FOXO3 Selectively Amplifies Enhancer Activity to Establish Target Gene Regulation

Forkhead box O (FOXO) transcription factors regulate diverse cellular processes, affecting tumorigenesis, metabolism, stem cell maintenance, and lifespan. We show that FOXO3 transcription regulation mainly proceeds through the most active subset of enhancers. In addition to the general distinction between “open” and “closed” chromatin, we show that the level of activity marks (H3K27ac, RNAPII, enhancer RNAs) of these open chromatin regions prior to FOXO3 activation largely determines FOXO3 DNA binding. Consequently, FOXO3 amplifies the levels of these activity marks and their absolute rather than relative changes associate best with FOXO3 target gene regulation. The importance of preexisting chromatin state in directing FOXO3 gene regulation, as shown here, provides a mechanism whereby FOXO3 can regulate cell-specific homeostasis. Genetic variation is reported to affect these chromatin signatures in a quantitative manner, and, in agreement, we observe a correlation between cancer-associated genetic variations and the amplitude of FOXO3 enhancer binding

The expression of the tumor suppressor HBP1 is down-regulated by growthfactors via the PI3K – PKB – FOXO pathway

Growth factors inactivate the Forkhead-box O (FOXO) transcription factors through phosphatidylinositol-3 kinase (PI3K) and protein kinase B (PKB). By comparing microarray data from multiple model systems, we identified the HMG-box protein 1 (HBP1) as a novel downstream target of this pathway. HBP1 mRNA was down-regulated by PDGF, FGF, PI3K and PKB, while it was up-regulated by FOXO factors. This observation was confirmed in human and murine fibroblasts as well as in cell lines derived from leukemia, breast adenocarcinoma and colon carcinoma. Bioinformatics analysis led to the identification of a conserved consensus FOXO-binding site in the HBP1 promoter. By luciferase activity assay and chromatin immunoprecipitation, we demonstrated that FOXO bound to this site and regulated the HBP1 promoter activity in a PI3K-dependent manner. Silencing of HBP1 by small-hairpin RNA (shRNA) increased human fibroblasts proliferation in response to growth factors, suggesting that HBP1 limits cell growth. Finally, by analyzing a transcriptomics data set from The Cancer Genome Atlas, we observed that HBP1 expression was lower in breast tumors that had lost FOXO expression. In conclusion, HBP1 is a novel target of the PI3K - FOXO pathway and controls cell proliferation in response to growth factor

Inhibition of the Phosphoinositide 3-Kinase Pathway Induces a Senescence-like Arrest Mediated by p27Kip1

A senescence-like growth arrest is induced in mouse primary embryo fibroblasts by inhibitors of phosphoinositide 3-kinase (PI3K). We observed that senescence-like growth arrest is correlated with an increase in p27Kip1 but that down-regulation of other cyclin-dependent kinase (CDK) inhibitors, including p15INK4b, p16INK4a, p19 INK4d, and p21Cip1 as well as other negative cell cycle regulators such as p53 and p19ARF, implies that this senescence-related growth arrest is independent of the activity of p53, p19ARF, p16INK4a, and p21Cip1, which are associated with replicative senescence. The p27Kip1 binds to the cyclin/CDK2 complexes and causes a decrease in CDK2 kinase activity. We demonstrated that ectopic expression of p27Kip1 can induce permanent cell cycle arrest and a senescence-like phenotype in wild-type mouse embryo fibroblasts. We also obtained results suggesting that the kinase inhibitors LY294002 and Wortmannin arrest cell growth and induce a senescence-like phenotype, at least partially, through inhibition of PI3K and protein kinase B/Akt, activation of the forkhead protein AFX, and up-regulation of p27Kip1expression. In summary, these observations taken together suggest that p27Kip1 is an important mediator of the permanent cell cycle arrest induced by PI3K inhibitors. Our data suggest that repression of CDK2 activity by p27Kip1 is required for the PI3K-induced senescence, yet mouse embryo fibroblasts derived fromp27 Kip1−/− mice entered cell cycle arrest after treatment with LY294002. We show that this is due to a compensatory mechanism by which p130 functionally substitutes for the loss of p27Kip1. This is the first description that p130 may have a role in inhibiting CDK activity during senescence.Predoctoral fellows supported by the Spanish Ministry of Education and Culture (I. G.-C.) and the Department of Immunology and Oncology (M. B.). g A junior research fellow supported by the National Funds for Scientific Research, Belgium. h Supported by the Ludwig Institute for Cancer Research and the Leukemia Research Fund. k Supported by the Spanish Research Council (CSIC), the Spanish Ministry of Education and Culture, and a core grant to the Department of Immunology and Oncology from the consortium between Pharmacia and Upjohn and CSIC.Peer reviewe

A FOXO-dependent replication checkpoint restricts proliferation of damaged cells

DNA replication is challenged by numerous exogenous and endogenous factors that can interfere with the progression of replication forks. Substantial accumulation of single-stranded DNA during DNA replication activates the DNA replication stress checkpoint response that slows progression from S/G2 to M phase to protect genomic integrity. Whether and how mild replication stress restricts proliferation remains controversial. Here, we identify a cell cycle exit mechanism that prevents S/G2 phase arrested cells from undergoing mitosis after exposure to mild replication stress through premature activation of the anaphase promoting complex/cyclosome (APC/CCDH1). We find that replication stress causes a gradual decrease of the levels of the APC/CCDH1 inhibitor EMI1/FBXO5 through Forkhead box O (FOXO)-mediated inhibition of its transcription factor E2F1. By doing so, FOXOs limit the time during which the replication stress checkpoint is reversible and thereby play an important role in maintaining genomic stability

Dysregulated miRNome of plasmacytoid dendritic cells from patients with Sjogren's syndrome is associated with processes at the centre of their function

Objective: A considerable body of evidence supports a role for type-I IFN in the pathogenesis of primary SS (pSS). As plasmacytoid dendritic cells (pDCs) are a major source of type-I IFN, we investigated their molecular regulation by measuring expression of a large set of miRNAs. Methods: pDCs were isolated from peripheral blood of pSS patients (n = 30) and healthy controls (n = 16) divided into two independent cohorts (discovery and replication). Screening of 758 miRNAs was assessed by an OpenArray quantitative PCR-based technique; replication of a set of identified miRNAs was performed by custom array. Functional annotation of miRNA targets was performed using pathway enrichment. Novel targets of miR-29a and miR-29c were identified using a proteomic approach (stable isotope labelling with amino acids in cell culture). Results: In the discovery cohort, 20 miRNAs were differentially expressed in pSS pDCs compared with healthy control pDCs. Of these, differential expression of 10 miRNAs was confirmed in the replication cohort. The dysregulated miRNAs were involved in phosphoinositide 3-kinase-Ak strain transforming and mammalian target of rapamycin signalling, as well as regulation of cell death. In addition, a set of novel protein targets of miR-29a and miR-29c were identified, including five targets that were regulated by both miRs. Conclusion: The dysregulated miRNome in pDCs of patients with pSS is associated with aberrant regulation of processes at the centre of pDC function, including type-I IFN production and cell death. As miR-29a and miR-29c are pro-apoptotic factors and several of the novel targets identified here are regulators of apoptosis, their downregulation in patients with pSS is associated with enhanced pDC survival