CRISPR/Cas9 Screens Reveal Multiple Layers of B cell CD40 Regulation

Cell Reports
Chang JiangBenjamin E Gewurz

Abstract

CD40 has major roles in B cell development, activation, and germinal center responses. CD40 hypoactivity causes immunodeficiency whereas its overexpression causes autoimmunity and lymphomagenesis. To systematically identify B cell autonomous CD40 regulators, we use CRISPR/Cas9 genome-scale screens in Daudi B cells stimulated by multimeric CD40 ligand. These highlight known CD40 pathway components and reveal multiple additional mechanisms regulating CD40. The nuclear ubiquitin ligase FBXO11 supports CD40 expression by targeting repressors CTBP1 and BCL6. FBXO11 knockout decreases primary B cell CD40 abundance and impairs class-switch recombination, suggesting that frequent lymphoma monoallelic FBXO11 mutations may balance BCL6 increase with CD40 loss. At the mRNA level, CELF1 controls exon splicing critical for CD40 activity, while the N6-adenosine methyltransferase WTAP negatively regulates CD40 mRNA abundance. At the protein level, ESCRT negatively regulates activated CD40 levels while the negative feedback phosphatase DUSP10 limits downstream MAPK responses. These results serve as a resource for future studies and highlight potential therapeutic targets.

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Datasets Mentioned

BETA
GSE101666

Methods Mentioned

BETA
fluorescence-activated cell sorting
FACS
co-immunoprecipitation
PCR
immunoprecipitation
pull-down
RNA-immunoprecipitation
FCS
transfection
Knockout

Software Mentioned

Gene Set Enrichment Analysis ( GSEA
GSEAPreranked
Zeiss ZEN Lite (
GeneMANIA
STARSs
GSEA
ZEN Lite
STARs
GraphPad Prism7
SRAMP

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