MRNA-seq based gene expression profiling of differentiating murine brown preadipocytes (IBA)
ABSTRACT: mRNA-sequencing was used to profile temporal changes in gene expression during in vitro differentiation of IBA at five different time points: Day 0 (confluence), 2h (post induction), Day 1, Day 2 and Day 4 (mature brown adipocytes), performed in two biological replicates (with two technical replicates in the first biological replicate).
Brown adipocytes regulate energy expenditure via mitochondrial uncoupling, which makes them attractive therapeutic targets to tackle obesity. However, the regulatory mechanisms underlying brown adipogenesis are still poorly understood. To address this, we profiled the transcriptome and chromatin state during mouse brown fat cell differentiation, revealing extensive gene expression changes and chromatin remodeling, especially during the first day post-differentiation. To identify putatively causa ...[more]
Project description:ChIP sequencing was used to generate genome-wide maps of the histone mark H3K27ac during in vitro differentiation of a murine brown preadipocyte cell line (IBA) at five different time points, in two biological replicates: Day 0 (confluence), 2h (post induction), Day 1, Day 2 and Day 4 (mature brown adipocytes). Additionally, transcription factor (TF) localisation maps of the Nuclear Factor 1 (NFI) were generated using ChIP sequencing at two time points: Day 0 and Day 4.
Project description:Loss of function assays were performed in differentiating IBAs using short hairpin RNAs (shRNAs) against a set of candidate transcription factors: NFIA, DBP, HOXA4, ZFP467, SOX18, ELF3, YBX2 and PPARa. Gene expression profiles of the perturbed and non perturbed (controls including non transduced cells, cells transduced with scrambled and GFP constructs) transcriptomes were measured at Day 6 of differentiation using a novel multiplex, bulk mRNA-seq barcoding method which employs a 3' sequencing approach. This method allowed the measurement of the deviation of the perturbed transcriptome from the non-perturbed transcriptome.
Project description:Background: We here show that inhibitors of mitochondrial complex I promote physical activity, stress resistance as well as lifespan of Caenorhabditis elegans despite normal food uptake, i.e. in the absence of DR. Dietary restriction (DR) extends lifespan and promotes metabolic health in evolutionary distinct species. The RNA-seq data comprises 4 age groups (1, 5, 10 and 20 days after L4) and 2 different conditions (rotenone and normal feeding (DMSO)) 22 samples: mRNA profiles of 1-day, 5-day and 10-day old worms as triplicates for each, rotenone and solvent control treatment; mRNA profiles of 20-day old worms as duplicates for each, rotenone and solvent control treatment
Project description:We performed RNA sequencing on several clones of bovine mesenchymal stem cells from the same donor. Bulk mRNA sequencing in 17 different clones from the same donor and a heterogeneous population from the same donor.
Project description:Foxp3-expressing regulatory T (Treg) cells are essential regulators in the immune system; molecular mechanisms underlying Treg cell expansion and function are still not well understood. SUMOylation is an important post-translational modification characterized by covalent attachment of SUMO moieties to lysine within proteins. UBC9 is the only E2 conjugation enzyme involved in this process and loss of UBC9 completely impairs the SUMOylation pathway. Here we report that selective deletion of Ubc9 within the Treg cell lineage resulted in fatal early-onset autoimmunity as the Foxp3 mutant mice. Ubc9-deficient Treg cells exhibited severe defects in TCR-driven homeostatic proliferation, accompanied by impaired activation and compromised suppressor function. Importantly, TCR-enhanced SUMOylation of IRF4, a critical regulator of Treg cell function downstream of TCR signals, regulates its stability in Treg cells. Our data thus have demonstrated an essential role of SUMOylation in the expansion and function of Treg cells. RNA-seq library was generated using mRNA of CD4+ YFP+ Treg cells sorted from lymph nodes and spleen of Foxp3cre/wtUbc9fl/wt or Foxp3cre/wtUbc9fl/fl mice, each sample contained pooled Treg cells from 5~10 mice.
