Transcriptomic Differences in Peripheral Monocyte Populations in Septic Patients Based on Outcome.

ABSTRACT: Post-sepsis early mortality is being replaced by survivors who experience either a rapid recovery and favorable hospital discharge or the development of chronic critical illness (CCI) with suboptimal outcomes. The underlying immunological response that determines these clinical trajectories remains poorly defined at the transcriptomic level. As classical and non-classical monocytes are key leukocytes in both the innate and adaptive immune systems, we sought to delineate the transcriptomic response of these cell types. Using single-cell RNA sequencing and pathway analyses, we identified gene expression patterns between these two groups that are consistent with differences in TNFα production based on clinical outcome. This may provide therapeutic targets for those at risk for CCI in order to improve their phenotype/endotype, morbidity, and long-term mortality.

The post-septic peripheral myeloid compartment reveals unexpected diversity in myeloid-derived suppressor cells.

Introduction: Sepsis engenders distinct host immunologic changes that include the expansion of myeloid-derived suppressor cells (MDSCs). These cells play a physiologic role in tempering acute inflammatory responses but can persist in patients who develop chronic critical illness. Methods: Cellular Indexing of Transcriptomes and Epitopes by Sequencing and transcriptomic analysis are used to describe MDSC subpopulations based on differential gene expression, RNA velocities, and biologic process clustering. Results: We identify a unique lineage and differentiation pathway for MDSCs after sepsis and describe a novel MDSC subpopulation. Additionally, we report that the heterogeneous response of the myeloid compartment of blood to sepsis is dependent on clinical outcome. Discussion: The origins and lineage of these MDSC subpopulations were previously assumed to be discrete and unidirectional; however, these cells exhibit a dynamic phenotype with considerable plasticity.

Single-molecule long-read methylation profiling reveals regional DNA methylation regulated by Elongator Complex Subunit 2 in Arabidopsis roots experiencing spaceflight.

BACKGROUND: The Advanced Plant Experiment-04 - Epigenetic Expression (APEX-04-EpEx) experiment onboard the International Space Station examined the spaceflight-altered cytosine methylation in two genetic lines of Arabidopsis thaliana, wild-type Col-0 and the mutant elp2-5, which is deficient in an epigenetic regulator Elongator Complex Subunit 2 (ELP2). Whole-genome bisulfite sequencing (WGBS) revealed distinct spaceflight associated methylation differences, presenting the need to explore specific space-altered methylation at single-molecule resolution to associate specific changes over large regions of spaceflight related genes. To date, tools of multiplexed targeted DNA methylation sequencing remain limited for plant genomes. RESULTS: To provide methylation data at single-molecule resolution, Flap-enabled next-generation capture (FENGC), a novel targeted multiplexed DNA capture and enrichment technique allowing cleavage at any specified sites, was applied to survey spaceflight-altered DNA methylation in genic regions of interest. The FENGC capture panel contained 108 targets ranging from 509 to 704 nt within the promoter or gene body regions of gene targets derived from spaceflight whole-genome data sets. In addition to genes with significant changes in expression and average methylation levels between spaceflight and ground control, targets with space-altered distributions of the proportion of methylated cytosines per molecule were identified. Moreover, trends of co-methylation of different cytosine contexts were exhibited in the same DNA molecules. We further identified significant DNA methylation changes in three previously biological process-unknown genes, and loss-of-function mutants of two of these genes (named as EMO1 and EMO2 for ELP2-regulated Methylation in Orbit 1 and 2) showed enhanced root growth rate. CONCLUSIONS: FENGC simplifies and reduces the cost of multiplexed, targeted, single-molecule profiling of methylation in plants, providing additional resolution along each DNA molecule that is not seen in population-based short-read data such as WGBS. This case study has revealed spaceflight-altered regional modification of cytosine methylation occurring within single DNA molecules of cell subpopulations, which were not identified by WGBS. The single-molecule survey by FENGC can lead to identification of novel functional genes. The newly identified EMO1 and EMO2 are root growth regulators which may be epigenetically involved in plant adaptation to spaceflight.

Exertional heat stroke causes long-term skeletal muscle epigenetic reprogramming, altered gene expression, and impaired satellite cell function in mice.

