Genome-wide analysis of FOXO3 mediated transcription regulation through RNA polymerase II profiling.
ABSTRACT: 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: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: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: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: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:Eukaryotic gene transcription is regulated at many steps, including RNA polymerase II (Pol II) recruitment, transcription initiation, promoter-proximal Pol II pause release, and transcription termination; however, mechanisms regulating transcription during productive elongation remain poorly understood. Enhancers, which activate gene transcription, themselves undergo Pol II-mediated transcription, but our understanding of enhancer transcription and enhancer RNAs (eRNAs) remains incomplete. Here we show that transcription at intragenic enhancers interferes with and attenuates host gene transcription during productive elongation. While the extent of attenuation correlates positively with nascent eRNA expression, the act of intragenic enhancer transcription alone, but not eRNAs, explains the attenuation. Through CRISPR/Cas9-mediated deletions, we demonstrate a physiological role for intragenic enhancer-mediated transcription attenuation in cell fate determination. We propose that intragenic enhancers not only enhance transcription of one or more genes from a distance but also fine-tune transcription of their host gene through transcription interference, facilitating differential utilization of the same regulatory element for disparate functions.
Project description:Although FoxO and Pax proteins represent two important families of transcription factors in determining cell fate, they had not been functionally or physically linked together in mediating regulation of a common target gene during normal cellular transcription programs. Here, we identify MyoD, a key regulator of myogenesis, as a direct target of FoxO3 and Pax3/7 in myoblasts. Our cell-based assays and in vitro studies reveal a tight codependent partnership between FoxO3 and Pax3/7 to coordinately recruit RNA polymerase II and form a preinitiation complex (PIC) to activate MyoD transcription in myoblasts. The role of FoxO3 in regulating muscle differentiation is confirmed in vivo by observed defects in muscle regeneration caused by MyoD downregulation in FoxO3 null mice. These data establish a mutual interdependence and functional link between two families of transcription activators serving as potential signaling sensors and regulators of cell fate commitment in directing tissue specific MyoD transcription.
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:Enhancers govern transcription through multiple mechanisms, including the regulation of elongation by RNA polymerase II (RNAPII). We characterized the dynamics of looped enhancer contacts during synchronous transcription elongation. We found that many distal enhancers form stable contacts with their target promoters during the entire interval of elongation. Notably, we detected additional dynamic enhancer contacts throughout the gene bodies that track with elongating RNAPII and the leading edge of RNA synthesis. These results support a model in which the gene body changes its position relative to a stable enhancer-promoter complex, which has broad ramifications for enhancer function and architectural models of transcriptional elongation.
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.