Motivated by this hypothesis, we developed a gene activation tool by repurposing nascent RNAs to recruit abundant aTFs at their transcription sites. We reasoned that artificially concentrating high-density of transcriptional factors at transcription sites might be able to induce maximal activation of target genes. SEs are therefore able to drive higher levels of transcription than typical enhancers and thus regulate genes with especially important roles in cell identity 37, 38. The density of TFs and co-factors assembled at SEs is estimated to be approximately tenfold the density of the same component at typical enhancers. Furthermore, m6A-marked nascent RNAs (including pre-mRNAs and eRNAs) can recruit reader proteins to regulate transcription 35, 36. For example, eRNAs at enhancers can trap transcription factors such as yin and yang 1 (YY1) and enhance their binding to enhancers 34. BRD4 is recruited by recognizing the acetylated lysine mediated by p300 and promotes transcriptional elongation of SE-associated pluripotency genes 26, 29, 30, 31.Īctive enhancers and super-enhancers can produce transcripts termed enhancer RNAs (eRNAs), which have been suggested to bring transcriptional activators to the promoters of neighboring protein-coding genes 32, 33. The coactivator p300 and its paralog CREB-binding protein (CBP) active transcription by facilitating transcriptional machinery assembly and by acetylating histones and other factors 26, 27, 28. MED1 (also known as TRAP220) is a key subunit of the Mediator complex, which forms a bridge between the RNA polymerase II and transcriptional activators 24, 25. Super-enhancers (SEs) are clusters of enhancers that are occupied by exceptionally high densities of interacting factors, including TFs, co-factors (e.g., p300, BRD4, and MED1), RNA polymerase II, and non-coding RNAs 23. This could be highly desirable for some biological processes, such as the direct conversion of cell types and industrial applications 17, 18.Įnhancers are cis-regulatory elements (small segments of DNA) bound by TFs and other components of the transcription apparatus that modulate the expression of cell identity genes 19, 20, 21, 22. Thus, a new gene activation strategy based on this principle may effectively activate genes that are inaccessible to current CRISPRa methods. However, the use of multiple sgRNAs tiled across the target gene promoter can significantly improve CRISPRa efficiency, suggesting that recruiting as many TFs as possible may be crucial for activating gene expression with wider dynamic ranges 9, 10, 11, 16. The ability of CRISPRa to activate target genes by using single sgRNAs enables genome-wide transcriptional activation screens 14, 15. Based on this principle, the recently developed DNA-targeting platform, CRISPR-Cas9, has enabled the recruitment of artificial transcription factors (aTFs) to any specific genomic site to induce endogenous gene activation, termed CRISPR activation (CRISPRa) 9, 10, 11, 12, 13. TFs typically consist of two subdomains, a DNA-binding domain (DBD) and an activation domain (AD) which interacts with coactivator complexes to modulate transcriptional level 6, 7, 8. 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. 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.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |