Transcriptional profiling of IRF8 KO B cell differentiation
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE208254 | GEO | 2023/06/20
REPOSITORIES: GEO
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Project description:Primary murine B cells were stimulated with IL2, IL5, LPS to induce B cell differentiation for 24 hours. RNA was extracted from control and differentiated B cell to compare gene expression. Murine B cells (CD43-) were purfied from 5 weeks old mice splenocytes. And purified B cells were stimulated with IL2, IL5, LPS for 24 hours.
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Project description:Upon antigen recognition B cells undertake a bifurcated response in which some cells rapidly differentiate into plasmablasts while others undergo affinity maturation in germinal centers (GC). We uncover a double negative feedback loop between interferon regulatory factors IRF4 and IRF8, which regulates the initial bifurcation of activated B cells as well as the GC response. IRF8 dampens BCR signaling, facilitates antigen specific interaction with helper T cells, and promotes selection of high affinity clones while antagonizing IRF4 driven plasmablast differentiation. Genomic analysis reveals concentration dependent action of IRF4 and IRF8 in regulating distinctive gene expression programs. Stochastic modeling suggests that the double negative feedback is sufficient to initiate bifurcating B cell developmental trajectories. Naïve B cells were isolated from Irf8+/+ (wild type, WT) mice spleen and activated in vitro with 10μg/ml LPS (Sigma). CD138hi IgMhi (IRF4hi*) cells and CD138lo IgMlo (IRF8hi*) cells were sorted by flow cytometry at 72 hours. Total RNA was prepared by using Rneasy Mini kit (Qiagen) and sequenced with Illumina HiSeq2500. Alignment was performed with Taphat2, and transcript abundance quantification using Cuffdiff function from Cufflinks. GC B cells were sorted from CD19cre/+ Irf8+/+ (Ctrl) or CD19cre/+ Irf8flox/flox (Irf8 cKO) mice on dpi 13 post NP-KLH immunization. Total RNA was prepared by using Rneasy Mini kit (Qiagen) and amplified with with Ovation RNA-Seq System v2 (NuGEN). The data were analyzed by Wardrobe. More details are provided in the manuscript.
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Project description:Primary murine B cells were stimulated with IL2, IL5, LPS to induce B cell differentiation for 24 hours. RNA was extracted from control and differentiated B cell to compare gene expression.
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Project description:Upon antigen recognition B cells undertake a bifurcated response in which some cells rapidly differentiate into plasmablasts while others undergo affinity maturation in germinal centers (GC). We uncover a double negative feedback loop between interferon regulatory factors IRF4 and IRF8, which regulates the initial bifurcation of activated B cells as well as the GC response. IRF8 dampens BCR signaling, facilitates antigen specific interaction with helper T cells, and promotes selection of high affinity clones while antagonizing IRF4 driven plasmablast differentiation. Genomic analysis reveals concentration dependent action of IRF4 and IRF8 in regulating distinctive gene expression programs. Stochastic modeling suggests that the double negative feedback is sufficient to initiate bifurcating B cell developmental trajectories. Naïve B cells were isolated from wild type (WT) mice spleen and activated in vitro with 10μg/ml LPS (Sigma). ChIP was performed by using anti-IRF4, -IRF8 antibodies (Santa Cruz Biotech). For massively parallel sequencing, 10-20 μg of chromatin fragments from indicated samples were immunoprecipitated by using anti-IRF-4 and anti-IRF8 antibodies, and DNA libraries were prepared with Illumina Kit. DNA was sequenced by using the Illumina HiSeq2500. Reads were aligned to the mouse genome (mm9) by using Taphat2 and peak calling were performed by homer 2. GC B cells were sorted from WT mice on dpi 13 post NP-KLH immunization. Cells were flash frozen immediately and process by Active Motif for IRF8 ChIP-Seq. Reads were aligned to mm9 by using BAM and peak calling were performed by using MACS. More details are provided in the manuscript.
2015-08-25 | E-GEOD-70710 | biostudies-arrayexpress
Project description:The transcription factor (TF) interferon regulatory factor 8 (IRF8) controls both developmental and inflammatory stimulus-inducible genes in macrophages, but the mechanisms underlying these two different functions are largely unknown. One possibility is that these different roles are linked to the ability of IRF8 to bind alternative DNA sequences. We found that IRF8 is recruited to distinct sets of DNA consensus sequences before and after lipopolysaccharide (LPS) stimulation. In resting cells, IRF8 was mainly bound to composite sites together with the master regulator of myeloid development PU.1. Basal IRF8M-bM-^@M-^SPU.1 binding maintained the expression of a broad panel of genes essential for macrophage functions (such as microbial recognition and response to purines) and contributed to basal expression of many LPS-inducible genes. After LPS stimulation, increased expression of IRF8, other IRFs, and AP-1 family TFs enabled IRF8 binding to thousands of additional regions containing low-affinity multimerized IRF sites and composite IRFM-bM-^@M-^SAP-1 sites, which were not premarked by PU.1 and did not contribute to the basal IRF8 cistrome. While constitutively expressed IRF8-dependent genes contained only sites mediating basal IRF8/PU.1 recruitment, inducible IRF8-dependent genes contained variable combinations of constitutive and inducible sites. Overall, these data show at the genome scale how the same TF can be linked to constitutive and inducible gene regulation via distinct combinations of alternative DNA-binding sites. Chromatin immuno-precipitations of transcription factors IRF8, IRF1, PU.1, STAT1, STAT2 and of H3 lysine 27 acetylated followed by multiparallel sequencing, performed in bone marrow-derived macrophages from wild type (WT) and BXH2/TyJ mice. Cells were treated with lipopolysaccharide (LPS) for 2 or 4 hours, or interferon b (IFNb) for 30 or 60 minutes, 2 or 4 hours, or left unstimulated.
