Wei Du, Ph.D.

Function and Regulation of the Retinoblastoma Family of Proteins

Research Summary

We are interested in understanding how cell proliferation is regulated, particularly how environmental and developmental signals are integrated and translated into a decision of cell cycle control. In mammalian cells, the decision to proliferate or to remain quiescent is made primarily during G1 phase of the cell cycle, and the inactivation of the retinoblastoma (RB) family of proteins (pRB, p107, and p130) by phosphorylation at late G1 is believed to be a critical step for G1/S transition. The RB family of proteins regulate the transcription factor E2F. The inactivation of pRB function and the subsequent deregulation of E2F activity is thought to contribute to the uncontrolled proliferation of many tumor cells.

To study how the RB family of proteins normally regulate cell cycle progression and cell differentiation, and how their functions are regulated during development, we used Drosophila as a model system because of the simplicity of this system and because of the ease of combining genetic and molecular approaches to dissect biochemical and cellular processes. Using a modified yeast two hybrid system, we have isolated the Drosophila homolog of the RB protein family (RBF). RBF combines many of the structural and functional features of pRB, p107 and p130. RBF associates with dE2F and dDP in vivo and is a stoichiometric component of E2F DNA-binding complexes. Furthermore, RBF specifically represses E2F transcriptional activity. These findings show that the structure and function of RB-related proteins is conserved between mammals and flies. To analyze the effect of RBF and E2F on cell cycle control, we studied the effect of ectopic expression of RBF, dE2F, and dDP in the developing eye. Expression of RBF or the co-expression of dDP and dE2F disrupted normal eye development resulting in abnormal patterns of bristles, cone cells and photoreceptors. Expression of dE2F and dDP drives normally postmitotic cells back into cell cycle, and induces increased apoptosis, coexpression of RBF together with dE2F and dDP completely suppressed the biological consequences of the dE2F and dDP ectopic expression. Importantly, these phenotypes provide sensitized backgrounds to carry out genetic screens to identify interacting genes.

The following are the studies that we are or will be pursuing:

1. Studying the function of RBF in regulating the cell cycle and differentiation during Drosophila development. We have generated loss of function RBF mutant by P element mediated mutagenesis. Studies are in progress to characterize the detailed mutant phenotype, and to test the genetic interactions with other cell cycle mutants.
2. Identifying genes that interact with the RB pathway through genetic screens. Overexpression of RBF in the eye results in the loss of bristles and fused ommatidia (due to missing pigment cells). This provides a sensitized background to identify genes that regulate RBF. We have screened for mutations that modify the effects of RBF overexpression in the developing eye. Enhancers and suppressors of this phenotype have been identified following EMS mutagenesis , and are currently being characterized.
3. Since the RB-E2F pathway is conserved during evolution, it is likely that their regulators and the interactions between these regulators are also conserved. Our long term goal is to apply the information derived from Drosophila to mammalian systems, to study whether similar regulations exist in mammalian system, and whether/how perturbation of these processes affect cell cycle control and result in human disease.

Selected Papers

Du W and Maniatis T. (1992). An ATF/CREB binding site is required for virus induction of the human interferon ß gene. Proc. Natl. Acad. Sci. USA, 89:2150-2154.

Maniatis T, Whittemore LA, Du W, Fan CM, Keller A, Palombella V and Thanos D. (1992). Positive and negative control of human interferon-ß gene expression. in Transcription Regulation, edited by McKnight, S. & Yamamoto, K., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1193-1220.

Du W, Thanos D and Maniatis T. (1993). Mechanisms of Transcriptional Synergism between Distinct Virus Inducible Enhancer Elements. Cell, 74:887-898.

Du W and Maniatis T. (1994). The high Mobility group protein HMG I(Y) can stimulate or inhibit DNA binding of distinct transcription factor ATF-2 isoforms. Proc. Natl. Acad. Sci. USA, 91:11318-11322.

Du W, Vidal M, Xie J and Dyson N. (1996). RBF, a novel RB-related gene that regulates E2F activity and interacts with cyclin E in Drosophila. Genes & Dev., 10:1206-1218.

Du W, Xie J and Dyson N. (1996). Ectopic expression of dE2F and dDP induces cell proliferation and death in the Drosophila eye. EMBO J., 15:3684-3692.

Du W*. and Dyson, N. (1999). The role of RBF in the introduction of G1 regulation during Drosophila embryogenesis. EMBO J. 18, 916-925. (*corresponding author).

Du W. (2000). Suppression of the rbf null mutants by a de2f1 allele that lacks transactivation domain. Development. 127, 367-379.

Meyer CA, Jacobs HW, Datar SA, Du W, Edgar BA, Christian F, Lehner CF. (2000). Drosophila Cdk4 stimulates growth and is dispensable for cell cycle progression. EMBO J. 19, 4533-4542.

Bosco G, Du W, Orr-Weaver T. (2001). DNA replication control through interaction of E2FRB and the origin recognition complex. Nature Cell Biology, 3, 289-295.

Xu J, Xin S and Du W. (2001). Drosophila Chk2 is required for DNA damage-mediated cell cycle arrest and apoptosis. FEBS Letters 508, 394-398.

Xin S, Weng L, Xu J and Du W. (2002). The role of RBF in developmentally regulated cell proliferation in the eye disc and in Cyclin D/Cdk4 induced cellular growth. Development. 129, 1345-1356.

Duman-Scheel M, Weng L, Xin S, and Du W. (2002). Hedgehog signaling controls cell proliferation and growth through upregulation of Cyclin D and Cyclin E. Nature, 417, 299-304.

Xu J and Du W. (2003). Drosophila chk2 plays an important role in a mitotic checkpoint in syncytial embryos. FEBS letter, 545, 209-212.

Weng L, Zhu C, Xu J and Du W. (2003). Critical role of active repression by E2F and Rb proteins in endoreplication during Drosophila development. EMBO J., 22, 3865-3875.

Du W. (2003). Endocycle and E2F-dependent transcriptional activation and repression. Cell Cycle, 2, 515-516.

Duman-Scheel M, Johnston L and Du W. (2004). Repression of dMyc expression by Wingless promotes RBF-induced G1 arrest in the Presumptive Drosophila Wing Margin. PNAS, 101, 3857-3862.

Pogoriler J, Millen K, Utset M, and Du W. (2006). Loss of cyclin D1 impairs cerebellar development and suppresses medulloblastoma formation.  Development, 133, 3929-3937.

Du W, and Pogoriler J. (2006). Retinoblastoma family genes.  Oncogene, 25, 5190-5200.

Shafaee Z, Schmidt H, Du W, Posner M, and Weichselbaum R. (2006). Cyclopamine increases the cytotoxic effects of taxol and radiation in Hedgehog expressing pancreatic cancer.  Cancer Chemother Pharmacol.  Mar 22; [Epub ahead of print].

Du W, and Pogoriler J. (2006). Rb and E2F family of proteins in Gene Expression and Regulation, edited by Jun Ma, Higher Education Press & Springer, pp 207-217.