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

Peters A and Storb U. (1996). Somatic hypermutation of immunoglobulin genes is linked to transcription initiation. Immunity, 4:57-65.

Storb U. (1996). Molecular mechanism of somatic hypermutation of Ig genes. Curr. Opin. Immunol., 8:206-214.

Shen HM, Peters A, Baron B, Zhu X and Storb U. (1998). Mutation of BCL-6 gene in normal B cells by the process of somatic hypermutation of Ig genes. Science, 280:1750-1752.

Storb U, Peters A, Kim N, Shen HM, Bozek G, Michael N, Hackett J, Klotz E, Loeb L, and Martin T. (1999). Molecular aspects of somatic hypermutation of Ig genes. Cold Spring Harbor Lab. Symp. Quant. Biol., 64:227-234.

Engler, P. and Storb, U. (2000). A linkage map of distal mouse chromosome 4 in the vicinity of Ssm1, Mammalian Genome, 11: 694-695.

Shen, H., Michael, N., Kim, N., and Storb, U. (2000). The TATA binding protein, c-Myc, and survivin genes are not somatically hypermutated, while Ig and BCL6 genes are hypermutated in human memory B cells, Internatl. Immunol., 12: 1085-1093.

Longacre A and Storb U. (2000). Minireview: A novel cytidine deaminase affects antibody diversity, Cell 102: 541-544.

Storb U, Shen H, Michael N, Kim N. (2001). Somatic hypermutation of immunoglobulin and non-immunoglobulin genes. Philosoph. Transacts. Roy. Soc. London B , 356: 13-19.

Wang Z, Engler P, Longacre A and Storb U. (2001). An efficient method for high fidelity BAC/PAC retrofitting with a selectable marker for mammalian cell transfections. Genome Res. 11: 137-142.

Sun T and Storb U. (2001). Insertion of phosphoglycerine kinase (PGK)- neo 5' of Jl1 dramatically enhances VJl1 rearrangement. J. Exp. Med. 193: 699-711.

Shen H, Peters A, Kao D and Storb U. (2001). The 3' Igk enhancer contains RNA polymerase II promoters. Internatl. Immunol. 13: 665-674.

Storb U. (2001). DNA polymerases in immunity: profiting from errors. Nature Immunol. 2: 484-485.

Michael N, Martin T, Nicolae D, Kim N, Padjen K, Zhan P, Nguyen H, Pinkert C and Storb U. (2002). Effects of sequence and structure on the hypermutatbility of immunoglobulin genes, Immunity, 16: 123-134.

Sun T, Clark M 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: 245-255.

Fu Y-X and Storb U. (2002). Autoreactive B cells migrate into T cell territory. Science 297: 2006-2008.

Storb U and Stavnezer J. (2002). Immunoglobulin genes: generating diversity with AID and UNG, Curr. Biol., 12: R725-R727.

Diaz M and Storb U. (2003). A novel cytidine deaminase AIDs in the delivery of error prone polymerases to immunoglobulin genes, DNA Repair, 2: 623-627

Kim N, Martin TE, Simon MC and Storb U. (2003). The transcription factor Spi-B is not required for somatic hypermutation, Mol. Immunol. 39: 577-583.

Longacre A, Sun T, Goldsby R, Preston B and Storb U. (2003). Ig gene somatic hypermutation in mice defective for DNA polymerase delta proofreading, Internatl. Immunol., 15: 477-481.

Michael N, Shen HM, 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.

Shen HM and Storb U. (2004). The cytidine deaminase, AID, can target both DNA strands when the DNA is supercoiled, Proc. Natl. Acad. Sci. USA, 101:12997-13002.

Longerich S and Storb U. (2005). Contested role of uracil DNA glycosylase UNG in immunoglobulin gene diversification. In press, Trends in Genetics

Faculty and Research

Programs

Cancer Biology

Immunology

Microbiology

Molecular Metabolism
and Nutrition

Molecular Pathogenesis and
Molecular Medicine