Real-time visualization of reconstituted transcription reveals RNA polymerase II activation mechanisms at single promoters

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2025, Номер unknown

Опубликована: Янв. 6, 2025

RNA polymerase II (RNAPII) is regulated by sequence-specific transcription factors (TFs) and the pre-initiation complex (PIC): TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, Mediator. TFs Mediator contain intrinsically-disordered regions (IDRs) form phase-separated condensates, but how IDRs control RNAPII function remains poorly understood. Using purified PIC factors, we developed a Real-time In-vitro Fluorescence Transcription assay (RIFT) for second-by-second visualization of at hundreds promoters simultaneously. We show rapid activation IDR-dependent, without condensate formation. For example, MED1-IDR can functionally replace native TF, activating with similar (not identical) kinetics; however, squelches as condensate, activates single-protein. cooperatively activate bursting re-initiation surprisingly, drive TF-promoter recruitment, TF-DNA binding. Collectively, RIFT addressed questions largely intractable cell-based methods, yielding mechanistic insights about IDRs, enhancer-promoter communication, that complement live-cell imaging data.

Язык: Английский

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Identification of molecular determinants of gene-specific bursting patterns by high-throughput imaging screens

Molecular Cell, Год журнала: 2025, Номер unknown

Опубликована: Фев. 1, 2025

Язык: Английский

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Comparative single-cell analysis of transcriptional bursting reveals the role of genome organization in de novo transcript origination

Proceedings of the National Academy of Sciences, Год журнала: 2025, Номер 122(18)

Опубликована: Апрель 30, 2025

Spermatogenesis is a key developmental process underlying the origination of newly evolved genes. However, rapid cell type–specific transcriptomic divergence Drosophila germline has posed significant technical barrier for comparative single-cell RNA-sequencing studies. By quantifying surprisingly strong correlation between species- and in three closely related species, we apply statistical procedure to identify core set 198 genes that are highly predictive type identity while remaining robust species-specific differences span over 25 30 My evolution. We then utilize classifications based on 198-gene show how transcriptional increases throughout spermatogenic time. After validating these cross-species using RNA fluorescence situ hybridization imaging, investigate influence genome organization molecular evolution spermatogenesis vis-a-vis bursting. first altering burst size contributes premeiotic transcription bursting frequency postmeiotic expression. report global autosomal vs. X chromosomal may arise stage preceding full testis organogenesis by showing evolutionarily conserved decreases X-linked kinetics all examined somatic types. Finally, provide evidence supporting cultivator model de novo gene demonstrating appearance testis-specific transcripts potentially provides short-range regulation neighboring genes’ properties during stages spermatogenesis.

Язык: Английский

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Regulation of RNA polymerase II transcription through re-initiation and bursting

Molecular Cell, Год журнала: 2025, Номер 85(10), С. 1907 - 1919

Опубликована: Май 1, 2025

Язык: Английский

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Doxorubicin resistance involves modulation of interferon signaling, transcriptional bursting, and gene co-expression patterns of U-ISGF3-related genes

Neoplasia, Год журнала: 2024, Номер 58, С. 101071 - 101071

Опубликована: Окт. 13, 2024

Язык: Английский

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Targeting lysine acetylation readers and writers

Nature Reviews Drug Discovery, Год журнала: 2024, Номер unknown

Опубликована: Ноя. 21, 2024

Язык: Английский

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