature is difficult because absorption is not a background-free measurement and is often complicated by sample scattering. Here we report ground-state depletion microscopy for ultrasensitive detection of absorption contrast. We image 20 nm gold nanoparticles as an initial demonstration of this microscopy. We then demonstrate the detection of an absorption signal from a single chromophore molecule at room temperature. This is accomplished by using two tightly focused collinear continuous-wave laser beams at different wavelengths, both within a molecular absorption band, one of which is intensity modulated at a high frequency (>MHz). The transmission of the other beam is found to be modulated at the same frequency due to ground state depletion. The signal of single chromophore molecules scanned across the common laser foci can be detected with shot-noise limited sensitivity. This measurement represents the ultimate detection sensitivity of nonlinear optical spectroscopy at room temperature.", "doi": "10.1021/jz1014289", "issn": "1948-7185", "publisher": "American Chemical Society", "publication": "Journal of Physical Chemistry Letters", "publication_date": "2010-12-02", "series_number": "23", "volume": "1", "issue": "23", "pages": "3316-3322" }, { "id": "authors:pd0vh-g2b62", "collection": "authors", "collection_id": "pd0vh-g2b62", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210902-233956011", "type": "article", "title": "Label-free imaging of heme proteins with two-photon excited photothermal lens microscopy", "author": [ { "family_name": "Lu", "given_name": "Sijia", "clpid": "Lu-Sijia" }, { "family_name": "Min", "given_name": "Wei", "orcid": "0000-0003-2570-3557", "clpid": "Min-Wei" }, { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Holtom", "given_name": "Gary R.", "clpid": "Holtom-Gary-R" }, { "family_name": "Xie", "given_name": "X. Sunney", "clpid": "Xie-X-Sunney" } ], "abstract": "Heme proteins, such as hemoglobins and cytochromes, play important roles in various biological processes. Here we employ the two-photon excited photothermal effect as a contrast mechanism to map heme proteins distribution. Particularly, both a thermal lens scheme and a high-frequency modulation are utilized to enhance the signal-to-noise ratio. We demonstrate label-free imaging of individual red blood cells, subcellular distribution of cytochromes in live mammalian cells, and the microvascular networks in mouse ear tissue and in a zebrafish gill.", "doi": "10.1063/1.3308485", "issn": "0003-6951", "publisher": "American Institute of Physics", "publication": "Applied Physics Letters", "publication_date": "2010-03-15", "series_number": "11", "volume": "96", "issue": "11", "pages": "Art. No. 113701" }, { "id": "authors:xxn52-m4c03", "collection": "authors", "collection_id": "xxn52-m4c03", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210902-233954894", "type": "article", "title": "Imaging chromophores with undetectable fluorescence by stimulated emission microscopy", "author": [ { "family_name": "Min", "given_name": "Wei", "orcid": "0000-0003-2570-3557", "clpid": "Min-Wei" }, { "family_name": "Lu", "given_name": "Sijia", "clpid": "Lu-Sijia" }, { "family_name": "Chong", "given_name": "Shasha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Roy", "given_name": "Rahul", "clpid": "Roy-Rahul" }, { "family_name": "Holtom", "given_name": "Gary R.", "clpid": "Holtom-Gary-R" }, { "family_name": "Xie", "given_name": "X. Sunney", "clpid": "Xie-X-Sunney" } ], "abstract": "Fluorescence, that is, spontaneous emission, is generally more sensitive than absorption measurement, and is widely used in optical imaging. However, many chromophores, such as haemoglobin and cytochromes, absorb but have undetectable fluorescence because the spontaneous emission is dominated by their fast non-radiative decay. Yet the detection of their absorption is difficult under a microscope. Here we use stimulated emission, which competes effectively with the nonradiative decay, to make the chromophores detectable, and report a new contrast mechanism for optical microscopy. In a pump\u2013probe experiment, on photoexcitation by a pump pulse, the sample is stimulated down to the ground state by a time-delayed probe pulse, the intensity of which is concurrently increased. We