Complex Relationship between Mismatch Repair Proteins and MBD4 during Immunoglobulin Class Switch Recombination

Abstract

Mismatch repair (MMR) safeguards against genomic instability and is required for efficient Ig class switch recombination (CSR). Methyl CpG binding domain protein 4 (MBD4) binds to MutL homologue 1 (MLH1) and controls the post-transcriptional level of several MMR proteins, including MutS homologue 2 (MSH2). We show that in WT B cells activated for CSR, MBD4 is induced and interacts with MMR proteins, thereby implying a role for MBD4 in CSR. However, CSR is in the normal range in Mbd4 deficient mice deleted for exons 2–5 despite concomitant reduction of MSH2. We show by comparison in Msh2+/− B cells that a two-fold reduction of MSH2 and MBD4 proteins is correlated with impaired CSR. It is therefore surprising that CSR occurs at normal frequencies in the Mbd4 deficient B cells where MSH2 is reduced. We find that a variant Mbd4 transcript spanning exons 1,6–8 is expressed in Mbd4 deficient B cells. This transcript can be ectopically expressed and produces a truncated MBD4 peptide. Thus, the 3′ end of the Mbd4 locus is not silent in Mbd4 deficient B cells and may contribute to CSR. Our findings highlight a complex relationship between MBD4 and MMR proteins in B cells and a potential reconsideration of their role in CSR.

MBD4 Facilitates Immunoglobulin Class Switch Recombination

“…Diminished levels of MMR proteins are found in activated splenic B cells and murine embryonic fibroblasts (MEF) from Mbd4 Δ2-5/Δ2-5 mice (16,17). Western blot analyses indicate that MLH1, PMS2, and MSH2 levels are unchanged in CIT-activated control and Mbd4 KO cells (Fig.…”

“…Murine MBD4 is a 554-amino-acid (554-aa) protein that is expressed in the cytoplasm and imported into the nuclei of splenic B cells and CH12 cells that are induced to undergo CSR (17). MBD4 contains a methyl-CpG-binding domain (MBD), encoded by exons 2 and 3, and a DNA glycosylase domain, encoded by exons 5 to 8 (20,21).…”

“…Our earlier studies showed that Mbd4-deficient mice lacking exons 2 to 5 (16), referred to here as Mbd4 Δ2-5/Δ2-5 mice, supported efficient CSR (17). However, the Mbd4 Δ2-5/Δ2-5 locus retains the puromycin resistance gene and the phosphoglycerate kinase (PGK) promoter, which could support the transcription of the residual 3= exons (16).…”

“…We were intrigued by the functional association of MBD4 and AID in active DNA demethylation in zebrafish (15). Moreover, MBD4 interacts with MLH1 (16) and PMS2 (17) and might directly contribute to the removal of U-G mismatches in collaboration with the MMR pathway (18). Unexpectedly, CSR was not affected by targeted deletion of Mbd4 exon 3 (19) or exons 2 to 5 (termed Mbd4 Δ2-5/Δ2-5 ) (17) in mice.…”

“…Moreover, MBD4 interacts with MLH1 (16) and PMS2 (17) and might directly contribute to the removal of U-G mismatches in collaboration with the MMR pathway (18). Unexpectedly, CSR was not affected by targeted deletion of Mbd4 exon 3 (19) or exons 2 to 5 (termed Mbd4 Δ2-5/Δ2-5 ) (17) in mice. However, we now show that targeted deletion of Mbd4 5= exons permits variant transcript expression from the intact 3= end of the locus.…”