2015-01-30 | E-GEOD-56121 | biostudies-arrayexpress
Project description:IThe transcription factor (TF) interferon regulatory factor 8 (IRF8) controls both developmental and inflammatory stimulus-inducible genes in macrophages, but the mechanisms underlying these two different functions are largely unknown. One possibility is that these different roles are linked to the ability of IRF8 to bind alternative DNA sequences. We found that IRF8 is recruited to distinct sets of DNA consensus sequences before and after lipopolysaccharide (LPS) stimulation. In resting cells, IRF8 was mainly bound to composite sites together with the master regulator of myeloid development PU.1. Basal IRF8M-bM-^@M-^SPU.1 binding maintained the expression of a broad panel of genes essential for macrophage functions (such as microbial recognition and response to purines) and contributed to basal expression of many LPS-inducible genes. After LPS stimulation, increased expression of IRF8, other IRFs, and AP-1 family TFs enabled IRF8 binding to thousands of additional regions containing low-affinity multimerized IRF sites and composite IRFM-bM-^@M-^SAP-1 sites, which were not premarked by PU.1 and did not contribute to the basal IRF8 cistrome. While constitutively expressed IRF8-dependent genes contained only sites mediating basal IRF8/PU.1 recruitment, inducible IRF8-dependent genes contained variable combinations of constitutive and inducible sites. Overall, these data show at the genome scale how the same TF can be linked to constitutive and inducible gene regulation via distinct combinations of alternative DNA-binding sites. Bone marrow cells isolated from C57BL/6 or BXH2/TyJ mice were plated in 10 cm plates in 5ml of BM-medium (high glucose DMEM supplemented with 20% low endotoxin fetal bovine serum, 30% L929-conditioned medium, 1% glutamine, 1%, Pen/Strep, 0.5% Sodium Pyruvate, 0.1% M-NM-2-mercaptoethanol). Cultures were fed with 2.5 ml of fresh medium every two days. Macrophages were subjected to different treatment (see individual samples for details). Total RNA was extracted from 5x106 cells using RNAeasy kit (Qiagen). Libraries were prepared from 1-2 M-BM-5g of RNA, after oligo-dT selection, using the TruSeq RNA sample preparation kit (Illumina).
2015-01-30 | E-GEOD-56122 | biostudies-arrayexpress
Project description:Upon antigen recognition B cells undertake a bifurcated response in which some cells rapidly differentiate into plasmablasts while others undergo affinity maturation in germinal centers (GC). We uncover a double negative feedback loop between interferon regulatory factors IRF4 and IRF8, which regulates the initial bifurcation of activated B cells as well as the GC response. IRF8 dampens BCR signaling, facilitates antigen specific interaction with helper T cells, and promotes selection of high affinity clones while antagonizing IRF4 driven plasmablast differentiation. Genomic analysis reveals concentration dependent action of IRF4 and IRF8 in regulating distinctive gene expression programs. Stochastic modeling suggests that the double negative feedback is sufficient to initiate bifurcating B cell developmental trajectories.
2015-08-25 | GSE70711 | GEO
Project description:Upon antigen recognition B cells undertake a bifurcated response in which some cells rapidly differentiate into plasmablasts while others undergo affinity maturation in germinal centers (GC). We uncover a double negative feedback loop between interferon regulatory factors IRF4 and IRF8, which regulates the initial bifurcation of activated B cells as well as the GC response. IRF8 dampens BCR signaling, facilitates antigen specific interaction with helper T cells, and promotes selection of high affinity clones while antagonizing IRF4 driven plasmablast differentiation. Genomic analysis reveals concentration dependent action of IRF4 and IRF8 in regulating distinctive gene expression programs. Stochastic modeling suggests that the double negative feedback is sufficient to initiate bifurcating B cell developmental trajectories.
2015-08-25 | GSE70710 | GEO
Project description:IThe transcription factor (TF) interferon regulatory factor 8 (IRF8) controls both developmental and inflammatory stimulus-inducible genes in macrophages, but the mechanisms underlying these two different functions are largely unknown. One possibility is that these different roles are linked to the ability of IRF8 to bind alternative DNA sequences. We found that IRF8 is recruited to distinct sets of DNA consensus sequences before and after lipopolysaccharide (LPS) stimulation. In resting cells, IRF8 was mainly bound to composite sites together with the master regulator of myeloid development PU.1. Basal IRF8–PU.1 binding maintained the expression of a broad panel of genes essential for macrophage functions (such as microbial recognition and response to purines) and contributed to basal expression of many LPS-inducible genes. After LPS stimulation, increased expression of IRF8, other IRFs, and AP-1 family TFs enabled IRF8 binding to thousands of additional regions containing low-affinity multimerized IRF sites and composite IRF–AP-1 sites, which were not premarked by PU.1 and did not contribute to the basal IRF8 cistrome. While constitutively expressed IRF8-dependent genes contained only sites mediating basal IRF8/PU.1 recruitment, inducible IRF8-dependent genes contained variable combinations of constitutive and inducible sites. Overall, these data show at the genome scale how the same TF can be linked to constitutive and inducible gene regulation via distinct combinations of alternative DNA-binding sites.
2015-01-30 | GSE56122 | GEO