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| Status |
Public on Aug 18, 2024 |
| Title |
Optimization of the Irf8 +32 kb enhancer disrupts dendritic cell lineage segregation [scRNA-seq] |
| Organism |
Mus musculus |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Autoactivation of lineage-determining transcription factors (TFs) mediates bistable expression to generate distinct cell phenotypes essential for complex body plans. Classical dendritic cells type 1 (cDC1) and type 2 (cDC2) provide non-redundant functions required for defense against distinct immune challenges. Interferon Regulatory Factor 8 (IRF8), the cDC1 lineage-determining TF, undergoes autoactivation in cDC1 progenitors to establish cDC1 identity, yet its expression is downregulated during cDC2 differentiation by an unknown mechanism. This study reveals that the Irf8 +32 kb enhancer, responsible for IRF8 autoactivation, has been tuned to possess low-affinity IRF8 binding sites. Incorporation of multiple high-affinity IRF8 binding sites into the Irf8 +32 kb enhancer induces erroneous IRF8 autoactivation in specified cDC2 progenitors, causing their redirection towards cDC1 and a novel hybrid DC subset with mixed lineage phenotypes. These developmental alterations critically impair both cDC1- and cDC2-dependent arms of immunity. Collectively, our findings underscore the significance of enhancer suboptimization in the developmental segregation of classical dendritic cells required for normal immune function.
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| Overall design |
BM and spleens were harvested from two WT and two Irf8 +32H/H mice. The BM samples were depleted of lin+ cells expressing CD3e, CD8b, CD11b, CD19, B220, ly-6G, TER-119, and NK1.1. Each sample was stained with fluorochrome-conjugated antibodies and labeled with a unique Antibody Capture TotalSeq B antibody (Biolegend) before sorting for lin- Flt3+ Kitint-lo cells. Two samples from each genotype were pooled and resuspended in PBS with 0.04% BSA at a final concentration of approximately 1,000 cells/mL. The spleen samples were enriched for CD11c-expressing cells, similarly stained with fluorochrome-conjugated antibodies and labeled with a unique Antibody Capture TotalSeq B antibody (Biolegend), before further sort-purification for CD11c+ cells. All four samples were pooled and resuspended in PBS with 0.04% BSA at approximately 1,000 cells/mL. These were loaded on a 10× Genomics Chromium Single Cell Controller at GTAC@MGI at Washington University School of Medicine. Library preparation was performed using the 10× Genomics Next GEM Single Cell 3’ Reagents Kit v3. Libraries were sequenced using an Illumina Novaseq 6000 sequencer, producing 150 bp paired-end reads. Sequencing targeted depths of 1B reads for the gene expression (GEX) library and 100M reads for the hashtag oligo (HTO) library, respectively. The Cell Ranger pipeline v8.0.0 was used for aligning reads to the mm10 reference genome, single cell counting, barcode processing, and sample demultiplexing. scRNA-seq downstream analysis was conducted using Seurat v5.0.3 in R. After removing unwanted cells, UMI counts in each cell were normalized using log transformation with a scale factor of 10,000. For each dataset, 2,000 highly variable genes were identified and used for PCA. For the BM dataset, cell cycle phase scores were calculated, and the difference between G2M and S phase scores was regressed out. Dimensional reduction and clustering were performed using the FindNeighbors, RunUMAP, and FindClusters functions, utilizing the top 30 PCs. The clustering resolution was set at 0.6 for the BM dataset and 0.5 for the spleen dataset.
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| Contributor(s) |
Ou F, Murphy KM |
| Citation(s) |
39375550 |
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| Submission date |
Jun 17, 2024 |
| Last update date |
Nov 18, 2024 |
| Contact name |
Feiya Ou |
| E-mail(s) |
feiya.ou@wustl.edu
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| Organization name |
Washington University in St. Louis
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| Department |
Pathology & Immunology
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| Lab |
Murphy Lab
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| Street address |
4939 Children's Place
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| City |
St. Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
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| Platforms (1) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
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| Samples (6)
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| Relations |
| BioProject |
PRJNA1124930 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE270060_HH_BM_per_biological_replicate_outs.tar.gz |
217.7 Mb |
(ftp)(http) |
TAR |
| GSE270060_Spleen_per_biological_replicate_outs.tar.gz |
116.2 Mb |
(ftp)(http) |
TAR |
| GSE270060_WT_BM_per_biological_replicate_outs.tar.gz |
217.1 Mb |
(ftp)(http) |
TAR |
| GSE270060_feature_README.txt |
419 b |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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