Project description:The spatial and temporal control of gene expression during development requires the concerted actions of sequence-specific transcriptional regulators and epigenetic chromatin modifiers, which are thought to function within precise nuclear compartments. However, how these activities are coordinated within the dynamic context of the nuclear environment is still largely unresolved. Here we show that transcriptional repression by the Msx1 homeoprotein coordinates recruitment of Polycomb to genomic targets with localization to the nuclear periphery. Using genome-wide ChIP-Seq analyses to identify genomic binding sites for Msx1, we find that repressed target genes are enriched at the nuclear periphery in myoblast cells. We further show that the interaction of Msx1 with the Polycomb repressive complex PRC2 is required for transcriptional repression and regulation of myoblast differentiation, and promotes increased tri-methylation of lysine 27 on histone H3 (H3K27me3) at Msx1 target genes. Furthermore, Msx1 genomic binding promotes the dynamic spatial redistribution of the H3K27me3 repressive mark to the nuclear periphery in developing embryos in vivo. Thus, our findings suggest a hitherto unappreciated spatial coordination of transcription factor binding, Polycomb recruitment, and subnuclear localization in regulation of developmentgene expression programs. In order to identify genes regulated by Msx1, we infected C2C12 myoblast cells with a retrovirus expressing a tamoxifen-regulated Msx1 protein, Msx1-ER (or with empty vector as a control), followed by induction with 0.2 nM of tamoxifen or vehicle (DMSO) for 6 hours. Regulated genes were identified as those that changed in expression upon tamoxifen induction of the Msx1-ER protein but did not change in the empty vector control. Experiments were performed in triplicate for each of the four experimental conditions (Msx1-ER + tamoxifen, Msx1-ER - tamoxifen, empty vector control + tamoxifen, empty vector control - tamoxifen), for a total of 12 independent array samples. This submission represents the transcriptome component of the study.
Project description:We newly identified skeletal muscle differentiation-associated miRNAs by comparing miRNA expression profile between C2C12 cell and Wnt4-overexpressing C2C12 cell. miR-487b, miR-3963 and miR-6412 are significantly down-regulated in differentiating C2C12 cells, and transfection of their mimics resulted in reduced expression of myogenic differentiation markers including Troponin T, myosin heavy chain fast and slow type. Single analysis for each condition (proliferating C2C12 cells, differentiating C2C12 cells, proliferating Wnt4-overexpressing C2C12 subline cells
Project description:In lymphocytes, NFATc1 is the most prominent NFAT transcription factor which play a crucial role in the fate and activity of peripheral T and B cells. NFATc1 is synthesized in two prominent isoforms, the inducible short isoform NFATc1/aA and the constitutively expressed long isoform NFATc1/C. Several lines of evidence suggested that both isoforms differ markedly in their function. It was speculated that NFATc1/aA supports the proliferation and survival of lymphocytes, whereas NFATc1/C should support apoptosis and anergy induction. To proof this hypothesis we established WEHI 231 B lymphoma cells that stably (over-) express either NFATc1/aA or NFATc1/C. In preliminary experiments we could should that WEHI cells overexpressing NFATc1/aA were protected against apoptosis induction, while cells overexpressing NFATc1/C should a higher apoptosis rate. Transcriptom analysis of WEHI-231 cells overexpressing either NFATc1/aA or NFATc1/C were performed, along with a control group of WEHI-231 cells overexpressing the E.coli enzyme BirA Ligase (which is also present in all target cell lines since for further molecular assays the NFATc1 proteins were expressed as chimeric protein containing C-terminal bio-tags. The experimental results obtained indicate that the both NFATc1 proteins, NFATc1/aA and NFATc1/C, differ tremendously in their transcriptional properties.
Project description:To comprehensively profile early neurodevelopmental alterations in individuals with ASD, we harnessed a time series approach to monitor patient-derived induced pluripotent stem cells (iPSCs) throughout the recapitulation of cortical development. This dataset consists of patient derived neurons that go through all consecutive developmental stages (NSC-derived neurons) as well as a comparative set of iPSC-iNs (neurons generated from the same patients that bypass early NSC-like stages using an Ngn2-transgene approach). For this, we first used fluorescence-activated cell sorting (FACS) to purify a homogeneous population of NSCs based on the expression of the cell-surface markers CD184+/CD271-/CD44-/CD24-/CD15+. To trace ASD and control neurons over time, we performed a series of retroviral lineage-tracing experiments to trace the progenies of dividing NSCs using a retroviral vector expressing a membrane-tagged enhanced green fluorescent protein (eGFP) (CAG::LckN-eGFP). As differentiating neurons express PSA-NCAM on the cell surface, we established a FACS-based protocol for purification of defined subpopulations of retrovirally labeled eGFP+/PSA-NCAM+ double-positive neurons after 2, 4, 7 and 14 days of differentiation. IPSCs were sorted based on the expression of SSEA-4 and TRA1-81 and maturing iPSC-iNs were collected at the indicated days after induction by sorting for eGFP (indicative for the Ngn2 transgene)- and PSA-NCAM-positive cells.