The effect of exertional heat stroke (EHS) exposure on skeletal muscles is incompletely understood. Muscle weakness is an early symptom of EHS but is not considered a major target of multiorgan injury. Previously, in a preclinical mouse model of EHS, we observed the vulnerability of limb muscles to a second EHS exposure, suggesting hidden processes contributing to declines in muscle resilience. Here, we evaluated the possible molecular origins of EHS-induced declines in muscle resilience. Female C57BL/6 mice [total n = 56; 28/condition, i.e., EHS and exercise control (EXC)] underwent forced wheel running at 37.5°C/40% relative humidity until symptom limitation (unconsciousness). EXC mice exercised identically at room temperature (22-23°C). After 1 mo of recovery, the following were assessed: 1) specific force and caffeine-induced contracture in soleus (SOL) and extensor digitorum longus (EDL) muscles; 2) transcriptome and DNA methylome responses in gastrocnemius (GAST); and 3) primary satellite cell function (proliferation and differentiation). There were no differences in specific force in either SOL or EDL from EXC. Only EHS solei exhibited lower caffeine sensitivity. EHS GAST exhibited higher RNA expression of genes encoding structural proteins of slow fibers, heat shock proteins, and myogenesis. A total of ∼2,500 differentially methylated regions of DNA that could potentially affect many cell functions were identified. Primary satellite cells exhibited suppressed proliferation rates but normal differentiation responses. Results demonstrate long-term changes in skeletal muscles 1 mo after EHS that could contribute to declines in muscle resilience. Skeletal muscle may join other, more recognized tissues considered vulnerable to long-term effects of EHS.NEW & NOTEWORTHY Exertional heat stroke (EHS) in mice induces long-term molecular and functional changes in limb muscle that could reflect a loss of "resilience" to further stress. The phenotype was characterized by altered caffeine sensitivity and suppressed satellite cell proliferative potential. This was accompanied by changes in gene expression and DNA methylation consistent with ongoing muscle remodeling and stress adaptation. We propose that EHS may induce a prolonged vulnerability of skeletal muscle to further stress or injury.

Long-term epigenetic and metabolomic changes in the mouse ventricular myocardium after exertional heat stroke.

Evidence from human epidemiological studies suggests that exertional heat stroke (EHS) results in an elevated risk of long-term cardiovascular and systemic disease. Previous results using a preclinical mouse model of EHS demonstrated severe metabolic imbalances in ventricular myocardium developing at 9-14 days of recovery. Whether this resolves over time is unknown. We hypothesized that the long-term effects of EHS on the heart reflect retained maladaptive epigenetic responses. In this study, we evaluated genome-wide DNA methylation, RNA-Seq, and metabolomic profiles of the left ventricular myocardium in female C57BL/6 mice, 30 days after EHS (exercise in 37.5°C; n = 7-8), compared with exercise controls. EHS mice ran to loss of consciousness, reaching core temperatures of 42.4 ± 0.2°C. All mice recovered quickly. After 30 days, the left ventricles were rapidly frozen for DNA methyl sequencing, RNA-Seq, and untargeted metabolomics. Ventricular DNA from EHS mice revealed >13,000 differentially methylated cytosines (DMCs) and >900 differentially methylated regions (DMRs; ≥5 DMCs with ≤300 bp between each CpG). Pathway analysis using DMRs revealed alterations in genes regulating basic cell functions, DNA binding, transcription, and metabolism. Metabolomics and mRNA expression revealed modest changes that are consistent with a return to homeostasis. Methylation status did not predict RNA expression or metabolic state at 30 days. We conclude that EHS induces a sustained DNA methylation memory lasting over 30 days of recovery, but ventricular gene expression and metabolism return to a relative homeostasis at rest. Such long-lasting alterations to the DNA methylation landscape could alter responsiveness to environmental or clinical challenges later in life.

Methylscaper: an R/Shiny app for joint visualization of DNA methylation and nucleosome occupancy in single-molecule and single-cell data.

SUMMARY: Differential DNA methylation and chromatin accessibility are associated with disease development, particularly cancer. Methods that allow profiling of these epigenetic mechanisms in the same reaction and at the single-molecule or single-cell level continue to emerge. However, a challenge lies in jointly visualizing and analyzing the heterogeneous nature of the data and extracting regulatory insight. Here, we present methylscaper, a visualization framework for simultaneous analysis of DNA methylation and chromatin accessibility landscapes. Methylscaper implements a weighted principal component analysis that orders DNA molecules, each providing a record of the chromatin state of one epiallele, and reveals patterns of nucleosome positioning, transcription factor occupancy, and DNA methylation. We demonstrate methylscaper's utility on a long-read, single-molecule methyltransferase accessibility protocol for individual templates (MAPit-BGS) dataset and a single-cell nucleosome, methylation, and transcription sequencing (scNMT-seq) dataset. In comparison to other procedures, methylscaper is able to readily identify chromatin features that are biologically relevant to transcriptional status while scaling to larger datasets. AVAILABILITY AND IMPLEMENTATION: Methylscaper, is implemented in R (version > 4.1) and available on Bioconductor: https://bioconductor.org/packages/methylscaper/, GitHub: https://github.com/rhondabacher/methylscaper/, and Web: https://methylscaper.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Exertional heat stroke leads to concurrent long-term epigenetic memory, immunosuppression and altered heat shock response in female mice.

