Analysis of chromatin accessibility upon NODAL/Activin signalling reveal mechanisms of SMAD2-regulated transcription
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ABSTRACT: In this experiment patterns of chromatin accessibility were compared in P19 mouse embryonic teratoma cells treated with either the Nodal/Activin singalling inhibitor SB-431542, or with Activin for 1hr (acute signalling) and 8 hr (prolonged signalling), or left untreated (chronic signalling)
Project description:This experiment was designed to probe the function of Activin/Nodal signalling in the deposition of m6A in human pluripotent stem cells (hPSCs). hPSCs were cultured in presence of Activin or subjected to short-term inhibition of Activin/Nodal signalling for 2h using the receptor antagonist SB-431542 (IP). The global abundance of m6A was then measured by nuclear-enriched methylated RNA immunoprecipitation followed by deep sequencing (NeMeRIP-seq). Pre-NeMeRIP input RNA was used as control to normalise for the changes in gene expression in the two conditions.
Project description:This experiment was designed to monitor the gene expression changes induced by the knockdown of WTAP in human pluripotent stem cells (hPSCs) during the exit from pluripotency induced by the inhibition of Activin/Nodal signalling. hPSCs carrying a tetracycline-inducible short hairpin RNA (shRNA) against WTAP were cultured in control conditions or in presence of tetracycline (TET) to drive gene knockdown. Cells were monitored by RNA-seq in the pluripotent state (culture conditions containing Activin), or at various time points after inhibition of Activin/Nodal signalling with the receptor antagonist SB-431542 (SB). Samples were collected after 2h, 8h and 32h.
Project description:In differentiated mouse ESCs, most of the nodal/activin responsive genes are dependent on both Smad4 and Trim33, some are solely dependent on Smad4, and some are dependent on Trim33. Ebs at Day2.5 from WT, Smad4 null, and Trim33 knock-down ESCs, were treated with activin or SB 431542 for 2 h.
Project description:The Nodal/Activin morphogens are secreted signaling molecules that form concentration gradients during early embryogenesis providing stem cells with positional information and differentiation instructions important for embryonic patterning. The molecular basis driving stem cell interpretation of signaling gradients and the undertaking of distinct cell fate decisions remains poorly understood. We show that perturbation of endogenous Nodal/Activin signaling in ES cells leads to exit from self renewal towards mesendodermal differentiation at high signaling and trophectoderm induction during low signaling. ChIP-seq of Phospho-Smad2, the downstream transcription factor of the Nodal/Activin pathway reveals binding to distinct subsets of target genes in a dose dependent manner including the promoter region of the Oct4 master regulator of stemness. Consequently, both Oct4 mRNA and protein levels are directly driven by graded Nodal/Activin signaling. Hence stem cells interpret and carry out differential Nodal/Activin signaling instructions via a corresponding gradient of Smad2 phosphorylation that selectively titers self renewal against alternative differentiation programs. 3 pSmad2 ChIP samples corresponding to ES cells pretreated for 6 hours in 10uM SB followed by 18 hours in 25ng/ml Activin, 1/5000 DMSO and 10uM SB in 20% KSR media. Controls include the corresponding input DNA for each treatment.
Project description:The purpose of this RNA-seq experiment was to identify Activin A target genes that were differentially expressed in wild type and Arkadia null mouse embryonic stem cells (ESC). We performed the RNA sequencing experiment in two different wt and Arkadia-/- ESC under conditions of signalling inhibition SB431542 (SB; T0) and signalling stimulation with Activin A for 3 and 6 hours (T3 and T6 respectively).
Project description:Activin signalling controls the pluripotency of human embryonic stem cells (hESCs) by inducing the formation of nuclear Smad2/3-Nanog complexes that bind to and regulate several downstream targets. We aimed to clarify the transcriptional dynamics to the inhibition of Activin in hPSCs. To this end we performed microarray analysis of hPSCs treated with SB431542 (SB), an antagonist of the ALK4 and ALK7 type I Activin receptors, for early (2h, 4h and 8h) and late (24h and 48h) time points. Moreover, given our finding that Activin signalling regulates H3K4me3 of its target genes, we tested the transcriptional effect to the knock-down of the Mll/Set1 complex subunit Dpy30 by using shRNA. Finally, we also evaluated the transcriptional response to the knock-down of the Smad2/3 cofactor Nanog by using shRNA.
