Role ofDrosophilaretinoblastoma protein instability element in cell growth and proliferation

Abstract

The RB tumor suppressor, a regulator of the cell cycle, apoptosis, senescence, and differentiation, is frequently mutated in human cancers. We recently described an evolutionarily conserved C-terminal "instability element" (IE) of the Drosophila Rbf1 retinoblastoma protein that regulates its turnover. Misexpression of wild-type or nonphosphorylatable forms of the Rbf1 protein leads to repression of cell cycle genes. In contrast, overexpression of a defective form of Rbf1 lacking the IE (DIE), a stabilized but transcriptionally less active form of the protein, induced ectopic S phase in cell culture. To determine how mutations in the Rbf1 IE may induce dominant effects in a developmental context, we assessed the impact of in vivo expression of mutant Rbf1 proteins on wing development. DIE expression resulted in overgrowth of larval wing imaginal discs and larger adult wings containing larger cells. In contrast, a point mutation in a conserved lysine of the IE (K774A) generated severely disrupted, reduced wings. These contrasting effects appear to correlate with control of apoptosis; expression of the pro-apoptotic reaper gene and DNA fragmentation measured by acridine orange stain increased in flies expressing the K774A isoform and was suppressed by expression of Rbf1DIE. Intriguingly, cancer associated mutations affecting RB homologs p130 and p107 may similarly induce dominant phenotypes.

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

“…These flies were maintained in the lab and used for crossing to homozygous gRNA flies described below. nub-GAL4>UAS:dCas9-Rbf1 homozygous flies had a notched wing phenotype, consistent with Rbf1 overexpression in the wing (Elenbaas et al 2015). nub-GAL4>UAS:dCas9-Rbf2 did not have a wing phenotype, and neither did any of the other homozygous fly lines.…”

“…An Rbf1 ΔIE mutant can still interact with E2F1 and E2F2, but is unable to repress cell cycle promoters such as PCNA (Acharya et al 2010;Raj et al 2012a). Overexpression phenotypes in the fly eye and wing suggests that it may be a hypomorph (Acharya et al 2010;Elenbaas et al 2015). Indeed, according to RNA-seq, there is limited repression of various genes after overexpression of Rbf1 ΔIE in wing discs, in comparison to the WT Rbf1 (Figure S6A, B).…”

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

“…To further examine the significance of RBf1 association with ribosomal protein gene promoters, we performed RNA-seq of larval wing discs that were engineered to overexpress RBf1 ( Elenbaas et al 2014 ). Globally, a majority of the ribosomal protein genes showed modest reductions in expression, with only a few showing an increase ( Figure S6 ).…”

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

“…On the other hand, for Rbf, the increased rate plays a critical role in the regulation of cell proliferation [45]. 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].…”