The retinoblastoma (RB) tumor suppressor and its family members, p107 and p130, function by repressing E2F transcription factor activity to limit the expression of genes required for cell cycle progression. Traditionally, it is thought that the RB family proteins repress E2F target gene expression through complexing with E2F at gene promoters. However, whereas chromatin immunoprecipitation experiments have demonstrated p107 and p130 at E2F-responsive promoters, RB chromatin association has not been reliably observed. Here we used green fluorescent protein-tagged proteins to rigorously explore the mechanism of RB-mediated transcriptional repression relative to its p107 and p130 family members. The use of live cell fluorescent imaging demonstrated that RB, p107, and p130 exhibit similar nuclear dynamics. Although these findings suggest a similar engagement with nuclear structures, chromatin immunoprecipitation approaches with multiple independent antibodies failed to detect the association of RB with target gene promoters. However, by employing antibodies directed against green fluorescent protein, we could utilize the same antibody to assess RB, p107, and p130 engagement. This approach demonstrated RB association with target gene promoters in a fashion analogous to p107 and p130. Extension of this technology demonstrated that direct RB phosphorylation disrupts promoter association to regulate transcription. Thus, RB is associated with promoters in a manner similar to p107/p130 and that association is modulated by phosphorylation during cell cycle progression.
"It has been reported that pRb controls the G1 to S transition by repressing the transcriptional activity of the E2F protein family that is required for the expression of genes necessary for DNA synthesis and cell cycle progression , . By inhibiting the phosphorylation of pRb, we found that both pathways that regulate pRb phosphorylation ultimately converge on cell-cycle gene regulation. "
[Show abstract][Hide abstract] ABSTRACT: The retinoblastoma protein (pRb) is required for cell-cycle exit of embryonic mammalian hair cells but is not required for hair cell fate determination and early differentiation, and this provides a strategy for hair cell regeneration by manipulating the pRb pathway. To reveal the mechanism of pRb functional modification in the inner ear, we compared the effects of attenuated pRb phosphorylation by an inhibitor of the Mitogen-Activated Protein (MAP) kinase pathway and an inhibitor of the Rb-Raf-1 interaction on cultured chicken otocysts. We demonstrated that the activity of pRb is correlated with its phosphorylation state, which is regulated by a newly established cell cycle-independent pathway mediated by the physical interaction between Raf-1 and pRb. The phosphorylation of pRb plays an important role during the early stage of inner ear development, and attenuated phosphorylation in progenitor cells leads to cell cycle arrest and increased apoptosis along with a global down-regulation of the genes involved in cell cycle progression. Our study provides novel routes to modulate pRb function for hair cell regeneration.
PLoS ONE 12/2013; 8(12):e83726. DOI:10.1371/journal.pone.0083726 · 3.23 Impact Factor
"One possible mechanism for how LIN-35 might be specifically inhibited from binding in the germline comes from mammalian studies that have shown that Rb is largely refractory to ChIP analysis in transformed cells (reviewed in ). The phosphorylation status of Rb apparently alters its association with chromatin: phosphorylated Rb shows poor binding, while a phosphorylation-defective mutant has increased binding . Therefore, one possibility is that post-translational regulation of LIN-35/Rb, perhaps by phosphorylation, limits its association with chromatin in a germline-specific manner. "
[Show abstract][Hide abstract] ABSTRACT: Background
The tumor suppressor Rb/E2F regulates gene expression to control differentiation in multiple tissues during development, although how it directs tissue-specific gene regulation in vivo is poorly understood.
We determined the genome-wide binding profiles for Caenorhabditis elegans Rb/E2F-like components in the germline, in the intestine and broadly throughout the soma, and uncovered highly tissue-specific binding patterns and target genes. Chromatin association by LIN-35, the C. elegans ortholog of Rb, is impaired in the germline but robust in the soma, a characteristic that might govern differential effects on gene expression in the two cell types. In the intestine, LIN-35 and the heterochromatin protein HPL-2, the ortholog of Hp1, coordinately bind at many sites lacking E2F. Finally, selected direct target genes contribute to the soma-to-germline transformation of lin-35 mutants, including mes-4, a soma-specific target that promotes H3K36 methylation, and csr-1, a germline-specific target that functions in a 22G small RNA pathway.
In sum, identification of tissue-specific binding profiles and effector target genes reveals important insights into the mechanisms by which Rb/E2F controls distinct cell fates in vivo.
[Show abstract][Hide abstract] ABSTRACT: The majority of estrogen receptor (ER)-positive breast cancers are treated with endocrine therapy. While this is effective, acquired resistance to therapies targeted against ER is a major clinical challenge. Here, model systems of ER-positive breast cancers with differential susceptibility to endocrine therapy were employed to define common nodes for new therapeutic interventions. These analyses revealed that cell cycle progression is effectively uncoupled from the activity and functional state of ER in these models. In this context, cyclin D1 expression and retinoblastoma tumor suppressor protein (RB) phosphorylation are maintained even with efficient ablation of ER with pure antagonists. These therapy-resistant models recapitulate a key feature of deregulated RB/E2F transcriptional control. Correspondingly, a gene expression signature of RB-dysfunction is associated with luminal B breast cancer, which exhibits a relatively poor response to endocrine therapy. These collective findings suggest that suppression of cyclin D-supported kinase activity and restoration of RB-mediated transcriptional repression could represent a viable therapeutic option in tumors that fail to respond to hormone-based therapies. Consistent with this hypothesis, a highly selective CDK4/6 inhibitor, PD-0332991, was effective at suppressing the proliferation of all hormone refractory models analyzed. Importantly, PD-0332991 led to a stable cell cycle arrest that was fundamentally distinct from those elicited by ER antagonists, and was capable of inducing aspects of cellular senescence in hormone therapy refractory cell populations. These findings underscore the clinical utility of downstream cytostatic therapies in treating tumors that have experienced failure of endocrine therapy.
Endocrine Related Cancer 03/2011; 18(3):333-45. DOI:10.1530/ERC-10-0262 · 4.81 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.