Ursula Storb - US grants

The organization and control of expression of immunoglobulin genes are being investigated. Specifically, we are studying the evolution and organization of immunoglobulin lambda genes in mice. We have cloned all of the variable (V) and constant (C) genes of this locus, which we are now trying to link up by "chromosome walking." We are analyzing the specifics of lambda gene expression in SJL mice, which have a defect in lambda 1 synthesis. A nucleotide change in C lambda 1 is genetically linked with the defect. Additional DNA cloning and RNA and protein studies are aimed at determining the exact basis of the defect. Furthermore, we are studying the mechanisms of allelic exclusion and V-C gene rearrangement by gene transfection experiments. Finally, we are trying to determine the basis for the very localized somatic mutations in V genes that are expressed. (AB)

DESCRIPTION (investigator's abstract): This application is for the renewal of two previous grants to study the regulation of B cell development. Our previous work has shown that the expression of Ig-gamma genes in developing B cells blocks their maturation and that substituting with the CH1 and transmembrane domains of mu Ig does not alleviate the block. It is planned to create and study another mu substitution and to investigate molecular/cellular events induced normally by signaling through a mu containing pre-B cell receptor. The expression of light chains is also tightly controlled in B cell development. Normally lambda1 light chains are in excess of lambda 2 and 3. In the SJL mouse strain, lambda1 expression is dramatically reduced. Previous findings suggest that a point mutation in the lambda1 constant region changing a glycine to a valine codon may be responsible. It is planned to knock-in a valine codon into a wildtype lambdal locus, If this change causes the defect, its molecular and cellular basis will be investigated by cell signaling experiments with various lambda1 mutant chains and by x-ray crystallographic comparison of the wildtype and SJL lambda light chains. Furthermore, based on unexpected results with a control serine knock-in mouse, the mechanism of hyper-activation of the lambda 1 locus by insertion of PGK-neo will be investigated. Understanding the development of B cells and its relationship to Ig gene rearrangement and Ig expression s of basic importance and clinical relevance. The regulation of Ig gene expression in B cells is one of the best studied systems of differentiation, Its complete unraveling will no doubt also give clues for the control of differentiation in general. On the clinical side, many immunological disorders involve B cells, such as autoimmunities, allergies, immunodeficiencies and, probably, susceptibility to cancer. Unraveling B cell development should help in understanding the basis of these diseases.

These experiments will study the genes responsible for certain T-lymphocyte-specific functions. These will include genes coding for T-cell receptors as well as for the cytolytic activity of T-killer cells. With the new findings by several laboratories on the proteins and genes that correspond to putative T-cell receptors, more defined questions about T-cell receptors can now be asked. We have begun a collaboration with Drs. R. Rich and J. Allison, University of Texas, to clone the receptor genes from a cytolytic T-cell line. The approach will be to produce cDNA clones that have been made T cell specific after elimination of cDNAs that hybridize with the RNA of B cells or other cells. The combined expertise in cellular immunology of Dr. Rich's laboratory and in immunochemistry of Dr. Allison's laboratory will allow the definitive functional testing of the T-cell receptors encoded by the genes we are about to clone. (SR)

Experiments are proposed to study early B cell development, particularly as it relates to the expression of immunoglobulin (Ig) genes. The following plans are described in this proposal: During the current grant period it was found that gamma2b heavy chain transgenes inhibit the development of B cells, because gamma2b causes strong feedback inhibition of H gene rearrangement, but cannot replace mu in nurturing preB cell maturation. The molecular and cellular basis of the roles of gamma2b and mu in B cell development will be studied by comparing gamma2b with mu in cell signaling; making new transgenic mice with gamma2b/mu hybrid genes to determine the domain of the mu molecule responsible for B cell maturation; comparing the numbers and distribution of apoptotic B cells in bone marrow of gamma2b transgenic and normal mice; crossing gamma2b transgenics with mice which express a Bcl-2 transgene in preB cells. While all gamma2b transgenic mice in several labs show the B cell defect, a unique gamma2b transgenic mouse line, the C line, arose in which mu is not required for preB cell development. Apparently, the transgene at its insertion site activates another gene whose function replaces mu in preB cell development. It is expected that this mouse line will provide novel insights into B cell development and the following experiments are proposed: the C line will be crossed with a RAG-1 knockout line to determine if T cells are required for early B cell development in the C line; the kinetics of pro/preB cell development, the levels of mRNAs important in early B cell development, and the rearrangement patterns of Ig genes will be compared with conventional gamma2b transgenic and normal mice; the molecules associated with gamma2b will be determined; feedback inhibition of VD and DJ rearrangement will be compared in C line and normal mice to determine if, as postulated, gamma2b has a strong heavy chain feedback effect; the V(D)J recombinase feedback inhibition by gamma2b/L chain will be compared to that by mu/L chain; the candidate gene postulated to be responsible for the phenotype of this mouse line will be cloned and characterized; new transgenic mice will be produced with the candidate gene to assess whether its expression under the control of the heavy chain enhancer can induce a C-phenotype; when a candidate gene has been proven to be responsible for the C phenotype, mice with homozygous deletion of this gene will be produced to determine if it is essential for B cell development. It is expected that these experiments will contribute to the understanding of the control of B cell development and Ig gene expression, and will provide new insights into immunological disorders with a B cell component.

