New insights into genome folding by loop extrusion from inducible degron technologies

Nature Reviews Genetics, Год журнала: 2022, Номер 24(2), С. 73 - 85

Опубликована: Сен. 30, 2022

Язык: Английский

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Non-canonical functions of UHRF1 maintain DNA methylation homeostasis in cancer cells

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Апрель 5, 2024

Abstract DNA methylation is an essential epigenetic chromatin modification, and its maintenance in mammals requires the protein UHRF1. It yet unclear if UHRF1 functions solely by stimulating DNMT1, or it has important additional functions. Using degron alleles, we show that depletion causes a much greater loss of than DNMT1 depletion. This not caused passive demethylation as UHRF1-depleted cells proliferate more slowly DNMT1-depleted cells. Instead, bioinformatics, proteomics genetics experiments establish UHRF1, besides activating interacts with DNMT3A DNMT3B promotes their activity. In addition, antagonizes active TET2. Therefore, non-canonical roles contribute importantly to homeostasis; these findings have practical implications for epigenetics health disease.

Язык: Английский

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Genetic gradual reduction of OGT activity unveils the essential role of O-GlcNAc in the mouse embryo

PLoS Genetics, Год журнала: 2025, Номер 21(1), С. e1011507 - e1011507

Опубликована: Янв. 9, 2025

The reversible glycosylation of nuclear and cytoplasmic proteins (O-GlcNAcylation) is catalyzed by a single enzyme, namely O-GlcNAc transferase (OGT). mammalian Ogt gene X-linked, it essential for embryonic development the viability proliferating cells. We perturbed OGT’s function in vivo creating murine allelic series four amino acid substitutions, reducing catalytic activity to range degrees. severity lethality was proportional extent impairment catalysis, demonstrating that modification itself required early development. identified hypomorphic alleles perturb homeostasis while being compatible with embryogenesis. analysis transcriptomes mutant embryos at different developmental stages suggested sexually-dimorphic delay caused decrease O-GlcNAc. Furthermore, mild reduction enzymatic sufficient loosen silencing endogenous retroviruses .

Язык: Английский

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Rapid and efficient degradation of endogenous proteins in vivo identifies stage-specific roles of RNA Pol II pausing in mammalian development

Developmental Cell, Год журнала: 2022, Номер 57(8), С. 1068 - 1080.e6

Опубликована: Апрель 1, 2022

Язык: Английский

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Rapid and specific degradation of endogenous proteins in mouse models using auxin-inducible degrons

eLife, Год журнала: 2022, Номер 11

Опубликована: Июнь 23, 2022

Auxin-inducible degrons are a chemical genetic tool for targeted protein degradation and widely used to study function in cultured mammalian cells. Here, we develop CRISPR-engineered mouse lines that enable rapid highly specific of tagged endogenous proteins vivo. Most but not all cell types competent degradation. By combining ligand titrations with crosses generate animals different allelic combinations, show kinetics depend upon the dose protein, ligand, E3 ligase substrate receptor TIR1. Rapid condensin I II - two essential regulators mitotic chromosome structure revealed both complexes individually required division precursor lymphocytes, their differentiated peripheral lymphocyte derivatives. This generalisable approach provides unprecedented temporal control over models, implications studying biological pathways modelling drug activity tissues.

Язык: Английский

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Control of cardiac contractions using Cre-lox and degron strategies in zebrafish

Proceedings of the National Academy of Sciences, Год журнала: 2024, Номер 121(3)

Опубликована: Янв. 9, 2024

Cardiac contractions and hemodynamic forces are essential for organ development homeostasis. Control over cardiac can be achieved pharmacologically or optogenetically. However, these approaches lack specificity require direct access to the heart. Here, we compare two genetic control by modulating levels of sarcomeric protein Tnnt2a in zebrafish. We first recombine a newly generated tnnt2a floxed allele using multiple lines expressing Cre under cardiomyocyte-specific promoters, show that it does not recapitulate tnnt2a/silent heart mutant phenotype embryos. this early contraction defects is due, at least part, long half-life mRNA, which masks gene deletion effects until larval stages. then generate an endogenous Tnnt2a-eGFP fusion line use together with zGRAD system efficiently degrade all cardiomyocytes. Using single-cell transcriptomics, find depletion leads phenotypes similar those observed mutants, loss blood pericardial flow-dependent cell types. Furthermore, achieve conditional degradation splitting into fragments that, when combined cpFRB2-FKBP system, reassembled upon rapamycin treatment. Thus, enables non-invasive high spatial temporal will help further understand how they shape