extract the miniscule intensity increase with shot-noise-limited sensitivity by using a lock-in amplifier and intensity modulation of the pump beam at a high megahertz frequency. The signal is generated only at the laser foci owing to the nonlinear dependence on the input intensities, providing intrinsic three-dimensional optical sectioning capability. In contrast, conventional one-beam absorption measurement exhibits low sensitivity, lack of three-dimensional sectioning capability, and complication by linear scattering of heterogeneous samples. We demonstrate a variety of applications of stimulated emission microscopy, such as visualizing chromoproteins, non-fluorescent variants of the green fluorescent protein, monitoring lacZ gene expression with a chromogenic reporter, mapping transdermal drug distributions without histological sectioning, and label-free microvascular imaging based on endogenous contrast of haemoglobin. For all these applications, sensitivity is orders of magnitude higher than for spontaneous emission or absorption contrast, permitting nonfluorescent reporters for molecular imaging.", "doi": "10.1038/nature08438", "issn": "0028-0836", "publisher": "Nature Publishing Group", "publication": "Nature", "publication_date": "2009-10-22", "series_number": "7267", "volume": "461", "issue": "7267", "pages": "1105-1109" }, { "id": "authors:j002j-efv70", "collection": "authors", "collection_id": "j002j-efv70", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210902-233953133", "type": "article", "title": "An Extensive Ylide Thermodynamic Stability Scale Predicted by First-Principle Calculations", "author": [ { "family_name": "Fu", "given_name": "Yao", "clpid": "Fu-Yao" }, { "family_name": "Wang", "given_name": "Hua-Jing", "clpid": "Wang-Hua-Jing" }, { "family_name": "Chong", "given_name": "Sha-Sha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Guo", "given_name": "Qing-Xiang", "clpid": "Guo-Qing-Xiang" }, { "family_name": "Liu", "given_name": "Lei", "clpid": "Liu-Lei" } ], "abstract": "Thermodynamic stabilities of ylides are measured by the ease of the carbanion formation through the removal of a proton from their precursors. A full-spectrum scale of ylide thermodynamic stability is important to understand the reactivities and selectivities in ylide chemistry. In the present study is reported the first theoretical protocol for predicting the acidities of structurally unrelated ylide precursors in DMSO whose reliability has been tested against almost all the available experimental data. The ONIOM/G3B3//HF//CPCM/Bondi method is found to be the optimal protocol to handle the N-, P-, and S-containing ylides, whereas the B3LYP/6-311+G(3df,2p)//B3LYP/6-31G(d)//HF//CPCM/Bondi method can be used to deal with those systems for which the ONIOM/G3B3 method is not feasible. Extensive calculations on about 80 experimentally characterized ylide precursors show that this theoretical protocol can reliably predict the pK_a values of diverse structurally unrelated ylide precursors in DMSO with an error bar of ca. 1.6\u22121.9 pK_a units. With the authorized theoretical protocol in hand, we have developed an extensive scale of ylide thermodynamic stability that may find applications in synthetic organic chemistry.", "doi": "10.1021/jo802128w", "issn": "0022-3263", "publisher": "American Chemical Society", "publication": "Journal of Organic Chemistry", "publication_date": "2009-01-16", "series_number": "2", "volume": "74", "issue": "2", "pages": "810-819" }, { "id": "authors:qx9w8-9vh75", "collection": "authors", "collection_id": "qx9w8-9vh75", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210902-233952044", "type": "article", "title": "Ring Opening versus Ring Expansion in Rearrangement of Bicyclic Cyclobutylcarbinyl Radicals", "author": [ { "family_name": "Shi", "given_name": "Jing", "clpid": "Shi-Jing" }, { "family_name": "Chong", "given_name": "Sha-Sha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Fu", "given_name": "Yao", "orcid": "0000-0003-2282-4839", "clpid": "Fu-Yao" }, { "family_name": "Guo", "given_name": "Qing-Xiang", "clpid": "Guo-Qing-Xiang" }, { "family_name": "Liu", "given_name": "Lei", "clpid": "Liu-Lei" } ], "abstract": "Ring opening and expansion of multicyclic cyclobutylcarbinyl radicals provides an