Abstract
The retinoblastoma tumor suppressor (Rb) is a potent and ubiquitously expressed cell cycle regulator, but patients with a germline Rb mutation develop a very specific tumor spectrum. This surprising observation raises the possibility that mechanisms that compensate for loss of Rb function are present or activated in many cell types. In particular, p107, a protein related to Rb, has been shown to functionally overlap for loss of Rb in several cellular contexts. To investigate the mechanisms underlying this functional redundancy between Rb and p107 in vivo, we used gene targeting in embryonic stem cells to engineer point mutations in two consensus E2F binding sites in the endogenous p107 promoter. Analysis of normal and mutant cells by gene expression and chromatin immunoprecipitation assays showed that members of the Rb and E2F families directly bound these two sites. Furthermore, we found that these two E2F sites controlled both the repression of p107 in quiescent cells and also its activation in cycling cells, as well as in Rb mutant cells. Cell cycle assays further indicated that activation of p107 transcription during S phase through the two E2F binding sites was critical for controlled cell cycle progression, uncovering a specific role for p107 to slow proliferation in mammalian cells. Direct transcriptional repression of p107 by Rb and E2F family members provides a molecular mechanism for a critical negative feedback loop during cell cycle progression and tumorigenesis. These experiments also suggest novel therapeutic strategies to increase the p107 levels in tumor cells.
Highlights
The retinoblastoma gene retinoblastoma tumor suppressor (Rb) was initially identified as a prototypic tumor suppressor through its association with hereditary retinoblastoma; mutations in Rb or in genes that play a role in the regulation of Rb function are found in virtually all types of human cancers
The best-described function of Rb is to act as a transcriptional co-factor: Rb regulates the activities of numerous transcription factors and recruits chromatin remodeling complexes to control the expression of genes involved in the control of cell cycle progression, differentiation, and senescence
Two consensus E2F binding sites in the mouse p107 promoter contribute to activation and repression of transcription in reporter assays To understand the functions of the tandem E2F consensus binding sites in the mouse p107 promoter, we first generated a series of four luciferase constructs in which inactivating point mutations [33] were introduced into the two consensus E2F sites, either individually or together, into a construct containing 900 bp of the mouse p107 promoter (Figure 1B). These constructs were transfected into wild-type mouse embryonic stem cells, which contain high levels of activating E2F transcription factors as well as limited Rb family function, due to hyperphosphorylation of the Rb family proteins through high activity of Cyclin/Cdk complexes [34,35,36]
Summary
The retinoblastoma gene Rb was initially identified as a prototypic tumor suppressor through its association with hereditary retinoblastoma; mutations in Rb or in genes that play a role in the regulation of Rb function are found in virtually all types of human cancers. The best-described function of Rb is to act as a transcriptional co-factor: Rb regulates the activities of numerous transcription factors and recruits chromatin remodeling complexes to control the expression of genes involved in the control of cell cycle progression, differentiation, and senescence. Rb is expressed in most cell types [4], patients and mice carrying heterozygous mutations for the Rb gene are not strongly predisposed to a broad range of tumors [5,6,7,8,9]. The ability of p107 to compensate for loss of Rb has since been observed in numerous cell types, beyond the mouse retina [13,14,15,16,17,18,19]
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