A genome-wide RNA interference screen identifies putative chromatin regulators essential for E2F repression

Abstract

Regulation of chromatin structure is critical in many fundamental cellular processes. Previous studies have suggested that the Rb tumor suppressor may recruit multiple chromatin regulatory proteins to repress E2F, a key regulator of cell proliferation and differentiation. Taking advantage of the evolutionary conservation of the E2F pathway, we have conducted a genome-wide RNAi screen in cultured Drosophila cells for genes required for repression of E2F activity. Among the genes identified are components of the putative Domino chromatin remodeling complex, as well as the Polycomb Group (PcG) protein-like fly tumor suppressor, L3mbt, and the related CG16975/dSfmbt. These factors are recruited to E2F-responsive promoters through physical association with E2F and are required for repression of endogenous E2F target genes. Surprisingly, their inhibitory activities on E2F appear to be independent of Rb. In Drosophila, domino mutation enhances cell proliferation induced by E2F overexpression and suppresses a loss-of-function cyclin E mutation. These findings suggest that potential chromatin regulation mediated by Domino and PcG-like factors plays an important role in controlling E2F activity and cell growth.

E2F-associated chromatin modifiers and cell cycle control

“…It will be interesting to determine whether this is a general mechanism through which the E2F-pRB pathway functions to modulate gene expression during the cell cycle and whether other transcriptional repressors share this ability to recruit L3MBTL1. Interestingly, the fly tumor suppressor L(3)mbt was also identified in a genome-wide RNAi screen for genes required for E2F-dependent repression [4]. L(3)mbt was recruited to many target genes in an E2F-dependent, but RBFindependent, manner, suggesting that an E2F-L3MBT chromatin modification and repression pathway may be functionally conserved from flies to humans.…”

“…However, the identification of eight E2F family members and the development of new functional genomic approaches have accelerated our progress and proven that the function of the E2F family is substantially more complex. In addition, genome-wide approaches in mammalian cells, flies, and worms have indicated that this family controls expression of genes beyond the S phase transition and indeed beyond the cell cycle by promoting differentiation and specification of distinct tissues [1][2][3][4][5][6].…”

L3MBTL1 polycomb protein, a candidate tumor suppressor in del(20q12) myeloid disorders, is essential for genome stability

“…We, and others, have found L3MBTL1 complexed with Rb/ E2F proteins repressed the activation of some E2F target genes, including c-myc and Cyclin E1, two established oncogenes (7,8,19,20). We tested whether these genes were up-regulated in L3MBTL1 knockdown cells, but noted no changes in their levels at 48 h following lentiviral infection with shRNAs against L3MBTL1 (Fig.…”

Essential Role of p400/mDomino Chromatin-remodeling ATPase in Bone Marrow Hematopoiesis and Cell-cycle Progression

“…Impairment of Cell-cycle Regulatory Gene Expression in mDomino-deficient Cells-The p400/mDomino chromatin-remodeling complex is known to interact physically or functionally with cell-cycle-regulatory transcription factors, including c-Myc, E1A, p53, and E2F (3,(17)(18)(19)(20). To gain insight into the molecular function of mDomino in cell-cycle regulation, we characterized the gene expression profiles of mDom fl/fl ;CreER MEFs treated or untreated with OHT by DNA microarray analysis (n ϭ 3 for both untreated and OHT-treated cells), which revealed that 770 genes were differentially (Ն2.0-fold) expressed by mDomino depletion, including 191 down-regulated genes (supplemental Table 1) and 579 up-regulated genes (data not shown).…”

“…This histone-exchanging activity plays a key role in the epigenetic regulation of gene expression as well as in DNA repair (12)(13)(14)(15)(16). p400/mDomino is known to interact physically and/or functionally with growth-regulating transcription factors, such as Myc, p53, E2F, and adenovirus E1A (3,(17)(18)(19)(20). In primary human fibroblasts and osteosarcoma-derived U2OS cells, the knockdown of p400/mDomino results in cell-cycle arrest at the G 1 phase with the induction of p21 expression (21,22).…”