Inhibition of pluripotency networks by the Rb tumor suppressor restricts reprogramming and tumorigenesis

Mutations in the retinoblastoma tumor suppressor gene Rb are involved in many forms of human cancer. In this study, we investigated the early consequences of inactivating Rb in the context of cellular reprogramming. We found that Rb inactivation promotes the reprogramming of differentiated cells to a pluripotent state. Unexpectedly, this effect is cell cycle independent, and instead reflects direct binding of Rb to pluripotency genes, including Sox2 and Oct4, which leads to a repressed chromatin state. More broadly, this regulation of pluripotency networks and Sox2 in particular is critical for the initiation of tumors upon loss of Rb in mice. These studies therefore identify Rb as a global transcriptional repressor of pluripotency networks, providing a molecular basis for previous reports about its involvement in cell fate pliability, and implicate misregulation of pluripotency factors such as Sox2 in tumorigenesis related to loss of Rb function

Inhibition of pluripotency networks by the Rb tumor suppressor restricts reprogramming and tumorigenesis

Mutations in the retinoblastoma tumor suppressor gene Rb are involved in many forms of human cancer. In this study, we investigated the early consequences of inactivating Rb in the context of cellular reprogramming. We found that Rb inactivation promotes the reprogramming of differentiated cells to a pluripotent state. Unexpectedly, this effect is cell cycle independent, and instead reflects direct binding of Rb to pluripotency genes, including Sox2 and Oct4, which leads to a repressed chromatin state. More broadly, this regulation of pluripotency networks and Sox2 in particular is critical for the initiation of tumors upon loss of Rb in mice. These studies therefore identify Rb as a global transcriptional repressor of pluripotency networks, providing a molecular basis for previous reports about its involvement in cell fate pliability, and implicate misregulation of pluripotency factors such as Sox2 in tumorigenesis related to loss of Rb function

Smyd3 regulates cancer cell phenotypes and catalyzes histone H4 lysine 5 methylation

Smyd3 is a lysine methyltransferase implicated in chromatin and cancer regulation. Here we show that Smyd3 catalyzes histone H4 methylation at lysine 5 (H4K5me). This novel histone methylation mark is detected in diverse cell types and its formation is attenuated by depletion of Smyd3 protein. Further, Smyd3-driven cancer cell phenotypes require its enzymatic activity. Thus, Smyd3, via H4K5 methylation, provides a potential new link between chromatin dynamics and neoplastic disease

Notch signaling inhibits hepatocellular carcinoma following inactivation of the RB pathway

Mice lacking all three Rb genes in the liver develop tumors resembling specific subgroups of human hepatocellular carcinomas, and Notch activity appears to suppress the growth and progression of these tumors

Tandem E2F Binding Sites in the Promoter of the p107 Cell Cycle Regulator Control p107 Expression and Its Cellular Functions

The retinoblastoma tumor suppressor (Rb) is a potent and ubiquitously expressed cell cycle regulator, but patients with a germline Rb mutation develop a very specific tumor spectrum. This surprising observation raises the possibility that mechanisms that compensate for loss of Rb function are present or activated in many cell types. In particular, p107, a protein related to Rb, has been shown to functionally overlap for loss of Rb in several cellular contexts. To investigate the mechanisms underlying this functional redundancy between Rb and p107 in vivo, we used gene targeting in embryonic stem cells to engineer point mutations in two consensus E2F binding sites in the endogenous p107 promoter. Analysis of normal and mutant cells by gene expression and chromatin immunoprecipitation assays showed that members of the Rb and E2F families directly bound these two sites. Furthermore, we found that these two E2F sites controlled both the repression of p107 in quiescent cells and also its activation in cycling cells, as well as in Rb mutant cells. Cell cycle assays further indicated that activation of p107 transcription during S phase through the two E2F binding sites was critical for controlled cell cycle progression, uncovering a specific role for p107 to slow proliferation in mammalian cells. Direct transcriptional repression of p107 by Rb and E2F family members provides a molecular mechanism for a critical negative feedback loop during cell cycle progression and tumorigenesis. These experiments also suggest novel therapeutic strategies to increase the p107 levels in tumor cells

Organ Size Control Is Dominant over Rb Family Inactivation to Restrict Proliferation In Vivo

In mammals, a cell’s decision to divide is thought to be under the control of the Rb/E2F pathway. We previously found that inactivation of the Rb family of cell cycle inhibitors (Rb, p107, and p130) in quiescent liver progenitors leads to uncontrolled division and cancer initiation. Here, we show that, in contrast, deletion of the entire Rb gene family in mature hepatocytes is not sufficient for their long-term proliferation. The cell cycle block in Rb family mutant hepatocytes is independent of the Arf/p53/p21 checkpoint but can be abrogated upon decreasing liver size. At the molecular level, we identify YAP, a transcriptional regulator involved in organ size control, as a factor required for the sustained expression of cell cycle genes in hepatocytes. These experiments identify a higher level of regulation of the cell cycle in vivo in which signals regulating organ size are dominant regulators of the core cell cycle machinery

Altered <i>p107</i> expression affects cellular proliferation.

