Foxd3 promotes the exit from naïve pluripotency and prevents germline specification through enhancer decommissioning [ChIP-Seq]
ABSTRACT: Following implantation, mouse epiblast cells transit from a naïve to a primed state in which they are competent for both somatic and primordial germ cell (PGC) specification. Using mouse embryonic stem cells (mESC) as an in vitro model to study the transcriptional regulatory principles orchestrating peri-implantation development, here we show that the transcription factor Foxd3 is necessary for the exit from naïve pluripotency and the progression to a primed pluripotent state. During this transition, Foxd3 acts as a repressor that dismantles a significant fraction of the naïve pluripotency expression program through the decommissioning of active enhancers associated with key naïve pluripotency and early germline genes. Subsequently, Foxd3 needs to be silenced in primed pluripotent cells to allow the reactivation of relevant genes required for proper PGC specification. Our findings uncover a wave of activation-deactivation of Foxd3 as a crucial step for the exit from naïve pluripotency and subsequent PGC specification. Genome-wide binding profiles for Foxd3 were investigated in mouse embryonic stem cells (mESC). A mESC line (FH-Foxd3 mESC line) expressing exogenous Foxd3 tagged with Flag and HA epitope (FH-Foxd3) at nearly endogenous levels was generated. ChIPs were performed against FH-Foxd3 using anti-HA or anti-Flag antibodies.
Project description:Following implantation, mouse epiblast cells transit from a naïve to a primed state in which they are competent for both somatic and primordial germ cell (PGC) specification. Using mouse embryonic stem cells (mESC) as an in vitro model to study the transcriptional regulatory principles orchestrating peri-implantation development, here we show that the transcription factor Foxd3 is necessary for the exit from naïve pluripotency and the progression to a primed pluripotent state. During this transition, Foxd3 acts as a repressor that dismantles a significant fraction of the naïve pluripotency expression program through the decommissioning of active enhancers associated with key naïve pluripotency and early germline genes. Subsequently, Foxd3 needs to be silenced in primed pluripotent cells to allow the reactivation of relevant genes required for proper PGC specification. Our findings uncover a wave of activation-deactivation of Foxd3 as a crucial step for the exit from naïve pluripotency and subsequent PGC specification. mRNA profiles were generated by RNA-seq in duplicates for each of the following mESC lines: Foxd3fl/fl;Cre-ER mESC maintained in "Serum+LIF" (SL) treated with TM for three days (SL Foxd3-/-); untreated Foxd3fl/fl;Cre-ER SL mESC (SL Foxd3fl/fl); tetON Foxd3 SL mESC treated with Dox for three days; WT SL mESC treated with Dox for three days; Foxd3fl/fl;Cre-ER mESC maintained in "2i+LIF" (2i) treated with TM for three days (2i Foxd3-/-); untreated Foxd3fl/fl;Cre-ER 2i mESC (2i Foxd3fl/fl).
Project description:Prdm14 is a sequence-specific transcriptional regulator of embryonic stem cell (ESC) pluripotency and primordial germ cell (PGC) formation. It exerts its function, at least in part, through repressing genes associated with epigenetic modification and cell differentiation. Here, we show that this repressive function is mediated through an ETO-family co-repressor Mtgr1, which tightly binds to the pre-SET/SET domains of Prdm14 and co-occupies its genomic targets in mouse ESCs. Structure-guided point mutants abrogated the Prdm14-Mtgr1 association and disrupted Prdm14's function in mESC gene expression and PGC formation in vitro. Altogether, our work uncovers the molecular mechanism underlying Prdm14-mediated repression. Examination of Prdm14 and Mtgr1 occupancy by ChIP-seq and effects on gene expression in mouse embryonic stem cells
Project description:The transcription factor forkhead box D3 (FoxD3) is a transcriptional factor which belongs to forkhead box (Fox) transcription factor family. The functions of FOXD3 in embryogenesis and in the development of neural crest cells have been clearly defined. Its tumor suppressor function hasbeen found in many types of cancer in recent years. However, the study about its roles in lung cancerdevelopment is still lacking. Our study found that deficiency of FoxD3 in lung cancer enhanced cell growth and cell invasion. RNA-sequence analysis demonstrated that loss of FoxD3 mainly affected cell cycle progression related gene expression. Knockdown of FoxD3 led to G2-M cell accumulation with up-regulation of DNA replication licensing factor MCM5 and MCM4 as well as cell cycle regulator polo-like kinase-1 (PLK1) and CDC6. Over-expression of those genesin lung cancer was associated with poor clinical outcomes of lung cancer patients. We also identified high mobility group box-1(HMGB1), Hras and Ephrin B1 gene expression increase after FoxD3 silencing which may participate in enhanced lung cancer cell invasion. Our study identifiedtumor suppressor function of FoxD3 in lung cancer andwe did the first comprehensive analysis of the genes regulated by FoxD3 for cell proliferation and invasion in lung cancer. The identified genes regulated by FoxD3 through our analysis will provide valuable information to uncover the mechanism of FoxD3 tumor suppressor function. Three control and three FOXD3 knockdown A549 cell lines were subjected to RNA sequencing.
