PRJNA691303: The role of XIST in X-inactivation maintenance in B cells (RNA-seq)
概要: 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
项目整体设计: 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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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. |
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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 |
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For RNAseq, RNA was extracted in TRIzol using Quick-RNA Miniprep Kit following the manufacturer's protocol with on-column Dnase digestion (Zymo Research). |
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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. |
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Poly(A)+ RNA |
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PAIRED |
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Forward |
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ILLUMINA |
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Illumina HiSeq 4000 |
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Specific |
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B cell-specific XIST complex enforces X-inactivation and restrains atypical B cells.
Cell . 2021-03-17 [PMID:
33735607]
