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Foxd3 promotes the exit from naïve pluripotency and prevents germline specification through enhancer decommissioning [RNA-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. 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).

ORGANISM(S): Mus musculus

SUBMITTER: Alvaro Rada-Iglesias

PROVIDER: E-GEOD-70546 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Foxd3 promotes the exit from naïve pluripotency and prevents germline specification through enhancer decommissioning [ChIP-Seq]

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. 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.

2015-09-14 | E-GEOD-70545 | biostudies-arrayexpress

PRDM14 Ensures a Naïve Pluripotency by a Dual Mechanism Involving Signaling and Epigenetic Pathways in Mouse Embryonic Stem Cells

Project description:Mouse embryonic stem cells (mESCs) fluctuate between a naïve inner cell mass (ICM)-like state and a primed epiblast-like state of pluripotency in serum, but are harnessed exclusively in a distinctive, apparently more naïve state of pluripotency (the ground state) with inhibitors for mitogen-activated protein kinase (MAPK) and glycogen synthase kinase 3 pathways (2i). Understanding the mechanism ensuring a naïve state of pluripotency would be critical in realizing a full potential of ESCs. We show here that PRDM14, a PR domain-containing transcriptional regulator, ensures a naïve pluripotency by a dual mechanism: Antagonizing fibroblast growth factor receptor (FGFR) signaling that is activated paradoxically by the core transcriptional circuitry for pluripotency and directs a primed state and repressing de novo DNA methyltransferases that create a primed epiblast-like epigenome. PRDM14 exerts these functions by recruiting polycomb repressive complex 2 (PRC2) specifically to key targets and repressing their expression. Mouse Embryonic Stem Cells (mESCs) or mESC-like cells with different Prdm14 genotypes {Prdm14(+/+), Prdm14(-/-), and Prdm14(-/-) rescued with Avitag-EGFP-Prdm14 transgene [Prdm14(-/-)+AGP14]} are cultured on MEF in different medium [2i, Serum(day 2), Serum+MEK inhibitor (PD0325901) (day 2), Serum without LIF (day2)].

2013-01-18 | E-GEOD-42580 | biostudies-arrayexpress

Foxd3 promotes the exit from naïve pluripotency and prevents germline specification through enhancer decommissioning

Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

2015-09-14 | E-GEOD-70547 | biostudies-arrayexpress

Foxd3 binding at enhancers controls developmental timing of gene expression by regulation of histone acetylation [ChIP-seq]

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

2015-12-08 | E-GEOD-58407 | biostudies-arrayexpress

Genome-wide transcriptome profiles in pluripotent mouse Embryonic Stem Cells and during Retinoic Acid-induced differentiation

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.

2015-05-01 | E-GEOD-65697 | biostudies-arrayexpress

Transcription profiling of mouse B16-F10 melanoma cells transfected with FoxD3

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.

2008-10-26 | E-GEOD-13129 | biostudies-arrayexpress

FOXD3 is a novel tumor suppressor in lung cancer

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.

2015-12-24 | E-GEOD-64513 | biostudies-arrayexpress

Microarray profiling of mESCs differentiation time course toward neuronal lineage

Project description:mESC adapted to 2i/LIF conditions over four passages (8 days) before differentiation towards the neuronal lineage was induced using experimental conditions described in the literature (PMID: 12524553).

2017-09-27 | E-MTAB-5861 | biostudies-arrayexpress

Transcriptome of mouse pluripotent embryonic stem cells (mESC) cultured in R2i, 2i, PD and SB conditions

Project description:In this study we have analyzed the global gene expression of naïve mouse embryonic stem cells in different culture conditions including R2i (PD0325901+SB431542), 2i (PD0325901+CHIR99021), and also PD0325901+LIF and SB431542+LIF to show the similarities and differences between the conditions in maintaining pluripotency. Since the first generation of mouse embryonic stem (ES) cells, extrinsic regulation of pluripotency has been at the focus of attention. Here we show that suppression of transforming growth factor β (TGFβ) signaling could have impressive effects on self-renewal and pluripotency of mouse ES cells. We introduce a chemically defined medium with inhibitors of TGFβ and mitogen-activated protein kinase (MAPK) kinase, designated here as R2i (Royan 2 inhibitors), for highly efficient establishment of ES cell lines from various mouse strains. R2i also supports homogenous expression of Nanog and Dppa3 proteins in ES cells and shows minimal differentiation leakage. Our results uncover an appropriate condition for ES cell generation from single blastomeres of various cleavage-stage embryos. Further, the high accuracy of genome integrity after long-term cultivation in R2i illustrates an optimal condition for ES cell culture. Global transcriptomic analysis of R2i cells demonstrates that bone morphogenetic protein 4 (BMP4) signaling becomes overrepresented pursuant to TGFβ repression, which may confer further robustness to pluripotency through shielding cells from differentiation stimuli. These findings point to a new facet of ground state pluripotency by blocking differentiation pathways, without influencing the pluripotency regulatory circuitry of mouse ES cells. Whole gene expression study of 4 different mouse embryonic stem cell lines maintained under 4 different chemically defined conditions: R2i, 2i, PD and SB

2013-10-21 | E-GEOD-43682 | biostudies-arrayexpress

Conversion of mES to cXEN cells

Project description:The inner cell mass of the mouse pre-implantation blastocyst is comprised of epiblast progenitor and primitive endoderm cells of which cognate embryonic (mESCs) or extra-embryonic (XEN) stem cell lines can be derived. Importantly, each stem cell type retains the defining properties and lineage restriction of their in vivo tissue of origin. Recently, we demonstrated that XEN-like cells arise within mESC cultures. This raises the possibility that mESCs can generate self-renewing XEN cells without the requirement for gene manipulation. We have developed a novel approach to convert mESCs to XEN cells (cXEN) using growth factors. We confirm that the down-regulation of the pluripotency transcription factor Nanog and the expression of primitive endoderm-associated genes Gata6, Gata4, Sox17 and Pdgfra are necessary for cXEN cell derivation. This approach highlights an important function for Fgf4 in cXEN cell derivation. Paracrine FGF-signalling compensates for the loss of endogenous Fgf4, which is necessary to exit mESC self-renewal, but not for XEN cell maintenance. Our cXEN protocol also reveals that distinct pluripotent stem cells respond uniquely to differentiation promoting signals. cXEN cells can be derived from mESCs cultured with Erk- and Gsk3-inhibitors (2i) and LIF, similarly to conventional mESCs. However, we find that epiblast stem cells (EpiSCs) derived from the post-implantation embryo are refractory to cXEN cell establishment, consistent with the hypothesis that EpiSCs represent a pluripotent state distinct from mESCs. In all, these findings suggest that the potential of mESCs includes the capacity to give rise to both extra-embryonic and embryonic lineages. A total of eight samples were analyzed. Three mouse embryonic stem cell (mESC) lines were used as a negative control, 2 embryo-derived extraembryonic endoderm (XEN) cell lines were used as a positive control, 3 converted XEN (cXEN) cells were used as the experiemental cell lines.

2012-06-04 | E-GEOD-38477 | biostudies-arrayexpress

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