Project description:The RING domain protein Arkadia/RNF111 is a ubiquitin ligase in the transforming growth factor beta (TGFβ) pathway. We previously identified Arkadia as a small ubiquitin-like modifier (SUMO)-binding protein with clustered SUMO-interacting motifs (SIMs) that together form a SUMO-binding domain (SBD). However, precisely how SUMO interaction contributes to the function of Arkadia was not resolved. Through analytical molecular and cell biology, we found that the SIMs share redundant function with Arkadia's M domain, a region distinguishing Arkadia from its paralogs ARKL1/ARKL2 and the prototypical SUMO-targeted ubiquitin ligase (STUbL) RNF4. The SIMs and M domain together promote both Arkadia's colocalization with CBX4/Pc2, a component of Polycomb bodies, and the activation of a TGFbeta pathway transcription reporter. Transcriptome profiling through RNA sequencing showed that Arkadia can both promote and inhibit gene expression, indicating that Arkadia's activity in transcriptional control may depend on the epigenetic context, defined by Polycomb repressive complexes and DNA methylation [Sun, Liu, and Hunter (2014) Mol Cell Biol 34(16):2981-2995]. To determine the role of Arkadia in TGFβ signaling at the transcriptome level, the profiles of TGFβ-stimulated gene expression were examined in Ark+/+ (Ark_WT), Ark-/- (Ark_null), and Arkadia (WT and sim mutant)-reconstituted Ark-/- MEFs. RNA sequencing was carried out using poly(A)-enriched RNA samples from unstimulated cells and cells treated with TGFβ for 1h, 4h, or 16h as indicated. Two batches of sequencing data for a total of 16 independent samples were submitted.
Project description:The spatial and temporal control of gene expression during development requires the concerted actions of sequence-specific transcriptional regulators and epigenetic chromatin modifiers, which are thought to function within precise nuclear compartments. However, how these activities are coordinated within the dynamic context of the nuclear environment is still largely unresolved. Here we show that transcriptional repression by the Msx1 homeoprotein coordinates recruitment of Polycomb to genomic targets with localization to the nuclear periphery. We used genome-wide ChIP-Seq analyses to identify genomic binding sites for Msx1 in C2C12 murine myoblast cells. C2C12 myoblast cells were seeded at a density of 2.5 x 10^5 cells / 10-cm plate one day prior to infection, and infected with a retrovirus containing a Flag-Msx1 construct for two consecutive days. The resulting cells were then crosslinked with formaldehyde, and DNA was enriched by chromatin immunoprecipitation (ChIP) with an anti-Flag antibody and analyzed by Solexa sequencing. Enriched regions were identified using a Poissonian background model, and were further compared to an additional background of sequences from a sample of Flag-immunoprecipitated DNA from C2C12 cells infected with an empty vector to determine enrichment. ChIP was performed using an antibody against the Flag epitope (Sigma M2, F3165).
Project description:The effect of CD151 expression onto the kinome of Jurkat T cells was assessed using kinome analysis. CD151 was expressed in Jurkat T cells by retroviral transduction based on a pMSCV vector. Entrez Gene: 977 UniProtKB: P48509 Jurkat T cells were transduced with the MSCV-CD151 vector and successfully transduced cells were selected using puromycin. For the kinome array experiments 3 independent samples of Jurkat cells and three independent samples of J-CD151 cells were collected. To minimize unspecific background signals, lysates from Jurkat and J-CD151 T cells harvested at different growth stages, which were then pooled to provide one sample prior to loading on the Kinexus antibody microarrays.
Project description:Induction of the Arf tumor suppressor in response to hyperproliferative stress following oncogene activation activates a p53-dependent transcriptional program that limits the expansion of incipient cancer cells. Although Arf is not expressed in most tissues of fetal or young adult mice, it is physiologically expressed in the fetal yolk sac, a tissue derived from the extraembryonic endoderm. We demonstrate that expression of the mouse p19Arf protein marks late stages of extraembryonic endoderm differentiation in cultured embryoid bodies derived from either embryonic stem cells or induced pluripotent stem cells, and that Arf inactivation specifically delays the differentiation of the extraembryonic endoderm lineage, but not the formation of other germ cell lineages from pluripotent progenitors. Arf is required for the timely induction of extraembryonic endodermal cells in response to Ras/Erk signaling and, in turn, acts through p53 to ensure extraembryonic endoderm lineage development, but not maintenance. Remarkably, a significant temporal delay in extraembryonic endoderm differentiation detected during the maturation of Arf-null embryoid bodies is rescued by enforced expression of miR-205, a micro-RNA up-regulated by p19Arf and p53. Introduction of miR-205 into Arf-null embryonic stem cells rescues defective ExEn formation and elicits a program of gene expression that controls the migration and adhesion of embryonic endodermal cells. This occurs, at least in part, through atypical regulation of genes that control the epithelial-to-mesenchymal transition in cancer cells. Our findings suggest that noncanonical and canonical roles of Arf in extraembryonic endoderm development and tumor suppression, respectively, may be conceptually linked through mechanisms that govern cell-to-cell attachment and migration. 4 samples total two each at two time points in ESC development At each time point one sample was treted with miR-205 and the other with a GFP control vector