Biomedical subjects

Nicholas J Dyson

Publications and source records attributed to Nicholas J Dyson.

16 recordsLinked to original sources

PubMed2006

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 p27(Kip1) through an unknown mechanism. Degradation of p27(Kip1) is mediated by ubiquitin-dependent targeting of p27(Kip1) by SCF -Skp2 (ref. 4). Here, we report a novel interaction between pRB and the anaphase-promoting complex/cyclosome (APC/C) that controls p27(Kip1) 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.

Anaphase↗

PubMed2006

Functional identification of Api5 as a suppressor of E2F-dependent apoptosis in vivo.

Retinoblastoma protein and E2-promoter binding factor (E2F) family members are important regulators of G1-S phase progression. Deregulated E2F also sensitizes cells to apoptosis, but this aspect of E2F function is poorly understood. Studies of E2F-induced apoptosis have mostly been carried out in tissue culture cells, and the analysis of the factors that are important for this process has been restricted to the testing of a few candidate genes. Using Drosophila as a model system, we have generated tools that allow genetic modifiers of E2F-dependent apoptosis to be identified in vivo and developed assays that allow effects on E2F-induced apoptosis to be studied in cultured cells. Genetic interactions show that dE2F1-dependent apoptosis in vivo involves dArk/Apaf1 apoptosome-dependent activation of both initiator and effector caspases and is sensitive to levels of Drosophila inhibitor of apoptosis-1 (dIAP1). Using these approaches, we report the surprising finding that apoptosis inhibitor-5/antiapoptosis clone-11 (Api5/Aac11) is a critical determinant of dE2F1-induced apoptosis in vivo and in vitro. This functional interaction occurs in multiple tissues, is specific to E2F-induced apoptosis, and is conserved from flies to humans. Interestingly, Api5/Aac11 acts downstream of E2F and suppresses E2F-dependent apoptosis without generally blocking E2F-dependent transcription. Api5/Aac11 expression is often upregulated in tumor cells, particularly in metastatic cells. We find that depletion of Api5 is tumor cell lethal. The strong genetic interaction between E2F and Api5/Aac11 suggests that elevated levels of Api5 may be selected during tumorigenesis to allow cells with deregulated E2F activity to survive under suboptimal conditions. Therefore, inhibition of Api5 function might offer a possible mechanism for antitumor exploitation.

Amino Acid Sequence↗

PubMed2006

The retinoblastoma protein regulates pericentric heterochromatin.

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 pRb's 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 pRb's 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 G(1)-to-S-phase transition.

Aneuploidy↗

PubMed2005

Retinoblastoma family 2 is required in vivo for the tissue-specific repression of dE2F2 target genes.

In higher eukaryotes, the Retinoblastoma and E2F families of proteins control the transcription of a large number of target genes. Here, we have mutated the second Drosophila Retinoblastoma family gene (Rbf2), and contrasted the in vivo molecular functions of RBF2 with dE2F2, the only E2F partner of RBF2. Previous studies failed to uncover a unique role for RBF2 in E2F regulation. Here we find that RBF2 functions in concert with dE2F2 in vivo to repress the expression of differentiation markers in ovaries and embryos where RBF2 is highly expressed. We have compared the profiles of transcripts that are mis-expressed in ovaries, embryos and S2 cells where RBF2 function has been ablated and find that RBF2 and dE2F2 control strikingly different transcriptional programs in each situation. In vivo promoter occupancy studies point to the redistribution of dE2F/RBF complexes to different promoters in different cell types as one mechanism governing the tissue-specific regulation of dE2F/RBF target genes. These results demonstrate that RBF2 has a unique function in repressing E2F-regulated differentiation markers and that dE2F2 and RBF2 are required to regulate different sets of target genes in different tissues.

Alleles↗

PubMed2005

The E2F transcriptional network: old acquaintances with new faces.

The E2 factor (E2F) family of transcription factors are downstream targets of the retinoblastoma protein. E2F factors have been known for several years to be important regulators of S-phase entry. Recent studies have improved our understanding of the molecular mechanisms of action used by this transcriptional network. In addition, they have given us an appreciation of the fact that E2F has functions that reach beyond G1/S control and impact cell proliferation in several different ways. The discovery of new family members with unusual properties, the unexpected phenotypes of mutant animals, a diverse collection of biological activities, a large number of new putative target genes and the new modes of transcriptional regulation have all contributed to an increasingly complex view of E2F function. In this review, we will discuss these recent developments and describe how they are beginning to shape a new and revised picture of the E2F transcriptional program.