Язык: Английский

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Auxin-Inducible Degron System: An Efficient Protein Degradation Tool to Study Protein Function

BioTechniques, Год журнала: 2023, Номер 74(4), С. 186 - 198

Опубликована: Апрель 1, 2023

Targeted protein degradation, with its rapid depletion kinetics, allows the measurement of acute changes in cell. The auxin-inducible degron (AID) system, rapidly degrades AID-tagged proteins only presence auxin. AID system being inducible makes study essential genes and dynamic processes like cell differentiation, cycle genome organization feasible. degradation has been adapted to yeast, protozoans, C. elegans, Drosophila, zebrafish, mouse mammalian lines. Using researchers have unveiled novel functions for at developmental stages that were previously difficult investigate due early lethality. This comprehensive review discusses development, advancements, applications drawbacks compares it other available systems.

Язык: Английский

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Degron tagging for rapid protein degradation in mice

Disease Models & Mechanisms, Год журнала: 2024, Номер 17(4)

Опубликована: Апрель 1, 2024

Degron tagging allows proteins of interest to be rapidly degraded, in a reversible and tuneable manner, response chemical stimulus. This provides numerous opportunities for understanding disease mechanisms, modelling therapeutic interventions constructing synthetic gene networks. In recent years, many laboratories have applied degron successfully cultured mammalian cells, spurred by rapid advances the fields genome editing targeted protein degradation. this At Glance article, we focus on efforts apply mouse models, discussing distinct set challenges posed vivo environment.

Язык: Английский

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State-of-the-art in engineering small molecule biosensors and their applications in metabolic engineering

SLAS TECHNOLOGY, Год журнала: 2023, Номер 29(2), С. 100113 - 100113

Опубликована: Ноя. 1, 2023

Genetically encoded biosensors are crucial for enhancing our understanding of how molecules regulate biological systems. Small molecule biosensors, in particular, help us understand the interaction between chemicals and processes. They also accelerate metabolic engineering by increasing screening throughput eliminating need sample preparation through traditional chemical analysis. Additionally, they offer significantly higher spatial temporal resolution cellular analyte measurements. In this review, we discuss recent progress vivo control systems-biosensor-based controllers-for engineering. We specifically explore protein-based that utilize less commonly exploited signaling mechanisms, such as protein stability induced degradation, compared to more prevalent transcription factor allosteric regulation mechanism. propose these lesser-used mechanisms will be significant eukaryotic systems slower-growing prokaryotic where turnover may facilitate rapid reliable measurement current state. Lastly, emphasize utilization cutting-edge state-of-the-art techniques development achieved rational design, directed evolution, collaborative approaches.

Язык: Английский

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Rapid and sustained degradation of the essential centrosome protein CEP192 in live mice using the AID2 system

Science Advances, Год журнала: 2025, Номер 11(9)

Опубликована: Фев. 28, 2025

Studying essential genes required for dynamic processes in live mice is challenging as genetic perturbations are irreversible and limited by slow protein depletion kinetics. The auxin-inducible degron (AID) system a powerful tool analyzing inducible loss vitro, but it toxic to mice. Here, we use an optimized second-generation AID achieve the conditional reversible of centrosomal CEP192 We show that auxin derivative 5-phenyl-indole-3-acetic acid well tolerated over 2 weeks drives near-complete degradation less than 1 hour vivo. did not affect centriole duplication decreased γ-tubulin recruitment centrosomes impairing mitotic spindle assembly. Sustained vivo led cell division failure death proliferative tissues. Thus, suited rapid and/or sustained study functions

Язык: Английский

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