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SAFB associates with nascent RNAs and can promote gene expression in mouse embryonic stem cells [ChIP-seq]

ABSTRACT: SAFB has roles in gene repression, and we asked whether PRC2 was involved in this process

ORGANISM(S): Mus musculus

PROVIDER: GSE211905 | GEO | 2023/08/31

REPOSITORIES: GEO

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SAFB associates with nascent RNAs and can promote gene expression in mouse embryonic stem cells

Project description:SAFB associates with nascent RNAs and can promote gene expression in mouse embryonic stem cells

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SAFB associates with nascent RNAs and can promote gene expression in mouse embryonic stem cells

Project description:This SuperSeries is composed of the SubSeries listed below.

2023-08-31 | GSE211920 | GEO

SAFB associates with nascent RNAs and can promote gene expression in mouse embryonic stem cells [RNA-seq]

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SAFB associates with nascent RNAs and can promote gene expression in mouse embryonic stem cells [RIP-seq]

Project description:SAFB associates with nascent RNAs and can promote gene expression in mouse embryonic stem cells [RIP-seq]

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SAFB associates with nascent RNAs and can promote gene expression in mouse embryonic stem cells [RIP-seq]

Project description:RNA-IPs in WT and SAFB1/2 DKO mESCs.

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SAFB associates with nascent RNAs and can promote gene expression in mouse embryonic stem cells [RNA-seq]

Project description:We performed gene expression profiling analysis using data obtained from RIP-seq inputs of 7 different cell lines.

2023-08-31 | GSE211915 | GEO

Identification of SAFB and HnrnpU interactors

Project description:Scaffold Attachment Factor B (SAFB) is a conserved RNA Binding Protein (RBP) that is essential for early mammalian development. However, the RNAs that associate with SAFB in mouse embryonic stem cells have not been characterized. Here, we addressed this unknown using RNA-seq and SAFB RNA immunoprecipitation followed by RNA-seq (RIP-seq) in wild-type mouse embryonic stem cells (ESCs) and in ESCs in which SAFB and SAFB2 were knocked out. The transcript most enriched in SAFB association was the lncRNA Malat1, which contains a series of purine-rich motifs in its 5 end. Beyond Malat1, SAFB predominantly associated with introns of protein-coding genes also through purine-rich motifs. Knockout of SAFB/2 led to down- and upregulation of genes in multiple biological pathways. The nascent transcripts of many downregulated genes associated with high levels of SAFB in wild-type cells, implying that SAFB binding promotes the expression of these genes. Reintroduction of SAFB into double-knockout cells restored gene expression towards wild-type levels, an effect that was again observable at the level of nascent transcripts. Proteomic analyses indicate an enrichment of nuclear speckle-associated, SR proteins in FLAG-tagged SAFB immunoprecipitated samples. Comparison to immunoprecipitates made from FLAG-tagging of another nuclear-enriched RNA-binding protein called HNRNPU (also known as SAF-A) identified both similarities and differences. Perhaps most notably, we observed a stronger enrichment for speckle-associated proteins in SAFB immunoprecipitations and a strong enrichment for paraspeckle-associated proteins in HNRNPU immunoprecipitations. Our findings suggest that among other potential functions in mouse embryonic stem cells, SAFB directly promotes the expression of a subset of genes through its ability to bind purine regions in nascent RNA.

2023-07-20 | PXD038103 | Pride

A monoclonal antibody raised against human EZH2 cross-reacts with the RNA-binding protein SAFB

Project description:The Polycomb Repressive Complex 2 (PRC2) is a conserved enzyme that tri-methylates Lysine 27 on Histone 3 (H3K27me3) to promote gene silencing. PRC2 is remarkably responsive to the expression of certain long noncoding RNAs (lncRNAs). In the most notable example, PRC2 is recruited to the X-chromosome shortly after expression of the lncRNA Xist begins during X-chromosome inactivation. However, the mechanisms by which lncRNAs recruit PRC2 to chromatin are not yet clear. We report that a broadly used rabbit monoclonal antibody raised against human EZH2, a catalytic subunit of PRC2, cross-reacts with an RNA-binding protein called Scaffold Attachment Factor B (SAFB) in mouse embryonic stem cells (ESCs) under buffer conditions that are commonly used for chromatin immunoprecipitation (ChIP). Knockout of EZH2 in ESCs demonstrated that the antibody is specific for EZH2 by western blot (no cross-reactivity). Likewise, comparison to previously published datasets confirmed that the antibody recovers PRC2-bound sites by ChIP-Seq. However, RNA-IP from formaldehyde-crosslinked ESCs using ChIP wash conditions recovers distinct peaks of RNA association that co-localize with peaks of SAFB and whose enrichment disappears upon knockout of SAFB but not EZH2. IP and mass spectrometry-based proteomics in wild-type and EZH2 knockout ESCs confirm that the EZH2 antibody recovers SAFB in an EZH2-independent manner. Our data highlight the importance of orthogonal assays when studying interactions between chromatin-modifying enzymes and RNA.

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A monoclonal antibody raised against human EZH2 cross-reacts with the RNA-binding protein SAFB [ChIP-seq]

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