Antibody Diversity Section
Patricia J. Gearhart, Ph.D., Chief
Somatic hypermutation of immunoglobulin genes occurs at a frequency that is a million times greater than mutation in other genes. We have used biochemical and genetic techniques to study this fascinating process that is initiated by activation-induced deaminase (AID) to deaminate cytosine in DNA to uracil. AID is targeted to variable regions in germinal center B cells by transcription. We found that RNA polymerase II accumulates for an extended distance of 1.2 kb from the TATA box, along with the Spt5 transcription factor and AID. The data support a model where mutations occur when the initiating form of RNA polymerase is retained. However, it is not known what causes RNA polymerase to pause in variable regions. During repair of the uracils, low-fidelity DNA polymerases introduce nucleotide substitutions. Although DNA polymerase eta is the major polymerase, other polymerases contribute. We determined that polymerase zeta introduces tandem mutations at a low frequency. Our results support a model where polymerase iota occasionally accesses the replication fork to generate a first mutation, and polymerase zeta extends the mismatch with a second mutation.
To study aging, we sequenced VH and VK rearranged genes from old mice, and found that the age-associated B cell subset had a heterogeneous repertoire with significant somatic hypermutation. The results indicate that these cells are antigen-experienced and accumulate over time to diverse antigens. To study inflammation, we are characterizing the role of B cells in atherosclerosis. Using ApoE -/- mice, we are determining how IgG antibodies exacerbate atherosclerosis by exploring their biological roles. We then cloned antibodies produced at the site of aortic plaques, which will be used to identify antigens. The long-term goal is to create potential vaccines for disease prevention.
- Targeting of activation-induced deaminase to variable regions by transcription.
- Role of low-fidelity DNA polymerases in somatic hypermutation.
- Characterization of B cells associated with aging.
- Role of B cells and antibodies in atherosclerosis.
Findings and Publications
Rajagopal, D., Maul, R.W., Ghosh, A., Chakraborty, T., Khamlichi, A.A., Sen, R., and Gearhart, P.J. Immunoglobulin switch mu sequence causes RNA polymerase II accumulation and reduces dA hypermutation. J. Exp. Med. 206:1237-1244, 2009 (PMC ID# 2715057).
Maul, R.W., Saribasak, H., Martomo, S.A., McClure, R.L., Yang, W., Vaisman, A., Gramlich, H.S., Schatz, D.G., Woodgate, R., Wilson III, D.M., and Gearhart, P.J. Uracil residues dependent on the deaminase AID in immunoglobulin gene variable and switch regions. Nature Immunol. 12:70-76, 2011 (PMC ID# 3653439).
Maul, R.W., Cao, Z., Venkataraman, L., Giorgetti, C.A., Press, J.L., Denizot, Y., Du, H., Sen, R. and Gearhart, P.J. Spt5 accumulation at variable genes distinguishes somatic hypermutation in germinal center B cells from ex vivo-activated cells. J. Exp. Med. 211:2297-2306, 2014 (PMC ID# 4203944).
Maul, R.W., MacCarthy, T., Frank, E.G., Donigan, K.A., McLenigan, M.P., Yang, W., Saribasak, H., Huston, D.E., Lange, S.S., Woodgate, R., and Gearhart, P.J. DNA polymerase iota functions in the generation of tandem mutations during somatic hypermutation of antibody genes. J. Exp. Med. 213:1675-1683, 2016 (PMC ID# 4995076).
Russell Knode, L.M., Naradikian, M.S., Myles, A., Scholz, J.L., Hao, Y., Liu, D., Ford, M.L., Tobias, J.W., Cancro, M.P., and Gearhart, P.J.: Age-associated B cells express a diverse repertoire of VH and VK genes with somatic hypermutation. J. Immunol.198:1921-1927, 2017 (PMC ID# 5322232).