The RNF8/RNF168 ubiquitin ligase cascade facilitates class switch recombination

Abstract

An effective immune response requires B cells to produce several classes of antibodies through the process of class switch recombination (CSR). Activation-induced cytidine deaminase initiates CSR by deaminating deoxycytidines at switch regions within the Ig locus. This activity leads to double-stranded DNA break formation at the donor and recipient switch regions that are subsequently synapsed and ligated in a 53BP1-dependent process that remains poorly understood. The DNA damage response E3 ubiquitin ligases RNF8 and RNF168 were recently shown to facilitate recruitment of 53BP1 to sites of DNA damage. Here we show that the ubiquitination pathway mediated by RNF8 and RNF168 plays an integral part in CSR. Using the CH12F3-2 mouse B cell line that undergoes CSR to IgA at high rates, we demonstrate that knockdown of RNF8, RNF168, and 53BP1 leads to a significant decrease in CSR. We also show that 53BP1-deficient CH12F3-2 cells are protected from apoptosis mediated by the MDM2 inhibitor Nutlin-3. In contrast, deficiency in either E3 ubiquitin ligase does not protect cells from Nutlin-3-mediated apoptosis, indicating that RNF8 and RNF168 do not regulate all functions of 53BP1.53BP1 | activation-induced cytidine deaminase | DNA damage response P art of an effective immune response requires the production of antibodies of different classes, each of which mediates a different effector function. This process is initiated by activationinduced cytidine deaminase (AID), which induces class switch recombination (CSR) by deaminating deoxycytidines within Ig switch regions (1, 2). The recognition and subsequent processing of the mutated residues by base excision repair and/or mismatch repair machineries generates double-stranded DNA breaks (DSBs) at switch regions (3). In an attempt to mend the ensuing breaks, B cells mount a damage response similar to that signaled by irradiated cells (1). Ultimately, AID-induced DSBs are repaired predominantly by nonhomologous end-joining (4, 5) and to a lesser extent by an alternative end-joining pathway (6).Generally, DSBs are readily recognized by sensor-kinase protein complexes, including the Mre11-Rad50-Nbs1 (MRN)/ataxiatelangiectasia mutated (ATM), Ku70/Ku80-DNA-PKcs, and ataxiatelangiectasia and Rad3-related (ATR)-ATR-interacting protein complexes (7). Upon binding to a DSB site, these factors are activated to trigger a cascade of events that culminates in cell cycle delay and/or DNA repair (7). During this process, sequential chromatin modifications around the break sites appear to be crucial for adequate resolution of the DNA breaks. Active chromatin modification is initiated by ATM-dependent phosphorylation of the histone variant H2AX to the γH2AX form (8-10). γ-H2AX then preferentially binds the tandem-BRCA1 C-terminal domain (BRCT) motifs of mediator of DNA damage checkpoint 1 (MDC1) (11, 12), which in turn recruits more MRN/ATM, thereby amplifying the γ-H2AX signal (11, 12). Recently, it was shown that ATM also phosphorylates MDC1 at a TQxF consensus, allowing for the recruitmen...

Characterizing RNF20 and RNF40 in the Class Switching of B Cells1

“…In order to effectively neutralize and clear pathogens and toxins, B cells must produce and secrete antibodies of higher affinity and of different classes. [2] The process of increasing the affinity of antibodies is known as somatic hypermutation. In somatic hypermutation, mutations are introduced into the variable region of the antibody gene, which encodes the binding region of the antibody.…”

“…Class switch recombination (CSR) is induced by activation-induced cytidine deaminase (AID), which leads to CSR by deaminating deoxycytidines within immunoglobulin switch regions. [2] The detection and processing of the mutated residues by base excision repair and/or mismatch repair machineries generates double-stranded DNA breaks (DSBs) at switch regions. [2] The DSBs cause B cells to signal a damage repair response, similar to that signaled by irradiated cells, in attempts to mend the resultant breaks.…”

“…[2] The detection and processing of the mutated residues by base excision repair and/or mismatch repair machineries generates double-stranded DNA breaks (DSBs) at switch regions. [2] The DSBs cause B cells to signal a damage repair response, similar to that signaled by irradiated cells, in attempts to mend the resultant breaks. CSR involves the removal of portions in the antibody heavy chain locus from the chromosome and the religation of surrounding gene segments to generate a different antibody gene that produces antibodies of a different isotype.…”

“…CSR involves the removal of portions in the antibody heavy chain locus from the chromosome and the religation of surrounding gene segments to generate a different antibody gene that produces antibodies of a different isotype. [2] Segments of the heavy chain locus, (ȏ, Ч, ɣ3) are removed by enzyme activity between switch regions ( Figure 1). This is followed by non-homologous end joining of the flanking regions.…”

“…This is followed by non-homologous end joining of the flanking regions. [2] Consequently, the B cell expresses antibodies of a different isotype. The DSBs are commonly recognized by sensor-kinase protein complexes, such as the Mre11-Rad50-Nbs1 (MRN)/ ataxia telangiectasia mutated (ATM), Ku70/Ku80-DNA-PKcs, and ataxia-telangiectasia and Rad3-related (ATR)-ATR-interacting protein complexes.…”