A suppressor screen in chlamydomonas identifies novel components of the retinoblastoma tumor suppressor pathway

The retinoblastoma (RB) protein is a eukaryotic tumor suppressor and negative cell-cycle regulator. Chlamydomonas reinhardtii cells that lack the RB homolog MAT3 show loss of size checkpoint control and deregulated cell-cycle progression leading to the production of tiny cells. We carried out an insertional mutagenesis screen to isolate bypass suppressors of mat3 (smt mutants) that reverted the mat3 cell-size defect. Previously we reported that the loci encoding Chlamydomonas homologs of E2F and DP were frequently disrupted in this screen, indicating that the architecture of the canonical RB pathway is conserved in Chlamydomonas with MAT3/RB acting as a negative regulator upstream of E2F/DP. Here, we describe four novel smt mutants that moderately suppressed the cell-size checkpoint and cell-cycle phenotypes of mat3. As single mutants, three of the smt strains displayed no obvious phenotypes, and one had a slightly small phenotype. Strikingly, several smt double-mutant combinations synergized to cause enhanced suppression of mat3 and even to cause a large-cell phenotype that is comparable to that caused by loss of DP1. Molecular characterization of one smt mutant revealed that suppression is due to a defect in a gene encoding a putative small ubiquitin-like modifier (SUMO) peptidase. Our results reveal a complex genetic network that lies downstream of MAT3/RB and implicate protein sumoylation as an important step for cell-cycle progression in cells that are missing MAT3/RB.     

Regulation of the Chlamydomonas cell cycle by a stable, chromatin-associated retinoblastoma tumor suppressor complex

We examined the cell cycle dynamics of the retinoblastoma (RB) protein complex in the unicellular alga Chlamydomonas reinhardtii that has single homologs for each subunit-RB, E2F, and DP. We found that Chlamydomonas RB (encoded by MAT3) is a cell cycle-regulated phosphoprotein, that E2F1-DP1 can bind to a consensus E2F site, and that all three proteins interact in vivo to form a complex that can be quantitatively immunopurified. Yeast two-hybrid assays revealed the formation of a ternary complex between MAT3, DP1, and E2F1 that requires a C-terminal motif in E2F1 analogous to the RB binding domain of plant and animal E2Fs. We examined the abundance of MAT3/RB and E2F1-DP1 in highly synchronous cultures and found that they are synthesized and remain stably associated throughout the cell cycle with no detectable fraction of free E2F1-DP1. Consistent with their stable association, MAT3/RB and DP1 are constitutively nuclear, and MAT3/RB does not require DP1-E2F1 for nuclear localization. In the nucleus, MAT3/RB remains bound to chromatin throughout the cell cycle, and its chromatin binding is mediated through E2F1-DP1. Together, our data show that E2F-DP complexes can regulate the cell cycle without dissociation of their RB-related subunit and that other changes may be sufficient to convert RB-E2F-DP from a cell cycle repressor to an activator.     

p130RB2 positively contributes to ATR activation in response to replication stress via the RPA32-ETAA1 axis

Ataxia-telangiectasia mutated and Rad3-related (ATR) kinase is a crucial regulator of the cell cycle checkpoint and activated in response to DNA replication stress by two independent pathways via RPA32-ETAA1 and TopBP1. However, the precise activation mechanism of ATR by the RPA32-ETAA1 pathway remains unclear. Here, we show that p130RB2, a member of the retinoblastoma protein family, participates in the pathway under hydroxyurea-induced DNA replication stress. p130RB2 binds to ETAA1, but not TopBP1, and depletion of p130RB2 inhibits the RPA32-ETAA1 interaction under replication stress. Moreover, p130RB2 depletion reduces ATR activation accompanied by phosphorylation of its targets RPA32, Chk1, and ATR itself. It also causes improper re-progression of S phase with retaining single-stranded DNA after cancelation of the stress, which leads to an increase in the anaphase bridge phenotype and a decrease in cell survival. Importantly, restoration of p130RB2 rescued the disrupted phenotypes of p130RB2 knockdown cells. These results suggest positive involvement of p130RB2 in the RPA32-ETAA1-ATR axis and proper re-progression of the cell cycle to maintain genome integrity.     

