Project description:We performed SMAD2/3 ChIP-seq analysis in MCF10A MII cells. To validate whether the changes in SMAD2/3 binding to the genome indeed resulted in changes in target gene expression, we performed RNA-seq transcriptome analysis after short and long periods of TGFβ stimulation (0, 1.5h and 16h) in MII cells. In addition, we revealed that JUNB is a critical AP1 component for SMAD2/3 binding after TGFβ stimulation. To assess the significance of JUNB for TGFβ-SMAD-target genes on a genome-wide scale, we also performed RNA-seq transcriptome analysis in JUNB-knock-downed MII cells.
Project description:The transforming growth factor beta (TGFβ) related signaling is one of the most important signaling pathways regulating early developmental events. Smad2 and Smad3 are structurally similar and it is mostly considered that they are equally important in mediating TGFβ signals. Here, we show that Smad3 is an insensitive TGFβ transducer as compared with Smad2. Smad3 preferentially localizes within the nucleus and is thus sequestered from membrane signaling. The ability of Smad3 in oligomerization with Smad4 upon agonist stimulation is also impaired given its unique linker region. Smad2 mediated TGFβ signaling plays a crucial role in epiblast development and patterning of three germ layers. However, signaling unrelated nuclear localized Smad3 is dispensable for TGFβ signaling-mediated epiblast specification, but important for early neural development, an event blocked by TGFβ/Smad2 signaling. Both Smad2 and Smad3 bind to the conserved Smads binding element (SBE), but they show nonoverlapped target gene binding specificity. We conclude that Smad2 and Smad3 possess differential sensitivities in relaying TGFβ signaling and have distinct roles in regulating early developmental events.
Project description:To further explore the role of these two factors in SMC reprogramming, we treated human aortic smooth muscle (HASMCs) in vitro with TGFβ and mapped binding of Smad2/3, which reflects TGFβ activity, and Pol2-Ser2p, which reflects transcriptional activity, by ChIP-seq analysis. Smad2/3 bound to numerous regulatory regions in the genome differentially regulating expression of numerous genes as demonstrate by alterations in Pol2-Serp2 binding profile and analysis of bulk RNA sequencing. Examination of the top 20 transcription factors identified by sequencing of Smad2/3 binding regulatory regions identified KLF4 gene as one of the most prominently regulated genes. Further analysis showed Smad2/3 binding to multiple KLF4 gene regulatory elements that increased following TGFβ treatment, indicating that KLF4 is a direct target of Smad2/3. At the same time, the amount of bound Pol2-Ser2p decreased, pointing to reduced transcriptional activity. In contrast, Smad2/3 did not bind to KLF2 or KLF5 regulatory elements, demonstrating that these genes are not directly regulated by TGFβ.
Project description:We performed SMAD2 ChIP-seq analysis in T47D cells with/without Palbociclib treatment. To validate whether the changes in SMAD2 binding to the genome indeed resulted in changes in target gene expression, we performed RNA-seq transcriptome analyses in T47D with/without ActA stimulation and Palbo treatment.
Project description:The aim of this experiment was to investigate the role of TGFβ signalling pathway in human pluripotency, through ChIP-seq analysis of its main downstream effector SMAD2/3 in naïve and primed human pluripotent stem cells (hPSCs).
Project description:In pancreatic cancer, two distinct transcriptomic subtypes were identified with a high prognostic relevance: the classical and basal-like subtype. Therefore, in this study, we wanted to use an unbiased method to investigate the global chromatin accessibility in subtype-defined pancreatic cancer cell lines, as well as define the binding profile of the highly subtype-dependent JUN/AP1 transcription factors JUNB (classical) and cJUN (basal). Hence, we performed ATAC-seq in two classical and basal-like cells, as well as ChIP-seq for JUNB in classical CAPAN1 cells and for cJUN in basal-like PANC1 cells.
Project description:A small toolkit of morphogens is used repeatedly to direct development, raising the question of how context dictates interpretation of the same cue. One example is the TGFβ pathway that in human embryonic stem cells fulfills two opposite functions: pluripotency maintenance and mesendoderm (ME) specification. Using proteomics coupled to analysis of genome occupancy, we uncover a regulatory complex comprised of transcriptional effectors of the Hippo pathway (TAZ/YAP/TEAD), the TGFβ pathway (SMAD2/3) and the pluripotency regulator OCT4 (TSO). TSO collaborates with NuRD repressor complexes to buffer pluripotency gene expression, while suppressing ME genes. Importantly, the SMAD DNA binding partner FOXH1, a major specifier of ME, is found near TSO elements and upon fate specification we show that TSO is disrupted with subsequent SMAD-FOXH1 induction of ME. These studies define switch enhancer elements and provide a framework to understand how cellular context dictates interpretation of the same morphogen signal in development. ChIP experiment, a total of 8 samples: 4 samples DMSO treated (Input ctr, IgG ctr, SMAD2 precipitation, TEAD4 precipitation), 4 samples SB431542 (Input ctr, IgG ctr, SMAD2 precipitation, TEAD4 precipitation)
Project description:In this study, we define the epithelial-specific contribution of SMAD2/3 to endometrial function by conditionally ablating the Smad2 and Smad3 transcription factors using Lactoferrin-iCre (Ltf-cre). Using epithelial organoid cultures from the endometrium, we uncover the signaling mechanisms downstream of TGFβ that control endometrial cell regeneration and are critical for endometrial regeneration and homeostasis.
Project description:In this study, we define the epithelial-specific contribution of SMAD2/3 to endometrial function by conditionally ablating the Smad2 and Smad3 transcription factors using Lactoferrin-iCre (Ltf-cre). Using epithelial organoid cultures from the endometrium, we uncover the signaling mechanisms downstream of TGFβ that control endometrial cell regeneration and are critical for endometrial regeneration and homeostasis.