appealing method for the construction of heavily substituted ring systems in a stereocontrollable fashion. Here we conducted the first, systematic study on the regioselectivity in the rearrangement of various synthetically relevant cyclobutylcarbinyl radicals. It was found that a two-layer ONIOM method, namely ONIOM(QCISD(T)/6-311+G(2d,2p):B3LYP/6-311+G(2df,2p)), could accurately predict the free energy barriers of the ring openings of cyclobutylcarbinyl radicals with a precision of 0.3 kcal/mol. By using this powerful tool we found that the regiochemistry for the ring opening of monocyclic cyclobutylcarbinyl radicals could be easily predicted by the relative stability of the two possible carbon radical products. A linear correlation was found between the activation and reaction free energies. This observation indicated that the ring opening of cyclobutylcarbinyl radicals was strongly affected by the thermodynamic factors. On the basis of the above results we extended our study to the rearrangement of bicyclic cyclobutylcarbinyl radicals that could undergo both ring opening and expansion. It was found that for bicyclic cyclobutylcarbinyl radicals whose radical center was located at the bridge methyl group, ring expansion was the favored rearrangement pathway unless a strongly radical-stabilizing substituent was placed in the cyclobutyl ring adjacent to the bridge methyl group. On the other hand, for bicyclic cyclobutylcarbinyl radicals whose radical center was located at the 2-position, ring opening was the favored rearrangement pathway unless a strongly radical-stabilizing substituent was placed in the cyclobutyl ring at the bridge position.", "doi": "10.1021/jo702164r", "issn": "0022-3263", "publisher": "American Chemical Society", "publication": "Journal of Organic Chemistry", "publication_date": "2008-02-01", "series_number": "3", "volume": "73", "issue": "3", "pages": "974-982" }, { "id": "authors:hex91-0vq73", "collection": "authors", "collection_id": "hex91-0vq73", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20210902-233950994", "type": "article", "title": "O\u2212H Bond Dissociation Enthalpies of Oximes:\u00a0 A Theoretical Assessment and Experimental Implications", "author": [ { "family_name": "Chong", "given_name": "Sha-Sha", "orcid": "0000-0002-5372-311X", "clpid": "Chong-Shasha" }, { "family_name": "Fu", "given_name": "Yao", "orcid": "0000-0003-2282-4839", "clpid": "Fu-Yao" }, { "family_name": "Liu", "given_name": "Lei", "clpid": "Liu-Lei" }, { "family_name": "Guo", "given_name": "Qing-Xiang", "clpid": "Guo-Qing-Xiang" } ], "abstract": "By using a multilayer composite ab initio method ONION-G3B3, we calculated O\u2212H bond dissociation enthalpies (BDEs) of 58 oximes that were measured experimentally. Experimental BDEs derived from thermal decomposition kinetics and calorimetric measurements were found to be consistent with the theory. However, the electrochemical method was found to give questionably high BDEs possibly due to errors in the measurement of pK_a's or redox potentials. Subsequently, the performances of a variety of DFT functionals including B3LYP, B3P86, B3PW91, BHandH, BHandHLYP, BMK, PBE1PBE, MPW1KCIS, mPWPW91, MPW1B95, and MPW1K were tested to calculate oxime O\u2212H BDEs, where ROBHandHLYP was found to be the most accurate. By using this method, we calculated O\u2212H BDEs of over 140 oximes in a systematic fashion. All of the calculated O\u2212H BDEs fell in the range from 76.8 to 89.8 kcal/mol. An amino group on the azomethine carbon was found to strengthen the O\u2212H bond, whereas bulky alkyl substituents on oximes decreased O\u2212H BDEs due to their large steric-strain-relieving effects in the process of O\u2212H bond cleavage. Para substituents had little effect on the BDEs of benzaldoximes and phenyl methyl ketoximes. Finally, on the basis of a spin distribution calculation, aryl-, alkyl-, and carbonyl-substituted iminoxyl radicals were found to be \u03c3-radicals, whereas amino-substituted iminoxyl radicals were of \u03c0-structure.", "doi": "10.1021/jp075699a", "issn": "1089-5639", "publisher": "American Chemical Society", "publication": "Journal of Physical Chemistry A", "publication_date": "2007-12-20", "series_number": "50", "volume": "111", "issue": "50", "pages": "13112-13125" } ]