Retinoblastoma protein activity revealed by CRISPRi study of divergent Rbf1 and Rbf2 paralogs.

IF 2.1 3区 生物学 Q3 GENETICS & HEREDITY G3: Genes|Genomes|Genetics Pub Date : 2024-10-04 DOI:10.1093/g3journal/jkae238
Ana-Maria Raicu, Patricia Castanheira, David N Arnosti
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Abstract

Retinoblastoma tumor suppressor proteins (Rb) are highly conserved metazoan transcriptional corepressors involved in regulating the expression of thousands of genes. The vertebrate lineage and the Drosophila genus independently experienced an Rb gene duplication event, leading to the expression of several Rb paralogs whose unique and redundant roles in gene regulation remain to be fully explored. Here, we used a novel CRISPRi system in Drosophila to identify the significance of paralogy in the Rb family. We engineered dCas9 fusions to the fly Rbf1 and Rbf2 paralogs and deployed them to gene promoters in vivo, studying them in their native chromatin context. By directly querying the in vivo response of dozens of genes to Rbf1 and Rbf2 targeting, using both transcriptional as well as sensitive developmental readouts, we find that Rb paralogs function as "soft repressors" and have highly context-specific activities. Our comparison of targeting endogenous genes to reporter genes in cell culture identified striking differences in activity, underlining the importance of using CRISPRi effectors in a physiologically relevant context to identify paralog-specific activities. Our study uncovers the complexity of Rb-mediated transcriptional regulation in a living organism, and serves as a stepping stone for future CRISPRi development in Drosophila.

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通过对不同的 Rbf1 和 Rbf2 paralogs 进行 CRISPRi 研究,揭示视网膜母细胞瘤蛋白的活性。
视网膜母细胞瘤肿瘤抑制蛋白(Rb)是高度保守的后生动物转录核心抑制因子,参与调控数千个基因的表达。脊椎动物系和果蝇属独立经历了一次 Rb 基因复制事件,导致多个 Rb 旁系亲属的表达,它们在基因调控中的独特和冗余作用仍有待充分探索。在这里,我们在果蝇中使用了一种新型的 CRISPRi 系统来鉴定 Rb 家族中旁系亲属的重要性。我们将 dCas9 融合到果蝇的 Rbf1 和 Rbf2 旁系亲属上,并将它们部署到体内的基因启动子上,在它们的原生染色质上下文中对它们进行研究。通过使用转录和灵敏的发育读数直接查询数十个基因对 Rbf1 和 Rbf2 靶向的体内反应,我们发现 Rb 旁系亲属作为 "软抑制因子 "发挥作用,并具有高度特异性的活性。我们对细胞培养中靶向内源基因和报告基因进行了比较,发现了活性上的显著差异,强调了在生理相关背景下使用 CRISPRi 效应子来鉴定旁系亲属特异性活性的重要性。我们的研究揭示了 Rb 介导的转录调控在活生物体中的复杂性,并为果蝇未来的 CRISPRi 开发奠定了基础。
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来源期刊
G3: Genes|Genomes|Genetics
G3: Genes|Genomes|Genetics GENETICS & HEREDITY-
CiteScore
5.10
自引率
3.80%
发文量
305
审稿时长
3-8 weeks
期刊介绍: G3: Genes, Genomes, Genetics provides a forum for the publication of high‐quality foundational research, particularly research that generates useful genetic and genomic information such as genome maps, single gene studies, genome‐wide association and QTL studies, as well as genome reports, mutant screens, and advances in methods and technology. The Editorial Board of G3 believes that rapid dissemination of these data is the necessary foundation for analysis that leads to mechanistic insights. G3, published by the Genetics Society of America, meets the critical and growing need of the genetics community for rapid review and publication of important results in all areas of genetics. G3 offers the opportunity to publish the puzzling finding or to present unpublished results that may not have been submitted for review and publication due to a perceived lack of a potential high-impact finding. G3 has earned the DOAJ Seal, which is a mark of certification for open access journals, awarded by DOAJ to journals that achieve a high level of openness, adhere to Best Practice and high publishing standards.
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