The retinoblastoma gene: a prototypic and multifunctional tumor suppressor

Genome instability has been implicated in the generation of multiple somatic mutations that underlie cancer. Germline mutation in the retinoblastoma (RB) gene leads to tumor formation in both human and experimental animal models, and reintroduction of wild-type RB is able to suppress neoplastic phenotypes. Rb governs the passage of cells through the G1 phase-restriction point and this control is lost in most cancer cells. Rb has also been shown to promote terminal differentiation and prevent cell cycle reentry. Recent studies implicate Rb in mitotic progression, faithful chromosome segregation, checkpoint control, and chromatin remodeling, suggesting that Rb may function in the maintenance of genome integrity. It is likely that Rb suppresses tumor formation by virtue of its multiple biological activities. A single protein capable of performing multiple antioncogenic functions may be a common characteristic of other tumor suppressors including p53 and BRCA1/2.     

Compensation of BRG-1 function by Brm: insight into the role of the core SWI-SNF subunits in retinoblastoma tumor suppressor signaling.

The BRG-1 subunit of the SWI-SNF complex is involved in chromatin remodeling and has been implicated in the action of the retinoblastoma tumor suppressor (RB). Given the importance of BRG-1 in RB function, germ line BRG-1 mutations in tumorigenesis may be tantamount to RB inactivation. Therefore, in this study we assessed the behavior of cells harboring discrete BRG-1 alleles for the RB-signaling pathway. Using p16ink4a, an upstream activator of endogenous RB, or a constitutively active RB construct (PSM-RB), we determined that the majority of tumor lines with germ line defects in BRG-1 were sensitive to RB-mediated cell cycle arrest. By contrast, A427 (lung carcinoma) cells were resistant to expression of p16ink4a and PSM-RB. Analysis of the SWI-SNF subunits in the different tumor lines revealed that A427 are deficient for BRG-1 and its homologue, Brm, whereas RB-sensitive cell lines retained Brm expression. Similarly, the RB-resistant SW13 and C33A cell lines were also deficient for both BRG-1/Brm. Reintroduction of either BRG-1 or Brm into A427 or C33A cells restored RB-mediated signaling to cyclin A to cause cell cycle arrest. Consistent with this compensatory role, we observed that Brm could also drive expression of CD44. We also determined that loss of these core SWI-SNF subunits renders SW13 cells resistant to activation of the RB pathway by the chemotherapeutic agent cisplatin, since reintroduction of either BRG-1 or Brm into SW13 cells restored the cisplatin DNA-damage checkpoint. Together, these data demonstrate that Brm can compensate for BRG-1 loss as pertains to RB sensitivity.     

E2F and p53 induce apoptosis independently during Drosophila development but intersect in the context of DNA damage

In mammalian cells, RB/E2F and p53 are intimately connected, and crosstalk between these pathways is critical for the induction of cell cycle arrest or cell death in response to cellular stresses. Here we have investigated the genetic interactions between RBF/E2F and p53 pathways during Drosophila development. Unexpectedly, we find that the pro-apoptotic activities of E2F and p53 are independent of one another when examined in the context of Drosophila development: apoptosis induced by the deregulation of dE2F1, or by the overexpression of dE2F1, is unaffected by the elimination of dp53; conversely, dp53-induced phenotypes are unaffected by the elimination of dE2F activity. However, dE2F and dp53 converge in the context of a DNA damage response. Both dE2F1/dDP and dp53 are required for DNA damage-induced cell death, and the analysis of rbf1 mutant eye discs indicates that dE2F1/dDP and dp53 cooperatively promote cell death in irradiated discs. In this context, the further deregulation in the expression of pro-apoptotic genes generates an additional sensitivity to apoptosis that requires both dE2F/dDP and dp53 activity. This sensitivity differs from DNA damage-induced apoptosis in wild-type discs (and from dE2F/dDP-induced apoptosis in un-irradiated rbf1 mutant eye discs) by being dependent on both hid and reaper. These results show that pro-apoptotic activities of dE2F1 and dp53 are surprisingly separable: dp53 is required for dE2F-dependent apoptosis in the response to DNA damage, but it is not required for dE2F-dependent apoptosis caused simply by the inactivation of rbf1.     

