Frederick A. Dick

Molecular mechanisms underlying RB protein function

Inactivation of the RB protein is one of the most fundamental events in cancer. Coming to a molecular understanding of its function in normal cells and how it impedes cancer development has been challenging. Historically, the ability of RB to regulate the cell cycle placed it in a central role in proliferative control, and research focused on RB regulation of the E2F family of transcription factors. Remarkably, several recent studies have found additional tumour-suppressor functions of RB, including alternative roles in the cell cycle, maintenance of genome stability and apoptosis. These advances and new structural studies are combining to define the multifunctionality of RB.

Retinoblastoma protein and anaphase-promoting complex physically interact and functionally cooperate during cell-cycle exit

The retinoblastoma protein (pRB) negatively regulates the progression from G1 to S phase of the cell cycle, in part, by repressing E2F-dependent transcription. pRB also possesses E2F-independent functions that contribute to cell-cycle control — for example, during pRB-mediated cell-cycle arrest pRB associates with Skp2, the F-box protein of the Skp1–Cullin–F-box protein (SCF) E3 ubiquitin ligase complex, and promotes the stability of the cyclin-dependent kinase-inhibitor p27Kip1 through an unknown mechanism. Degradation of p27Kip1 is mediated by ubiquitin-dependent targeting of p27Kip1 by SCF –Skp2 (ref. 4). Here, we report a novel interaction between pRB and the anaphase-promoting complex/cyclosome (APC/C) that controls p27Kip1 stability by targeting Skp2 for ubiquitin-mediated degradation. Cdh1, an activator of APC/C, not only interacts with pRB but is also required for a pRB-induced cell-cycle arrest. The results reveal an unexpected physical convergence between the pRB tumour-suppressor protein and E3 ligase complexes, and raise the possibility that pRB may direct APC/C to additional targets during pRB-mediated cell-cycle exit.

pRB contains an E2F1-specific binding domain that allows E2F1-induced apoptosis to be regulated separately from other E2F activities.

The interaction between pRB and E2F is critical for control of the cell cycle and apoptosis. Here we report that pRB contains two distinct E2F binding sites. The previously identified E2F binding site on pRB is necessary for stable association with E2Fs on DNA. A second E2F interaction site is located entirely within the C-terminal domain of pRB and is specific for E2F1. E2F1/pRB complexes formed through this site have low affinity for DNA, but the interaction is sufficient for pRB to regulate E2F1-induced apoptosis, and E2F1 loses the ability to interact with this site following DNA damage. These results show that pRB interacts with individual E2F proteins in different ways and suggest that pRBs regulation of E2F1-induced apoptosis is physically separable from its transcriptional control of other E2F proteins.

The retinoblastoma family of proteins and their regulatory functions in the mammalian cell division cycle.

The retinoblastoma (RB) family of proteins are found in organisms as distantly related as humans, plants, and insects. These proteins play a key role in regulating advancement of the cell division cycle from the G1 to S-phases. This is achieved through negative regulation of two important positive regulators of cell cycle entry, E2F transcription factors and cyclin dependent kinases. In growth arrested cells transcriptional activity by E2Fs is repressed by RB proteins. Stimulation of cell cycle entry by growth factor signaling leads to activation of cyclin dependent kinases. They in turn phosphorylate and inactivate the RB family proteins, leading to E2F activation and additional cyclin dependent kinase activity. This propels the cell cycle irreversibly forward leading to DNA synthesis. This review will focus on the basic biochemistry and cell biology governing the regulation and activity of mammalian RB family proteins in cell cycle control.

Loss of ATRX leads to chromosome cohesion and congression defects

αThalassemia/mental retardation X linked (ATRX) is a switch/sucrose nonfermenting-type ATPase localized at pericentromeric heterochromatin in mouse and human cells. Human ATRX mutations give rise to mental retardation syndromes characterized by developmental delay, facial dysmorphisms, cognitive deficits, and microcephaly and the loss of ATRX in the mouse brain leads to reduced cortical size. We find that ATRX is required for normal mitotic progression in human cultured cells and in neuroprogenitors. Using live cell imaging, we show that the transition from prometaphase to metaphase is prolonged in ATRX-depleted cells and is accompanied by defective sister chromatid cohesion and congression at the metaphase plate. We also demonstrate that loss of ATRX in the embryonic mouse brain induces mitotic defects in neuroprogenitors in vivo with evidence of abnormal chromosome congression and segregation. These findings reveal that ATRX contributes to chromosome dynamics during mitosis and provide a possible cellular explanation for reduced cortical size and abnormal brain development associated with ATRX deficiency.

Mitotic chromosome condensation mediated by the retinoblastoma protein is tumor-suppressive

Condensation and segregation of mitotic chromosomes is a critical process for cellular propagation, and, in mammals, mitotic errors can contribute to the pathogenesis of cancer. In this report, we demonstrate that the retinoblastoma protein (pRB), a well-known regulator of progression through the G1 phase of the cell cycle, plays a critical role in mitotic chromosome condensation that is independent of G1-to-S-phase regulation. Using gene targeted mutant mice, we studied this aspect of pRB function in isolation, and demonstrate that it is an essential part of pRB-mediated tumor suppression. Cancer-prone Trp53(-/-) mice succumb to more aggressive forms of cancer when pRBs ability to condense chromosomes is compromised. Furthermore, we demonstrate that defective mitotic chromosome structure caused by mutant pRB accelerates loss of heterozygosity, leading to earlier tumor formation in Trp53(+/-) mice. These data reveal a new mechanism of tumor suppression, facilitated by pRB, in which genome stability is maintained by proper condensation of mitotic chromosomes.

