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.

Keywords: Drosophila; Rbf1; Rbf2; repressor; retinoblastoma; transcription.

Fig. 1.

Sequence conservation of retinoblastoma proteins in Drosophila and humans. (A) Multiple sequence alignment of Rbf1 in 12 Drosophila species; conservation is observed in C-terminal IE region and A–B pocket and spacer, as well as N-terminal regions. (B) Multiple sequence alignment of Rbf2 in 12 Drosophila species, showing lower conservation in C-terminus and in A–B pocket and spacer, as well as N-terminal regions. (C) Pairwise alignment of Drosophila Rbf1 and Rbf2, showing higher conservation in central pocket domains, and lower in C-terminus. For (A), (B), and (C), the y axis represents alignment scores generated by ClustalW, which takes into consideration both identity and chemical similarity (see Materials and Methods). Higher bars indicate more conservation. The functional domains are denoted including the cyclin-fold domain, A pocket, B pocket, and the instability element (IE) in the C-terminus. (D) Multiple sequence alignment of C-termini of Drosophila and mammalian retinoblastoma proteins. The yellow color represents conserved residues and gray represents similar residues with respect to p107. Specific portions of the C-terminus involved in direct contacts with E2F/DP1 proteins are highlighted; the RbC^nter and RbC^core are shown on top of the figure, and the p107^core is shown at the bottom (Rubin et al. 2005; Liban et al. 2017). The IE, from residue 728–786 of Rbf1, is denoted by the red arrows. Triangles represent residues that make contacts with E2F/DP marked box domains for both Rb and p107. The asterisk denotes conserved serine residues within SP motifs that are targeted for phosphorylation. The K774 residue within the SPAK motif is denoted by a square.

Fig. 2.

Overexpression of Rbf2 results in profound effects on gene expression in embryos. (A) A heatmap generated by unsupervised clustering of RNA-seq data from Rbf1 and Rbf2 overexpressing embryos, and control embryos. Values represent log transformed RPKM reads that are mean centered for each gene. Blue indicates reads below the mean, black equal to the mean, and yellow, above the mean. Values represent average of three biologic replicates. RPKM <1 was excluded from the analysis. Only genes bound by Rbf1 or Rbf2 in vivo are included. The heatmap is divided into five major clusters based on Euclidean distance. (B) Gene ontology analysis of the five clusters based on the DAVID annotation tool. P values represent significance of enrichment for each category, the count represents the number of genes in the cluster belonging to each category, and total shows number of genes in the GO category. (C) Relative gene expression of ribosomal- and mitochondrial-related genes in Rbf1 or Rbf2 overexpressing embryos, relative to control embryos. Values represent average of three biologic replicates.

Fig. 3.

Rbf2 mutant alleles and longevity phenotype. (A) Schematic representation of the CRISPR targeting of Rbf2 and the alleles generated. gRNA1 produced Δ1 and Δ15C alleles, and gRNA2 produced Δ38, Δ41, Δ46, and Δ15N alleles. (B) Western blot indicating loss of Rbf2 protein from Rbf2 mutant ovaries from Rbf2^Δ1/Rbf2^Δ38, Rbf2^Δ1/Rbf2^Δ41, and Rbf2^Δ1/Rbf2^Δ46 flies. Anti-CtBP is used as a control. Asterisk indicates nonspecific band. (C) Survivorship curve for Rbf2^Δ1/Rbf2^Δ46 and Rbf2^Δ38/Rbf2^Δ46 females and (D) males in comparison to yw flies. The curves from the mutants were significantly different from the yw flies curves for both females and males according to log-rank test with a P value <0.0001.

Fig. 4.

Rbf2 effects on egg laying, ovariole numbers, and Pi3K92E expression. (A) Egg laying for introgressed Rbf2^Δ1 allele for the mutant males or females. The measurements represent average 24 h egg count for 4 days for each single female fly. Mutant or wild-type males were crossed to a single yw female, and mutant or wild-type females were crossed to three yw males. (B) Ovariole counts of individual adult ovaries for the following genotypes: yw (n = 56), Rbf2^Δ1/TM6B, Tb (n = 68), Rbf2^Δ38/TM6B, Tb (n = 20), Rbf2^Δ46/TM6B, Tb (n = 20), Rbf2^Δ15C/TM6B, Tb (n = 24), Rbf2^Δ1/Rbf2^Δ38 (n = 10), Rbf2^Δ1/Rbf2^Δ41 (n = 10), and Rbf2^Δ1/Rbf2^Δ46 (n = 10). (C) Difference in ovariole number between ovaries of each female for the following genotypes: yw (n = 36), Rbf2^Δ1/TM6B, Tb (n = 48), Rbf2^Δ15C/TM6B, Tb (n = 24), Rbf2^Δ1/Rbf2^Δ38 (n = 10), Rbf2^Δ1/Rbf2^Δ41 (n = 10), and Rbf2^Δ1/Rbf2^Δ46 (n = 10). (D) Box plot representing relative gene expression from ovaries of Rbf2^Δ1/Rbf2^Δ46 flies in comparison to control yw flies. Data represent six biologic replicates. For (B), (C), and (D), (*) indicates P value <0.05. (E) Images of ovary from yw and Rbf2^Δ15C/Rbf2^Δ15C_. Images were taken at 10× magnification.

Fig. 5.

Specific regulation of PCNA and CycB by Rbf1 and Rbf2. (A) Schematic representation of CycB, PCNA, chimeric CycB-PCNA, and PCNA-CycB luciferase reporter genes. Black bars indicate E2f motifs, and gray bars DREF motifs, often located in cell cycle-related genes. (B) Regulation of PCNA and CycB by Rbf1 and Rbf2. (C) Regulation of PCNA and CycB by E2F1 and E2F2. (D and E) Combined action of Rbf and E2F proteins on PCNA and CycB promoters. Luciferase measurements were normalized to expression of the reporters in cells cotransfected with the empty expression vectors (no Rbf or E2F genes). Fold changes represented on a log₂ scale plot. Values represent at least three biologic replicates and error bars represent SDs. (*) indicates P value <0.05.

Fig. 6.

The CycB core promoter drives responsiveness to Rbf2. (A) Luciferase reporter assays of chimeric reporters CycB-PCNA and PCNA-CycB in response to expression of Rbf1 or Rbf2. (B) Effect of expression of E2F1 or E2F2 on chimeric reporters. (C and D) Expression of CycB-PCNA or PCNA-CycB in response to coexpression of Rbf and E2F proteins. Luciferase measurements are normalized to expression of the reporters in cells cotransfected with the empty expression vector (no Rbf or E2F genes; red horizontal line). Values represent at least three biologic replicates and error bars represent SDs. (*) indicates P value <0.05.

Fig. 7.

Model for evolved functions of Rbf proteins. (A) Subfunctionalization of Rbf proteins into cell cycle and cell growth control. Ancestral Rbf proteins (gray circle) regulate cell cycle- and cell growth-related genes. Duplication of Rbf proteins resulted in subfunctionalization where the more derived Rbf2 protein (black circle) assumes regulation of cell growth-related genes. Rbf2 protein is subject to more rapid evolution within different Drosophila species to provide optimal growth control and fecundity. (B) Model for specific action of Rbf2 from the core promoter position, as in CycB; repression is weaker from 5′ positions. (C) Rbf2 competes with Rbf1 binding on E2F2-regulated genes, leading to partial derepression and optimal gene regulation on certain classes of genes.