Histone chaperone Spt6 is required for class switch recombination but not somatic hypermutation

Abstract

Activation-induced cytidine deaminase (AID) is shown to be essential and sufficient to induce two genetic alterations in the Ig loci: class switch recombination (CSR) and somatic hypermutation (SHM). However, it is still unknown how a single-molecule AID differentially regulates CSR and SHM. Here we identified Spt6 as an AIDinteracting protein by yeast two-hybrid screening and immunoprecipitation followed by mass spectrometry. Knockdown of Spt6 resulted in severe reduction of CSR in both the endogenous Ig locus in B cells and an artificial substrate in fibroblast cells. Conversely, knockdown of Spt6 did not reduce but slightly enhanced SHM in an artificial substrate in B cells, indicating that Spt6 is required for AID to induce CSR but not SHM. These results suggest that Spt6 is involved in differential regulation of CSR and SHM by AID.T he Ig genes in antigen-stimulated B lymphocytes are diversified by two distinct genetic alteration mechanisms, namely somatic hypermutation (SHM) and class switch recombination (CSR) (1, 2). SHM causes the accumulation of point mutations in the rearranged variable (V) region genes, leading to generation of antibodies with higher affinity after cellular selection by a limited amount of antigen (1). CSR replaces the heavy chain constant region (C H ) gene proximal to the V H gene, namely Cμ with one of the downstream C H genes by recombination between the switch (S) regions located 5′ to each C H gene, thereby producing antibodies with diverse effector functions without changing their antigen specificity (2).Both SHM and CSR require activation-induced cytidine deaminase (AID), which is specifically expressed in activated B cells (3). It is well accepted that AID initiates single-strand DNA breaks essential for SHM and CSR through its cytidine deaminase activity. AID can also introduce mutations in such nonIg loci as c-myc, Pim1, Pax5, bcl-6, and RhoH (4, 5). The number of target loci of AID seems to be larger than expected but still limited (5). Although extensive analyses have been done to uncover the exact molecular mechanism how AID induces DNA strand breaks at restricted loci, it is unknown how a singlemolecule AID differentially regulates CSR and SHM or how the Ig genes and other target loci are preferentially targeted in the whole genome. To answer these questions, extensive studies were carried out to identify cofactor(s) that may account for the target specificity of the AID function. Several AID-interacting proteins have been reported, including RNA polymerase II (6), replication protein A (7), protein kinase A (8, 9), DNA-PKcs (10), MDM2 (11), CTNNBL1 (12), Spt5 (13), and PTBP2 (14). Unfortunately, however, none of these proteins could show any functional correlation to support the target specificity of AID. There is no clear mechanism to limit the number of target loci. Most of the proteins like RNA polymerase II, protein kinase A, Spt5, and PTBP2 are rather ubiquitous and interact with many proteins other than AID. PTBP2 is a splicing factor, and Spt5 is one of the tra...

Accumulation of the FACT complex, as well as histone H3.3, serves as a target marker for somatic hypermutation

“…In addition, hypermutation of the reporter transgene may not fully mimic the SHM at the V(D)J region, as we reported previously that Spt6 reduced SHM of the V(D)J region but not of the HygGFP transgene (8,12). Moreover, reduction of the GFP reporter expression by elongation factor knockdown could perturb the SHM assay results.…”

“…In addition, we found that Spt5 promotes an end-joining repair process during CSR (11). Spt6 is another elongation factor that we found promotes CSR (12). We originally identified Spt6 based on its physical interaction with AID but later found that their interaction is not important for SHM or CSR (8).…”

Cytoplasmic activation-induced cytidine deaminase (AID) exists in stoichiometric complex with translation elongation factor 1α (eEF1A)

“…Although its primary and ancestral role is in protein synthesis (where it delivers aminoacyl-tRNAs to the translating ribosome), eEF1A is abundant in the cytosol and has frequently been detected during purification of other cytosolic proteins, where it is often viewed as a contaminant. Indeed, Okazaki et al (18) very recently noted the presence of eEF1A along with other proteins in a partially purified sample of tagged AID obtained from AID-overexpressing cells; bands with a mobility corresponding to that of eEF1A can also be discerned in other studies of overexpressed AID (17,19). However, the results obtained in this work showing the presence of eEF1A at stoichiometric abundance in samples of AID prepared without overexpression along with the mutagenesis experiments reveal that the AID/eEF1A interaction is indeed physiological.…”

Activation-Induced Cytidine Deaminase in Antibody Diversification and Chromosome Translocation

“…Consistent with this, the C-terminus of AID is involved in cooperative binding of AID, UNG and mismatch repair proteins MSH2-MSH6 to the switch regions (Ranjit et al, 2011). Other AID interacting factors including KAP1 and Spt6 have been found to be critical for CSR but not SHM (Jeevan-Raj et al, 2011; Okazaki et al, 2011). Altogether this suggests that AID itself plays an important role in dictating the downstream processing of the U:G mismatches it creates, likely by a physically interacting with CSR specific factors.…”