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Cell Rep. 2019 Jul 30;28(5):1307-1322.e8. doi: 10.1016/j.celrep.2019.06.079.

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

Author information

1
Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, 181 Longwood Avenue, Boston, MA 02115, USA; Department of Microbiology, Harvard Medical School, Boston, MA 02115, USA.
2
Department of Pathology, Children's Hospital Boston, Harvard Medical School, Boston, MA 02115, USA.
3
Department of Biostatistics and Bioinformatics, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA.
4
Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, 181 Longwood Avenue, Boston, MA 02115, USA; Department of Microbiology, Harvard Medical School, Boston, MA 02115, USA; Graduate Program in Virology, Division of Medical Sciences, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
5
Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, 181 Longwood Avenue, Boston, MA 02115, USA; Department of Microbiology, Harvard Medical School, Boston, MA 02115, USA; Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China.
6
Department of Pathology, Children's Hospital Boston, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA.
7
Biosit, Université de Rennes 1, 35043 Rennes, France; Centre National de la Recherche Scientifique UMR 6290, Institut de Génétique et Développement de Rennes, 35043 Rennes, France.
8
Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, 181 Longwood Avenue, Boston, MA 02115, USA.
9
Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA.
10
Department of Pathology, Children's Hospital Boston, Harvard Medical School, Boston, MA 02115, USA; Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy.
11
Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital, 181 Longwood Avenue, Boston, MA 02115, USA; Department of Microbiology, Harvard Medical School, Boston, MA 02115, USA; Department of Biostatistics and Bioinformatics, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA; Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA. Electronic address: bgewurz@bwh.harvard.edu.

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.

KEYWORDS:

B cell activation; CRISPR screen; ESCRT; MAP kinase; N6-Methyladenosine; NF-kappaB; TNF receptor superfamily; germinal center; humoral immunity; immunodeficiency

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