Animals↗

PubMed2005

Drosophila E2F1 has context-specific pro- and antiapoptotic properties during development.

E2F transcription factors are generally believed to be positive regulators of apoptosis. In this study, we show that dE2F1 and dDP are important for the normal pattern of DNA damage-induced apoptosis in Drosophila wing discs. Unexpectedly, the role that E2F plays varies depending on the position of the cells within the disc. In irradiated wild-type discs, intervein cells show a high level of DNA damage-induced apoptosis, while cells within the D/V boundary are protected. In irradiated discs lacking E2F regulation, intervein cells are largely protected, but apoptotic cells are found at the D/V boundary. The protective effect of E2F at the D/V boundary is due to a spatially restricted role in the repression of hid. These loss-of-function experiments demonstrate that E2F cannot be classified simply as a pro- or antiapoptotic factor. Instead, the overall role of E2F in the damage response varies greatly and depends on the cellular context.

Animals↗

PubMed2005

dDP is needed for normal cell proliferation.

To gain insight into the essential functions of E2F, we have examined the phenotypes caused by complete inactivation of E2F and DP family members in Drosophila. Our results show that dDP requires dE2F1 and dE2F2 for DNA-binding activity in vitro and in vivo. In tissue culture cells and in mutant animals, the levels of dE2F and dDP proteins are strongly interdependent. In the absence of dDP, the levels of dE2F1 and dE2F2 decline dramatically, and vice versa. Accordingly, the cell cycle and transcriptional phenotypes caused by targeting dDP mimic the effects of targeting both dE2F1 and dE2F2 and are indistinguishable from the effects of inactivating all three proteins. Although trans-heterozygous dDP mutant animals develop to late pupal stages, the analysis of somatic mutant clones shows that dDP mutant cells are at a severe proliferative disadvantage when compared directly with wild-type neighbors. Strikingly, the timing of S-phase entry or exit is not delayed in dDP mutant clones, nor is the accumulation of cyclin A or cyclin B. However, the maximal level of bromodeoxyuridine incorporation is reduced in dDP mutant clones, and RNA interference experiments show that dDP-depleted cells are prone to stall in S phase. In addition, dDP mutant clones contain reduced numbers of mitotic cells, indicating that dDP mutant cells have a defect in G2/M-phase progression. Thus, dDP is not essential for developmental control of the G1-to-S transition, but it is required for normal cell proliferation, for optimal DNA synthesis, and for efficient G2/M progression.

Animals↗

PubMed2004

Molecular mechanisms of E2F-dependent activation and pRB-mediated repression.

Alterations in transcription of genes regulated by members of the E2F family of transcription factors can be viewed as a measure of the ebb and flow in a constantly evolving battle between repressor and activator complexes. Various chromatin regulatory complexes have been linked to Rb/E2F proteins, and changes in histone modifications correlate with states of E2F-dependent transcription. E2F has traditionally been viewed in the context of cell-cycle control. However, several recent studies have revealed a new aspect of E2F function in which pRB/E2F-family proteins confer stable repression of transcription. Such repression is evident in both actively proliferating cells and in cells that have withdrawn from the cell cycle.

Acetyltransferases↗

PubMed2004

p55, the Drosophila ortholog of RbAp46/RbAp48, is required for the repression of dE2F2/RBF-regulated genes.

Many proteins have been proposed to be involved in retinoblastoma protein (pRB)-mediated repression, but it is largely uncertain which cofactors are essential for pRB to repress endogenous E2F-regulated promoters. Here we have taken advantage of the stream-lined Drosophila dE2F/RBF pathway, which has only two E2Fs (dE2F1 and dE2F2), and two pRB family members (RBF1 and RBF2). With RNA interference (RNAi), we depleted potential corepressors and looked for the elevated expression of groups of E2F target genes that are known to be directly regulated by RBF1 and RBF2. Previous studies have implicated histone deacetylase (HDAC) and SWI/SNF chromatin-modifying complexes in pRB-mediated repression. However, our results fail to support the idea that the SWI/SNF proteins are required for RBF-mediated repression and suggest that a requirement for HDAC activities is likely to be limited to a subset of targets. We found that the chromatin assembly factor p55/dCAF-1 is essential for the repression of dE2F2-regulated targets. The removal of p55 deregulated the expression of E2F targets that are normally repressed by dE2F2/RBF1 and dE2F2/RBF2 complexes in a cell cycle-independent manner but had no effect on the expression of E2F targets that are normally coupled with cell proliferation. The results indicate that the mechanisms of RBF regulation at these two types of E2F targets are different and suggest that p55, and perhaps p55's mammalian orthologs RbAp46 and RbAp48, have a conserved function in repression by pRB-related proteins.

