A genetic screen for dominant modifiers of a cyclin E hypomorphic mutation identifies novel regulators of S-phase entry in Drosophila

Abstract

Cyclin E together with its kinase partner Cdk2 is a critical regulator of entry into S phase. To identify novel genes that regulate the G1- to S-phase transition within a whole animal we made use of a hypomorphic cyclin E mutation, DmcycEJP, which results in a rough eye phenotype. We screened the X and third chromosome deficiencies, tested candidate genes, and carried out a genetic screen of 55,000 EMS or X-ray-mutagenized flies for second or third chromosome mutations that dominantly modified the DmcycEJP rough eye phenotype. We have focused on the DmcycEJP suppressors, S(DmcycEJP), to identify novel negative regulators of S-phase entry. There are 18 suppressor gene groups with more than one allele and several genes that are represented by only a single allele. All S(DmcycEJP) tested suppress the DmcycEJP rough eye phenotype by increasing the number of S phases in the postmorphogenetic furrow S-phase band. By testing candidates we have identified several modifier genes from the mutagenic screen as well as from the deficiency screen. DmcycEJP suppressor genes fall into the classes of: (1) chromatin remodeling or transcription factors; (2) signaling pathways; and (3) cytoskeletal, (4) cell adhesion, and (5) cytoarchitectural tumor suppressors. The cytoarchitectural tumor suppressors include scribble, lethal-2-giant-larvae (lgl), and discs-large (dlg), loss of function of which leads to neoplastic tumors and disruption of apical-basal cell polarity. We further explored the genetic interactions of scribble with S(DmcycEJP) genes and show that hypomorphic scribble mutants exhibit genetic interactions with lgl, scab (alphaPS3-integrin--cell adhesion), phyllopod (signaling), dEB1 (microtubule-binding protein--cytoskeletal), and moira (chromatin remodeling). These interactions of the cytoarchitectural suppressor gene, scribble, with cell adhesion, signaling, cytoskeletal, and chromatin remodeling genes, suggest that these genes may act in a common pathway to negatively regulate cyclin E or S-phase entry.

Figure 1.—

The identified dominant suppressors of DmcycE^JP: scanning electron-micrographs of adult eyes and BrdU labeling of eye imaginal discs from DmcycE^JP individuals heterozygous for the identified suppressor alleles. Genotypes are as indicated: wild type (WT); DmcycE^JP; DmcycE^JP; 43S2/+; DmcycE^JP; zn72D/+; DmcycE^JP, 2.2-39S2/+; DmcycE^JP, phyl²²⁴⁵/+; DmcycE^JP; 2S1/+; DmcycE^JP; trio^M89/+; DmcycE^JP, 2.5-42S11/+; DmcycE^JP, dEB1-l(2)04524/+; DmcycE^JP, 2.11-62S9/+; and DmcycE^JP, 2.11-l(2)01288/+.

Figure 2.—

Scanning-electron micrographs of adult eyes and BrdU labeling of eye imaginal discs from lgl, scrib, or dlg heterozygotes in a DmcycE^JP background. Genotypes are as indicated: DmcycE^JP; 2.1-23S9/+, DmcycE^JP; 2.1-27S3/+, DmcycE^JP; 2.1-E2S31/+, DmcycE^JP; 2.1-E6S2/+, DmcycE^JP; DmcycE^JP; scrib-63S15/+; DmcycE^JP; scrib¹/+; and dlg⁶/+; DmcycE^JP.

Figure 3.—

Cyclin E protein levels in eye imaginal discs from third instar larvae. Genotypes are as indicated: wild type (WT); DmcycE^JP; 2.1-23S9/+, DmcycE^JP; 2.1-E6S2/+, DmcycE^JP; lgl⁴/+, DmcycE^JP; and DmcycE^JP; scrib¹/+.

Figure 4.—

Possible pathways connecting cyclin E-interacting genes. Interactors identified in our cyclin E screen are shaded. Direct protein interactions between cyclin E interactors or other relevant proteins are indicated by the double-headed arrows. Arrows indicate positive interactions while barred lines indicate negative interactions. Not shown are interactions between Scab and the Dpp pathway, between E-cadherin and the Egfr pathway, between Fat and Atrophin (a nuclear corepressor), and between Expanded/Merlin and the Dpp and Egfr pathways. *, genes that genetically interact with scribble. See the text for details.