FOXO3 regulates gene expression from distal enhancers
ABSTRACT: FOXO transcription factors are key players in diverse cellular responses affecting tumorigenesis, stem cell maintenance and lifespan. To gain insight into mechanisms of FOXO regulated gene expression, we studied genome-wide effects of FOXO3 activation. Profiling RNA polymerase II (RNAPII) changes shows FOXO3 regulates gene expression through transcription initiation. Correlative analysis of FOXO3 and RNAPII ChIP-seq profiles demonstrates FOXO3 to act as a transcriptional activator. Furthermore, this analysis reveals a significant part of FOXO3 gene regulation proceeds through enhancer regions. FOXO3 binds to and activates enhancers as shown by the presence of and changes in enhancer-specific histone modifications and RNAPII occupancy. In addition, FOXO3-mediated enhancer regulation correlates with regulation of adjacent genes and existence of chromatin loops between FOXO3 bound enhancers and regulated genes. Combined, our data elucidate how FOXOs regulate gene transcription and provide insight into mechanisms by which FOXOs can induce different gene expression programs depending on chromatin architecture. seven 4C view point were analyzed on DLD1 colon carcinoma cells containing 4OH-Tamoxifen inducible FOXO3A3-ER (DL23 cells, Kops et al., 2002, Mol Cell Biol), to investigate 3D topology around FOXO3 bound regions and FOXO3 regulated genes before and 4 hours after addition of tamoxifen. 4C procedure, as published before (Splinter et al., 2001, Genes Dev). Cells are cross linked using 1% formaldehyde for 10min at room temperature, nuclei are isolated, after which chromatin is digested with DpnII and subsequently ligated under diluted conditions. After reversal of the cross links the DNA is purified and treated with the second restriction enzyme treatment (Csp). After a second re-ligation step the sample is purified and ligated fragments are analyzed by inverse PCR.
Project description:Forkhead box O (FOXO) transcription factors are key players in diverse cellular processes affecting tumorigenesis, stem cell maintenance and lifespan. To gain insight into the mechanisms of FOXO-regulated target gene expression, we studied genome-wide effects of FOXO3 activation. Profiling RNA polymerase II changes shows that FOXO3 regulates gene expression through transcription initiation. Correlative analysis of FOXO3 and RNA polymerase II ChIP-seq profiles demonstrates FOXO3 to act as a transcriptional activator. Furthermore, this analysis reveals a significant part of FOXO3 gene regulation proceeds through enhancer regions. FOXO3 binds to pre-existing enhancers and further activates these enhancers as shown by changes in histone acetylation and RNA polymerase II recruitment. In addition, FOXO3-mediated enhancer activation correlates with regulation of adjacent genes and pre-existence of chromatin loops between FOXO3 bound enhancers and target genes. Combined, our data elucidate how FOXOs regulate gene transcription and provide insight into mechanisms by which FOXOs can induce different gene expression programs depending on chromatin architecture.
Project description:FOXO transcription factors are key players in diverse cellular processes affecting tumorigenesis, stem cell maintenance and lifespan. To gain insight into mechanisms of FOXO regulated target gene expression, we studied genome-wide effects of FOXO3 activation. Profiling RNA polymerase II changes shows FOXO3 regulates gene expression through transcription initiation. Correlative analysis of FOXO3 and RNA polymerase II ChIP-seq profiles demonstrates FOXO3 to act as a transcriptional activator. Furthermore, this analysis reveals a significant part of FOXO3 gene regulation proceeds through enhancer regions. FOXO3 binds to pre-existing enhancers and further activates these enhancers as shown by changes in histone acetylation and RNA polymerase II recruitment. In addition, FOXO3-mediated enhancer activation correlates with regulation of adjacent genes and pre-existence of chromatin loops between FOXO3 bound enhancers and target genes. Combined, our data elucidate how FOXOs regulate gene transcription and provide insight into mechanisms by which FOXOs can induce different gene expression programs depending on chromatin architecture. Paper with published gene expression: PMID 22139133. Examination of FOXO3 binding and changes in RNAPII occupancy (0, 4, 24 hours after induction) in colorectal cell line
