APE2 Promotes AID-Dependent Somatic Hypermutation in Primary B Cell Cultures That Is Suppressed by APE1

Area of Science:

• Immunology
• Molecular Biology
• Genetics

Background:

• Somatic hypermutation (SHM) is essential for antibody diversification in germinal center (GC) B cells, involving DNA repair of lesions induced by activation-induced cytidine deaminase.
• GC B cells exhibit low levels of apurinic/apyrimidinic endonuclease 1 (APE1) and high levels of its homolog APE2, with prior studies suggesting APE2's role in promoting SHM.

Purpose of the Study:

• To investigate the distinct roles of APE1 and APE2 in regulating SHM and class-switch recombination (CSR).
• To test the hypothesis that APE2 promotes SHM while APE1 suppresses it.
• To elucidate how APE1/APE2 expression dynamics during B cell activation impact DNA repair pathways.

Main Methods:

• Analysis of APE1 and APE2 expression levels in primary murine spleen B cells during activation and in response to stimulation.
• Genetic manipulation of APE1 (apex1+/-) and APE2 overexpression to mimic GC B cell expression patterns.
• Assessment of activation-induced cytidine deaminase-dependent VDJH4 intron SHM and CSR in engineered B cell cultures.
• Investigation of APE2 C-terminal interactions with proliferating cell nuclear antigen (PCNA) and the ATR-Chk1 pathway.

Main Results:

• APE1 and APE2 expression levels change dynamically during B cell activation, influencing both SHM and CSR.
• Early activation favors CSR with high APE1/APE2 levels; sustained stimulation leads to decreased APE1 and increased APE2.
• Engineered GC-like APE1/APE2 expression (low APE1, high APE2) enabled detectable SHM in primary B cells.
• APE2 promotes SHM and CSR, but only when APE1 levels are reduced; APE1 suppresses SHM while promoting CSR.

Conclusions:

• Downregulation of APE1 in GC B cells is a prerequisite for SHM.
• APE2 promotes SHM, and its function is enhanced by reduced APE1 levels.
• The balance of APE1 and APE2 expression dictates the fidelity of DNA repair, balancing CSR and SHM during B cell maturation.