A Retinoblastoma Allele That Is Mutated at Its Common E2F Interaction Site Inhibits Cell Proliferation in Gene-Targeted Mice

Abstract

fThe retinoblastoma protein (pRB) is best known for regulating cell proliferation through E2F transcription factors. In this report, we investigate the properties of a targeted mutation that disrupts pRB interactions with the transactivation domain of E2Fs. Mice that carry this mutation endogenously (Rb1 ⌬G ) are defective for pRB-dependent repression of E2F target genes. Except for an accelerated entry into S phase in response to serum stimulation, cell cycle regulation in Rb1 ⌬G/⌬G mouse embryonic fibroblasts (MEFs) strongly resembles that of the wild type. In a serum deprivation-induced cell cycle exit, Rb1⌬G/⌬G MEFs display a magnitude of E2F target gene derepression similar to that of Rb1 ؊/؊ cells, even though Rb1 ⌬G/⌬G cells exit the cell cycle normally. Interestingly, cell cycle arrest in Rb1 ⌬G/⌬G MEFs is responsive to p16 expression and gamma irradiation, indicating that alternate mechanisms can be activated in G 1 to arrest proliferation. Some Rb1 ⌬G/⌬G mice die neonatally with a muscle degeneration phenotype, while the others live a normal life span with no evidence of spontaneous tumor formation. Most tissues appear histologically normal while being accompanied by derepression of pRB-regulated E2F targets. This suggests that non-E2F-, pRB-dependent pathways may have a more relevant role in proliferative control than previously identified.T he retinoblastoma tumor suppressor protein (pRB) has a central role in the regulation of the G 1 -to-S-phase transition. Inactivation of its control over cell cycle progression is one of the most common events in cancer (1). The RB protein is thought to regulate entry into S phase through its ability to repress E2F-dependent transcription (2). In the G 1 phase of the cell cycle, a direct interaction between the large pocket domain of pRB (RBLP) and the transactivation domain of E2Fs blocks transcription and recruits chromatin regulators that maintain the cell in G 1 (3). Activation of cyclin-dependent kinases (CDKs) results in the phosphorylation of pRB and the release of E2F transcription factors (4). Free E2Fs then activate a transcriptional program that drives the cell into S phase (3). This model of pRB regulation of E2F dominates our understanding of G 1 -to-S-phase control. Much of our knowledge of this model was derived from studies using viral oncoproteins encoded by small DNA tumor viruses (5, 6). Of particular note, the human papillomavirus E7 protein has been shown to compete for pRB-E2F interactions to deregulate proliferation (7, 8). However, E7 must also target pRB for degradation in order to induce proliferation (8). Thus, the experimental system that gave rise to the pRB-E2F regulatory axis in cell cycle control also suggests that pRB may engage other growth-suppressing activities beyond E2F regulation. By comparison with the pRB-E2F pathway, we know very little about pRB's non-E2F-dependent growth control mechanisms and their relative contribution to cell cycle regulation and tumor suppressor activities.The minimal growth-suppressive region of pRB...

Interchangeable Roles for E2F Transcriptional Repression by the Retinoblastoma Protein and p27KIP1–Cyclin-Dependent Kinase Regulation in Cell Cycle Control and Tumor Suppression

“…Compound mutant mice display viability at weaning similar to that of the Rb1 G/G genotype alone and without obvious anatomical defects, suggesting the combination of Rb1 G/G and Cdkn1b Ϫ/Ϫ deficiency is no different than either single mutant alone ( Fig. 2A and Table 1) (30). While doublemutant Rb1 G/G ; Cdkn1b Ϫ/Ϫ mice show normal development, we aged cohorts of double-and single-mutant mice and discovered that Rb1 G/G ; Cdkn1b Ϫ/Ϫ mice succumb to pituitary tumors with an average tumor-free survival of 214 days (Fig.…”

“…Our previously published analysis of Rb1 G/G primary fibroblasts and mice indicates that loss of pRB-E2F repression fails to bypass cell cycle exit signals (30). Figure 1 shows an example of a serum starvation arrest in which wild-type, Rb1 G/G , and knockout cells were serum starved for 60 h. Under these culture conditions, wild-type and Rb1 G/G cells reduce bromodeoxyuridine (BrdU) incorporation equivalently, while Rb1 Ϫ/Ϫ cells are defective (Fig.…”

“…In order to study E2F-independent functions of pRB at an endogenous level, we developed a mutant mouse model in which pRB binding to E2Fs is disrupted by R461E and K542E mutations (called Rb1 G ) (30). Importantly, the Rb1 G mutant protein is expressed at wild-type (WT) levels and makes normal interactions with LXCXE motifcontaining proteins (30).…”

“…Importantly, the Rb1 G mutant protein is expressed at wild-type (WT) levels and makes normal interactions with LXCXE motifcontaining proteins (30). Surprisingly, we found that this mutation had little effect on control of cell proliferation, as Rb1 G/G fibroblasts are capable of responding to serum starvation, p16 expression, DNA damage, and myogenic differentiation and in all cases show wild-type responses (30). In this study, we find that p27 expression is higher in Rb1 G/G fibroblasts.…”

Haploinsufficiency of an RB–E2F1–Condensin II Complex Leads to Aberrant Replication and Aneuploidy

“…coding substitutions into the murine Rb1 gene, respectively ( 41,42 ). The generation of Rb1 ΔS/ΔS mice will be published elsewhere.…”