KEY POINTS: Exposure to exertional heat stroke (EHS) has been linked to increased long-term decrements of health. Epigenetic reprogramming is involved in the response to heat acclimation; however, whether the long-term effects of EHS are mediated by epigenetic reprogramming is unknown. In female mice, we observed DNA methylation reprogramming in bone marrow-derived (BMD) monocytes as early as 4 days of recovery from EHS and as late as 30 days compared with sham exercise controls. Whole blood, collected after 30 days of recovery from EHS, exhibited an immunosuppressive phenotype when challenged in vitro by lipopolysaccharide. After 30 days of recovery from EHS, BMD monocytes exhibited an altered in vitro heat shock response. The location of differentially methylated CpGs are predictive of both the immunosuppressive phenotype and altered heat shock responses. ABSTRACT: Exposure to exertional heat stroke (EHS) has been linked to increased susceptibility to a second heat stroke, infection and cardiovascular disease. Whether these clinical outcomes are mediated by an epigenetic memory is unknown. Using a preclinical mouse model of EHS, we investigated whether EHS exposure produces a lasting epigenetic memory in monocytes and whether there are phenotypic alterations that may be consistent with these epigenetic changes. Female mice underwent forced wheel running at 37.5°C/40% relative humidity until symptom limitation, characterized by CNS dysfunction. Results were compared with matched exercise controls at 22.5°C. Monocytes were isolated from bone marrow after 4 or 30 days of recovery to extract DNA and analyse methylation. Broad-ranging alterations to the DNA methylome were observed at both time points. At 30 days, very specific alterations were observed to the promoter regions of genes involved with immune responsiveness. To test whether these changes might be related to phenotype, whole blood at 30 days was challenged with lipopolysaccharide (LPS) to measure cytokine secretion; monocytes were also challenged with heat shock to quantify mRNA expression. Whole blood collected from EHS mice showed markedly attenuated inflammatory responses to LPS challenge. Furthermore, monocyte mRNA from EHS mice showed significantly altered responses to heat shock challenge. These results demonstrate that EHS leads to a unique DNA methylation pattern in monocytes and altered immune and heat shock responsiveness after 30 days. These data support the hypothesis that EHS exposure can induce long-term physiological changes that may be linked to altered epigenetic profiles.

Publisher Correction: Bcl11b is essential for licensing Th2 differentiation during helminth infection and allergic asthma.

In the originally published version of this Article, the affiliation details for Dorina Avram incorrectly included "Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida, 2015 SW 16th Ave, Gainesville, FL, 32608, USA", instead of "UF Health Cancer Center, University of Florida, Gainesville, FL 32610, USA". This has now been corrected in both the PDF and HTML versions of the Article.

Bcl11b is essential for licensing Th2 differentiation during helminth infection and allergic asthma.

During helminth infection and allergic asthma, naive CD4+ T-cells differentiate into cytokine-producing Type-2 helper (Th2) cells that resolve the infection or induce asthma-associated pathology. Mechanisms regulating the Th2 differentiation in vivo remain poorly understood. Here we report that mice lacking Bcl11b in mature T-cells have a diminished capacity to mount Th2 responses during helminth infection and allergic asthma, showing reduced Th2 cytokines and Gata3, and elevated Runx3. We provide evidence that Bcl11b is required to maintain chromatin accessibility at Th2-cytokine promoters and locus-control regions, and binds the Il4 HS IV silencer, reducing its accessibility. Bcl11b also binds Gata3-intronic and downstream-noncoding sites, sustaining the Gata3 expression. In addition, Bcl11b binds and deactivates upstream enhancers at Runx3 locus, restricting the Runx3 expression and its availability to act at the Il4 HS IV silencer. Thus, our results establish novel roles for Bcl11b in the regulatory loop that licenses Th2 program in vivo.

High Fractional Occupancy of a Tandem Maf Recognition Element and Its Role in Long-Range ß-Globin Gene Regulation.