The aim of this proposal is to define the molecular mechanism of somatic hypermutation of immunoglobulin (Ig) genes. We have recently obtained conclusive evidence that V-region specific mutation of functional Ig genes can occur without ongoing rearrangement and at random chromosomal sites (O'Brien et al., submitted). Therefore, a special mutator must exist which alters previously rearranged Ig genes. The proposed work has the goal to determined the mechanisms responsible for the mutations. Specifically the following experiments are planned: 1) Identification of stably transformed cell lines with ongoing somatic mutations of Ig genes: A source of cells with ongoing mutations is required for the analysis of the enzymology involved in the mutations and to speed up the search for essential accessory sequences outside of the mutation targets. 2) Determination of whether the mutations are really VJ region restricted in situations where C-region mutations cannot be selected against: We will determine the exact regional limits of the mutations by sequencing nonfunctional K transgenes and the nonproductive H and L alleles of several myelomas and hybridomas where the productive alleles are known to have mutations in the V region. If these experiments show that the mutations are restricted to the V region and vicinity we will construct a K gene where a C region is inserted into the middle of the V region (VCV), to determine whether it is location or sequence that determines that normally only V regions are mutated. 3) Accessory DNA sequences outside of the mutator target which are required for the mutations: Is a V gene alone sufficient or are Ig promoters and/or Ig enhancers required? 4) Mutations due to recombination by short stretches of sequence from multiple donor genes will be investigated in transgenic mice with two different, but related and experimentally, distinguishable K genes. Exchange of sequences will be studied and the relative frequencies of recombinational and other mutations will be compared. 5) Nuclear extracts of mutating cells will be analyzed for the presence of proteins which specifically bind to V-region DNA. In future experiments it may then be possible to purify such proteins and to reconstruct a cell- free mutation sytem.

This application proposes the continuation of studies on somatic hypermutation of immunoglobulin (Ig) genes. During the previous funding period a connection between somatiC hypermutation and initiation of transcription was found, leading to a model of transcription coupled repair initiated by a specific mutator factor. Decisive experiments to test this model are proposed, most of them based on the use of transgenic mice carrying various test transgenes. These studies are important for the understanding of the creation of the varied repertoire of variable Ig genes with the potential of reaCting against any foreign antigenic determinant, including perhaps tumor cell antigens. Furthermore, somatic hypermutation has been implicated in autoimmune diseases. It is likely that understanding the components involved in somatic mutation will aid in understanding the genetic and environmental causes of autoimmunity.

DESCRIPTION (Adapted from the Investigator's abstract): This application proposes to: 1) Analyze of a novel lymphoid specific transcription factor, Pip which is required for lambda gene expression and whose binding to the lambda enhancers depends on the interaction with the proto-oncogene encoded protein PU.l. The genomic organization and expression of Pip will be analyzed and Pip deficient mice will be generated. 2) Examine the relationship between light chain gene expression and interferons. The Pip protein sequence shows that it is a new member of the interferon response factor (IRF) family. The IRF gene most closely related to Pip is ICSBP, an inhibitor of other IRFs. ICSBP may compete with Pip in the interaction with the lambda enhancers and in so doing, regulate lambda gene expression. 3) To clone of the gene(s) encoding proteins that interact with the lambda A motif of the lambda enhancers. Two of the enhancers, LEI and LE2, are similar to DNA motifs found in the Ig kappa and heavy chain- enhancers. A third one, LB, has now been shown by us to bind PU.1 and Pip. The genes that encode factors that interact with the essential LA motif will be cloned. When the factors binding to all four motifs are known, it should be possible to understand how the expression of lambda is controlled and especially, how the silencing activity of the V lambda promoter is overcome. 4) analyze the lambda-1 defect int he SJL mouse strain. The low level of Lambda-1 containing lgs in the SJL mouse strain is most likely due to inefficient B-cell activation. Preliminary evidence has been obtained showing that the defect is caused by a point mutation in the constant region of Lambda-1 which changes a wildtype glycine to a valine. Transgenic mice that carry the SJL gene with either the gly or the val codon will be made to confirm this hypothesis. Future studies will investigate how this change of a single residue interferes with B-cell activation.

