Ursula Storb, M.D.
Molecular Immunogenetics; Control of Somatic Mutation of Immunoglobulin (Ig) Genes; DNA Methylation and Chromatin Modifications in Embryogenesis
In contrast to almost all other genes, antibody genes are in a nonfunctional conformation in all cells, except specific immune cells, named B and T lymphocytes. When a precursor lymphocyte arises that has the potential to become an antibody-producing cell (these cells reside in the bone marrow and are called pre B cells), it develops an enzymatic machinery to rearrange its antibody genes. Such gene rearrangements lead to a set of functional antibody genes, a different one in each B cell, thus creating a large repertoire of B cells with varying specificities. One of our studies is aimed at understanding the mechanism of somatic hypermutation (SHM). SHM is a process of cell differentiation in which B cells immunized with a particular antigen, e.g. during an infection, mutate the specific antibody genes they express. B cells that have increased the affinity of their antibody molecules for the antigen by mutations will be selected to produce large amounts of the specific antibodies. They also will become “memory B cells” which respond fast and strongly when the organism is again infected with the same microbe. The somatic hypermutation process can be ongoing in leukemia cells, allowing them to escape therapy directed against their antibody molecules.
Our model of somatic hypermutation of immunoglobulin genes (1998). The mutator factor (MuF) associates with RNA polymerase II (pol) that is initiating transcription at an Ig gene promoter. If pol encounters a block to transcription (e.g. a hairpin in the nascent transcript), it pauses and transfers MuF to the DNA. MuF causes a nick in the non-transcribed DNA strand, an exonuclease trims back the single stranded 3' end, a DNA polymerase fills in the gap creating mutation(s) (x) near the MuF associated base(s). An endonuclease (Fen1) removes the DNA flap bound to MuF and a DNA ligase seals the mutated DNA strand.
We have modified this model of 1998 since the MuF is now known to be a cytidine deaminase, AID (Honjo et al., 2000), that introduces uridines into both DNA strands (Shen and Storb, 2004). We have shown that somatic hypermutation is linked to transcription. Our studies are aided by the introduction of genes into cells and mice by transgenic and embryonic stem cell technologies. We are testing the model by determining whether AID is indeed directly associated with the transcription machinery.
In a related study in which an artificial DNA substrate for the Ig recombinase was introduced into transgenic mice, we discovered that the substrate is methylated and inactive when the transgene is present in certain inbred mouse strains, but becomes unmethylated and active upon two sequential crossings into certain other mouse strains. The methylation is controlled by a strain-specific modifier gene, Ssm-1b, that we have identified by positional cloning of DNA from crosses between methylating and non-methylating mice. Ssm1-b causes complete methylation and chromatin compaction of the target gene. It is a member of a family of KRAB-zinc finger genes that appear to be involved in the inactivation of certain DNA sequences during early embryogenesis. We are trying to understand how different mouse strains inactivate undesirable genomic elements and to assess implications for human development and disease.
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, 19:235-242. PMID: 12932357
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. USA, 101:12997-13002. PMCID: PMC516507 PMID: 15328407
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. PMCID: PMC1140615 PMID: 15899878
Shen, H., Ratnam, S., and Storb, U. (2005). Targeting of the activation-induced cytosine deaminase is strongly influenced by the sequence and structure of the targeted DNA, Mol. Cell. Biol. 25:10815-10821. PMCID: PMC1316976 PMID: 16314506
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. PMCID: PMC2212980 PMID: 16301749
Longerich, S., Basu, U., Alt, F., and Storb, U. (2006). AID in somatic hypermutation and class switch recombination. Curr. Op. Immunol., 18:164-174. PMID: 16464563
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. PMID: 17015724
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. PMCID: PMC2196218 PMID: 17681497
Longerich, S., Orelli, B., Martin, R., Bishop, D., and Storb, U. (2008). Brca1 in Ig gene conversion and somatic hypermutation. DNA Repair, 7: 253-266. PMCID: PMC2267027 PMID: 18036997
Shen, H.M., Poirier, M.G., Allen, M., North, J., Lal, R., Widom, J.,and Storb, U. (2009). The activation induced cytidine deaminase (AID) efficiently targets DNA in nucleosomes, but only during transcription. J.Exp Med., 206:1057-71. PMCID: PMC2715043 PMID: 19380635
Storb, U., Shen, H., and Nicolae, D. (2009). Somatic hypermutation: Processivity of the cytosine deaminase AID and error-free repair of the resulting uracils. Cell Cycle. 8 :3097-3101 PMID: 19738437
Tanaka, A., Shen, H., Ratnam, S., Kodgire, P. and Storb, U. (2010) Targeting of somatic hypermutation through CAGGTG cis-elements. J. Exp. Med. 207:405-415. PMCID: PMC2822603 PMID: 20100870
Ratnam, S., Bozek, G., Nicolae, D., and Storb, U. (2010). The pattern of somatic hypermutation of Ig genes is altered when p53 is inactivated. Mol. Immunol., 47:2611-8. Epub 2010 Aug 5
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