Project description:Renal epithelial cells are exposed to mechanical forces due to flow-induced shear stress within the nephrons. We applied RNA sequencing to get a comprehensive overview of fluid-shear regulated genes and pathways in the immortalized renal proximal tubular epithelial cell line. Cells were exposed to laminar fluid shear stress (1.9 dyn/cm2) in a cone-plate device and compared to static controls.
Project description:Pkd1-/- renal epithelial cells are exposed to mechanical forces due to flow-induced shear stress within the nephrons. We applied RNA sequencing to get a comprehensive overview of fluid-shear regulated genes and pathways in the immortalized Pkd1-/- renal proximal tubular epithelial cell line. Cells were exposed to laminar fluid shear stress (1.9 dyn/cm2) in a cone-plate device and compared to static controls.
Project description:We have found that thyroid hormones (THs), acting as soluble integrin αvβ3 ligands, activate growth-related signaling pathways in T-cell lymphomas (TCL). Specifically, TH-activated αvβ3 integrin signaling promotes TCL proliferation and angiogenesis, in part, via the up-regulation of VEGF. CUTLL1 cells were treated with T3- and T4-bound agarose or agarose alone for 24hrs. Total RNA was harvested from cells and used for expression profiling via RNA-seq.
Project description:C/EBPα induces transdifferentiation of B cells into macrophages at high efficiencies and enhances reprogramming into induced pluripotent stem cells (iPSCs) when co-expressed with Oct4, Sox2, Klf4 and Myc (OSKM). However, how C/EBPα accomplishes these effects is unclear. We now found that transient C/EBPα expression followed by OSKM activation induces a 100 fold increase in iPSC reprogramming efficiency, involving 95% of the cells. During this conversion pluripotency and epithelial-mesenchymal transition genes become dramatically up-regulated and 60% of the cells express Oct4 within 2 days. C/EBPα acts as a pathbreaker since it transiently makes the chromatin of pluripotency genes more accessible to DNase I. It also induces the expression of the dioxygenase Tet2 and promotes its translocation to the nucleus where it binds to regulatory regions of pluripotency genes that become demethylated following OSKM induction. In line with these findings, overexpression of Tet2 enhances OSKM‐induced B cell reprogramming. Since the enzyme is also required for efficient C/EBPα-induced immune cell conversion, our data suggest that Tet2 provides a mechanistic link between iPSC reprogramming and B cell transdifferentiation. The rapid iPS reprogramming approach described should help to fully elucidate the process and has potential clinical applications. Change in gene expression, comparing primary B-cells treated with estradiol for 18h to induce C/EBPa to untreated cells.
Project description:Enhancers play a central role in cell-type-specific gene expression and are marked by H3K4me1/2. Active enhancers are further marked by H3K27ac. However, the methyltransferases responsible for the deposition of H3K4me1/2 on enhancers remain elusive. Furthermore, the functions of these methyltransferases on enhancers and associated cell-type-specific gene expression are poorly understood. Here, we identify MLL4 (KMT2D) as a major H3K4 mono- and di-methyltransferase in mammalian cells. Using adipogenesis and myogenesis as model systems, we show that MLL4 exhibits cell-type- and differentiation-stage-specific genomic binding and is predominantly localized on enhancers. MLL4 co-localizes with lineage-determining transcription factors (TFs) on active enhancers during differentiation. Deletion of MLL4 dramatically decreases H3K4me1/2 and H3K27ac on enhancers and leads to severe defects in cell-type-specific gene expression and cell differentiation. Finally, we provide evidence that lineage-determining TFs recruit and require MLL4 to establish enhancers critical for cell-type-specific gene expression. Together, these results identify MLL4 as an H3K4 mono-/di-methyltransferase required for enhancer activation during cell differentiation. ChIP-Seq analyses of MLL4 binding profiles at D0 (day 0), D2 (day 2), and D7 (day 7) of adipogenesis in WT (MLL3-/-) and MLL4 KO (MLL3-/-;MLL4-/-) brown preadipocytes. D0 and D2 input DNA samples can be found in GSE50417.