Animals↗

PubMed2003

Cell cycle-dependent and cell cycle-independent control of transcription by the Drosophila E2F/RB pathway.

To determine which E2F/RB-family members are functionally important at E2F-dependent promoters, we used RNA interference (RNAi) to selectively remove each component of the dE2F/dDP/RBF pathway, and we examined the genome-wide changes in gene expression that occur when each element is missing. The results reveal a remarkable division of labor between family members. Classic E2F targets, encoding functions needed for cell cycle progression, are expressed in cycling cells and are primarily dependent on dE2F1and RBF1 for regulation. Unexpectedly, there is a second program of dE2F/RBF-dependent transcription, in which dE2F2/RBF1or dE2F2/RBF2 complexes repress gene expression in actively proliferating cells. These new E2F target genes encode differentiation factors that are transcribed in developmentally regulated and gender-specific patterns and not in a cell cycle-regulated manner. We propose that dE2F/RBF complexes should not be viewed simply as a cell cycle regulator of transcription. Instead, dE2F/RBF-mediated repression is exerted on genes that encode an assortment of cellular functions, and these effects are reversed on sets of functionally related genes in particular developmental contexts. As a result, dE2F/RBF regulation is used to link gene expression with cell cycle progression at some targets while simultaneously providing stable repression at others.

Adaptor Proteins, Vesicular Transport↗

PubMed2003

G1 cyclin-dependent kinases are insufficient to reverse dE2F2-mediated repression.

Here we show that the cell cycle defects of dE2F1-depleted cells depend on the cooperative effects of dE2F2 and DACAPO (DAP), an inhibitor of Cyclin E/cyclin-dependent kinase 2 (CycE/cdk2). The different properties of cells lacking dE2F1/dE2F2 and dE2F1/DAP lead to the surprising observation that dE2F2-mediated repression differs from retinoblastoma family protein 1 (RBF1) inhibition of dE2F1, and is resistant to both CycE/cdk2 and Cyclin D/cyclin-dependent kinase 4 (CycD/cdk4). This resistance occurs even though dE2F2/RBF1 complexes are disrupted by CycE/cdk2, and may explain why dE2F2 is so potent in the absence of de2f1. The implication of these results is that cells containing dE2F2 require dE2F1 to either prevent, or reverse, dE2F-mediated repression.

Alleles↗

PubMed2003

Dp1 is required for extra-embryonic development.

Release of E2F1/DP1 heterodimers from repression mediated by the retinoblastoma tumor suppressor (pRB) triggers cell cycle entry into S phase, suggesting that E2F1 and DP1 proteins must act in unison, either to facilitate or to suppress cell-cycle progression. In stark contrast to the milder phenotypes that result from inactivation of E2Fs, we report that loss of Dp1 leads to death in utero because of the failure of extra-embryonic development. Loss of Dp1 compromises the trophectoderm-derived tissues - specifically, the expansion of the ectoplacental cone and chorion, and endoreduplication in trophoblast giant cells. Inactivation of p53 is unable to rescue the Dp1-deficient embryonic lethality. Thus, DP1 is absolutely required for extra-embryonic development and consequently embryonic survival, consistent with E2F/DP1 normally acting to promote growth in vivo.

Animals↗

PubMed2002

Quantitatively determined survivin expression levels are of prognostic value in human gliomas.

PURPOSE: Survivin is a novel antiapoptotic gene that has been recently cloned and characterized. Its expression has been found to be of prognostic significance in several tumor types. This is the first study on the prognostic significance of survivin expression in human gliomas. MATERIALS AND METHODS: We used quantitative Western blot analysis with densitometry to determine survivin protein expression levels in 92 glioma cases for which frozen tissue was available for analysis. Survivin positivity and expression levels were correlated with histopathologic features of the tumors, apoptosis (as measured by cleaved, or activated, caspase 3 levels), and clinical outcome. RESULTS: Survivin expression has clear prognostic value in human gliomas. Patients with detectable survivin expression had significantly shorter overall survival times (P <.0001) compared with those without detectable expression when all glioma patients were considered. Although glioblastoma multiforme (GBM) patients had significantly higher rates of survivin positivity and higher levels of survivin expression (P <.0001) than their non-GBM counterparts, the prognostic value of survivin expression seemed to be independent of histology alone. Survivin-positive GBM patients had significantly shorter overall survival times compared with survivin-negative GBM patients (P <.0001). Likewise, survivin-positive non-GBM patients had shorter survival times compared with survivin-negative non-GBM patients (P =.029). Furthermore, increasing levels of survivin expression significantly correlated with reduced survival times when all glioma patients were considered, and markedly so for GBM patients (P <.0001). Increasing survivin levels significantly correlated with reduced expression of cleaved caspase 3, indicating its association with antiapoptotic activity. CONCLUSION: Survivin positivity and protein expression levels, as determined quantitatively, are of significant prognostic value in human gliomas and seem to be associated with reduced apoptotic capacity of these tumors.

