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

65 Pages Posted: 14 Jan 2019 Sneak Peek Status: Review Complete

See all articles by Chang Jiang

Chang Jiang

Harvard University - Division of Infectious Diseases; Harvard University - Department of Microbiology and Immunobiology

Stephen J. Trudeau

Harvard University - Department of Microbiology and Immunobiology; Harvard University - Division of Infectious Diseases

Taek-Chin Cheong

Harvard University - Department of Pathology

Mingxiang Teng

H. Lee Moffitt Cancer Center and Research Institute - Department of Biostatistics and Bioinformatics

Liang Wei Wang

Harvard University - Division of Infectious Diseases; Harvard University - Department of Microbiology and Immunobiology; Harvard University - Graduate Program in Virology

Chiara Pighi

Harvard University - Department of Pathology

Carole Gautier-Courteille

Université de Rennes 1 - Biosit; University of Angers - Institute of Genetics and Development of Rennes

Yijie Ma

Harvard University - Division of Infectious Diseases; Harvard University - Department of Microbiology and Immunobiology

Sizun Jiang

Harvard University - Division of Infectious Diseases; Harvard University - Department of Microbiology and Immunobiology; Harvard University - Graduate Program in Virology

Chong Wang

Harvard University - Division of Infectious Diseases

Bo Zhao

Harvard University - Division of Infectious Diseases

Luc Paillard

Université de Rennes 1 - Biosit; University of Angers - Institute of Genetics and Development of Rennes

John G. Doench

Masssachusetts Institute of Technology and Harvard University - Broad Institute

Roberto Chiarle

Harvard University - Department of Pathology

Benjamin E. Gewurz

Division of Infectious Disease, Brigham & Women's Hospital; Harvard Medical School Department of Microbiology; Harvard University - Graduate Program in Virology; Masssachusetts Institute of Technology and Harvard University - Broad Institute

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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 performed CRISPR/Cas9 genome-wide screens. These found CD40 pathway components and revealed mechanisms strongly influencing CD40 responses. At the transcriptional level, the ubiquitin ligase FBXO11 supported CD40 abundance by targeting the repressor CTBP1. FBXO11 knockout decreased primary B-cell CD40 expression and impaired class-switch recombination. Frequent lymphoma monoallelic FBXO11 mutations may therefore balance BCL6 increase with CD40 loss. At the mRNA level, CELF1 controlled exon splicing critical for CD40 protein expression, while the N6-adenosine methyltransferase WTAP controlled CD40 message abundance. At the protein level, ESCRT negatively-regulated activated CD40 levels while the negative feedback phosphatase DUSP10 downmodulated MAPK. These results reveal multiple layers of CD40 regulation, serve as a resource for future studies of this key co-receptor, and highlight potential therapeutic targets. 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 performed CRISPR/Cas9 genome-wide screens. These found CD40 pathway components and revealed mechanisms strongly influencing CD40 responses. At the transcriptional level, the ubiquitin ligase FBXO11 supported CD40 abundance by targeting the repressor CTBP1. FBXO11 knockout decreased primary B-cell CD40 expression and impaired class-switch recombination. Frequent lymphoma monoallelic FBXO11 mutations may therefore balance BCL6 increase with CD40 loss. At the mRNA level, CELF1 controlled exon splicing critical for CD40 protein expression, while the N6-adenosine methyltransferase WTAP controlled CD40 message abundance. At the protein level, ESCRT negatively-regulated activated CD40 levels while the negative feedback phosphatase DUSP10 downmodulated MAPK. These results reveal multiple layers of CD40 regulation, serve as a resource for future studies of this key co-receptor, and highlight potential therapeutic targets.

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

Suggested Citation

Jiang, Chang and Trudeau, Stephen J. and Cheong, Taek-Chin and Teng, Mingxiang and Wang, Liang Wei and Pighi, Chiara and Gautier-Courteille, Carole and Ma, Yijie and Jiang, Sizun and Wang, Chong and Zhao, Bo and Paillard, Luc and Doench, John G. and Chiarle, Roberto and Gewurz, Benjamin E., CRISPR/Cas9 Screens Reveal Multiple Layers of B Cell CD40 Regulation (January 12, 2019). Available at SSRN: https://ssrn.com/abstract=3314564 or http://dx.doi.org/10.2139/ssrn.3314564
This is a paper under consideration at Cell Press and has not been peer-reviewed.

Chang Jiang

Harvard University - Division of Infectious Diseases

181 Longwood Avenue
Boston, MA 02115
United States

Harvard University - Department of Microbiology and Immunobiology

77 Ave Louis Pasteur
Boston, MA 02115
United States

Stephen J. Trudeau

Harvard University - Department of Microbiology and Immunobiology

77 Ave Louis Pasteur
Boston, MA 02115
United States

Harvard University - Division of Infectious Diseases

181 Longwood Avenue
Boston, MA 02115
United States

Taek-Chin Cheong

Harvard University - Department of Pathology

Boston, MA 02115
United States

Mingxiang Teng

H. Lee Moffitt Cancer Center and Research Institute - Department of Biostatistics and Bioinformatics

Tampa, FL 33612
United States

Liang Wei Wang

Harvard University - Division of Infectious Diseases

181 Longwood Avenue
Boston, MA 02115
United States

Harvard University - Department of Microbiology and Immunobiology

77 Ave Louis Pasteur
Boston, MA 02115
United States

Harvard University - Graduate Program in Virology

77 Avenue Louis Pasteur
Boston, MA 02115
United States

Chiara Pighi

Harvard University - Department of Pathology

Boston, MA 02115
United States

Carole Gautier-Courteille

Université de Rennes 1 - Biosit

Rennes, 35043
France

University of Angers - Institute of Genetics and Development of Rennes

Rennes, 35043
France

Yijie Ma

Harvard University - Division of Infectious Diseases

181 Longwood Avenue
Boston, MA 02115
United States

Harvard University - Department of Microbiology and Immunobiology

77 Ave Louis Pasteur
Boston, MA 02115
United States

Sizun Jiang

Harvard University - Division of Infectious Diseases

181 Longwood Avenue
Boston, MA 02115
United States

Harvard University - Department of Microbiology and Immunobiology

77 Ave Louis Pasteur
Boston, MA 02115
United States

Harvard University - Graduate Program in Virology

77 Avenue Louis Pasteur
Boston, MA 02115
United States

Chong Wang

Harvard University - Division of Infectious Diseases

181 Longwood Avenue
Boston, MA 02115
United States

Bo Zhao

Harvard University - Division of Infectious Diseases

181 Longwood Avenue
Boston, MA 02115
United States

Luc Paillard

Université de Rennes 1 - Biosit

Rennes, 35043
France

University of Angers - Institute of Genetics and Development of Rennes

Rennes, 35043
France

John G. Doench

Masssachusetts Institute of Technology and Harvard University - Broad Institute

415 Main Street
Cambridge, MA 02142
United States

Roberto Chiarle

Harvard University - Department of Pathology

Boston, MA 02115
United States

Benjamin E. Gewurz (Contact Author)

Division of Infectious Disease, Brigham & Women's Hospital ( email )

181 Longwood Avenue
Boston, MA 02115
United States

Harvard Medical School Department of Microbiology ( email )

77 Ave Louis Pasteur
Boston, MA 02115
United States

Harvard University - Graduate Program in Virology ( email )

77 Avenue Louis Pasteur
Boston, MA 02115
United States

Masssachusetts Institute of Technology and Harvard University - Broad Institute ( email )

415 Main Street
Cambridge, MA 02142
United States

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