Project description:Activin/Nodal signalling is necessary to maintain pluripotency of human Embryonic Stem Cells (hESCs) and to induce their differentiation towards endoderm. However, the mechanisms by which Activin/Nodal signalling achieves these opposite functions remain unclear. To unravel these mechanisms, we examined the transcriptional network controlled in hESCs by Smad2 and Smad3 which represent the direct effectors of Activin/Nodal signalling. These analyses reveal that Smad2/3 participate in the control of the core transcriptional network characterising pluripotency which includes Oct-4, Nanog, FoxD3, Dppa4, Tert, Myc and UTF-1. In addition, similar experiments performed on endoderm cells confirm that a broad part of the transcriptional network directing differentiation is downstream of Smad2/3. Therefore, Activin/Nodal signalling appears to control divergent transcriptional networks in hESCs and in endoderm. Importantly, we observed an overlap between the transcriptional network downstream of Nanog and Smad2/3 in hESCs while functional studies showed that both factors cooperate to control the expression of pluripotency genes. Therefore, the effect of Activin/Nodal signalling on pluripotency and differentiation could be dictated by tissue specific Smad2/3 partners such as Nanog, explaining the mechanisms by which signalling pathways can orchestrate divergent cell fate decisions.
Project description:Activin/Nodal signalling is necessary to maintain pluripotency of human Embryonic Stem Cells (hESCs) and to induce their differentiation towards endoderm. However, the mechanisms by which Activin/Nodal signalling achieves these opposite functions remain unclear. To unravel these mechanisms, we examined the transcriptional network controlled in hESCs by Smad2 and Smad3 which represent the direct effectors of Activin/Nodal signalling. These analyses reveal that Smad2/3 participate in the control of the core transcriptional network characterising pluripotency which includes Oct-4, Nanog, FoxD3, Dppa4, Tert, Myc and UTF-1. In addition, similar experiments performed on endoderm cells confirm that a broad part of the transcriptional network directing differentiation is downstream of Smad2/3. Therefore, Activin/Nodal signalling appears to control divergent transcriptional networks in hESCs and in endoderm. Importantly, we observed an overlap between the transcriptional network downstream of Nanog and Smad2/3 in hESCs while functional studies showed that both factors cooperate to control the expression of pluripotency genes. Therefore, the effect of Activin/Nodal signalling on pluripotency and differentiation could be dictated by tissue specific Smad2/3 partners such as Nanog, explaining the mechanisms by which signalling pathways can orchestrate divergent cell fate decisions. Identification of Smad2/3 binding sites in pluripotent hESCs. 5 ChIP-Seq samples including 1 input control sample and 4 ChIP samples (two conditions x two replicates).
Project description:During development, key processes are orchestrated by Nodal/Activin signaling via SMAD2. Interplay between the SMADs, co-factors and chromatin determines cell-type specific responses, but the sequence of events underpinning SMAD2-mediated transcription is unknown. We performed RNA-and ChIP-sequencing for SMAD2, RNA Polymerase II and various histone modifications in different signaling states. Integration of these data reveals a dynamic transcriptional network downstream of Nodal/Activin signaling regulated by SMAD2 acting via multiple mechanisms. Upon ligand stimulation, SMAD2 can bind to pre-acetylated nucleosome-depleted sites, but also to unacetylated closed chromatin, where it induces nucleosome displacement and histone acetylation. Importantly, SMAD2 binding is highly dynamic and does not directly correlate with the transcriptional kinetics of target genes. Moreover, we show that SMAD2 initiates transcription by inducing RNA Polymerase II recruitment. We therefore define new paradigms for SMAD2-dependent transcription and provide a framework to understand how cells correctly execute gene expression programs in response to Nodal/Activin signaling.