Project description:We wished to examine the genes regulated by FoxD3 in pigment cells to gain understanding in how FoxD3 represses melanoblast specification in the neural crest. For technical reasons, we could not use neural crest cells, so we used melanoma cells, since they are derived from neural crest cells. To this end, we transfected B16-F10 mouse melanoma cells with constructs expressing FoxD3, or FoxD3-VP16, in which the C-terminal portion of FoxD3 (which contains the transcriptional repression domain) has been replaced by the VP16 transcriptional activation domain. Experiment Overall Design: The base vector used for these studies was pMES, which expresses the gene of interest under control of the chick beta-actin promoter. EGFP is also expressed from the bicistronic mRNA through the use of an IRES. Experiment Overall Design: B16-F10 cells were transfected with either empty pMES, pFoxD3 (which contains FoxD3 inserted into pMES), or pFoxD3-VP16 (similar to pFoxD3, except that the C-terminal portion of FoxD3, which contains the transcriptional repression domain, has been replaced by the transcriptional activation domain of VP16). Experiment Overall Design: 24 hours after transfection, EGFP-positive cells were collected by FACS and those cells were subjected to microarray analysis.
Project description:Transcription factor/enhancer interactions determine cell specific gene expression. Here, we followed enhancers during differentiations of embryonic stem (ESCs) to epiblast like cells (EpiLCs). There were highly dynamic changes in histone lysine 27 acetylation at enhancer sites throughout the genome. These sites were enriched for a Foxd3 binding motif, a forkhead transcription factor essential in early embryonic development. Surprisingly, Foxd3 occupied largely mutually exclusive sites in the ESCs versus EpiLCs. Foxd3 bound to nucleosome occupied regions, simultaneously evicting the histones while inhibiting full gene expression through the recruitment of histone deacetylases. Knockout of Foxd3 resulted in hyperacetylation and transcriptional upregulation of neighboring genes, many of which were further upregulated at later stages of differentiation. These data show that Foxd3 primes enhancer sites during pregastrulation by removing nucleosomes, yet suppresses neighboring histone hyperacetylation. Such a mechanism may be common to many transcription factors that prepare enhancers for later gene activation during development. ChIP-seq of H3K4me1, H3K27ac, H3K27me3, p300, H3K4me3, RNA Pol2 and Oct4 in four pluripotent states: embryonic stem cells (ESCs) day 1 ESC differentiation, Epi-like stem cells (EpiLCs), and epiblast stem cells (EpiSCs); ChIP-seq of 3XFlag tagged Foxd3 in ESCs and EpiLCs; ChIP-seq of H3K4me1, H3K27ac, H3K27me3, p300 and H3K4me3 in Foxd3 conditional knockout cells (tamoxifen-inducible) -/+ 36h Tamoxifen treatemnt. ChIP seq of Flag-Foxd3 (third replicate), ChIP-seq of HDAC1 and Brg1 in WT and Foxd3 KO cells and MNase-ChIP-seq of H3K4me1
Project description:To understand the transcriptional impact of FOXD3 in melanoma cells, we utilized a microarray approach. We collected RNA from three unrelated mutant B-RAF melanoma cell lines (WM115, WM793, and A375) that were engineered to inducibly express FOXD3 or the control gene, β-galactosidase (LacZ), after 5 days of transgene induction. This time point was chosen based on maximal phenotypic changes previously observed. Comparison of gene signatures between the 3 cell lines produced approximately 2,600 common genes differentially regulated by FOXD3-expressing cells compared to the LacZ controls. Three unrelated mutant B-RAF melanoma cell lines (WM115, WM793, and A375) were induced to express FOXD3 and compared against the same cell lines expressing the control gene, β-galactosidase (LacZ).
Project description:We utilized FAIRE-seq to identify accesible chromatin in mouse embryonic-, epiblast-, and neural- stem cells in addition to mouse embryonic fibroblasts. Analysis of these data sets reveal cell type specific chromatin signatures that differentiate naïve and primed pluripotency. Functional analysis of type-specific peaks revealed cell-type specific enhancers. FAIRE-seq of mESC, EpiSC, NSC and MEF
Project description:We report the genome-wide RNA expression levels in pluripotent mESC and as mESC differentiate towards a neuronal lineage in response to high levels of Retinoic Acid treatment in vitro. RNA-seq was performed to identify all RNAs expressed in both ESCs and neuronal cells. In total, In total, 14,443 expressed genes were detected, of which 1,834 were up-regulated and 1,477 down-regulated (fold change (FC) > -/+2.0 and p-value < 0.035) during RA-induced neuronal differentiation. The top down-regulated genes included members of the pluripotency core transcriptional network, including Klf4, Sox2, Oct4, Nanog, Suz12, Esrrb, Stat3 and Tcfcp2l1. The top up-regulated genes are important for neuronal differentiation (e.g. Pax3, Irx3, Rest and Foxd3) and reside in the RA-pathway (e.g. various homeobox genes), the retinoic acid receptors and the RA-degradation enzyme Cyp26a1. Examination, identification and comparision of mRNA expression profliles in two cellular states.
Project description:Fumarate hydratase (FH) is the enzyme in the Krebs cycle, which transforms fumarate to malate. Loss of fumarate hyrdragase leads to hereditary leiomyomatosis and renal cell cancer (HLRCC). The biallelic inactivation has been highly accumulated in FH-deficient cells and is considered a major pro-oncogenic factor for HLRCC tumorigenesis. We used microarrays to understand the global readaptation of cell metabolism of gene expression underlying the truncated Krebs cycle by loss of function of fumarate hydratase. The fumarate hydratase wild type and knock out cells were generated for RNA extraction and hybridization on Affymetrix microarrays.