Adult↗

PubMed2002

Insulin-like growth factor receptor I mediates resistance to anti-epidermal growth factor receptor therapy in primary human glioblastoma cells through continued activation of phosphoinositide 3-kinase signaling.

Overexpression of the epidermal growth factor receptor (EGFR) has been shown previously to correlate with enhanced malignant potential of many human tumor types, including glioblastoma multiforme (GBM). Anti-EGFR targeting has been demonstrated to enhance apoptosis and reduce both cellular invasion and angiogenic potential. It remains unclear whether absolute EGFR expression levels are sufficient to predict which tumors will respond best to anti-EGFR therapy. We have identified two primary GBM cell lines with equivalent EGFR expression levels with very different sensitivities to the EGFR receptor tyrosine kinase inhibitor, AG1478. This was apparent despite similar reductions in EGFR signaling in both cell lines, as measured by phospho-EGFR levels. AG1478 enhanced both spontaneous and radiation-induced apoptosis and reduced invasive potential in the GBM(S), but not in the GBM(R), cell line. The resistant GBM(R) cell line demonstrated an up-regulation of insulin-like growth factor receptor I (IGFR-I) levels on AG1478 administration. This resulted in sustained signaling through the phosphoinositide 3-kinase pathway, resulting in potent antiapoptotic and proinvasion effects. Cotargeting IGFR-I with EGFR greatly enhanced both spontaneous and radiation-induced apoptosis of the GBM(R) cells and reduced their invasive potential. Akt1 and p70(s6k) appeared to be important downstream targets of IGFR-I-mediated resistance to anti-EGFR targeting. These findings suggest that IGFR-I signaling through phosphoinositide 3-kinase may represent a novel and potentially important mechanism of resistance to anti-EGFR therapy.

Apoptosis↗

PubMed2002

A revised picture of the E2F transcriptional network and RB function.

New techniques have enhanced our picture of E2F regulation. These studies have shed light on the roles played by individual E2F and retinoblastoma family members and implicate these proteins in processes extending well beyond the G1/S transition. One thorny issue remains: do our current molecular models of E2F and retinoblastoma action explain all of the functions of these proteins in vivo?

Animals↗

PubMed2002

Three regions of the pRB pocket domain affect its inactivation by human papillomavirus E7 proteins.

A critical event in papillomavirus transformation of human cells is the inactivation of pRB by the E7 protein. E7, like many other viral oncoproteins, possesses a well-characterized LXCXE peptide motif that interacts with the pocket domain of pRB. Disruption of the LXCXE-binding cleft on pRB renders it resistant to E7 binding and inactivation. Such binding cleft mutants of pRB are capable of inducing a G(1) arrest in the human papillomavirus 18-transformed HeLa cell line. We show here that the efficient inactivation of pRB in HeLa cells does not simply depend on the integrity of the LXCXE-binding cleft. Multiple site-directed mutants that alter conserved surfaces of the pRB pocket domain cause HeLa cells to accumulate in G(1). We divide these mutants into two classes: those that can be bound by E7 and those that cannot. The E7 interacting mutants include changes in conserved residues that lie in a groove between the A and B halves of the pocket. Surprisingly, none of these mutants show a clear defect in any of the known mechanisms for pRB inactivation by E7. Analysis of mutants that are compromised for E7 binding reveals that this interaction depends on both the LXCXE-binding cleft and on a conserved group of lysines adjacent to the cleft. These basic amino acids on pRB define a discrete interaction point with E7. These residues most likely form ionic interactions with conserved acidic amino acids on E7 since a stable pRB/E7 interaction was restored when the lysine residues on pRB and the acidic residues on E7 were interchanged.

Amino Acid Motifs↗