Naturally death-resistant precursor cells revealed as the origin of retinoblastoma.

The molecular mechanisms and the cell-of-origin leading to retinoblastoma are not well defined. In this issue of Cancer Cell, Bremner and colleagues describe the first inheritable model of retinoblastoma, revealing that loss of the pocket proteins pRb and p107 deregulates cell cycle exit in retinal precursors. The authors show that a subset of these precursors contain an inherent resistance to apoptosis, and that while most terminally differentiate, some are likely to acquire additional mutations, leading to tumor formation. Thus, this work defines the cell-of-origin of retinoblastoma and suggests that mutations giving increased proliferative capacity are required for retinoblastoma development.

Characterization of E2F8, a novel E2F-like cell-cycle regulated repressor of E2F-activated transcription.

The E2F family of transcription factors are downstream effectors of the retinoblastoma protein, pRB, pathway and are essential for the timely regulation of genes necessary for cell-cycle progression. Here we describe the characterization of human and murine E2F8, a new member of the E2F family. Sequence analysis of E2F8 predicts the presence of two distinct E2F-related DNA binding domains suggesting that E2F8 and, the recently, identified E2F7 form a subgroup within the E2F family. We show that E2F transcription factors bind the E2F8 promoter in vivo and that expression of E2F8 is being induced at the G1/S transition. Purified recombinant E2F8 binds specifically to a consensus E2F-DNA-binding site indicating that E2F8, like E2F7, binds DNA without the requirement of co-factors such as DP1. E2F8 inhibits E2F-driven promoters suggesting that E2F8 is transcriptional repressor like E2F7. Ectopic expression of E2F8 in diploid human fibroblasts reduces expression of E2F-target genes and inhibits cell growth consistent with a role for repressing E2F transcriptional activity. Taken together, these data suggest that E2F8 has an important role in turning of the expression of E2F-target genes in the S-phase of the cell cycle.

The tumor suppressor gene hypermethylated in cancer 1 is transcriptionally regulated by E2F1.

The Hypermethylated in Cancer 1 (HIC1) gene encodes a zinc finger transcriptional repressor that cooperates with p53 to suppress cancer development. We and others recently showed that HIC1 is a transcriptional target of p53. To identify additional transcriptional regulators of HIC1, we screened a set of transcription factors for regulation of a human HIC1 promoter reporter. We found that E2F1 strongly activates the full-length HIC1 promoter reporter. Promoter deletions and mutations identified two E2F responsive elements in the HIC1 core promoter region. Moreover, in vivo binding of E2F1 to the HIC1 promoter was shown by chromatin immunoprecipitation assays in human TIG3 fibroblasts expressing tamoxifen-activated E2F1. In agreement, activation of E2F1 in TIG3-E2F1 cells markedly increased HIC1 expression. Interestingly, expression of E2F1 in the p53(-/-) hepatocellular carcinoma cell line Hep3B led to an increase of endogenous HIC1 mRNA, although bisulfite genomic sequencing of the HIC1 promoter revealed that the region bearing the two E2F1 binding sites is hypermethylated. In addition, endogenous E2F1 induced by etoposide treatment bound to the HIC1 promoter. Moreover, inhibition of E2F1 strongly reduced the expression of etoposide-induced HIC1. In conclusion, we identified HIC1 as novel E2F1 transcriptional target in DNA damage responses.

Bypass of senescence by the polycomb group protein CBX8 through direct binding to the INK4A-ARF locus.

The Polycomb group (PcG) proteins are essential for embryogenesis, and their expression is often found deregulated in human cancer. The PcGs form two major protein complexes, called polycomb repressive complexes 1 and 2 (PRC1 and PRC2) whose function is to maintain transcriptional repression. Here, we demonstrate that the chromodomain-containing protein, CBX8, which is part of one of the PRC1 complexes, regulates proliferation of diploid human and mouse fibroblasts through direct binding to the INK4A-ARF locus. Furthermore, we demonstrate that CBX8 is limiting for the regulation of INK4A-ARF, and that ectopic expression of CBX8 leads to repression of the Ink4a-Arf locus and bypass of senescence, leading to cellular immortalization. Gene expression and location analysis demonstrate that besides the INK4A-ARF locus, CBX8 also regulates a number of other genes important for cell growth and survival. On the basis of these results, we conclude that CBX8 is an essential component of one of the PRC1 complexes, which directly regulate the expression of numerous target genes, including the INK4A-ARF locus, involved in cell-fate decisions.

Selective E2F-dependent gene transcription is controlled by histone deacetylase activity during neuronal apoptosis.

The alteration of chromatin through histone acetylation and deacetylation participates in the regulation of gene expression. We have investigated the effects of histone deacetylase inhibition on neuronal fate. We show that treatment of primary neurones with trichostatin A (TSA) or sodium butyrate (NaBu) induces typical features of apoptosis, a cell death that relies on specific genetic programmes. We have further explored the molecular mechanisms implicated in the TSA response and demonstrated that TSA-induced apoptosis is partly dependent on the activation of the transcription factor E2F-1, which has pro-apoptotic functions in these neurones. Furthermore, the increased e2f-1 transcriptional response is probably the result of mechanisms occurring through E2F-responsive elements. Histone acetylation also takes place at the e2f-1 promoter, but this modification is neither required nor by itself sufficient to induce increased transcription at the e2f-1 promoter. Activation might thus occur through acetylation of non-histone proteins binding this regulatory element. Finally, we show that TSA induces the transcription of E2F-dependent genes, such as its cell cycle target cyclin E, but also pro-apoptotic genes, such as Apaf1. Taken together, our results suggest that, in neuroprotective conditions, histone deacetylase activity allows a constitutive repression of the e2f-1 gene in mature neurones in order to ensure survival. Deregulation of this repression will ultimately lead to an E2F-dependent cell death.