Project description:Transcription factors FOXOs (1, 3, 4) are essential for the maintenance of haematopoietic stem cells. FOXOs are evolutionary conserved substrates of the AKT serine threonine protein kinase that are also phosphorylated by several kinases other than AKT. Specifically, phosphorylation by AKT is known to result in the cytosolic localization of FOXO and subsequent inhibition of FOXO transcriptional activity. In addition to phosphorylation, FOXOs are regulated by a number of other post-translational modifications including acetylation, methylation, redox modulation, and ubiquitination that altogether determine these factors' output. Cumulating evidence raises the possibility that in stem cells, including in haematopoietic stem cells, AKT may not be the dominant regulator of FOXO. To address this question in more detail, we examined gene expression, subcellular localization, and response to AKT inhibition of FOXO1 and FOXO3, the main FOXO expressed in HSPCs (haematopoietic stem and progenitor cells). Here we show that while FOXO1 and FOXO3 transcripts are expressed at similar levels, endogenous FOXO3 protein is mostly nuclear compared to the cytoplasmic localization of FOXO1 in HSPCs. Furthermore, inhibition of AKT does not enhance nuclear localization of FOXO1 nor FOXO3. Nonetheless AKT inhibition in the context of loss of NAD-dependent SIRT1 deacetylase modulates FOXO3 localization in HSPCs. Together, these data suggest that FOXO3 is more active than FOXO1 in primitive haematopoietic stem and multipotent progenitor cells. In addition, they indicate that upstream regulators other than AKT, such as SIRT1, maintain nuclear FOXO localization and activity in HSPCs.
Project description:Activated phosphoinositide 3-kinase (PI3K)-AKT signaling appears to be an obligate event in the development of cancer. The highly related members of the mammalian FoxO transcription factor family, FoxO1, FoxO3, and FoxO4, represent one of several effector arms of PI3K-AKT signaling, prompting genetic analysis of the role of FoxOs in the neoplastic phenotypes linked to PI3K-AKT activation. While germline or somatic deletion of up to five FoxO alleles produced remarkably modest neoplastic phenotypes, broad somatic deletion of all FoxOs engendered a progressive cancer-prone condition characterized by thymic lymphomas and hemangiomas, demonstrating that the mammalian FoxOs are indeed bona fide tumor suppressors. Transcriptome and promoter analyses of differentially affected endothelium identified direct FoxO targets and revealed that FoxO regulation of these targets in vivo is highly context-specific, even in the same cell type. Functional studies validated Sprouty2 and PBX1, among others, as FoxO-regulated mediators of endothelial cell morphogenesis and vascular homeostasis.
Project description:The gene FOXO3, encoding the transcription factor forkhead box O-3 (FoxO3), is one of only two for which genetic polymorphisms have exhibited consistent associations with longevity in diverse human populations.Here, we review the multitude of actions of FoxO3 that are relevant to health, and thus healthy ageing and longevity.The study involved a literature search for articles retrieved from PubMed using FoxO3 as keyword.We review the molecular genetics of FOXO3 in longevity, then current knowledge of FoxO3 function relevant to ageing and lifespan. We describe how FoxOs are involved in energy metabolism, oxidative stress, proteostasis, apoptosis, cell cycle regulation, metabolic processes, immunity, inflammation and stem cell maintenance. The single FoxO in Hydra confers immortality to this fresh water polyp, but as more complex organisms evolved, this role has been usurped by the need for FoxO to control a broader range of specialized pathways across a wide spectrum of tissues assisted by the advent of as many as 4 FoxO subtypes in mammals. The major themes of FoxO3 are similar, but not identical, to other FoxOs and include regulation of cellular homeostasis, particularly of stem cells, and of inflammation, which is a common theme of age-related diseases. Other functions concern metabolism, cell cycle arrest, apoptosis, destruction of potentially damaging reactive oxygen species and proteostasis.The mechanism by which longevity-associated alleles of FOXO3 reduce age-related mortality is currently of great clinical interest. The prospect of optimizing FoxO3 activity in humans to increase lifespan and reduce age-related diseases represents an exciting avenue of clinical investigation. Research strategies directed at developing therapeutic agents that target FoxO3, its gene and proteins in the pathway(s) FoxO3 regulates should be encouraged and supported.
