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M.D. Scharff
Albert Einstein College of Medicine
Bronx, NY

Somatic V Region Hypermutation in Cultured Antibody-Forming Cells

We have developed an in vitro system for studying antibody variable (V) region hypermutation in which heavy (H) chain Ig genes are stably transfected into antibody-forming cell lines. In this model system, the V regions contain a nonsense mutation which when mutated results in the reversion of the nonsense mutation and expression and secretion of the H chain. These revertants are then detected by the ELISA spot assay, and rates of mutation are quantified by fluctuation analysis. Constructs containing the H chain promoter, VDJ, Em, and the g2a constant (C) region, and their associated intervening and flanking sequences, undergo high rates of mutation in the NSO and S107 plasmacytoma cell lines and in the 18.81 pre-B cell line but have a low background rate of mutation in the J558 plasmacytoma cell line. These same constructs have low rates of V region mutation in non-B cells such as mouse L cells and CHO cells, even though the H chains are highly transcribed in all of these cell lines. Reversion is due to single base changes that are more often transitions and occur 20 to 30 times more frequently in hot spot than in non-spot motifs. In addition, the same H chain constructs that are studied in vitro also undergo high rates of mutation in vivo in the PNA high cells of transgenic mice. In the NSO plasmacytoma cell line, constructs containing the g2a C region mutate at a 1000-fold higher rate than the same construct in which the g2a C region has been replaced by Cm. Deletion analysis has been used to identify the cis-acting sequences responsible for this difference (see poster by Hong and Green). These studies, and sequential transfection with the m and g2a constructs, reveal that sequences in the CH1 domain of Cg2a can transactivate the rate of mutation of the V associated with the m C region (see poster by Poltoratsky et al.). The transactivation of mutation of Vm by the g2a C region requires the presence of an AP1/GCN4 DNA binding motif in the coding exon of the CH1 domain of the g2a C region and is lost when that motif is mutated. Nuclear extracts from the cells that are transactivated for the mutation of Vm form complexes in EMSA with the AP1/GCN4 motif. These complexes do not appear to contain DNA binding proteins, such as jun or fos, that frequently bind AP1 sites. However, one of these complexes is supershifted by antibodies to the transcriptional adaptor protein GCN5, which contains histone acetylase activity. The overexpression of human GCN5 transactivates the mutation of Vm 350-fold, but a mutant of hGCN5 that lacks histone acetylase activity does not transactivate Vm. CBP, another histone acetylase, also does not transactivate mutation of Vm (see poster by Poltoratsky et al.). Taken together, these results show that Ig genes can constitutively undergo high rates of mutation in cultured cells. Furthermore, they illustrate how this model in vitro system can be used to discover cis-acting sequences and trans-acting factors that are involved in the regulation of V region mutation in vitro. Finally, they suggest that chromatin remodeling may play an important role in the regulation and targeting of mutations to Ig genes in cultured B cells and could be important in vivo.

These studies were carried out by Nancy Green, Mark Lin, Vladimir Poltoratsky, and Margrit Wiesendanger of the Albert Einstein College of Medicine, in collaboration with Nicholai Barlev and Shelley Berger of the Wistar Institute.