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The activity of the E2F transcription factor is controlled by physical association with the retinoblastoma protein (pRB) and two related proteins, p107 and p130. The pRB family members are thought to control different aspects of E2F activity, but it has been unclear what the respective functions of these proteins might be. To dissect the specific functions of pRB, p107, and p130 we have investigated how the expression of E2F-regulated genes is changed in cultures of primary cells lacking each of these family members. Whereas no changes were found in the expression of E2F-target genes in cells lacking either p107 or p130, deregulated expression of E2F targets was seen in cells lacking pRB and in cells lacking both p107 and p130. Surprisingly, the genes that were disregulated in these two settings were completely different. These findings show that pRB and p107/p130 indeed provide different functions in E2F regulation and identify target genes that are dependent on pRB family proteins for their normal expression.

The pl30 protein shares extensive sequence similarity with pRB, the product of the retinoblastoma gene, and is a major E2F-associated protein in quiescent cells. To investigate its biological function, we have mutated pl30 via gene targeting in the mouse. Homozygous mutation of pl30 had little discernible effect on development or on the growth of mouse embryo fibroblasts in culture. Much of the E2F activity that normally associates with pl30 in serum-starved mouse embryo fibroblasts associated instead with the highly related pl07 protein. To determine whether pl30 and pl07 have overlapping biological roles, we produced mice having simultaneous inactivation of the pl30 and pl07 genes. Such mice exhibited deregulated chondrocyte growth, defective endochondral bone development, shortened limbs, and neonatal lethality. These findings indicate that pl30 and pl07 play an important role in limb development through their abilities to control chondrocyte proliferation. Thus, in certain settings pl07 and pl30 perform growth-regulatory functions that are not fulfilled by pRB.

Retinoblastoma is a rare cancer of the infant retina, which forms when both RB1 alleles mutate in a susceptible retinal cell, likely a cone photoreceptor precursor. Loss of the tumour suppressor functions of the retinoblastoma protein, pRB, leads to uncontrolled cell division and recurrent genomic changes during tumour progression. Although pRB is expressed in virtually all tissues, cone precursors have biochemical and molecular features that may sensitize to RB1 loss to enable tumourigenesis. Retinoblastoma is diagnosed in ~8,000 children each year worldwide. Patient survival is >95% in high-income countries, but <30% globally. However, outcomes are improving through increasing awareness for earlier diagnosis, new guidelines and sharing of expertise. Intra-arterial and intravitreal chemotherapy have emerged as promising methods to salvage eyes. Ongoing international collaborations will replace the multiple different classifications of eye involvement with standardized definitions to consistently assess eligibility, efficacy and safety of treatment options. Life-long follow-up is warranted since survivors of heritable retinoblastoma are at risk for developing second cancers. Defining the molecular consequences of RB1 loss in diverse tissues may open new avenues for treatment and prevention of retinoblastoma as well as second cancers in patients with germline RB1 mutations.

Association of the E2F transcription factor with the pRb and p107 proteins appears to regulate the activity of E2F and, in turn, affect cell cycle progression. We found, however, that pRb and p107 are only minor E2F-associated proteins in Go/G 1 mouse fibroblasts, and we sought to identify the major E2F partner protein in these cells. Because the adenovirus E1A oncoprotein seemed able to bind to the Go E2F partner, we enriched for proteins that associated both with an E2F-binding site DNA column and with E1A. The major species in Go and early G1 fibroblasts detected with this approach had properties identical to the pRb-and pl07-related p130 protein. In serum-stimulated cells, p107 replaced p130 as the major E2F-associated protein near the G~/S border, concomitant with an increase in p107 protein levels, pl30-E2F complexes resembled pl07-E2F complexes in their ability to bind to cyclin-cdk kinases, and they appeared to be associated with the cyclin E-cdk2 kinase in late G~ cells. These observations indicate that E2F transcription factors are regulated by a succession of partner proteins with which they associate during defined stages of the cell cycle.

SUMMARY Retinoblastomas develop due to the loss of the Rb protein, yet the cell type in which Rb suppresses retinoblastoma, and the cellular circuitry that underlies the need for Rb are undefined. Here, we show that retinoblastoma cells express markers of post-mitotic cone precursors, but not markers of other retinal cell types. We also demonstrate that human cone precursors prominently express MDM2 and N-Myc, that retinoblastoma cells require both of these proteins for proliferation and survival, and that MDM2 is specifically needed to suppress ARF-induced apoptosis in cultured retinoblastoma cells. Interestingly, retinoblastoma cell MDM2 expression was regulated by the cone-specific RXRγ transcription factor and a human-specific RXRγ consensus binding site, and proliferation required RXRγ as well as the cone-specific thyroid hormone receptor-β2. These findings provide support for a cone precursor origin of retinoblastoma and suggest that human cone-specific signaling circuitry sensitizes to the oncogenic effects of RB1 mutations.

The E2F transcription factors play a role in regulating the expression of genes required for cell proliferation. Their activity appears to be regulated by association with the retinoblastoma protein (pRb) and the pRb-related proteins p107 and p130. In vivo, pRb is found in complex with a subset of E2F components-namely, E2F-1, E2F-2, and E2F-3. Here we describe the characterization of cDNAs encoding two unusual E2Fs, E2F-4 and E2F-5, each identified by the ability of their gene product to interact with p130 in a yeast two-hybrid system. E2F-4 and -5 share common sequences with E2F-1, E2F-2, and E2F-3 and, like these other E2Fs, the ability to heterodimerize with DP-1, thereby acquiring the ability to bind an E2F DNA recognition sequence with high affinity. However, in contrast to E2F-1, E2F-4 and E2F-5 fail to bind pRb in a two-hybrid assay. Moreover, they show a unique pattern of expression in synchronized human keratinocytes: E2F-4 and E2F-5 mRNA expression is maximal in mid-G1 phase before E2F-1 expression is detectable. These findings suggest that E2F-4 and E2F-5 may contribute to the regulation of early GI events including the Go/Gl transition. E2F/DP heterodimeric transcription factors are likely to be required for regulation of a large number of genes involved in cell proliferation (1, 2). An E2F consensus binding site has been demonstrated to be critical for the control of promoters activated at various different points in the cell cycle including the promoters of the c-myc (3, 4), DHFR (5), and cdc2 (6) genes. This wide spectrum of action may reflect the activities of several distinct E2F heterodimers, whose expression and function are regulated differentially, following distinct, cell cycle-specific schedules.A number of observations support this model. Cellular E2F activity is associated with several different protein species. Three distinct genes coding for E2Fs (7-11) and three for DPs (refs. 1 and 12; C. L. Wu and E. Harlow, personal communication) have already been identified. Moreover, the expression of the various E2Fs has been reported to be cell cycle dependent. For example, E2F-1 is expressed in the late G1 phase of the cell cycle (8, 13), clearly later than the induction of some E2F-responsive genes such as c-myc (3, 4). Finally, E2F activity appears to be directly and tightly regulated at several successive levels by the cell cycle machinery (14). For example, the E2F-1/DP-1 heterodimer appears to be inactivated through its binding to hypophosphorylated pRb in G, (15,16). Subsequently, phosphorylation of both pRb and E2F-1 (17) in late G1 results in the release of active E2F-1/ DP-1 transcription factors and in the transient expression of E2F-1-dependent genes. During the S and G2 phases that follow, the direct phosphorylation of E2F-1/DP-1 by cdk2/ cyclin A may then cause inactivation (14, 18). These processes may well explain the regulation of the three E2F subtypes (E2F-1, -2, and -3) that associate with pRb (11), but they do not address yet other aspects of E2F behavior. Thus, ...

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