CRISPR/Cas9 Screens Reveal Multiple Layers of B cell CD40 Regulation
Chang Jiang,
Stephen J. Trudeau,
Taek-Chin Cheong,
Rui Guo,
Mingxiang Teng,
Liang Wei Wang,
Zhonghao Wang,
Chiara Pighi,
Carole Gautier-Courteille,
Yijie Ma,
Sizun Jiang,
Chong Wang,
Bo Zhao,
Luc Paillard,
John G. Doench,
Roberto Chiarle,
Benjamin E. Gewurz
Affiliations
Chang Jiang
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
Stephen J. Trudeau
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
Taek-Chin Cheong
Department of Pathology, Children’s Hospital Boston, Harvard Medical School, Boston, MA 02115, USA
Rui Guo
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
Mingxiang Teng
Department of Biostatistics and Bioinformatics, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA
Liang Wei Wang
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
Zhonghao Wang
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
Chiara Pighi
Department of Pathology, Children’s Hospital Boston, Harvard Medical School, Boston, MA 02115, USA; Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
Carole Gautier-Courteille
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
Yijie Ma
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
Sizun Jiang
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
Chong Wang
Division of Infectious Diseases, Department of Medicine, Brigham and Women’s Hospital, 181 Longwood Avenue, Boston, MA 02115, USA
Bo Zhao
Division of Infectious Diseases, Department of Medicine, Brigham and Women’s Hospital, 181 Longwood Avenue, Boston, MA 02115, USA
Luc Paillard
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
John G. Doench
Broad Institute of Harvard and MIT, Cambridge, MA 02142, USA
Roberto Chiarle
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
Benjamin E. Gewurz
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; Corresponding author
Summary: 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. : CD40 is critical for B cell development, germinal center formation, somatic hypermutation, and class-switch recombination. Increased CD40 abundance is associated with autoimmunity and cancer, whereas CD40 hypoactivity causes immunodeficiency. Jiang et al. performed a genome-wide CRISPR/Cas9 screen to reveal key B cell factors that control CD40 abundance and that regulate CD40 responses. Keywords: B cell activation, NF-kappaB, MAP kinase, CRISPR screen, TNF receptor superfamily, humoral immunity, immunodeficiency, N6-Methyladenosine, ESCRT, germinal center
