Retinoblastoma/p107/p130 pocket proteins: Protein dynamics and interactions with target gene promoters
Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA. Journal of Biological Chemistry
(Impact Factor: 4.57).
04/2009; 284(29):19265-71. DOI: 10.1074/jbc.M808740200
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.
Available from: Zheng-Yi Chen
- "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. "
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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
Available from: PubMed Central
- "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. "
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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.
Genome biology 01/2013; 14(1):R5. DOI:10.1186/gb-2013-14-1-r5 · 10.81 Impact Factor
Available from: sciencedirect.com
- "RB Tumor Suppression Requires LXCXE Binding (Stengel et al., 2009). In contrast, both GFP-RB and GFP-N757F exhibited identical dynamic behavior that is consistent with the transient interaction of corepressors with chromatin (Figures 1C and S1A). "
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ABSTRACT: The LXCXE peptide motif facilitates interaction between the RB tumor suppressor and a large number of cellular proteins that are expected to impinge on diverse biological processes. In vitro and in vivo analyses demonstrated that LXCXE binding function is dispensable for RB promoter association and control of basal gene expression. Dependence on this function of RB is unmasked after DNA damage, wherein LXCXE binding is essential for exerting control over E2F3 and suppressing cell-cycle progression in the presence of genotoxic stress. Gene expression profiling revealed that the transcriptional program coordinated by this specific aspect of RB is associated with progression of human hepatocellular carcinoma and poor disease outcome. Consistent with these findings, biological challenge revealed a requirement for LXCXE binding in suppression of genotoxin-initiated hepatocellular carcinoma in vivo. Together, these studies establish an essential role of the LXCXE binding motif for RB-mediated transcriptional control, response to genotoxic insult, and tumor suppression.
Molecular cell 08/2011; 43(4):663-72. DOI:10.1016/j.molcel.2011.06.029 · 14.02 Impact Factor
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