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

Abstract

Retinoblastoma tumor suppressor proteins are highly conserved transcriptional corepressors, regulating the key transition from G1 to S phase of the cell cycle. The mammalian Rb family is composed of Rb, p107, and p130, which possess both overlapping and unique roles in gene regulation. Likewise, the Drosophila lineage experienced a gene duplication event, leading to the expression of the Rbf1 and Rbf2 paralogs. To uncover the significance of the multiplicity of the Rb family, and how gene regulatory roles have been apportioned between the family members, we made use of the CRISPRi system. We engineered dCas9 fusions to Rbf1 and Rbf2, and deployed them to gene promoters in developing Drosophila tissue to study their relative impact on target promoters. On some genes, both Rbf1 and Rbf2 are able to mediate potent repression, and this happens in a distance-dependent manner. In general, a greater repression effect is noted for promoter-proximal sites, suggesting that Rb proteins have short-range effects, and may directly impact function of the basal transcriptional machinery. Depending on context, Rbf1 or Rbf2 was more potent, suggesting that the paralogs possess non-identical functions, possibly due to primary sequence divergence. Notably, with this dCas9 tethering system, an Rbf1 mutant lacking the entire pocket domain, that has been generally assumed to be necessary for function, retained repression activity. Significantly, direct comparison of Rb activity on endogenous genes and transiently transfected reporters showed that only qualitative, but not key quantitative aspects of repression were conserved, indicating that the native chromatin environment is essential for understanding the context-specific effects of Rb activity. The data presented here points to the complexity of Rb-mediated transcriptional regulation in a living organism, undoubtedly impacted by the different promoter landscapes and the evolution of the Rb proteins themselves.

A regulatory role for the unstructured C-terminal domain of the CtBP transcriptional corepressor

“…Targeting the InR promoter produced adult wings with mild phenotypes, similar to those produced with the non-targeting QUAS gRNA control, so this effect is difficult to distinguish from a mild overexpression phenotype rather than specific InR targeting ( Figure 2C ). Clearly, positioning the CtBP chimeras near the InR transcriptional start site does not strongly affect the wing, although we know that positioning dCas9-Rb chimeras at this promoter does impact development and transcription [19]. This distinct effect is consistent with CtBP promoter selectivity, a property illustrated from recent high-throughput assays [16].…”

“…Here, we detail the effects of targeting the E2F2/Mpp6 bidirectional promoter, the insulin receptor ( InR ) promoter, and the promoter of Acf , a nucleosome remodeling subunit ( Figure 2 ). Targeting CtBP(S) to the divergent E2F2/Mpp6 promoter produced small wings with severe morphological defects, similar to that seen with dCas9-Rb fusions ( Figure 2B ) [19]. Intriguingly, CtBP(L) did not produce this phenotype, but instead produced much milder effects, including wings with ectopic veins and supernumerary bristles ( Figure 2B ).…”

“…These gRNAs were obtained as fly lines from the Bloomington Drosophila Stock Center ( Supplementary Table 1 ). We previously tested dCas9-Rb chimeras in L3 discs, where we observed gene-specific effects after targeting ∼30 different gene promoters; here, we targeted many of the same promoters with the CtBP isoforms ( Supplementary Table 1 ) [19].…”

“…We presume that ectopic CtBP, even when fused to dCas9, may interact with diverse endogenous CtBP binding sites on the genome, leading to these mild phenotypes. The QUAS control gRNAs used here did not produce phenotypes with dCas9-Rb corepressor fusions tested previously, so the phenotypic effect is CtBP-specific [19].…”

“…Effects on E2F2 were more modest, with only ∼10% decrease in E2F2 expression resulting from targeting by dCas9 and dCas9-CtBP(L), and no statistically significant change after targeting with dCas9-CtBP(S) ( Figure 3B ). The greater impact on Mpp6 is likely a reflection of the inherent short-range of action of many transcriptional repressors and corepressors, which may influence chromatin structure over a span of a nucleosome [19, 22].…”