Checkpoint kinase 1 negatively regulates somatic hypermutation

Abstract

Immunoglobulin (Ig) diversification by somatic hypermutation in germinal center B cells is instrumental for maturation of the humoral immune response, but also bears the risk of excessive or aberrant genetic changes. Thus, introduction of DNA damage by activation-induced cytidine deaminase as well as DNA repair by multiple pathways need to be tightly regulated during the germinal center response to prevent lymphomagenesis. In the present study, we show that DNA damage checkpoint signaling via checkpoint kinase 1 (Chk1) negatively regulates somatic hypermutation. Chk1 inhibition in human B cell lymphoma lines as well as inactivation of Chk1 alleles by gene targeting in DT40 B cells leads to increased somatic hypermutation. This is apparently due to changes in DNA repair pathways regulated by Chk1, such as a decreased homologous recombination efficiency that also leads to decreased Ig gene conversion in DT40. Our data show that Chk1 signaling plays a crucial role in regulation of Ig diversification and sheds unexpected light on potential origins of aberrant somatic hypermutation in B cell lymphomagenesis.

Checkpoint kinase 2 is required for efficient immunoglobulin diversification

“…Thus, it appears that Chk2 inactivation leads to a decreased somatic hypermutation in this model system, in contrast to an increased hypermutation detectable in the same assay in case of Chk1 inhibition. 27 To assess whether Chk2 inhibition also leads to decreased hypermutation of the endogenous Ig loci in Burkitt lymphoma cell lines, and to prevent potential EBV effects on checkpoint control, we used the well established EBV-negative RAMOS cell system. 33,34 Here, introduction of deleterious mutations into the Ig genes leads to a loss of surface IgM expression that can be measured by FACS analyses.…”

“…Thus, we con- 34 The positions of the primers used for PCR are indicated with arrows. To ensure adequate detection of decreased SHM, a substantially longer stretch of the IgH locus (marked with a black line) than in the previous study on Chk1 function 27 (marked in gray) was analyzed. The total number of sequences analyzed is indicated in the middle of the pie chart and the number of non-unique point mutations per DNA sequence is given in the periphery of the chart segments.…”

“…27 First, we generated a Chk2 knockout in the DT40CV ¡ cell line that lacks the pseudogenes required for Ig gene conversion and thus constitutively performs somatic hypermutation. 35 With a targeting strategy and vectors previously used for Chk2 inactivation in DT40 (Fig.…”

“…This is consistent with an increase in homologous recombination activity upon Chk2 inactivation, while a drop in AID activity or base excision repair would both have lead to a decreased Ig gene conversion activity. 39,40 Thus, Chk2 inactivation apparently leads to opposite effect as compared to Chk1 inactivation in diversification of the Ig variable region: while Chk1 inactivation causes increased somatic hypermutation and decreased Ig gene conversion, 27 Chk2 inactivation leads to decreased somatic hypermutation and increased Ig gene conversion. These effects might be explained if Chk1 and Chk2 were able to oppositely regulate homologous recombination, 7,38 as a major repair pathway determining the decision between hypermutation and Ig gene conversion.…”

“…We have previously shown that Chk1 negatively regulates somatic hypermutation via promotion of homologous recombination and Ig gene conversion. 27 This is intriguing in the context of the reported downregulation of the ATR/Chk1/p53 axis in hypermutating germinal center B cells by the transcription factor BCL-6, [28][29][30][31] and implies that such a regulation may be required for efficient hypermutation. On the other hand, though, deregulation of this pathway may lead to excessive genetic instability and lymphomagenesis.…”