Transcription factors (TFs) ensure this specificity by recognizing and binding to specific DNA sequences to modulate gene expression through their effector domains 4, 5. Regulation of gene expression requires that the transcriptional machinery be precisely and efficiently assembled at specific genomic loci 1, 2, 3. Overall, our work expands the gene activation toolbox for biomedical research. Quantitative imaging illustrated that nascent RNA-directed aTFs could induce the high-density assembly of coactivators at transcription sites, which may explain the larger transcriptional burst size induced by Narta. Moreover, Narta provides better activation potency of some expressed genes than CRISPRa and, when used in combination with CRISPRa, has an enhancing effect on gene activation. Importantly, the activation is reversible, tunable and specific. Using Narta, we demonstrate robust activation of a broad range of exogenous and endogenous genes in various cell types, including zebrafish embryos, mouse and human cells. In contrast to existing methods based on recruiting transcriptional modulators via DNA-binding proteins, we developed a strategy termed Narta ( nascent RNA-guided transcriptional activation) to achieve gene activation by recruiting artificial transcription factors (aTFs) to transcription sites through nascent RNAs of the target gene. Zhejiang Provincial Key Laboratory of Genetic & Developmental Disorders, Hangzhou 310058, China.Technologies for gene activation are valuable tools for the study of gene functions and have a wide range of potential applications in bioengineering and medicine. Zhejiang Laboratory for Systems & Precision Medicine, Zhejiang University Medical Center, Hangzhou 311121, China. Institute of Hematology, Zhejiang University & Zhejiang Engineering Laboratory for Stem Cell and Immunotherapy, Hangzhou 310058, China. Insititute of Translational Medicine, Zhejiang University, Hangzhou 310003, China. The Fourth Affiliated Hospital, Zhejiang University School of Medicine, Yiwu 322000, China. Institute of Natural Sciences, Shanghai Jiao Tong University, Shanghai 200240, China. School of Physics and Astronomy, Shanghai Jiao Tong University, Shanghai 200240, China. This error does not affect the conclusions of the article.ĭepartment of Cell Biology, and Bone Marrow Transplantation Center of the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China. ( J) Bar graph showing quantifications of transcriptional activity in daughter cells derived from three groups of mother cells with no, low and high transcription of LMNA gene, respectively. R 2 represents the coefficient of determination. Each dot represents a pair of the mother cell and one of its daughter cells, n = 90 pairs. ( I) Transcriptional activity of the mother cell and its daughter cells at LMNA loci was quantified to generate the scatter plot. ( H) Snapshots of the transcription activity of LMNA gene in HeLa cells at indicated time points through mitosis. ( G) Bar graph showing transcription states of two LMNA alleles in the same cell (case in A, n = 284 cells) or pairs of sister chromatids (case in D, n = 229 cells). Intensities of both transcription sites were plotted, with each dot representing a different pair of sister chromatids, n = 240 pairs. ( F) Sister chromatids of the same allele have correlated transcription activity. ( E) Line scan of intensity profiles showing dynamic labeling of DNA and nascent RNAs at corresponding sister chromatids pointed by arrows in (D). ( D) Representative images revealing three distinct transcriptional states of sister chromatid pairs identified by co-labeling of dCas9-GFP 14X and stdMCPtdTomato.
( B, C) Line scan of intensity profiles illustrating dynamic labeling of DNA (green) and nascent RNAs (red) of locus 1 (B) and locus 2 (C) indicated in (A). ( A) Fluorescent images of a cell showing DNA labeling of two LMNA alleles labeled by dCas9-GFP 14X and their transcriptional kinetics indicated by stdMCP-tdTomato at multiple time points. TriTag monitors allele-specific transcriptional bursting across the cell cycle.
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