A Novel Targeted Deletion of Mbd4 Reveals an Unappreciated Role in Class Switch Recombination
thesisposted on 28.06.2013 by Fernando Grigera
In order to distinguish essays and pre-prints from academic theses, we have a separate category. These are often much longer text based documents than a paper.
Methyl CpG binding domain protein 4 (Mbd4) is a mismatch DNA glycoslyase is involved in rectifying T/G and U/G mismatches that result from deamination of 5-methyl cytosine (5meC) and cytosine (C) in DNA, respectively. Activation induced cytidine deaminase (AID) initiates class switch recombination (CSR) and somatic hypermutation (SHM), two processes that occur in B cells critical for expanding the primary antibody repertoire, by deaminating C, thereby converting it to uracil (U) and creating a U/G mismatch in DNA. Key intermediates, double-stranded breaks (DSBs) and Phase II SHM mutations are formed downstream of the U/G lesion and are not wholly dependent on the activity of uracil N-glycosylase (UNG) in CSR and SHM, respectively, suggesting that other glycosylases fulfill these roles. The aim of this thesis was to determine whether Mbd4 supplies glycosylase and/or scaffolding activity during CSR. B cells harboring a deletion of exons 2-5 of the Mbd4 gene (Mbd4 2-52-5) do not manifest any CSR phenotype. An investigation of the mismatch repair proteins, Msh2, and Mlh1, revealed that these proteins are substantially reduced in this genetic background. The lack of CSR phenotype is surprising with respect to MMR proteins, because these genes have been shown to be happloinsufficient for CSR, suggesting a potential modulator role of Mbd4. In contradistinction, a CH12.F3 cell line that I designed where exons 6-8 of the Mbd4 gene have been deleted (Mbd4 6-86-8) show a reduced CSR phenotype, which is accompanied by reduced DSBs, a skewing of DSB positioning within S, and longer microhomologies at S-S junctions. In addition MMR proteins were also affected by Mbd4 deficiency in the CH12.F3 cell line, highlighting the only similarity between these different Mbd4 deficient models. I conclude that the Mbd4 is involved in CSR and it shares several features common to the MMR pathway. The discrepancy that exists between the different Mbd4 deletion models can be attributed to the incomplete nature of the Mbd4 deficiency in Mbd4 2-52-5 B cells. I determined that a residual transcript continues to be made in these cells and that it yields a stable truncated peptide that is likely protective for CSR.