05-379 Sigma-AldrichAnti-E2F-1 Antibody, clones KH20 and KH95

Host cell factor-1 (HCF-1) is a metazoan transcriptional coregulator essential for cell-cycle progression and cell proliferation. Current models suggest a mechanism whereby HCF-1 functions as a direct coregulator of E2F proteins, facilitating the expression of genes necessary for cell proliferation. In this report, we show that HCF-1 recruitment to numerous E2F-bound promoters is mediated by the concerted action of zinc finger transcription factors THAP11 and ZNF143, rather than E2F proteins directly. THAP11, ZNF143, and HCF-1 form a mutually dependent complex on chromatin, which is independent of E2F occupancy. Disruption of the THAP11/ZNF143/HCF-1 complex results in altered expression of cell-cycle control genes and leads to reduced cell proliferation, cell-cycle progression, and cell viability. These data establish a model in which a THAP11/ZNF143/HCF-1 complex is a critical component of the transcriptional regulatory network governing cell proliferation.

Colorectal cancer (CRC) is the third major cause of cancer related deaths in the world. 5-fluorouracil (5-FU) is widely used for the treatment of colorectal cancer but as a single-agent renders low response rates. Choline kinase alpha (ChoKα), an enzyme that plays a role in cell proliferation and transformation, has been reported overexpressed in many different tumors, including colorectal tumors. ChoKα inhibitors have recently entered clinical trials as a novel antitumor strategy.ChoKα specific inhibitors, MN58b and TCD-717, have demonstrated a potent antitumoral activity both in vitro and in vivo against several tumor-derived cell line xenografts including CRC-derived cell lines. The effect of ChoKα inhibitors in combination with 5-FU as a new alternative for the treatment of colon tumors has been investigated both in vitro in CRC-tumour derived cell lines, and in vivo in mouse xenografts models. The effects on thymidilate synthase (TS) and thymidine kinase (TK1) levels, two enzymes known to play an essential role in the mechanism of action of 5-FU, were analyzed by western blotting and quantitative PCR analysis. The combination of 5-FU with ChoKα inhibitors resulted in a synergistic effect in vitro in three different human colon cancer cell lines, and in vivo against human colon xenografts in nude mice. ChoKα inhibitors modulate the expression levels of TS and TK1 through inhibition of E2F production, providing a rational for its mechanism of action.Our data suggest that both drugs in combination display a synergistic antitumoral effect due to ChoKα inhibitors-driven modulation of the metabolization of 5-FU. The clinical relevance of these findings is strongly supported since TCD-717 has recently entered Phase I clinical trials against solid tumors.

Transcription factors (TFs) regulate gene expression by binding to short DNA sequence motifs, yet their binding specificities alone cannot explain how certain TFs drive a diversity of biological processes. In order to investigate the factors that control the functions of the pleiotropic TF STAT3, we studied its genome-wide binding patterns in four different cell types: embryonic stem cells, CD4(+) T cells, macrophages and AtT-20 cells. We describe for the first time two distinct modes of STAT3 binding. First, a small cell type-independent mode represented by a set of 35 evolutionarily conserved STAT3-binding sites that collectively regulate STAT3's own functions and cell growth. We show that STAT3 is recruited to sites with E2F1 already pre-bound before STAT3 activation. Second, a series of different transcriptional regulatory modules (TRMs) assemble around STAT3 to drive distinct transcriptional programs in the four cell types. These modules recognize cell type-specific binding sites and are associated with factors particular to each cell type. Our study illustrates the versatility of STAT3 to regulate both universal- and cell type-specific functions by means of distinct TRMs, a mechanism that might be common to other pleiotropic TFs.

