Whole transcriptome profiling of the steady state and Hh-treated (8 hrs) Alpk1+/- (heterozygous) and Alpk1-/- (knockout) BMDMs using RNA-Sequencing approach
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ABSTRACT:
PROVIDER: PRJEB27929 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR2713070_1.fastq.gz | Fastqsanger.gz | |||
| ERR2713070_2.fastq.gz | Fastqsanger.gz | |||
| ERR2713071_1.fastq.gz | Fastqsanger.gz | |||
| ERR2713071_2.fastq.gz | Fastqsanger.gz | |||
| ERR2713072_1.fastq.gz | Fastqsanger.gz |
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Project description:Alpk1-deficient mice demonstrate exacerbated colitis and increased IL-12/Th1 response upon challenge with an intestinal pathobiont, Helicobacter hepaticus (Hh). Hematopoietic compartment is driving the pathogenic phenotype in this animal model, and Alpk1 is highly expressed in myeloid cells (macrophages and dendritic cells). Alpk1 deficiency has a recessive phenotype, since Alpk1+/- (heterozygous) mice show the same phenotype as the wild type mice. Mouse bone-marrow derived macrophages (BMDMs) show elevated IL-12 production in Alpk1-/- mice in response to stimulation with Hh. Since the molecular mechanism of how Alpk1 deficiency affects macrophage response to Hh is unknown, we aimed to characterise global changes in gene expression in Alpk1+/- vs Alpk1-/- bone-marrow differentiated cells (BMDMs). Cells were isolated from bone marrow of Alpk1+/- and Alpk1-/- (mixture of bone marrows from three mice per genotype) and plated in BMDM differentiation medium (RPMI, 10% FCS, penicillin and streptomycin, 50 micro beta-mercapthoethanol, 20 ng/ml recombinant mouse GM-CSF(Peprotech)), 7 million cells in per 10 sm uncoated TC dish in 10 ml of medium for eight days, extra 10 ml of medium was added to plates at day 4, before collection and replating in 96-well plates, 150 thousand cells/200 microliters of differentiation medium per well in technical triplicates per genotype/stimulation condition (R1-R3 labels of the samples). The following day BMDMs were stimulated with MOI of 10 of Hh and 10ng/ml of mouse IFNg (Peprotech) (Alpk1+/- BMDMs – het _Hh_8h vs Alpk1-/- BMDMs – alpk1_Hh_8h) or IFNg only (het _nonstim_8h vs alpk1_nonstim_8h) before lysis for RNA extraction using Quick-RNA kit from Zymo Research. Purified RNA was submitted to the Welcome Trust Centre for Human Genetics (Oxford) for RNA-Sequencing
2018-07-30 | E-MTAB-7076 | biostudies-arrayexpress
Project description:Whole transcriptome profiling of the steady state and Hh-treated (8 hrs) Alpk1+/- (heterozygous) and Alpk1-/- (knockout) BMDMs using RNA-Sequencing approach
2018-09-17 | E-MTAB-7076 | ExpressionAtlas
Project description:Bone marrow was harvested from Rosa26CreER; Stk40+/+ (WT; n = 3) and Rosa26CreER; Stk40loxp/loxp (Stk40 KO; n = 3) mice and differentiated for 6 days in the presence of 100 nM 4-OHT to generate WT and Stk40 KO bone-marrow derived macrophages (BMDMs). 2. On day 7 following differentiation BMDMs were treated with 100 ng x ml-1 LPS and harvested at 0 hrs, 6 hrs, 16 hrs, and 32 hrs following LPS exposure. 3. The cells were snap-frozen at the time of harvest. RNA was extracted using the Qiagen RNeasy mini kit as per manufacturer’s protocol including the on-column DNase digestion. Groups: There are cells from 3 mice x 2 genotypes x 4 time points G1: WT 0 hr LPS G2: WT 6 hr LPS G3: WT 16 hr LPS G4: WT 32 hr LPS G5: Stk40 KO 0 hr LPS G6: Stk40 KO 6 hr LPS G7: Stk40 KO 16 hr LPS G8: Stk40 KO 32 hr LPS
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Project description:Paracrine Hedgehog (Hh) signaling regulates growth and patterning in many Drosophila organs. We mapped chromatin binding sites for Cubitus interruptus (Ci), the transcription factor that mediates outputs of Hh signal transduction, and we analyzed transcription profiles of control and mutant embryos to identify genes that are regulated by Hh. Putative targets we identified include several Hh pathway components, most previously identified targets, and many targets that are novel. Analysis of expression patterns of pathway components and target genes gave evidence of autocrine Hh signaling in the optic primordium of the embryo. And, every Hh target we analyzed that is not a pathway component appeared to be regulated by Hh in a tissue-specific manner. We present evidence that Hh-dependent tissue specificity is dependent upon transcription factors that are Hh-independent, suggesting that “pre-patterns” of transcription factors partner with Ci to make Hh-dependent gene expression position-specific.