<p>(A,B) Immortalized wild-type and <i>p107^{E2F-1*2*/1*2*}</i> MEFs were synchronized in G0 through at least three days of serum starvation. DMEM supplemented with 20% BGS was used to stimulate cell-cycle entry. Extracts were collected at the number of hours indicated post-serum stimulation. (A) RT-qPCR analysis of <i>Cdc6</i> mRNA in wild-type and <i>p107^{E2F-1*2*/1*2*}</i> MEFs. (n = 3) (B) Percentage of cells in S-phase, as determined by BrdU/PI staining, at the indicated time points. (n≥4) (C) Cell-cycle profiles of asynchronous primary wild-type, <i>p107^{E2F-1*2*/1*2*}</i>, and <i>p107^−/−</i> MEFs. Percentages of cells in each phase were determined by BrdU/PI staining. (n≥2) (D) Cellular proliferation of primary wild-type, <i>p107^{E2F-1*2*/1*2*}</i>, and <i>p107^−/−</i> MEFs. Equal numbers of cells were plated at day 0. Cells were then counted every other day from day 1 to day 9 post-plating. For statistical analysis, <i>p107^{E2F-1*2*/1*2*}</i> cells were compared to wild-type cells at each time point. (n≥13) (E) Model for the context-dependent regulation of <i>p107</i> transcription by E2F family members. In cycling mESCs, activating members of the E2F family such as E2F3 bind to the <i>p107</i> promoter mostly through the distal consensus E2F binding site (site 1). In quiescent MEFs, binding of the E2F4 repressor is also largely dependent on the presence of the distal consensus site. However, E2F4 may also be recruited to the <i>p107</i> promoter through interactions with other transcription factors and/or by binding to other DNA sequences. The size of the E2F boxes indicates the relative binding activity.</p

<i>p107</i> repression in quiescent MEFs is mediated by the two E2F binding sites.

<p>(A) mESCs targeted by the neomycin resistance cassette but retaining a wild-type <i>p107</i> promoter and mESCs targeted by homozygous mutations into the distal (1*/1*) or both E2F sites (1*2*/1*2*) were injected to generate chimeric embryos. Wild-type, <i>p107^E2F-1*/1*</i> and <i>p107^{E2F-1*2*/1*2*}</i> MEFs derived from chimeric embryos were selected for Neomycin resistance to generate pure populations. (B) RT-qPCR analysis of <i>p107</i> expression in quiescent wild-type and <i>p107^{E2F-1*2*/1*2*}</i> MEFs. (n≥9) (C) Immunoblot analysis of p107 in the same conditions. Tubulin expression is shown as a loading control. (D) Quantitative ChIP analysis of E2F4, p107, and p130 binding on the <i>p107</i> promoter in quiescent immortalized wild-type, <i>p107^E2F-1*/1*</i>, and <i>p107^{E2F-1*2*/1*2*}</i> MEFs. The <i>B-Myb</i> promoter is shown as a control. (n = 3) (E) Quantitative ChIP analysis of Rb binding to the <i>p107</i> and <i>Mcm3</i> promoters in cycling immortalized wild-type and <i>p107^{E2F-1*2*/1*2*}</i> MEFs. Mouse IgG antibodies serve as a negative control. (n≥3) For (D,E), fold enrichment is calculated over <i>actin</i> and the y-axis is plotted on a <i>log2</i> scale.</p

Regulation of the mouse <i>p107</i> promoter through E2F binding sites in reporter assays.

<p>(A) Conservation of the proximal <i>p107</i> promoter across mammalian species. The two tandem consensus E2F binding sites (BS1 and BS2) are each indicated by a box. (B) Schematic representation of wild-type (WT), p107-1*, p107-2*, and p107-1*2* luciferase vectors. Transcription factor binding sites contained in this promoter region, as identified by sequence analysis, are indicated, as is the transcription start site (arrow). Black rectangular boxes indicate E2F consensus sites; white boxes indicate E2F consensus sites that are mutated. The inset represents the mutations (aaa) introduced in each site. (C) Relative luciferase activity expressed by the four constructs, co-transfected with CMV-E2F3 (+) or empty pCDNA (−), in cycling mESCs. For statistical analysis, each mutant construct was compared to the wild-type one and the effect of E2F3 on each construct was analyzed. (n = 3) (D) Relative luciferase activity in quiescent MEFs. (n = 15) (E) Comparison of the models for the regulation of the human and mouse <i>p107</i> promoters by E2F based on reporter assays. Gradient triangles indicate the relative importance of each consensus E2F site to either activation or repression of <i>p107</i>.</p