This application is for a new grant to study the mechanism of somatic hypermutation of immunoglobulin (Ig) genes. Our previous work has shown that this process depends on initiation of transcription. The new aims are directed at determining how transcription relates to the somatic mutation process, what the role is of the primary sequence which is the target for mutation and what the cis-acting regulatory elements consist of. Finally, we plan to clone and identify the gene(s) that encode a postulated mutator factor. These studies are important to understand the creation of the lg repertoire with the potential of reacting against any foreign antigen, including tumor antigens. Also, somatic hypermutation has been implicated in autoimmunities. Finally, certain malignant lymphoid tumors arise during the lg gene somatic mutation process and understanding its mechanism will shed light on the tumorigenesis and, hopefully, its prevention.

The process of somatic hypermutation (SHM) of immunoglobulin (Ig) genes takes place in B lymphocytes after specific interaction with antigen and T lymphocytes. SHM has several steps and requirements. In our working model, the cell needs to produce a postulated mutator factor (MuF) which has to be targeted to Ig genes by transcription. The MuF may act as an endonuclease that creates a nick or double-strand break in the gene. The nick/break is believed to be repaired in an error-prone fashion, creating mutations. Mismatch repair may remove some of the mutations or fix them by "correcting" the wildtype nucleotide on the complementary DNA strand. Cell replication propagates the mutations to one or both daughter cells. The following investigations are proposed: 1) What is the role of a specific E-box site in SHM? Such a transctivator site was found to enhance SHM without enhancing transcription. 2) How does the position of nucleosomes affect the SHM process? SHM occurs in clusters that suggest involvement of nucleosomes in the targeting of mutations. Mono-nucleosomal DNA from a mutable mouse transgene will be tested for phasing of nucleosomes over the SHM target relative to mutations. 3) Can a double-strand DNA break induce SHM? The possibility will be tested that introduction of a double strand break into a SHM target gene induces mutations without the need for the initiating events of SHM. 4) When during the cell cycle does SHM occur? It is still unknown if DNA replication of the genome is involved directly in SHM, and/or if recombination is required. Single cells will be isolated at various cell cycle stages and screened for molecular intermediates consistent with an ongoing mutation process. 5) Which mRNAs are modified by the cytidine deaminase, AID, in mutating B lymphocytes? The cytosine deaminase, AID, is required for SHM. The potential target mRNAs will be identified. The planned experiments are important for learning how the varied repertoire of Ig genes is created with the potential to react against any foreign antigenic determinant, including tumor cell antigens. Somatic hypermutation has also been implicated in autoimmune diseases. Furthermore, many B cell lymphomas arise apparently as a consequence of the somatic mutation process. It is likely that understanding the components involved in somatic mutation will aid in understanding the genetic and environmental causes of autoimmunity, and the treatment of infectious diseases and tumors. Surprisingly, the BCL6 proto-oncogene is highly mutated in human memory B cells. This is likely to be involved in tumorigenesis. Thus, a better understanding of the somatic mutation process may aid in the understanding and perhaps prevention, diagnosis and treatment of human B lymphomas related to BCL6 expression. PERFORMANCESITE(S) (organizationc,ity,state) The University of Chicago, Chicago, Illinois KEYPERSONNELS. eeinstructionsU. secontinuationpagesasneededtoprovidetherequiredinformatiointheformatshownbelow. StartwithPrincipalInvestigatorL. istallotherkeypersonneiln alphabeticaolrder,lastnamefirst. Name Organization RoleonProject Ursula B. Storb The University of Chicago Principal Investigator Randal Cox None Consultant Peter Engler The University of Chicago Research Associate Stephen Gasior The University of Chicago Research Associate Simone Longerich The University of Chicago Grad Res Assistant Nancy Michael The University of Chicago Research Associate Hong Ming Shen The University of Chicago Research Associate DisclosurePermission StatemenL ApplicabletoSBIR/STTROnly.Seeinstructions[.] Yes [] No [] PHS398(Rev.05/01) Page 2 FormPage2 [] _h,mhepr agesconsec,,tivela,,tthebottomtr_ro,,gho,t,hte applicationDonot,,ses,,ffi,,es,,chas"aa.,ah [] PrincipalInvestigator/ProgramDirector(Last,first, middle): Storb, Ursula The name of theprincipalinvestigator/programdirectormustbe providedatthetop of eachprintedpage andeachcontinuationpage. RESEARCH GRANT TABLE OF CONTENTS Page Numbers Face Page ............................................................................................................................................ 1 Description,

