Chicken MBD4 Regulates Immunoglobulin Diversification by Somatic Hypermutation.

Area of Science:

• Immunology
• Molecular Biology
• Genetics

Background:

• Immunoglobulin (Ig) diversification, crucial for adaptive immunity, involves somatic hypermutation (SHM) and class switch recombination (CSR).
• Activation-induced deaminase (AID) initiates these processes by converting cytosine to uracil in DNA, leading to mutations.
• Two main DNA repair pathways, base excision repair via uracil DNA glycosylase (UNG) and mismatch repair (MMR), process uracil in Ig genes.

Purpose of the Study:

• To investigate the function of Methyl binding domain protein 4 (MBD4), a uracil glycosylase, in the context of somatic hypermutation (SHM).
• To explore the potential role of MBD4 in immunoglobulin gene diversification due to its interaction with the MMR complex component MLH1.

Main Methods:

• Examined the conserved DNA uracil glycosylase domain of MBD4 across various species, noting striking structural conservation between human and chicken MBD4.
• Utilized CRISPR/Cas9 genome editing to construct structural variants of MBD4 in chicken DT40 B cells, which exhibit constitutive SHM.
• Assessed the impact of MBD4 disruption on SHM frequency using IgM loss assays.

Main Results:

• The DNA uracil glycosylase domain of MBD4 is highly conserved across diverse species, including mammals, birds, sharks, and insects.
• Disruption of the catalytic region of MBD4 in chicken DT40 B cells led to an increased frequency of somatic hypermutation (SHM).
• These findings suggest a regulatory role for MBD4 in the SHM process.

Conclusions:

• Methyl binding domain protein 4 (MBD4) is proposed to play a significant role in regulating somatic hypermutation (SHM) during immunoglobulin diversification.
• The catalytic activity of MBD4 appears to influence the rate of SHM, indicating its involvement in DNA repair pathways governing Ig gene diversification.