Raicu Ana-Maria, Castanheira Patricia, Arnosti David N
Cell and Molecular Biology Program, Michigan State University, East Lansing, MI, 48824, USA.
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI, 48824, USA.
G3 (Bethesda). 2024 Oct 4;14(12). doi: 10.1093/g3journal/jkae238.
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.
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共抑制因子，参与调控数千个基因的表达。脊椎动物谱系和果蝇属独立经历了一次Rb基因复制事件，导致几种Rb旁系同源物的表达，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CRISPR干扰系统来确定Rb家族中旁系同源性的重要性。我们构建了与果蝇Rbf1和Rbf2旁系同源物的dCas9融合体，并将它们应用于体内基因启动子，在其天然染色质环境中对它们进行研究。通过使用转录以及敏感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旁系同源物起“软抑制因子”的作用，并且具有高度上下文特异性的活性。我们在细胞培养中将靶向内源基因与报告基因进行比较，发现活性存在显著差异，强调了在生理相关背景下使用CRISPR干扰效应物来鉴定旁系同源物特异性活性的重要性。我们的研究揭示了生物体中Rb介导的转录调控的复杂性，并为果蝇未来的CRISPR干扰发展奠定了基础。