2010-11-03 | GSE24028 | GEO
Project description:Paracrine Hedgehog (Hh) signaling regulates growth and patterning in many Drosophila organs. We mapped chromatin binding sites for Cubitus interruptus (Ci), the transcription factor that mediates outputs of Hh signal transduction, and we analyzed transcription profiles of control and mutant embryos to identify genes that are regulated by Hh. Putative targets we identified include several Hh pathway components, most previously identified targets, and many targets that are novel. Analysis of expression patterns of pathway components and target genes gave evidence of autocrine Hh signaling in the optic primordium of the embryo. And, every Hh target we analyzed that is not a pathway component appeared to be regulated by Hh in a tissue-specific manner. We present evidence that Hh-dependent tissue specificity is dependent upon transcription factors that are Hh-independent, suggesting that “pre-patterns” of transcription factors partner with Ci to make Hh-dependent gene expression position-specific. Analysis of the expression profiles of loss of function mutantations in core components of the Hh signaling pathway. A total of 14 samples were analysed consisting of comparisons of hh-, ci-, smo-, ptc-, and Cim1-m4 (Activator) mis-expression embryos compared to wt sibling embryos.
2010-11-03 | E-GEOD-24028 | biostudies-arrayexpress
Project description:Reticuloendothelial macrophages engulf ~0.2 trillion senescent erythrocytes daily in a process called erythrophagocytosis (EP). This critical mechanism preserves systemic heme-iron homeostasis by regulating red blood cell (RBC) catabolism and iron recycling. Although extensive work has demonstrated the various effects on macrophage metabolic reprogramming by stimulation with proinflammatory cytokines, little is known about the impact of EP on the macrophage metabolome and proteome. Thus, we performed mass spectrometry-based metabolomics and proteomics analyses of bone marrow-derived macrophages (BMDMs) before and after EP of IgG-coated RBCs. Further, metabolomics was performed on BMDMs incubated with free IgG to ensure that changes to macrophage metabolism were due to opsonized RBCs and not to free IgG binding. Uniformly labeled tracing experiments were conducted on BMDMs in the presence and absence of IgG-coated RBCs to assess the flux of glucose through the pentose phosphate pathway (PPP). In this study, we demonstrate that EP significantly alters amino acid and fatty acid metabolism, the Krebs cycle, OXPHOS, and arachidonate-linoleate metabolism. Increases in levels of amino acids, lipids and oxylipins, heme products, and RBC-derived proteins are noted in BMDMs following EP. Tracing experiments with U-13C6 glucose indicated a slower flux through glycolysis and enhanced PPP activation. Notably, we show that it is fueled by glucose derived from the macrophages themselves or from the extracellular media prior to EP, but not from opsonized RBCs. The PPP-derived NADPH can then fuel the oxidative burst, leading to the generation of reactive oxygen species necessary to promote digestion of phagocytosed RBC proteins via radical attack. Results were confirmed by redox proteomics experiments, demonstrating the oxidation of Cys152 and Cys94 of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and hemoglobin-β, respectively. Significant increases in early Krebs cycle and C5-branched dibasic acid metabolites (α-ketoglutarate and 2-hydroxyglutarate, respectively) indicate that EP promotes the dysregulation of mitochondrial metabolism. Lastly, EP stimulated aminolevulinic acid (ALA) synthase and arginase activity as indicated by significant accumulations of ALA and ornithine after IgG-mediated RBC ingestion. Importantly, EP-mediated metabolic reprogramming of BMDMs does not occur following exposure to IgG alone. In conclusion, we show that EP reprograms macrophage metabolism and modifies macrophage polarization.
2020-05-27 | PXD017788 | Pride
Project description:Purpose: Most Hedgehog responsive gene expression is mediated through GLI de-repression. Additionally GLI -repression is proposed to play roles in limb pre-patterning before HH pathway activation. This study evaluates if GLI repression is established prior to HH pathway activation. Methods: To determine if GLI-repression is established prior to pathway activation, we used genomic approaches to study GLI-mediated repression using the mouse developing limb as a model. We identified pre-HH (E9.25, 21-23S) and post-HH (E10.5, 32-25S) GLI3 binding regions using CUT&RUN for endogenous FLAG-tagged GLI3 proteins. Using a combination of ChIP-seq, CUT&RUN, CUT&Tag, ATAC-seq and RNA-seq, we tested whether loss of Gli3 prior to HH signaling was able to de-repress genes and enhancers, as it does after HH signaling. Results: Prior to HH signaling, GLI3 binds to poised, accessible regions with histone deacetylase (HDAC) proteins, similar to post-HH signaling. Despite GLI3 binding to most regions as it does in the post-HH limb, loss of Gli3 is unable to prematurely active target genes or enhancers. Furthermore, we find that GLI3-dependent chromatin compaction does not occur until roughly 10 hours after HH signaling would have normally been induced. Collectively, these results support that GLI repressor proteins are inert prior to HH pathway activation.
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