Project description:FOXO family members (FOXOs: FOXO1, FOXO3, FOXO4 and FOXO6) are important transcription factors and tumor suppressors controlling cell homeostasis and cell fate. They are characterized by an extraordinary functional diversity, being involved in regulation of cell cycle, proliferation, apoptosis, DNA damage response, oxidative detoxification, cell differentiation and stem cell maintenance, cell metabolism, angiogenesis, cardiac and other organ's development, aging, and other critical cellular processes. FOXOs are tightly regulated by reversible phosphorylation, ubiquitination, acetylation and methylation. Interestingly, the known kinases phosphorylate only a small percentage of the known or predicted FOXOs phosphorylation sites, suggesting that additional kinases that phosphorylate and control FOXOs activity exist. In order to identify novel regulators of FOXO3, we have employed a proteomics screening strategy. Using HeLa cancer cell line and a Tandem Affinity Purification followed by Mass Spectrometry analysis, we identified several proteins as binding partners of FOXO3. Noteworthy, Polo Like Kinase 1 (PLK1) proto-oncogene was one of the identified FOXO3 binding partners. PLK1 plays a critical role during cell cycle (G2-M transition and all phases of mitosis) and in maintenance of genomic stability. Our experimental results presented in this manuscript demonstrate that FOXO3 and PLK1 exist in a molecular complex through most of the phases of the cell cycle, with a higher occurrence in the G2-M cell cycle phases. PLK1 induces translocation of FOXO3 from the nucleus to the cytoplasm and suppresses FOXO3 activity, measured by the decrease in the pro-apoptotic Bim protein levels and in the cell cycle inhibitor protein p27. Furthermore, PLK1 can directly phosphorylate FOXO3 in an in vitro kinase assay. These results present the discovery of PLK1 proto-oncogene as a binding partner and a negative regulator of FOXO3 tumor suppressor.
Project description:Forkhead box class O family member proteins (FoxOs) are evolutionarily conserved transcription factors for their highly conserved DNA-binding domain. In mammalian species, all the four FoxO members, FoxO1, FoxO3, FoxO4, and FoxO6, are expressed in different organs. In bone, the first three members are extensively expressed and more studied. Bone development, remodeling, and homeostasis are all regulated by multiple cell lineages, including osteoprogenitor cells, chondrocytes, osteoblasts, osteocytes, osteoclast progenitors, osteoclasts, and the intercellular signaling among these bone cells. The disordered FoxOs function in these bone cells contribute to osteoarthritis, osteoporosis, or other bone diseases. Here, we review the current literature of FoxOs for their roles in bone cells, focusing on helping researchers to develop new therapeutic approaches and prevent or treat the related bone diseases.
Project description:Spermatogonial stem cells (SSCs) capable of self-renewal and differentiation are the foundation for spermatogenesis. Although several factors important for these processes have been identified, the fundamental mechanisms regulating SSC self-renewal and differentiation remain unknown. Here, we investigated a role for the Foxo transcription factors in mouse spermatogenesis and found that Foxo1 specifically marks mouse gonocytes and a subset of spermatogonia with stem cell potential. Genetic analyses showed that Foxo1 was required for both SSC homeostasis and the initiation of spermatogenesis. Combined deficiency of Foxo1, Foxo3, and Foxo4 resulted in a severe impairment of SSC self-renewal and a complete block of differentiation, indicating that Foxo3 and Foxo4, although dispensable for male fertility, contribute to SSC function. By conditional inactivation of 3-phosphoinositide-dependent protein kinase 1 (Pdk1) and phosphatase and tensin homolog (Pten) in the male germ line, we found that PI3K signaling regulates Foxo1 stability and subcellular localization, revealing that the Foxos are pivotal effectors of PI3K-Akt signaling in SSCs. We also identified a network of Foxo gene targets--most notably Ret--that rationalized the maintenance of SSCs by the Foxos. These studies demonstrate that Foxo1 expression in the spermatogenic lineage is intimately associated with the stem cell state and revealed what we believe to be novel Foxo-dependent mechanisms underlying SSC self-renewal and differentiation, with implications for common diseases, including male infertility and testicular cancer, due to abnormalities in SSC function.
