Ursula Storb, M.D.

Molecular Immunogenetics; Control of B Lymphocyte Development; Regulation of Ig Gene Expression; Somatic Mutation of Ig Genes; DNA Methylation in Embryogenesis; Control of Expression, Rearrangement, and Somatic Hypermutation of Immunoglobulin Genes

Research Summary

We are studying B lymphocyte development and the control of immunoglobulin gene expression using transgenic and knockout mice, cell transfection, and other cellular and molecular tools. Our studies on the regulation of immunoglobulin (ig) gene rearrangement has led to a detailed understanding of the role of individual genetic elements in B cell differentiation and maturation. In contrast to most or all other genes, antibody genes are in a nonfunctional conformation in all cells, except specific immune cells. When a precursor cell 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 will eventually lead to a set of functional antibody genes in the particular B cell. One of our studies is aimed at understanding the mechanism of somatic hypermutation, a process of cell differentiation in which the antibody genes undergo mutational changes to increase the affinity of the antibody molecules for the antigen. The somatic hypermutation process is ongoing in leukemia cells and allows them to escape therapy directed against their antibody molecules. We have shown that somatic hypermutation is linked to transcription. Our laboratory is also concerned with the control mechanisms which enable pre B cells to rearrange its antibody genes. We have found, in transgenic mice, that developing B lymphocytes can sense the intracellular appearance of complete antibody molecules and react with cessation of further rearrangement of immunoglobulin genes. The feedback-complex must be bound to cellular membranes. Other studies are concerned with the structure and function of immunoglobulin gene enhancers, and the role of DNA methylation in development and Ig gene expression.

Our experimental system is the analysis of expression of immunoglobulin (Ig) genes.These genes encode antibody molecules and are induced to highest activity during encounter with agents foreign to the organism, such as microbes. Our studies are aided by the introduction of genes into cells and into mice by transgenic and embryonic stem cell technologies. In addition to transcriptional control, segments of Ig genes undergo rearrangement only in antibody-producing B lymphocytes, a step required to create a functional gene. During the development of B cells, an Ig gene-specific recombinase is produced. When the developing B cells have correctly rearranged one Ig heavy chain and one light chain gene, the recombinase is shut off. We are studying the function of the recombinase. We are also determining how Ig genes become accessible to the recombinase and how the shutoff after productive gene rearrangement is controlled. In studying the rearrangement of an artificial DNA substrate for the Ig recombinase in transgenic mice, we discovered that the substrate is completely 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-1, that maps to the distal end of mouse chromsome 4. We are attempting to identify the Ssm-1 gene by positional cloning in order to examine the role of this novel type of regulation. Finally, we are studying the molecular mechanism of somatic hypermutation of expressed immunoglobulin genes. We have evidence that transcription is required for this process. We are testing a model which proposes that a mutator factor is loaded onto the transcriptional complex at initiation, leading to point mutations during the elongation of transcripts (see figure). Surprisingly, the BCL6 gene which is involved in lymph cell cancers is also mutable by this process.

Model of somatic hypermutation of immunoglobulin genes. 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, exonuclease trims back the single stranded 3' end, a DNA polymerase fills in the gap creating mutation(s) (x) opposite the MuF associated base(s). An endonuclease (Fen1) removes the DNA end bound to MuF and a DNA ligase seals the mutated DNA strand (from Storb et al., J. Exp. Med. 188:689, 1998).

Selected Papers

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

Shen, H., Ratnam, S., and Storb, U.  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, 2005.

Longerich, S., Tanaka, A., Bozek, G., Nicolae, D. and Storb, U.  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, 2005.

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

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

Volgina, V., Sun, T., Bozek, G., Martin, T., and Storb, U.  Scarcity of l1 B cells in mice with a single point mutation in Cl1 is due to low BCR signal caused by misfolded lambda1 light chain.  Mol. Immunol., 44:1417-1428, 2007.

Storb, U., Shen, H., Longerich, S., Ratnam, S., Tanaka, A., Bozek, G., and Pylawaka, S. Targeting of AID to immunoglobulin genes, Adv. Exp. Med. Biol. 596:83-91, 2007.

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

Longerich, S., Orelli, B., Martin, R., Bisho, D., and Storb, U. (2008). Brca1 in Ig gene conversion and somatic hypermutation.  DNA Repair, 7: 253-266.

Longerich, S. and Storb, U.  (2008). 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. (2008). Expression of AID transgene is regulated in activated B cells but not in resting B cells and kidney, Mol. Immunol., 45: 1883-1892.