Enhancers and promoters assemble protein complexes that ultimately regulate the recruitment and activity of RNA polymerases. Previous work has shown that at least some enhancers form stable protein complexes, leading to the formation of enhanceosomes. We analyzed protein-DNA interactions in the murine ß-globin gene locus using the methyltransferase accessibility protocol for individual templates (MAPit). The data show that a tandem Maf recognition element (MARE) in locus control region (LCR) hypersensitive site 2 (HS2) reveals a remarkably high degree of occupancy during differentiation of mouse erythroleukemia cells. Most of the other transcription factor binding sites in LCR HS2 or in the adult ß-globin gene promoter regions exhibit low fractional occupancy, suggesting highly dynamic protein-DNA interactions. Targeting of an artificial zinc finger DNA-binding domain (ZF-DBD) to the HS2 tandem MARE caused a reduction in the association of MARE-binding proteins and transcription complexes at LCR HS2 and the adult ßmajor-globin gene promoter but did not affect expression of the ßminor-globin gene. The data demonstrate that a stable MARE-associated footprint in LCR HS2 is important for the recruitment of transcription complexes to the adult ßmajor-globin gene promoter during erythroid cell differentiation.

Integrated DNA methylation and chromatin structural analysis at single-molecule resolution.

Chromatin limits the accessibility of DNA to trans-acting factors in transcription, replication, and repair. Although transcriptional variation between cells in a population may contribute to survival and disease, most assays of chromatin structure recover only population averages. We have developed DNA methyltransferases (MTases) as probing agents of DNA accessibility in chromatin, either expressed in vivo in budding yeast or as recombinant enzymatic probes of nuclei isolated from mammalian cells. In this chapter, we focus on the use of recombinant MTase (M) M.CviPI to probe chromatin accessibility in nuclei isolated from mammalian cell lines and animal tissue. This technique, named methylation accessibility protocol for individual templates (MAPit), reports protein-DNA interactions at single-molecule resolution. The single-molecule readout allows identification of chromatin subpopulations and rare epigenetic variants within a cell population. Furthermore, the use of M.CviPI in mammalian systems gives a comprehensive view of both chromatin structure and endogenous DNA methylation in a single assay.

Human lymphoid translocation fragile zones are hypomethylated and have accessible chromatin.

Chromosomal translocations are a hallmark of hematopoietic malignancies. CG motifs within translocation fragile zones (typically 20 to 600 bp in size) are prone to chromosomal translocation in lymphomas. Here we demonstrate that the CG motifs in human translocation fragile zones are hypomethylated relative to the adjacent DNA. Using a methyltransferase footprinting assay on isolated nuclei (in vitro), we find that the chromatin at these fragile zones is accessible. We also examined in vivo accessibility using cellular expression of a prokaryotic methylase. Based on this assay, which measures accessibility over a much longer time interval than is possible with in vitro methods, these fragile zones were found to be more accessible than the adjacent DNA. Because DNA within the fragile zones can be methylated by both cellular and exogenous methyltransferases, the fragile zones are predominantly in a duplex DNA conformation. These observations permit more-refined models for why these zones are 100- to 1,000-fold more prone to undergo chromosomal translocation than the adjacent regions.

TRIM29 suppresses TWIST1 and invasive breast cancer behavior.

TRIM29 (ATDC) exhibits a contextual function in cancer, but seems to exert a tumor-suppressor role in breast cancer. Here, we show that TRIM29 is often silenced in primary breast tumors and cultured tumor cells as a result of aberrant gene hypermethylation. RNAi-mediated silencing of TRIM29 in breast tumor cells increased their motility, invasiveness, and proliferation in a manner associated with increased expression of mesenchymal markers (N-cadherin and vimentin), decreased expression of epithelial markers (E-cadherin and EpCAM), and increased expression and activity of the oncogenic transcription factor TWIST1, an important driver of the epithelial-mesenchymal transition (EMT). Functional investigations revealed an inverse relationship in the expression of TRIM29 and TWIST1, suggesting the existence of a negative regulatory feedback loop. In support of this relationship, we found that TWIST1 inhibited TRIM29 promoter activity through direct binding to a region containing a cluster of consensus E-box elements, arguing that TWIST1 transcriptionally represses TRIM29 expression. Analysis of a public breast cancer gene-expression database indicated that reduced TRIM29 expression was associated with reduced relapse-free survival, increased tumor size, grade, and metastatic characteristics. Taken together, our results suggest that TRIM29 acts as a tumor suppressor in breast cancer through its ability to inhibit TWIST1 and suppress EMT.

