Genome-Wide Analysis ofDrosophilaRBf2 Protein Highlights the Diversity of RB Family Targets and Possible Role in Regulation of Ribosome Biosynthesis

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.

Complex cis-regulatory landscape of the insulin receptor gene underlies the broad expression of a central signaling regulator

“…First, simple promoters, such as those driving ribosomal protein genes, might provide the high levels of activity needed for abundant transcripts, with a certain level of dynamic regulation, but the regulation of these genes may reflect only a few types of signaling input (e.g. TOR, cell cycle) (Powers and Walter, 1999;Martin et al, 2004;Wei et al, 2015). Second, InR expression, by contrast, is wired into many different signaling pathways and might need additional regulatory DNA to provide modules suitable for responses in different developmental settings, including growth control of larval tissues, stem cell niches in the larva and adult, and non-proliferating adult neurons.…”

“…The incoherent feed-forward properties of the InR gene, with simultaneous positive and negative effects, might ensure more precise changes in gene expression, preventing pleiotropic impacts Dashed lines indicate elements that are active in a cell type-specific manner. The dREAM complex proteins are associated with Rbf1 at the promoter, but their functional relevance for InR expression is unknown (Acharya et al, 2012;Georlette et al, 2007;Wei et al, 2015). Uncharacterized activator on the downstream signaling pathway.…”

“…More recently, we noted that the T1 proximal promoter region of the InR gene is occupied in vivo by the Rbf1 (Rbf -FlyBase) tumor suppressor protein, the homolog of mammalian Rb (Acharya et al, 2012;Korenjak et al, 2012;Wei et al, 2015). Binding of Rbf1 appears to be of functional significance, as a T1 promoter construct is repressed by Rbf1 expression in S2 cells (Raj et al, 2012).…”

“…An additional regulatory factor for the insulin receptor is the retinoblastoma co-repressor (Rb); this co-factor binds near the InR promoter, although the physiological role has yet to be investigated (Acharya et al, 2012;Korenjak et al, 2012;Wei et al, 2015). Rb tumor suppressor proteins are key regulators of the cell cycle; thus, coordinate regulation of the insulin receptor with these genes might provide a link between cell proliferation and growth (Du and Pogoriler, 2006;Giacinti and Giordano, 2006;Acharya et al, 2012).…”

Evaluation of Genotoxicity of Lufenuron and Chlorfluazuron Insecticides in Drosophila melanogaster Using a Germ-Line Cell Aneuploidy and Chromosomal Aberrations Test

“…Rb protein plays an important role in the regulation of cell division, namely, cell death, and its activity is altered in most human tumors [46,47]. It is able to modulate the action of E2F transcription factors and regulate cell cycle progression [48].…”