Abstract
RBf2 is a recently evolved retinoblastoma family member in Drosophila that differs from RBf1, especially in the C-terminus. To investigate whether the unique features of RBf2 contribute to diverse roles in gene regulation, we performed chromatin immunoprecipitation sequencing for both RBf2 and RBf1 in embryos. A previous model for RB−E2F interactions suggested that RBf1 binds dE2F1 or dE2F2, whereas RBf2 is restricted to binding to dE2F2; however, we found that RBf2 targets approximately twice as many genes as RBf1. Highly enriched among the RBf2 targets were ribosomal protein genes. We tested the functional significance of this finding by assessing RBf activity on ribosomal protein promoters and the endogenous genes. RBf1 and RBf2 significantly repressed expression of some ribosomal protein genes, although not all bound genes showed transcriptional effects. Interestingly, many ribosomal protein genes are similarly targeted in human cells, indicating that these interactions may be relevant for control of ribosome biosynthesis and growth. We carried out bioinformatic analysis to investigate the basis for differential targeting by these two proteins and found that RBf2-specific promoters have distinct sequence motifs, suggesting unique targeting mechanisms. Association of RBf2 with these promoters appears to be independent of dE2F2/dDP, although promoters bound by both RBf1 and RBf2 require dE2F2/dDP. The presence of unique RBf2 targets suggest that evolutionary appearance of this corepressor represents the acquisition of potentially novel roles in gene regulation for the RB family.
Highlights
RBf2 is a recently evolved retinoblastoma family member in Drosophila that differs from RBf1, especially in the C-terminus
A previous model for RB2E2F interactions suggested that RBf1 binds dE2F1 or dE2F2, whereas RBf2 is restricted to binding to dE2F2; we found that RBf2 targets approximately twice as many genes as RBf1
More than half of RBf1 target genes were bound by dE2F1 or dE2F2, whereas less than half of RBf2 target genes were bound by any E2F factor (Figure 1A)
Summary
RBf2 is a recently evolved retinoblastoma family member in Drosophila that differs from RBf1, especially in the C-terminus. The Drosophila retinoblastoma family members RBf1 and RBf2 are structurally similar to the vertebrate proteins and possess functionally conserved activities in control of cell cycle and developmental genes (reviewed in Du and Pogoriler 2006). It contains a distinct C-terminus that lacks the conserved instability element, which has been shown to control both stability and activity of RBf1 (Acharya et al 2010; Raj et al 2012) Both RBf1 and RBf2 mediate transcriptional repression; these proteins have different inherent ability to interact with E2F proteins; RBf1 has been found to functionally interact with both the activator dE2F1 as well as the repressor dE2F2, whereas RBf2 is found to interact with dE2F2 but not dE2F1 (Frolov et al 2001; Stevaux et al 2002). Considering the evolutionary conservation within the genus Drosophila of RBf2 and the pervasive cooccupancy of RBf1 and RBf2, the modest phenotype of RBf2 mutants presents a conundrum regarding the selection pressure for this gene over large evolutionary periods within Drosophila
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