Multiplex mapping of chromatin accessibility and DNA methylation within targeted single molecules identifies epigenetic heterogeneity in neural stem cells and glioblastoma.

Human tumors are comprised of heterogeneous cell populations that display diverse molecular and phenotypic features. To examine the extent to which epigenetic differences contribute to intratumoral cellular heterogeneity, we have developed a high-throughput method, termed MAPit-patch. The method uses multiplexed amplification of targeted sequences from submicrogram quantities of genomic DNA followed by next generation bisulfite sequencing. This provides highly scalable and simultaneous mapping of chromatin accessibility and DNA methylation on single molecules at high resolution. Long sequencing reads from targeted regions maintain the structural integrity of epigenetic information and provide substantial depth of coverage, detecting for the first time minority subpopulations of epigenetic configurations formerly obscured by existing genome-wide and population-ensemble methodologies. Analyzing a cohort of 71 promoters of genes with exons commonly mutated in cancer, MAPit-patch uncovered several differentially accessible and methylated promoters that are associated with altered gene expression between neural stem cell (NSC) and glioblastoma (GBM) cell populations. In addition, considering each promoter individually, substantial epigenetic heterogeneity was observed across the sequenced molecules, indicating the presence of epigenetically distinct cellular subpopulations. At the divergent MLH1/EPM2AIP1 promoter, a locus with three well-defined, nucleosome-depleted regions (NDRs), a fraction of promoter copies with inaccessible chromatin was detected and enriched upon selection of temozolomide-tolerant GBM cells. These results illustrate the biological relevance of epigenetically distinct subpopulations that in part underlie the phenotypic heterogeneity of tumor cell populations. Furthermore, these findings show that alterations in chromatin accessibility without accompanying changes in DNA methylation may constitute a novel class of epigenetic biomarker.

Local depletion of DNA methylation identifies a repressive p53 regulatory region in the NEK2 promoter.

Genome-scale mapping suggests that the function of DNA methylation varies with genomic context beyond transcriptional repression. However, the use of DNA-demethylating agents (e.g. 5-aza-2'-deoxycytidine (5aza-dC)) to study epigenetic regulation often focuses on gene activation and ignores repression elicited by 5aza-dC. Here, we show that repression of NEK2, which encodes the never in mitosis A (NIMA)-related kinase, by 5aza-dC is context-specific as NEK2 transcript levels were reduced in HCT116 colon cancer cells but not in isogenic p53(-/-) cells. Bisulfite sequencing showed that DNA methylation was restricted to the distal region of the NEK2 promoter. Demethylation by 5aza-dC was associated with increased accessibility to micrococcal nuclease, i.e. nucleosome depletion. Conversely, methyltransferase accessibility protocol for individual templates (MAPit) methylation footprinting showed that nucleosome occupancy and DNA methylation at the distal promoter were significantly increased in p53(-/-) cells, suggesting dynamic regulation of chromatin structure at this region by p53 in HCT116 cells. Stabilization of endogenous p53 by doxorubicin or ectopic expression of p53, but not a p53 DNA-binding mutant, decreased NEK2 expression. Chromatin immunoprecipitation demonstrated direct and specific association of p53 with the distal NEK2 promoter, which was enhanced by doxorubicin. Luciferase reporters confirmed that this region is required for p53-mediated repression of NEK2 promoter activity. Lastly, modulation of p53 abundance altered nucleosome occupancy and DNA methylation at its binding region. These results identify NEK2 as a novel p53-repressed gene, illustrate that its repression by 5aza-dC is specific and associated with nucleosome reorganization, and provide evidence that identification of partially methylated regions can reveal novel p53 target genes.

Microbiota impact on the epigenetic regulation of colorectal cancer.

Mechanisms of colorectal cancer (CRC) development can be generally divided into three categories: genetic, epigenetic, and aberrant immunologic signaling pathways, all of which may be triggered by an imbalanced intestinal microbiota. Aberrant gut microbial composition, termed 'dysbiosis', has been reported in inflammatory bowel disease patients who are at increased risk for CRC development. Recent studies indicate that it is feasible to rescue experimental models of colonic cancer by oral treatment with genetically engineered beneficial bacteria and/or their immune-regulating gene products. Here, we review the mechanisms of epigenetic modulation implicated in the development and progression of CRC, which may be the result of dysbiosis, and therefore may be amenable to therapeutic intervention.

