Research Summary

Gene expression is controlled by activation and repression. Repression can be caused by methylation of cytosine in the sequence 5'CpG3'. Preventing DNA methylation is embryonic lethal because it results in uncontrolled gene activation. Very little is known about how methylation is targeted. The methylation modifier, Ssm1, discovered by our laboratory, is a candidate for encoding such a novel targeting function. When a transgene, HRD, comes under the influence of Ssm1, it is highly methylated at CGs and not expressed. Ssm1 acts early in embryonic development. It may direct methyl-transferases to its target genes. Only after DNA methylation does the target gene adopt an inactive chromatin state and cease to be transcribed. We have mapped Ssm1 to a small interval in the mouse genome and are using positional cloning to identify the Ssm1 gene. The characterization of Ssm1 and the determination of its endogenous targets and effects throughout development will help to understand how genes are targeted for silencing in normal development and cancer.

Another project is the somatic hypermutation (SHM) of immunoglobulin genes that encode antibodies for immunity. Antibodies are produced by B lymphocytes. When these cells encounter a foreign substance, such as bacteria or viruses, they undergo a very high rate of SHM of the expressed antibody genes. Beneficial mutations that confer higher antibody affinity accumulate. SHM is initiated by a cytidine deaminase changing cytosines into uracils. In other genes, such uracils are repaired efficiently. However, in antibody genes during SHM, error-prone DNA polymerases introduce more errors into all four bases (A, C, G, T). In this fashion, the affinity of the antibodies vastly increases, aiding the destruction of infectious agents or cancer cells. The molecular details of the mutation mechanism, including transcription, error prone DNA repair, and the role of chromatin are a major focus of our laboratory.

Under the control of the modifier Ssm1, the HRD transgene undergoes strain-specific DNA methylation. Bisulfite analysis shows that CpG dinucleotides are almost completely methylated (red marks) in every transgene sequence in adult B6 (black mouse) but very little in D2 (beige mouse). Mice of the D2 strain express the transgene throughout life. B6 early embryos express the transgene, but starting around day 6 of embryogenesis, B6 mice cease expression when CpGs are completely methylated and inactivating chromatin changes have taken place.

Selected Publications

Longerich, S. and Storb, U. (2007). Somatic hypermutation in B lymphocytes. Encyclopedia of Life Sciences, in press.

Shen, H.M., Bozek, G., Pinkert, C., McBride, K., Wang, L., Kenter, A., and Storb, U. (2007) Expression of AID transgene is regulated in activated B cells but not in resting B cells and kidney, Mol Immunol. Dec 5; [Epub ahead of print]. (PubMed)

Longerich, S., Orelli, B., Martin, R., Bishop, D.K., and Storb, U. (2007). Brca1 in Ig gene conversion and somatic hypermutation. DNA Repair (Amst). Nov 22; [Epub ahead of print] (PubMed)

Volgina, V., Sun, T., Bozek, G., Martin, T., and Storb, U. (2007). Scarcity of lambda 1 B cells in mice with a single point mutation in C lambda 1 is due to a low BCR signal caused by misfolded lambda 1 light chain. Mol. Immunol., 44:1417-1428. (PubMed)

Longerich, S., Meira, S., Shaw, D. Samson, L., and Storb, U. (2007). Alkyladenine glycosylase (Aag) in somatic hypermutation and class switch recombination, DNA Repair, 6:1764-1773. (PubMed)

Shen H., Tanaka, A., Bozek, G., Nicolae, D., and Storb, U. (2006). Somatic hypermutation and class switch recombination in Msh6-/-Ung-/- double-knockout mice, J. Immunol., 177:5386-5392. (PubMed)

Longerich, S., Basu, U., Alt, F., and Storb, U. (2006). AID in somatic hypermutation and class switch recombination. Curr. Op. Immunol., 18:164-174. (PubMed)

Shen, H., Ratnam, S., and Storb, U. (2005). Targeting of the activation-induced cytosine deaminase (AID) is strongly influenced by the sequence and structure of the targeted DNA. Mol. Cell. Biol. 25:10815-10821. (PubMed)

Longerich, S., Tanaka, A., Bozek, G., Nicolae, D. and Storb, U. (2005). The very 5' end and the constant region of Ig genes are spared from somatic mutation because AID does not access these regions. J. Exp. Med. 202:1443-1454. (PubMed)

Longerich, S. and Storb, U. (2005). The contested role of uracil DNA glycosylase in immunoglobulin gene diversification. Trends in Genetics 21:253-256. (PubMed)

Padjen, K., Ratnam, S., and Storb, U. (2005). DNA methylation precedes chromatin modifications under the influence of the strain-specfici modifier Ssm1. Mol. Cell. Biol. 25: 4782-4791. (PubMed)

Shen, H. M. and Storb, U. (2004). "Activation-induced cytidine deaminase (AID) can target both DNA strands when the DNA is supercoiled." Proc Natl Acad Sci U S A 101: 12997-3002. (PubMed)

Michael, N., Shen, H. M., Longerich, S., Kim, N., Longacre, A. and Storb, U. (2003). "The E box motif CAGGTG enhances somatic hypermutation without enhancing transcription." Immunity 19: 235-42. (PubMed)

Sun, T., Clark, M. R. and Storb, U. (2002). "A point mutation in the constant region of Ig lambda1 prevents normal B cell development due to defective BCR signaling." Immunity 16: 245-55. (PubMed)

Michael, N., Martin, T. E., Nicolae, D., Kim, N., Padjen, K., Zhan, P., Nguyen, H., Pinkert, C. and Storb, U. (2002). "Effects of sequence and structure on the hypermutability of immunoglobulin genes." Immunity 16: 123-34. (PubMed)