Gene Expression
Nebulas
Gene Expression NebulasSummary: The long noncoding RNA (lncRNA) XIST establishes X chromosome inactivation (XCI) in female cells in early development and thereafter is thought to be largely dispensable. Here we show XIST is continually required in adult human B cells to silence a subset of X-linked immune genes such as TLR7. XIST-dependent genes lack promoter DNA methylation and require continual XIST-dependent histone deacetylation. XIST RNA-directed proteomics and CRISPRi screen reveal distinctive somatic cell-specific XIST complexes, and identify TRIM28 that mediates Pol II pausing at promoters of X-linked genes in B cells. XIST dysregylation, reflected by escape of XIST-dependent genes, occurs in CD11c+ atypical memory B cells across single-cell transcriptome data in patients with female-biased autoimmunity and COVID-19 infection. XIST inactivation with TLR7 agonism suffices to promote isotype-switched atypical B cells. These results suggest cell-type-specific diversification of lncRNA-protein complexes increase lncRNA functionalities, and expand roles for XIST in sex-differences in biology and medicine
Overall Design: RNA-seq of dCas9-Krab+ GM B cells transduced with guide RNA and treated with DNMT and EZH2 epigenetic inhibitors (sgNT+DMSO, sgXIST+DMSO, sgNT+inhibitors, sgXIST+inhibitors), Trim28_KO GM B cells, Spen_deltaRRM GM B cells, sgNT and sgXIST cells that are overexpressed with anti-CRISPR protein ( sgNT+ACRIIA4, sgXIST+ACRIIA4), dCas9-Krab+ IMR90 cells transduced with guide RNA (IMR90_sgNT, IMR90_sgXIST)
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| Growth Protocol: | GM related cell lines were cultured in RPMI 1640 (Thermo Fisher Scientific) with 10% FBS (Thermo Fisher Scientific) and 1% Penicillin-Streptomycin (Gibco). IMR90 cell line were cultured in EMEM (ATCC) with 10% FBS and 1% Penicillin-Streptomycin. |
| Treatment Protocol: | To inhibit DNA methylation and H3K27me3, B cell line was treated with 0.3 uM DNMT inhibitor 5-azacytidine and 2 uM EZH2 inhibitor EPZ-6438 for 7 days |
| Extract Protocol: | For RNAseq, RNA was extracted in TRIzol using Quick-RNA Miniprep Kit following the manufacturer's protocol with on-column Dnase digestion (Zymo Research). |
| Library Construction Protocol: | For RNAseq, at least 100ng RNA was used to prepare the RNA-seq library using TruSeq® Stranded mRNA Library Prep Kit ( Cat# 20020594, Illumina) for each sample following the manufacturer's instruction. |
| Molecule Type: | Poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 4000 |
| Strand-Specific: | Specific |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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