Inactivation of the retinoblastoma protein (pRb) by phosphorylation triggers uncontrolled cell proliferation. Accordingly, activation of cyclin-dependent kinase (CDK)/cyclin complexes or downregulation of CDK inhibitors appears as a common event in human cancer. Here we show that Pin1 (protein interacting with NIMA (never in mitosis A)-1), a peptidylprolyl isomerase involved in the control of protein phosphorylation, is an essential mediator for inactivation of the pRb. Our results indicate that Pin1 controls cell proliferation by altering pRb phosphorylation without affecting CDK and protein phosphatase 1 and 2 activity. We demonstrated that Pin1 regulates tumor cell proliferation through direct interaction with the spacer domain of the pRb protein, and allows the interaction between CDK/cyclin complexes and pRb in mid/late G1. Phosphorylation of pRb Ser 608/612 is the crucial motif for Pin1 binding. We propose that Pin1 selectively boosts the switch from hypo- to hyper-phosphorylation of pRb in tumor cells. In addition, we demonstrate that the CDK pathway is responsible for the interaction of Pin1 and pRb. Prospectively, our findings therefore suggest that the synergism among CDK and Pin1 inhibitors holds great promise for targeted pharmacological treatment of cancer patients, with the possibility of reaching high effectiveness at tolerated doses.

Apoptotic cell death is essential for mammalian development and tissue homeostasis. Dysregulation of apoptosis has been identified in pathologies including in pulmonary fibrotic remodeling. We previously reported that a key proapoptotic factor in fibrosis, angiotensin II (Ang II), mediates apoptosis in primary pulmonary artery endothelial cells (PAEC) via the AT(2) receptor and requires activation of AMP-regulated protein kinase (AMPK). We now demonstrate that Ang II induces E2F1 transcription factor binding to and activation of the promoter for the Bcl-2 homology 3 (BH3)-only protein Bim. In PAEC, Ang II treatment induced cyclin-dependent kinase 4 (Cdk4)-mediated hyperphosphorylation of retinoblastoma protein (Rb) and its disassociation from E2F1, a key step in facilitating E2F1-directed transcriptional activity. Indeed, ectopic expression of a dominant negative Cdk4 mutant inhibited Ang II-mediated hyperphosphorylation of Rb and Bim promoter activation. Our data also show that the β-subunit of AMPK was constitutively associated with Cdk4 in PAEC and that Ang II treatment induced AMPKβ phosphorylation and subsequent disassociation of this complex. Both Ang II-induced Rb hyperphosphorylation and Cdk4-AMPK disassociation were blocked by the AMPK inhibitor compound C. Together these findings illuminate a novel proapoptotic signaling pathway in endothelial cells, whereby Ang II triggers E2F1-mediated transcriptional upregulation of Bim via activation of AMPKβ1/2 and Cdk4.

As an inhibitor of cyclin-dependent kinases, p16(INK4A) is an important tumour suppressor and inducer of cellular senescence that is often inactivated during the development of cancer by promoter DNA methylation. Using newly established lymphoblastoid cell lines (LCLs) expressing a conditional EBNA3C from recombinant EBV, we demonstrate that EBNA3C inactivation initiates chromatin remodelling that resets the epigenetic status of p16(INK4A) to permit transcriptional activation: the polycomb-associated repressive H3K27me3 histone modification is substantially reduced, while the activation-related mark H3K4me3 is modestly increased. Activation of EBNA3C reverses the distribution of these epigenetic marks, represses p16(INK4A) transcription and allows proliferation. LCLs lacking EBNA3A express relatively high levels of p16(INK4A) and have a similar pattern of histone modifications on p16(INK4A) as produced by the inactivation of EBNA3C. Since binding to the co-repressor of transcription CtBP has been linked to the oncogenic activity of EBNA3A and EBNA3C, we established LCLs with recombinant viruses encoding EBNA3A- and/or EBNA3C-mutants that no longer bind CtBP. These novel LCLs have revealed that the chromatin remodelling and epigenetic repression of p16(INK4A) requires the interaction of both EBNA3A and EBNA3C with CtBP. The repression of p16(INK4A) by latent EBV will not only overcome senescence in infected B cells, but may also pave the way for p16(INK4A) DNA methylation during B cell lymphomagenesis.

