Diversification of Retinoblastoma Protein Function Associated with Cis and Trans Adaptations

Abstract

Retinoblastoma proteins are eukaryotic transcriptional corepressors that play central roles in cell cycle control, among other functions. Although most metazoan genomes encode a single retinoblastoma protein, gene duplications have occurred at least twice: in the vertebrate lineage, leading to Rb, p107, and p130, and in Drosophila, an ancestral Rbf1 gene and a derived Rbf2 gene. Structurally, Rbf1 resembles p107 and p130, and mutation of the gene is lethal. Rbf2 is more divergent and mutation does not lead to lethality. However, the retention of Rbf2 >60 My in Drosophila points to essential functions, which prior cell-based assays have been unable to elucidate. Here, using genomic approaches, we provide new insights on the function of Rbf2. Strikingly, we show that Rbf2 regulates a set of cell growth-related genes and can antagonize Rbf1 on specific genes. These unique properties have important implications for the fly; Rbf2 mutants show reduced egg laying, and lifespan is reduced in females and males. Structural alterations in conserved regions of Rbf2 gene suggest that it was sub- or neofunctionalized to develop specific regulatory specificity and activity. We define cis-regulatory features of Rbf2 target genes that allow preferential repression by this protein, indicating that it is not a weaker version of Rbf1 as previously thought. The specialization of retinoblastoma function in Drosophila may reflect a parallel evolution found in vertebrates, and raises the possibility that cell growth control is equally important to cell cycle function for this conserved family of transcriptional corepressors.

Widespread regulatory specificities between transcriptional co-repressors and enhancers in Drosophila

“…Rbf is a prototypical tumor suppressor of the retinoblastoma family (41) and a main transcriptional regulator of cell cycle genes as a part of the DREAM complex, where it counteracts E2F by binding E2F's transactivation domain (42)(43)(44). Rbf2 is a Drosophila-specific Rbf paralogue that has specialized and overlapping functions (34,45). Sin3A is required for repression and activation of target genes of various functions and likely mediates repression through its HDAC-interaction domain (46,47).…”

Soft repression: Subtle transcriptional regulation with global impact

“…[ 15 ] Similarly in Drosophila, paralogs Rbf1 and Rbf2 represent an ancient duplication event, where Rbf1 appears to have more roles in cell cycle regulation, while Rbf2 may interact with and regulate an extensive set of genes linked to growth control and metabolism, including ribosomal protein genes. [ 16 ] Below, we summarize basic properties of these proteins with a focus on work from Drosophila; vertebrate paralogs of Rb have been similarly examined in countless studies in the context of development and disease.…”

“…[ 19 ] Interestingly, specific promoters tested in transfection assays were differentially sensitive to the Rb paralogs – most, but not all, cell cycle genes being more sensitive to repression by Rbf1. [ 16,20,21 ] In contrast, Rbf2 has preferential action on certain ribosomal protein promoters, with which it is prominently associated in vivo, although the extent of regulation is much more modest than that seen for cell cycle genes. [ 12,16 ] Knockdown studies in human fibroblasts similarly illustrate that loss of each human Rb family member misregulates diverse classes of genes.…”

“…[ 16,20,21 ] In contrast, Rbf2 has preferential action on certain ribosomal protein promoters, with which it is prominently associated in vivo, although the extent of regulation is much more modest than that seen for cell cycle genes. [ 12,16 ] Knockdown studies in human fibroblasts similarly illustrate that loss of each human Rb family member misregulates diverse classes of genes. [ 22 ] Rb knockdown led to upregulation of DNA replication, DNA metabolism, and cell cycle genes; p107 knockdown led to downregulation of genes involved in oxidative phosphorylation, electron transport, and NADH dehydrogenase activity; genes involved with organelles were upregulated after p130 knockdown.…”

“…Rbf2—which has many targets within the soft targeting category—is dispensable for development but never lost over longer evolutionary time, likely due to pleiotropic effects. [ 16,20 ] Secondly, regulation of a variety of pathways that have been examined in detail show the significant phenotypic consequences of these less than all‐or‐nothing effects. For Drosophila Sin3A , RNAi knockdown upregulates multiple methionine catabolism genes by approximately two‐fold.…”