Characterization of mat A-2, mat A-3 and deltamatA mating-type mutants of Neurospora crassa.

The mating-type locus of Neurospora crassa regulates mating identity and entry into the sexual cycle. The mat A idiomorph encodes three genes, mat A-1, mat A-2, and mat A-3. Mutations in mat A-1 result in strains that have lost mating identity and vegetative incompatibility with mat a strains. A strain containing mutations in both mat A-2 and mat A-3 is able to mate, but forms few ascospores. In this study, we describe the isolation and characterization of a mutant deleted for mat (deltamatA), as well as mutants in either mat A-2 or mat A-3. The deltamatA strain is morphologically wild type during vegetative growth, but it is sterile and heterokaryon compatible with both mat A and mat a strains. The mat A-2 and mat A-3 mutants are also normal during vegetative growth, mate as a mat A strain, and produce abundant biparental asci in crosses with mat a, and are thus indistinguishable from a wild-type mat A strain. These data and the fact that the mat A-2 mat A-3 double mutant makes few asci with ascospores indicate that MAT A-2 and MAT A-3 are redundant and may function in the same pathway. Analysis of the expression of two genes (sdv-1 and sdv-4) in the various mat mutants suggests that the mat A polypeptides function in concert to regulate the expression of some sexual development genes.     

Radiation Dose-Dependent Maintenance of G2 Arrest Requires Retinoblastoma Protein

In response to ionizing radiation (IR), cell cycle checkpoints are activated to provide time for DNA repair. Several different checkpoint mechanisms have been elucidated. However, mechanisms that regulate the duration of cell cycle arrest are not understood. Previous studies have shown that the retinoblastoma tumor suppressor protein (RB) is required for radiation-induced G1 arrest. Working with primary fibroblasts derived from Rb+/+ and Rb-/- mouse embryos, we show that RB also regulates the duration of G2 arrest. The initial G2 checkpoint response is enhanced in Rb-/- cells due to a defect in G1 arrest. However, the permanent arrest in G2 induced by higher doses of IR does not occur in Rb-/- cells. Rb-/- cells either resumed proliferation or underwent apoptosis at IR doses that caused the majority of Rb+/+ cells to arrest permanently in G2. The prolongation of G2 arrest in Rb+/+ cells correlated with a gradual accumulation of hypophosphorylated RB. Thus, regulation of the RB function may be an important aspect in the maintenance of cell cycle checkpoints in DNA damage response.

Key Words:

RB phosphorylation, Ionizing radiation, DNA damage, G2 checkpoint, Mouse embryo fibroblasts     

DNA tumor virus oncoproteins and retinoblastoma gene mutations share the ability to relieve the cell's requirement for cyclin D1 function in G1

The retinoblastoma gene product (pRB) participates in the regulation of the cell division cycle through complex formation with numerous cellular regulatory proteins including the potentially oncogenic cyclin D1. Extending the current view of the emerging functional interplay between pRB and D-type cyclins, we now report that cyclin D1 expression is positively regulated by pRB. Cyclin D1 mRNA and protein is specifically downregulated in cells expressing SV40 large T antigen, adenovirus E1A, and papillomavirus E7/E6 oncogene products and this effect requires intact RB-binding, CR2 domain of E1A. Exceptionally low expression of cyclin D1 is also seen in genetically RB-deficient cell lines, in which ectopically expressed wild-type pRB results in specific induction of this G1 cyclin. At the functional level, antibody-mediated cyclin D1 knockout experiments demonstrate that the cyclin D1 protein, normally required for G1 progression, is dispensable for passage through the cell cycle in cell lines whose pRB is inactivated through complex formation with T antigen, E1A, or E7 oncoproteins as well as in cells which have suffered loss-of-function mutations of the RB gene. The requirement for cyclin D1 function is not regained upon experimental elevation of cyclin D1 expression in cells with mutant RB, while reintroduction of wild-type RB into RB-deficient cells leads to restoration of the cyclin D1 checkpoint. These results strongly suggest that pRB serves as a major target of cyclin D1 whose cell cycle regulatory function becomes dispensable in cells lacking functional RB. Based on available data including this study, we propose a model for an autoregulatory feedback loop mechanism that regulates both the expression of the cyclin D1 gene and the activity of pRB, thereby contributing to a G1 phase checkpoint control in cycling mammalian cells.     