APC/Cdh1 is a major cell cycle regulator and its function has been implicated in DNA damage repair; however, its exact role remains unclear. Using affinity purification coupled with mass spectrometry, we identified Claspin as a novel Cdh1-interacting protein and further demonstrated that Claspin is a novel Cdh1 ubiquitin substrate. As a result, inactivation of Cdh1 leads to activation of the Claspin/Chk1 pathway. Previously, we demonstrated that Rb interacts with Cdh1 to influence its ability to degrade Skp2. Here, we report that Cdh1 reciprocally regulates the Rb pathway through competing with E2F1 to bind the hypophosphorylated form of Rb. Although inactivation of Cdh1 in HeLa cells, with defective p53/Rb pathways, led to premature S phase entry, acute depletion of Cdh1 in primary human fibroblasts resulted in premature senescence. Acute loss of many other major tumor suppressors, including PTEN and VHL, also induces premature senescence in a p53- or Rb-dependent manner. Similarly, we showed that inactivation of the p53/Rb pathways by overexpression of SV40 LT-antigen partially reversed Cdh1 depletion-induced growth arrest. Therefore, loss of Cdh1 is only beneficial to cells with abnormal p53 and Rb pathways, which helps explain why Cdh1 loss is not frequently found in many tumors.

The transcription factor p110 CUX1 was shown to stimulate cell proliferation by accelerating entry into S phase. As p110 CUX1 can function as a transcriptional repressor or activator depending on promoter context, we investigated its mechanism of transcriptional activation using the DNA polymerase alpha gene promoter as a model system. Linker-scanning analysis revealed that a low-affinity E2F binding site is required for transcriptional activation. Moreover, coexpression with a dominant-negative mutant of DP-1 suggested that endogenous E2F factors are indeed needed for p110-mediated activation. Tandem affinity purification, coimmunoprecipitation, chromatin immunoprecipitation, and reporter assays indicated that p110 CUX1 can engage in weak protein-protein interactions with E2F1 and E2F2, stimulate their recruitment to the DNA polymerase alpha gene promoter, and cooperate with these factors in transcriptional activation. On the other hand, in vitro assays suggested that the interaction between CUX1 and E2F1 either is not direct or is regulated by posttranslational modifications. Genome-wide location analysis revealed that targets common to p110 CUX1 and E2F1 included many genes involved in cell cycle, DNA replication, and DNA repair. Comparison of the degree of enrichment for various E2F factors suggested that binding of p110 CUX1 to a promoter will favor the specific recruitment of E2F1, and to a lesser extent E2F2, over E2F3 and E2F4. Reporter assays on a subset of common targets confirmed that p110 CUX1 and E2F1 cooperate in their transcriptional activation. Overall, our results show that p110 CUX1 and E2F1 cooperate in the regulation of many cell cycle genes.

The KIR3DL1 gene is a member of killer immunoglobulin-like receptors family, which exhibits a variegated expression pattern in NK cells and subsets of CD4(+) and CD8(+) T cells. The E2F family of transcription factors plays a crucial role in the regulation of gene expression. The present study reports a naturally occurring point mutation (TTTGGCGC-->TTCGGCGC) within a putative E2F binding site in the KIR3DL1 promoter in K562 cells. Interestingly, this mutation introduces a new methylation site. This study shows for the first time that E2F1 binds to the KIR3DL1 promoter in vivo. This point mutation and concomitantly altered methylation pattern within the E2F1 binding site abolishes their binding and reduces the promoter activity, while elevated expression of E2F1 correlates with increased promoter activity. Therefore, E2F1 contributes to the transcriptional activation of the KIR3DL1 gene.

PTPRO is often silenced by DNA hypermethylation in primary human tumors and cancer cell lines and functions as a tumor suppressor. Here we show that PTPRO is a target of E2F1. In addition, the microRNA cluster miR-17-92, another target of E2F1, participates in PTPRO regulation. PTPRO mRNA was up-regulated during S phase in synchronized HeLa cells and in vitro PTPRO promoter activity is high in early S phase while the PTPRO 3'UTR reporter activity is low in late S phase. This study provides evidence that the PTPRO gene is co-regulated by both E2F1 and miR-17-92.