[unreadable] DESCRIPTION (provided by applicant): Despite the importance of CpG methylation and chromatin modifications in normal development and in disease, little is known about how methylation and chromatin patterns are established in mammals, and, specifically, how sequences are marked for inactivation. The investigator discovered a locus, Ssm1 (strain-specific modifier), on chromosome 4 of the mouse that has a major effect on the methylation and chromatin modification of a complex transgene, HRD, and certain derivatives. Methylation and inactivation occurs in a subset of mouse strains, including C57BL/6 (B6), but not other strains, including DBA/2 (D2). Methylation is dominant; (B6xD2) F1 mice methylate the target. [unreadable] [unreadable] Ssm1 is one of the very few mammalian loci shown to affect the methylation/chromatin status of specific target sequences. Determining how Ssm1 acts will significantly advance our understanding of epigenetic mechanisms in mammalian development and health. Ssm1 may be a member of a regulatory system to mark sequences for inactivation, involving complex interactions of allelic and non-allelic modifiers that are encoded by different as well as overlapping genes in different mouse strains and, by inference, different human individuals. The investigator has mapped Ssm1 to a narrow genomic interval on mouse chromosome 4 that is syntenic with human chromosome 1p36, a region involved in tumorigenesis and neurological defects. The defined genomic interval contains several potential Ssm1 candidates, all are KRAB-zinc finger genes that may encode proteins that bind their DNA target via the zinc finger, and recruit repressive proteins via the KRAB domain. She proposes to eliminate the B6 allele of Ssm1 candidates by gene replacement with a neo gene to identify Ssm1 as that gene whose elimination prevents inactivation of the HRD target. These experiments should elucidate how DNA methylation and chromatin modifications are regulated during early embryonic development and throughout life. Ssm1 orthologs in man may have clinical relevance, e.g., for epigenetic events in tumorigenesis and tumor progression, as well as for modification of MeCp2 function in neurological disorders. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The process of somatic hypermutation (SHM) of immunoglobulin genes is initiated by the cytidine deaminase AID creating cytidine (C) to uridine (U) transition mutations. Outside of SHM, U is faithfully repaired to C with the help of base excision repair (BER) and mismatch repair (MMR). During SHM, paradoxically, these repair mechanisms are recruited together with translesion DNA polymerases to create mutations at and near the U by error-prone repair. A critical hallmark of SHM is the restriction of the mutations to the 5'end of Ig genes, including the V(D)J region, but sparing the constant region. In this way, an enormous diversity of antigen-binding variable regions is created while protecting the biological functions of the antibody constant regions. We have considered two ways in which this V-region restriction may come about. First, AID may only be active in the 5'region, and second, AID may act throughout the gene but error-prone repair would only operate in the 5'region. In extensive experiments with mice that are defective in BER or MMR or both, we found the same pattern of mutations as in wild type mice, namely restriction to the variable region and its immediate flanks. This leads to the conclusion that AID does not act at the 3'portion of the Ig gene. We now wish to determine how exactly AID interacts with the sequences in which it deaminates Cs. Based on experiments where the constant region was mutated when the Ig gene promoter was duplicated in front of the constant region, we proposed the following model: AID associates with the transcription complex near the promoter, travels with the RNA polymerase during transcript elongation, deaminates Cs in single- stranded DNA arising in negative DNA supercoils behind the polymerase, and dissociates from the transcription complex within ~2kbp from the promoter due to limited affinity. We propose a novel and untried approach to test this model. The experiments will be carried out with a modified chicken B cell line, DT40 PseudoV-del, that suffers cytydine deaminations and subsequent SHM at a high rate. The modified line was produced by J.M. Buerstedde by deleting the pseudo V genes of the parent DT40 line so that the modified cells cannot carry out Ig gene conversion. In response to AID, the modified pseudoV-del DT40 cells undergo only SHM and do so at a high rate since AID- induced uridines are not directed toward gene conversion. They are therefore an ideal tool to study SHM. If the results of these exploratory experiments are clear, either supporting or rejecting the model of SHM described above, they will provide a platform and methods to unravel remaining questions, such as how AID is targeted only to Ig and some other genes, including some of known relevance to cancer and other diseases. PUBLIC HEALTH RELEVANCE: This is a proposal to study the hypermutation of antibody genes. This process is beneficial, because it can result in highly specific and efficient antibodies against pathogens and cancer. However, it is also dangerous, because it can cause cancer of lymphocytes and autoimmunity.