Radiation dose-dependent maintenance of G(2) arrest requires retinoblastoma protein

In response to ionizing radiation (IR), cell cycle checkpoints are activated to provide time for DNA repair. Several different checkpoint mechanisms have been elucidated. However, mechanisms that regulate the duration of cell cycle arrest are not understood. Previous studies have shown that the retinoblastoma tumor suppressor protein (RB) is required for radiation-induced G1 arrest. Working with primary fibroblasts derived from Rb+/+ and Rb-/- mouse embryos, we show that RB also regulates the duration of G2 arrest. The initial G2 checkpoint response is enhanced in Rb-/- cells due to a defect in G1 arrest. However, the permanent arrest in G2 induced by higher doses of IR does not occur in Rb-/- cells. Rb-/- cells either resumed proliferation or underwent apoptosis at IR doses that caused the majority of Rb+/+ cells to arrest permanently in G2. The prolongation of G2 arrest in Rb+/+ cells correlated with a gradual accumulation of hypophosphorylated RB. Thus, regulation of the RB function may be an important aspect in the maintenance of cell cycle checkpoints in DNA damage response.     

Mathematical model of the morphogenesis checkpoint in budding yeast

The morphogenesis checkpoint in budding yeast delays progression through the cell cycle in response to stimuli that prevent bud formation. Central to the checkpoint mechanism is Swe1 kinase: normally inactive, its activation halts cell cycle progression in G2. We propose a molecular network for Swe1 control, based on published observations of budding yeast and analogous control signals in fission yeast. The proposed Swe1 network is merged with a model of cyclin-dependent kinase regulation, converted into a set of differential equations and studied by numerical simulation. The simulations accurately reproduce the phenotypes of a dozen checkpoint mutants. Among other predictions, the model attributes a new role to Hsl1, a kinase known to play a role in Swe1 degradation: Hsl1 must also be indirectly responsible for potent inhibition of Swe1 activity. The model supports the idea that the morphogenesis checkpoint, like other checkpoints, raises the cell size threshold for progression from one phase of the cell cycle to the next.     

RB signaling prevents replication-dependent DNA double-strand breaks following genotoxic insult

Cell cycle checkpoints induced by DNA damage play an integral role in preservation of genomic stability by allowing cells to limit the propagation of deleterious mutations. The retinoblastoma tumor suppressor (RB) is crucial for the maintenance of the DNA damage checkpoint function because it elicits cell cycle arrest in response to a variety of genotoxic stresses. Although sporadic loss of RB is characteristic of most cancers and results in the bypass of the DNA damage checkpoint, the consequence of RB loss upon chemotherapeutic responsiveness has been largely uninvestigated. Here, we employed a conditional knockout approach to ablate RB in adult fibroblasts. This system enabled us to examine the DNA damage response of adult cells following acute RB deletion. Using this system, we demonstrated that loss of RB disrupted the DNA damage checkpoint elicited by either cisplatin or camptothecin exposure. Strikingly, this bypass was not associated with enhanced repair, but rather the accumulation of phosphorylated H2AX (γH2AX) foci, which indicate DNA double-strand breaks. The formation of γH2AX foci was due to ongoing replication following chemotherapeutic treatment in the RB-deficient cells. Additionally, peak γH2AX accumulation occurred in S-phase cells undergoing DNA replication in the presence of damage, and these γH2AX foci co-localized with replication foci. These results demonstrate that acute RB loss abrogates DNA damage-induced cell cycle arrest to induce γH2AX foci formation. Thus, secondary genetic lesions induced by RB loss have implications for the chemotherapeutic response and the development of genetic instability.