The transcription factors SOX9 and SOX5/SOX6 cooperate genome-wide through super-enhancers to drive chondrogenesis (RNA-Seq)
ABSTRACT: SOX9 is a transcriptional activator required for chondrogenesis, and SOX5 and SOX6 are closely related DNA-binding proteins that critically enhance its function. We used RNA-seq to charatierize a rat chondrosarcoma (RCS) cells as a faithful model for proliferating/early prehypertrophic growth plate chondrocytes and ChIP-seq to gain novel insights into the full spectrum of the target genes and modes of action of this chondrogenic trio. RNAs were isolated from three bioogical replicatse of rat chondrosarcoma (RCS) cells and rib samples for RNA-seq experiments.
Project description:SOX9 is a transcriptional activator required for chondrogenesis, and SOX5 and SOX6 are closely related DNA-binding proteins that critically enhance its function. We used RNA-seq to charatierize a rat chondrosarcoma (RCS) cells as a faithful model for proliferating/early prehypertrophic growth plate chondrocytes and ChIP-seq to gain novel insights into the full spectrum of the target genes and modes of action of this chondrogenic trio. ChIP-seq for SOX9, SOX6 and histone modifications were carried out using RCS cells
Project description:Tissue ChIP-seq was performed on transcription factor Sox5, Sox6 and Sox9 using limbs and tails from E13.5 mouse embryos to find their in vivo binding sites in the genome Overall design: Sox9 and Sox6 ChIP-seq was performed on wildtype mice whereas Sox5 ChIP-seq was done using transgenic wildtype mice.
Project description:Transgenic mice tagged with EGFP under the control of the endogenous Sox5, Sox6 and Sox9 promoter were used. E13.5 transgenic embryos were enriched for the skeletal elements and FACS was used to isolate the Sox5/6/9-expressing cells which were used for comparative expression profiling between wildtype and knockout. Overall design: Total RNA from sorted cells from Sox9 wildtype (3 biological replicates) and Sox9 chimeric knockout (2 biological replicates) embryos were used for comparative analysis. Each genotype had two technical replicates. Sox6 heterozygotes (4 biological replicates) and Sox5-Sox6 double null (2 biological replicates with 2 technical replicates) embryos at E13.5 were used for comparative analysis.
Project description:Sox9 acts together with Sox5 or Sox6 as a master regulator for chondrocyte differentiation; however, how these transcription factors functionally interact and collaborate to regulate chondrogenesis remains unclear. Here we show that the protein kinase MLTK plays an essential role in the onset of chondrogenesis through triggering the induction of Sox6 by Sox9. Knockdown of MLTK in Xenopus embryos results in drastic loss of craniofacial cartilages without defects in neural crest formation. We also find that Sox6 is specifically induced during craniofacial chondrogenesis and this induction is inhibited by MLTK knockdown. Remarkably, Sox6-knockdown embryos display essentially the same phenotype as the MLTK-knockdown embryos; the drastic loss of cartilages and the marked down-regulation of genes involved in chondrogenesis. Microarray analysis reveals a remarkable similarity between Sox6-depleted and MLTK-depleted embryos in their gene expression pattern. Moreover, we find that ectopic expression of MLTK can induce Sox6 expression in a Sox9-dependent manner. These results identify a novel, key regulator for chondrogenesis. We used microarrays to describe the genome-wide gene expression profiles of xMLTK-depleted and xSox6-depleted embryos. CoMO, xMLTK-MO, or xSox6-MO was injected into the animal pole of all blastomeres (10 ng/cell) at the four-cell stage. The heads of embryos were cut out and harvested at 72 hours after fertilization (corresponding to about stage 41). Total RNA was extracted using TRIsol reagent, treated with DNase (TURBO DNase, Ambion), and then purified using RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instruction. The quality of total RNA was assessed using the Agilent 2100 BioAnalyzer. Reverse transcription to synthesize first-strand cDNA, second-strand cDNA synthesis, in vitro transcription to synthesize Biotin-modified aRNA, aRNA purification and fragmentation of the labeled aRNA were performed using the GeneChip 3’ IVT Express Kit and hybridization to the Affymetrix GeneChip Xenopus laevis Genome 2.0 Array was performed using the GeneChip Hybridization Wash and Stain Kit according to the manufacturer’s instruction. Hybridized arrays were scanned using an Affymetrix GeneChip Scanner. Scanned Chip images were analyzed with GeneChip operating Software v.1.4 (GCOS) and GeneSpring GX 11.0.2. (Agilent technologies).
Project description:In order to understand how Sox6 coordinately regulates the transcription of multiple fiber type specific genes during muscle development, we have performed ChIP-seq analyses to identify Sox6 target genes as well as RNA polymerase II (Pol II) binding sites in mouse fetal myotubes. Examination of Sox6 and Pol II binding sites in mouse fetal primary myotubes
Project description:In order to understand how Sox6 coordinately regulates the transcription of multiple fiber type specific genes during muscle development, we have performed ChIP-seq analyses to identify Sox6 target genes as well as RNA polymerase II (Pol II) binding sites in mouse fetal myotubes. Overall design: Examination of Sox6 and Pol II binding sites in mouse fetal primary myotubes
Project description:The aim of the experiment is to identify target molecules of the SOX trio (SOX9, SOX5 and SOX6). hMSC were transfected with the adenoviruses carrying the SOX trio or the LacZ-control, and total RNA was isolated after 2 weeks of culture.
Project description:To gain an integrated view of the dynamic changes in gene expression during postnatal heart development at the organ level, time-series transcriptome analyses of the postnatal hearts of neonatal through adult mice (P1, P7, P14, P30, and P60) were performed using a newly developed bioinformatics pipeline. Additionally, the role of the transcription factor Sox6 in the postnatal maturation of cardiac muscle was investigated by differential transcriptome analyses between Sox6 knockout (KO) and control hearts. Overall design: Total RNA was extracted from cardiac ventricles of control (MCK-Cre) and Sox6 KO (Sox6loxp/loxp; MCK-Cre) mice at postnatal day 1 (P1), day 7 (P7), day 14 (P14), 1 month (P30), and 2 months (P60). Three biological replicates were prepared for each time point.
Project description:The developmental transcription factor SOX6 was found to regulate serum, as well as liver triglycerides in BL6 mice treated with SOX6 antisense gapmers compared to control gapmers. SOX6 was initially discovered as a gene over-expressed in mesenchymal stem cells derived from small for gestational age newborns. We characterized its metabolic function in BL6 mice using the above antisense knockdown approach. BL6 mice were treated with either SOX6 specific or control gapmer oligonucleotides for 48h. Knockdown efficiency was measured in liver and epididymal white adipose tissues and serum levels of triglycerides and cholesterol quantified.