Mutagenesis of the pRB pocket reveals that cell cycle arrest functions are separable from binding to viral oncoproteins.

ABSTRACT The pocket domain of pRB is required for pRB to arrest the cell cycle. This domain was originally defined as the region of the protein that is necessary and sufficient for pRBs interaction with adenovirus E1A and simian virus s40 large T antigen. These oncoproteins, and other pRB-binding proteins that are encoded by a variety of plant and animal viruses, use a conserved LXCXE motif to interact with pRB. Similar sequences have been identified in multiple cellular pRB-binding proteins, suggesting that the viruses have evolved to target a highly conserved binding site of pRB that is critical for its function. Here we have constructed a panel of pRB mutants in which conserved amino acids that are predicted to make close contacts with an LXCXE peptide were altered. Despite the conservation of the LXCXE binding site throughout evolution, pRB mutants that lack this site are able to induce a cell cycle arrest in a pRB-deficient tumor cell line. This G1 arrest is overcome by cyclin D-cdk4 complexes but is resistant to inactivation by E7. Consequently, mutants lacking the LXCXE binding site were able to induce a G1 arrest in HeLa cells despite the expression of HPV-18 E7. pRB mutants lacking the LXCXE binding site are defective in binding to adenovirus E1A and human papillomavirus type 16 E7 protein but exhibit wild-type binding to E2F or DP, and they retain the ability to interact with CtIP and HDAC1, two transcriptional corepressors that contain LXCXE-like sequences. Consistent with these observations, the pRB mutants are able to actively repress transcription. These observations suggest that viral oncoproteins depend on the LXCXE-binding site of pRB for interaction to a far greater extent than cellular proteins that are critical for cell cycle arrest or transcriptional repression. Mutation of this binding site allows pRB to function as a cell cycle regulator while being resistant to inactivation by viral oncoproteins.

The Retinoblastoma Protein Regulates Pericentric Heterochromatin

ABSTRACT The retinoblastoma protein (pRb) has been proposed to regulate cell cycle progression in part through its ability to interact with enzymes that modify histone tails and create a repressed chromatin structure. We created a mutation in the murine Rb1 gene that disrupted pRbs ability to interact with these enzymes to determine if it affected cell cycle control. Here, we show that loss of this interaction slows progression through mitosis and causes aneuploidy. Our experiments reveal that while the LXCXE binding site mutation does not disrupt pRbs interaction with the Suv4-20h histone methyltransferases, it dramatically reduces H4-K20 trimethylation in pericentric heterochromatin. Disruption of heterochromatin structure in this chromosomal region leads to centromere fusions, chromosome missegregation, and genomic instability. These results demonstrate the surprising finding that pRb uses the LXCXE binding cleft to control chromatin structure for the regulation of events beyond the G1-to-S-phase transition.

Structure-function analysis of the retinoblastoma tumor suppressor protein – is the whole a sum of its parts?

Biochemical analysis of the retinoblastoma proteins function has received considerable attention since it was cloned just over 20 years ago. During this time pRB has emerged as a key regulator of the cell division cycle and its ability to block proliferation is disrupted in the vast majority of human cancers. Much has been learned about the regulation of E2F transcription factors by pRB in the cell cycle. However, many questions remain unresolved and researchers continue to explore this multifunctional protein. In particular, understanding how its biochemical functions contribute to its role as a tumor suppressor remains to be determined. Since pRB has been shown to function as an adaptor molecule that links different proteins together, or to particular promoters, analyzing pRB by disrupting individual protein interactions holds tremendous promise in unraveling the intricacies of its function. Recently, crystal structures have reported how pRB interacts with some of its molecular partners. This information has created the possibility of rationally separating pRB functions by studying mutants that disrupt individual binding sites. This review will focus on literature that investigates pRB by isolating functions based on binding sites within the pocket domain. This article will also discuss the prospects for using this approach to further explore the unknown functions of pRB.

Inhibition of Pluripotency Networks by the Rb Tumor Suppressor Restricts Reprogramming and Tumorigenesis

Mutations in the retinoblastoma tumor suppressor gene Rb are involved in many forms of human cancer. In this study, we investigated the early consequences of inactivating Rb in the context of cellular reprogramming. We found that Rb inactivation promotes the reprogramming of differentiated cells to a pluripotent state. Unexpectedly, this effect is cell cycle independent, and instead reflects direct binding of Rb to pluripotency genes, including Sox2 and Oct4, which leads to a repressed chromatin state. More broadly, this regulation of pluripotency networks and Sox2 in particular is critical for the initiation of tumors upon loss of Rb in mice. These studies therefore identify Rb as a global transcriptional repressor of pluripotency networks, providing a molecular basis for previous reports about its involvement in cell fate pliability, and implicate misregulation of pluripotency factors such as Sox2 in tumorigenesis related to loss of Rb function.