Project description:PURPOSE:The aim of the present study is to investigate role of FoxO transcription factors in preimplantation embryo development by knocking down FoxO1, FoxO3, and FoxO4 genes and also to assess cell cycle arrest related proteins, p53 and p21, and apoptosis-related proteins, fas ligand (FASL), and cleaved caspase 3. METHODS:Knockdown of FoxOs using siRNA was confirmed utilizing RT-PCR and qRT-PCR in gene level and using immunofluorescence in protein level. Following knockdown of FoxO1, FoxO3, and FoxO4 in two-cell mouse embryos with or without resveratrol treatment; developmental competence of embryos and expression patterns of SIRT1, p53, p21, FASL, and CLEAVED CASPASE 3 proteins in embryos by immunofluorescence were assessed after 48 h. ROS levels were measured in knockdown embryos. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to determine resveratrol dose. RESULTS:Successful knockdown of FoxO genes in mouse embryos utilizing a non-invasive siRNA method was achieved. Significantly, knockdown of FoxO genes impaired preimplantation embryo development which cannot be prevented by resveratrol treatment. Immunofluorescence results showed that resveratrol could protect embryos from cell cycle arrest and apoptosis. FOXO proteins regulate apoptosis and cell cycle related proteins in mouse preimplantation embryos. Moreover, there might be an autofeedback mechanism where FOXO1, FOXO3, and FOXO4 regulate SIRT1 protein expression. CONCLUSIONS:These results suggest that FOXO transcription factors could contribute to mouse preimplantation embryo development, and it remains to investigate whether they have crucial roles in human preimplantation embryo and infertility.
Project description:The mammalian FoxO transcription factors - FoxO1, FoxO3, FoxO4 - function in the nucleus to direct transcription of specific gene targets governing cellular survival, proliferation, metabolism, differentiation and oxidative defense. Activation of PI3K by extracellular growth factors leads to AKT-mediated phosphorylation of FoxO1, FoxO3 and FoxO4, resulting in their sequestration in the cytoplasm such that they are unable to regulate their gene targets. Our study identified FoxOs as novel tumor suppressors in kidney cancer (Gan et al, 2010, Cancer Cell). To understand the tumor suppression function of FoxOs in kidney cancer cells, we performed gene expression profiling in human kidney cancer cells upon FoxO1 or FoxO3 reactivation in order to identify the key transcriptomic alterations mediating FoxO tumor suppression function in kidney cancer cells. Overall design: We generated RCC4 and UMRC2 cell lines (two human kidney cancer cells with low endogenous FoxO1 and FoxO3 expression) with stable expression of FoxO1(TA)ERT2 or FoxO3(TA)ERT2 construct, which expressed a fusion protein consisting of FoxO(TA) (containing three Ser/Thr AKT phosphorylation sites mutated to alanine) fused to the T2-modified estrogen receptor (ERT2) moiety. We documented that the FoxO(TA)ERT2 fusion protein sequestered FoxO(TA) in the cytoplasm and that 4OHT treatment resulted in rapid translocation of FoxO(TA)ERT2 into the nucleus. We also established stable cell lines with ERT2 expression as control cell lines. (For simplicity, ERT2, FoxO1(TA)ERT2 and FoxO3(TA)ERT2 cell lines will be referred to as EV (empty vector), FoxO1 and FoxO3, respectively, hereafter). We then conducted comparative transcriptome analysis (using the Human Genome U133 Plus 2.0 Array) of EV, FoxO1, or FoxO3-expressing RCC4 and UMRC2 cells at 12 hours with or without 100 nm 4OHT treatment (cultured in DMEM+10% FBS with puromycin selection). To enrich for more proximal actions of FoxO, we selected the 12 hour time point as time course studies revealed dramatic transcriptional changes of known FoxO targets (such as Cyclin D1), yet no discernable cellular phenotypes (apoptosis and cell cycle arrest). We generated 4 transcriptome datasets: FoxO1 RCC4, FoxO3 RCC4, FoxO1 UMRC2, and FoxO3 UMRC2 (by comparing transcriptome data with or without 4OHT treatment), and normalized these transcriptome data against 4OHT-treated EV cells, which show modest 4OHT-induced transcriptional changes.