Mosaic Genetic Screen for Suppressors of the de2f1 Mutant Phenotype in Drosophila

Abstract

The growth suppressive function of the retinoblastoma (pRB) tumor suppressor family is largely attributed to its ability to negatively regulate the family of E2F transcriptional factors and, as a result, to repress E2F-dependent transcription. Deregulation of the pRB pathway is thought to be an obligatory event in most types of cancers. The large number of mammalian E2F proteins is one of the major obstacles that complicate their genetic analysis. In Drosophila, the E2F family consists of only two members. They are classified as an activator (dE2F1) and a repressor (dE2F2). It has been previously shown that proliferation of de2f1 mutant cells is severely reduced due to unchecked activity of the repressor dE2F2 in these cells. We report here a mosaic screen utilizing the de2f1 mutant phenotype to identify suppressors that overcome the dE2F2/RBF-dependent proliferation block. We have isolated l(3)mbt and B52, which are known to be required for dE2F2 function, as well as genes that were not previously linked to the E2F/pRB pathway such as Doa, gfzf, and CG31133. Inactivation of gfzf, Doa, or CG31133 does not relieve repression by dE2F2. We have shown that gfzf and CG31133 potentiate E2F-dependent activation and synergize with inactivation of RBF, suggesting that they may act in parallel to dE2F. Thus, our results demonstrate the efficacy of the described screening strategy for studying regulation of the dE2F/RBF pathway in vivo.T HE family of E2F transcription factors and retinoblastoma (pRB) family tumor suppressor proteins play a pivotal role in control of cell proliferation(reviewed in Trimarchi and Lees 2002;Blais and Dynlacht 2004;Dimova and Dyson 2005;Degregori and Johnson 2006). Although E2F is involved in a variety of cellular activities, the best understood function of E2F is to regulate transcription of genes at the G 1 /S transition. Among E2F targets are genes that encode regulators of S-phase entry and components of the DNA replication machinery. In mammals, there are eight E2F genes. E2F-1 through E2F-6 function as heterodimers with a DP subunit, while E2F-7 and E2F-8 do not require DP to bind to DNA. In spite of structural similarities among E2F proteins, E2F-1, E2F-2, and E2F-3a are predominately involved in activation of gene expression while the group E2F-3b, E2F-4, E2F-5, E2F-6, E2F-7, and E2F-8 behave as repressors. In quiescent cells, when activity of cyclindependent kinases (cdks) is low, repressor E2Fs are complexed with the pRB family members (also called pocket proteins) and repress expression of E2F regulated genes. The prevailing mechanism of the repression is thought to be through the direct recruitment of histone deacetylases, histone methylases, and other corepressor complexes by pocket proteins to E2F regulated promoters. Upon entry into the cell cycle, mitogenic stimulation leads to an increase in the activity of G 1 cdks, which phosphorylate pRB family members and disrupt their interaction with E2Fs. This coincides with displacement of the repressor E2Fs, appearance of free ...

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