Increased Transcription Levels Induce Higher Mutation Rates in a Hypermutating Cell Line

Abstract

Somatic hypermutation, in addition to V(D)J recombination, is the other major mechanism that generates the vast diversity of the Ab repertoire. Point mutations are introduced in the variable region of the Ig genes at a million-fold higher rate than in the rest of the genome. We have used a green fluorescent protein (GFP)-based reversion assay to determine the role of transcription in the mutation mechanism of the hypermutating cell line 18-81. A GFP transgene containing a premature stop codon is transcribed from the inducible tet-on operon. Using the inducible promoter enables us to study the mutability of the GFP transgene at different transcription levels. By analyzing stable transfectants of a hypermutating cell line with flow cytometry, the mutation rate at the premature stop codon can be measured by the appearance of GFP-positive revertant cells. Here we show that the mutation rate of the GFP transgene correlates with its transcription level. Increased transcription levels of the GFP transgene caused an increased point mutation rate at the premature stop codon. Treating a hypermutating transfection clone with trichostatin A, a specific inhibitor of histone deacetylase, caused an additional 2-fold increase in the mutation rate. Finally, using Northern blot analysis we show that the activation-induced cytidine deaminase, an essential trans-factor for the in vivo hypermutation mechanism, is transcribed in the hypermutating cell line 18-81.

Biased dA/dT somatic hypermutation as regulated by the heavy chain intronic iEμ enhancer and 3′Eα enhancers in human lymphoblastoid B cells

“…Compared to that of P-VDJ-Cγ1, the increased mutation frequencies in P-VDJ-iEµ-Cγ1 and P-VDJ-iEµ-Cγ1-hs1,2 was unlikely due to possible transcription enhancing effects of iEµ or hs1,2 since the transcription level of V H 1-DXP′1-J H 5 could vary by as much as eight-fold among Ramos B cells with comparable mutation frequencies. This finding is in apparent contrast with a previous report suggesting that increased transcription levels induces higher mutation rates in 18-81 cells using an inducible GFP-transgene expression system (Bachl et al, 2001). This difference could be explained by the different cell lines used and/or promoters and genes analyzed.…”

“…If only unique mutations were counted, this yielded a mutation rate of 2.8 × 10 −5 mutation/bp/cell generation, which is comparable to the SHM rate previously reported in Ramos B cells (Sale and Neuberger, 1998;Zhang et al, 2001) and in the hypermutating pre-B cell line 18-81 (Bachl and Wabl, 1996;Bachl et al, 2001). Likewise, among 192 sequences of V H 1-DXP′1-J H 5 region in the P-VDJ-iEµ-Cγ1, 24 (12.5%) had one or more mutation and the mutational frequency was 4.4 × 10 −4 mutation/bp (Table 1), which yielded a mutation rate of 2.2 × 10 −5 mutation/bp/cell generation.…”

DNA cleavage in immunoglobulin somatic hypermutation depends onde novoprotein synthesis but not on uracil DNA glycosylase

“…NIR cells were established by transfection of NIH 3T3 cells with an AID fusion protein with the estrogen receptor hormine-binding domain (A IDER)-expressing construct, pAID-ER-BOSbsr (27), carrying an artificial SHM target gene (28,29). JP8BdelER (pFB-⌬C-…”

Activation-induced cytidine deaminase: structure–function relationship as based on the study of mutants

“…More recently, evidence has been provided for lesion and nicks occurring also on the DNA template strand [Basu et al, 2005;Bransteitter et al, 2003]. Transcription is also required for SHM [Bachl et al, 2001;Fukita et al, 1998], even if its triggering event has not been elucidated so far.…”