The ZEB1 pathway links glioblastoma initiation, invasion and chemoresistance.

Glioblastoma remains one of the most lethal types of cancer, and is the most common brain tumour in adults. In particular, tumour recurrence after surgical resection and radiation invariably occurs regardless of aggressive chemotherapy. Here, we provide evidence that the transcription factor ZEB1 (zinc finger E-box binding homeobox 1) exerts simultaneous influence over invasion, chemoresistance and tumourigenesis in glioblastoma. ZEB1 is preferentially expressed in invasive glioblastoma cells, where the ZEB1-miR-200 feedback loop interconnects these processes through the downstream effectors ROBO1, c-MYB and MGMT. Moreover, ZEB1 expression in glioblastoma patients is predictive of shorter survival and poor Temozolomide response. Our findings indicate that this regulator of epithelial-mesenchymal transition orchestrates key features of cancer stem cells in malignant glioma and identify ROBO1, OLIG2, CD133 and MGMT as novel targets of the ZEB1 pathway. Thus, ZEB1 is an important candidate molecule for glioblastoma recurrence, a marker of invasive tumour cells and a potential therapeutic target, along with its downstream effectors.

Epigenetic diversity of Kaposi's sarcoma-associated herpesvirus.

Spontaneous lytic reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) occurs at a low rate in latently infected cells in disease and culture. This suggests imperfect epigenetic maintenance of viral transcription programs, perhaps due to variability in chromatin structure at specific loci across the population of KSHV episomal genomes. To characterize this locus-specific chromatin structural diversity, we used MAPit single-molecule footprinting, which simultaneously maps endogenous CG methylation and accessibility to M.CviPI at GC sites. Diverse chromatin structures were detected at the LANA, RTA and vIL6 promoters. At each locus, chromatin ranged from fully closed to fully open across the population. This diversity has not previously been reported in a virus. Phorbol ester and RTA transgene induction were used to identify chromatin conformations associated with reactivation of lytic transcription, which only a fraction of episomes had. Moreover, certain chromatin conformations correlated with CG methylation patterns at the RTA and vIL6 promoters. This indicated that some of the diverse chromatin conformations at these loci were epigenetically distinct. Finally, by comparing chromatin structures from a cell line infected with constitutively latent virus, we identified products of lytic replication. Our findings show that epigenetic drift can restrict viral propagation by chromatin compaction at latent and lytic promoters.

DNA methyltransferase accessibility protocol for individual templates by deep sequencing.

A single-molecule probe of chromatin structure can uncover dynamic chromatin states and rare epigenetic variants of biological importance that bulk measures of chromatin structure miss. In bisulfite genomic sequencing, each sequenced clone records the methylation status of multiple sites on an individual molecule of DNA. An exogenous DNA methyltransferase can thus be used to image nucleosomes and other protein-DNA complexes. In this chapter, we describe the adaptation of this technique, termed Methylation Accessibility Protocol for individual templates, to modern high-throughput sequencing, which both simplifies the workflow and extends its utility.

Fibroblast growth factor receptor 2 homodimerization rapidly reduces transcription of the pluripotency gene Nanog without dissociation of activating transcription factors.

Nanog or Gata6-positive cells co-exist and are convertible within the inner cell mass of murine blastocysts and embryonic stem (ES) cells. Previous studies demonstrate fibroblast growth factor receptor 2 (FGFR2) triggers Nanog gene down-regulation and differentiation to primitive endoderm (PE); however, the underlying mechanisms responsible for reversible and fluctuating cell fate are poorly understood. Using an inducible FGFR2 dimerization system in ES cells, we demonstrate that FGFR2 activation rapidly down-regulated Nanog gene transcription through activation of the Mek pathway and subsequently differentiated ES cells into PE cells. FGFR2 rather selectively repressed the Nanog gene with minimal effect on other pluripotency genes, including Oct4 and Sox2. We determined the Nanog promoter region containing minimum Oct4/Sox2 binding sites was sufficient for this transcriptional down-regulation by FGFR2, when the reporter transgenes were integrated with insulators. Of interest, FGFR2-mediated Nanog transcriptional reduction occurred without dissociation of RNA polymerase II, p300, Oct4, Sox2, and Tet1 from the Nanog proximal promoter region and with no increase in repressive histone methylation marks or DNA methylation, implying the gene repression is in the early and transient phase. Furthermore, addition of a specific FGFR inhibitor readily reversed this Nanog repression status. These findings illustrate well how FGFR2 induces rapid but reversible Nanog repression within ES cells.