Project description:Genome-wide occupancy of biotinylated Jmjd2b, Jmjd2c from mESCs, as well as occupancy of selected factors and histone marks from wild-type mESCs, Anti-GFP KD, Jmj2b KD and Jmjd2c KD mESCs genome To identify genome-wide binding target sites of Jmjd2b and Jmjd2c in the mESCs genome, and genome-wide binding sites for selected factors and histone marks from Anti-GFP KD, Jmjd2b KD and Jmjd2c KD mESCs
Project description:We used microarray to determine the changes in gene expression profile after KD of Jmjd2b and Jmjd2c compared to Anti-GFP KD from mES cells Mouse ES cells were infected with AntiGFP, Jmjd2b and Jmjd2c shRNAs lentivirus, puromycin selected, passage them 2-3 times, collected for RNA isolation and gene expression
Project description:Self-renewal and pluripotency of embryonic stem cells (ESCs) are established by multiple regulatory pathways operating at several levels. The roles of histone demethylases (HDMs) in these programs are incompletely defined. We conducted a functional RNAi screen for HDMs and identified five potential HDMs essential for mouse ESC identity. In-depth analyses demonstrate that the closely related HDMs Jmjd2b and Jmjd2c are necessary for self-renewal of ESCs and induced pluripotent stem cell generation. Genome-wide occupancy studies reveal that Jmjd2b unique, Jmjd2c unique, and Jmjd2b-Jmjd2c common target sites belong to functionally separable Core, Polycomb repressive complex (PRC), and Myc regulatory modules, respectively. Jmjd2b and Nanog act through an interconnected regulatory loop, whereas Jmjd2c assists PRC2 in transcriptional repression. Thus, two HDMs of the same subclass exhibit distinct and combinatorial functions in control of the ESC state. Such complexity of HDM function reveals an aspect of multilayered transcriptional control.
Project description:We used microarray to determine the changes in gene expression profile after KD of Jmjd2b and Jmjd2c compared to Anti-GFP KD from mES cells
Project description:Genome-wide occupancy of biotinylated Jmjd2b, Jmjd2c from mESCs, as well as occupancy of selected factors and histone marks from wild-type mESCs, Anti-GFP KD, Jmj2b KD and Jmjd2c KD mESCs genome
Project description:Transcription factors (TFs) bind specific sequences in promoter-proximal and distal DNA elements in order to regulate gene transcription. RNA is transcribed from both promoter-proximal and distal DNA elements, and some DNA-binding TFs have also been shown to bind RNA. These obsevations led us to postulate that RNA transcribed from regulatory elements contributes to stable TF occupancy at these regulatory elements. We show here that the ubiquitously expressed TF YY1 binds to both proximal and distal regulatory elements and to the RNA species associated with these elements near active genes in embryonic stem cells. Inhibition of transcription from these elements reduces YY1 occupancy. In contrast, tethering of RNA species near YY1 DNA binding sites enhances YY1 occupancy. We propose that RNA acts as trap to maintain certain TFs at active enhancer and promoter-proximal regulatory elements. Thus, transcriptional control generally involves a positive feedback loop, where YY1 and other TFs stimulate local transcription, and newly transcribed nascent RNA reinforces local TF occupancy. This model helps explain why TFs occupy only the small fraction of their consensus motifs in the mammalian genome where transcription is detected. CLIP-Seq for YY1 in mouse embryonic stem cells
Project description:By applying RNA-ISH and RNAseq to circulating tumor cells (CTCs), the study provides definitive evidence of epithelial to mesenchymal transition (EMT) across all histological types of breast cancer, identifying mediators such as FOXC1 and TGF-β signaling, and demonstrating dynamic treatment-associated changes in EMT within clusters of CTCs. Epithelial to mesenchymal transition (EMT) has been postulated to contribute to the migration and dissemination of cancer cells, but supporting histopathological evidence is limited. We used a microfluidic device to isolate circulating tumor cells (CTCs), combined with multiplex fluorescent RNA-in-situ hybridization (ISH) and RNA sequencing, to quantify and characterize EMT in breast cancer cells within the bloodstream. Whereas only rare (0.1-10%) cells in the primary tumor expressed both mesenchymal and epithelial markers, such biphenotypic as well as purely mesenchymal cells were enriched among CTCs, across all histological subtypes of breast cancer. In an index patient followed longitudinally, fluctuation in epithelial and mesenchymal states was observed as a function of initial response and subsequent resistance to therapy. Mesenchymal markers were predominant in clusters of tumor cells, many of which had adherent platelets. Finally, RNA sequencing of CTC clusters identified TGF-β and other EMT-related signatures, which were absent from more epithelial CTCs. FOXC1, a known regulator of EMT, was abundantly expressed in mesenchymal CTCs and was detectable within localized regions of the primary breast tumor. Together, these data support a role for EMT in the blood-borne dissemination of breast cancer and point to the dynamic nature of this cell fate change.