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David G Schatz

Publications and source records attributed to David G Schatz.

At least 19 recordsLinked to original sources
PubMed2006

Control of gene conversion and somatic hypermutation by immunoglobulin promoter and enhancer sequences.

It is thought that gene conversion (GCV) and somatic hypermutation (SHM) of immunoglobulin (Ig) genes occur in two steps: the generation of uracils in DNA by activation-induced cytidine deaminase, followed by their subsequent repair by various DNA repair pathways to generate sequence-diversified products. It is not known how either of the two steps is targeted specifically to Ig loci. Because of the tight link between transcription and SHM, we have investigated the role of endogenous Ig light chain (IgL) transcriptional control elements in GCV/SHM in the chicken B cell line DT40. Promoter substitution experiments led to identification of a strong RNA polymerase II promoter incapable of supporting efficient GCV/SHM. This surprising finding indicates that high levels of transcription are not sufficient for robust GCV/SHM in Ig loci. Deletion of the IgL enhancer in a context in which high-level transcription was not compromised showed that the enhancer is not necessary for GCV/SHM. Our results indicate that cis-acting elements are important for Ig gene diversification, and we propose that targeting specificity is achieved through the combined action of several Ig locus elements that include the promoter.

Actins↗
PubMed2006

Roles of the Ig kappa light chain intronic and 3' enhancers in Igk somatic hypermutation.

Somatic hypermutation (SHM) of the rearranged Ig genes is required for the affinity maturation of Abs. SHM is almost exclusively targeted to the rearranged Ig loci, but the mechanism of this gene-specific targeting remains unclear. The Ig kappa L chain locus contains multiple enhancers, including the MAR/intronic (iE(kappa)) and 3' enhancers (3'E(kappa)). Previous transgenic studies indicate that both kappa enhancers are individually necessary for SHM of Igk. In contrast, later studies of Ag-selected V(kappa) genes in 3'E(kappa)(-/-) mice found no absolute requirement for 3'E(kappa) in kappa SHM. To address the roles of the two kappa enhancers in SHM in a physiological context, we analyzed SHM of the endogenous Igk in mice with a targeted deletion of either iE(kappa) or 3'E(kappa) in Peyer's patch germinal center B cells. Our findings indicate that, although 3'E(kappa) is quantitatively important for SHM of Igk, iE(kappa) is not required for kappa SHM. In addition, a reduction of kappa mRNA levels is also detected in activated 3'E(kappa)(-/-) B cells. These findings suggest that iE(kappa) and 3'E(kappa) play distinct roles in regulating Igk transcription and SHM.

3' Untranslated Regions↗
PubMed2006

AID and Igh switch region-Myc chromosomal translocations.

Chromosomal translocations involving Ig heavy chain switch regions and an oncogene, like Myc, represent early initiating events in the development of many B cell malignancies. These translocations are widely believed to result from aberrant class switch recombination (CSR). Recent reports have produced conflicting models for the role of activation-induced cytidine deaminase (AID) in this process. Here, we discuss possible roles of AID, CSR, and somatic hypermutation in generating chromosomal translocations and in tumor progression.

Animals↗
PubMed2006

Origins of peripheral B cells in IL-7 receptor-deficient mice.

The interleukin 7 (IL-7) signaling pathway is critical for early lymphoid differentiation. We found dramatic perturbations in fetal liver B cell development and confirmed a complete absence of developing B cells in the adult bone marrow in mice lacking the IL-7 receptor alpha (IL-7Ralpha) gene. We show that peripheral B-2 and B-1 cell populations are deficient in IL-7Ralpha-/- mice. B-2 follicular cell and peritoneal B-1 cell percentages are reduced, while B-2 marginal zone cell percentages are increased. A comparison of bone marrow and splenic populations at different ages revealed that the splenic B cell populations seen in adult IL-7Ralpha-/- mice first appear during neonatal development. We have measured N-nucleotide addition at the joints of V(D)J rearrangements in splenic B cells and have used it as a somatic marker to define and separate bone marrow-derived B cells from fetal liver-derived B cells. B cells isolated from the bone marrow and spleen of adult and neonatal IL-7Ralpha-deficient mice harbor high levels of N-nucleotide additions similar to those found in equivalent wild-type B cell populations. We conclude that the majority of splenic B cells in IL-7Ralpha-deficient mice originate from the bone marrow and not the fetal liver.

Age Factors↗
PubMed2006

Targeting of somatic hypermutation.

Somatic hypermutation (SHM) introduces mutations in the variable region of immunoglobulin genes at a rate of approximately 10(-3) mutations per base pair per cell division, which is 10(6)-fold higher than the spontaneous mutation rate in somatic cells. To ensure genomic integrity, SHM needs to be targeted specifically to immunoglobulin genes. The rare mistargeting of SHM can result in mutations and translocations in oncogenes, and is thought to contribute to the development of B-cell malignancies. Despite years of intensive investigation, the mechanism of SHM targeting is still unclear. We review and attempt to reconcile the numerous and sometimes conflicting studies on the targeting of SHM to immunoglobulin loci, and highlight areas that hold promise for further investigation.

Animals↗
PubMed2006

Mobilization of RAG-generated signal ends by transposition and insertion in vivo.

In addition to their essential roles in V(D)J recombination, the RAG proteins have been found to catalyze transposition in vitro, but it has been difficult to demonstrate transposition by the RAG proteins in vivo in vertebrate cells. As genomic instability and chromosomal translocations are common outcomes of transposition in other species, it is critical to understand if the RAG proteins behave as a transposase in vertebrate cells. To facilitate this, we have developed an episome-based assay to detect products of RAG-mediated transposition in the human embryonic kidney cell line 293T. Transposition events into the target episome, accompanied by characteristic target site duplications, were detected at a low frequency using RAG1 and either truncated "core" RAG2 or full-length RAG2. More frequently, insertion of the RAG-generated signal end fragment into the target was accompanied by deletions or more complex rearrangements, and our data indicate that these events occur by a mechanism that is distinct from transposition. An assay to detect transposition from an episome into the human genome failed to detect bona fide transposition events but instead yielded chromosome deletion and translocation events involving the signal end fragment mobilized by the RAG proteins. These assays provide a means of assessing RAG-mediated transposition in vivo, and our findings provide insight into the potential for the products of RAG-mediated DNA cleavage to cause genome instability.

Base Sequence↗
PubMed2005

Expression of activation-induced cytidine deaminase is regulated by cell division, providing a mechanistic basis for division-linked class switch recombination.

Class switch recombination (CSR) is the process by which B cells alter the effector function properties of their Ig molecules. The decision to switch to a particular Ig isotype is determined primarily by the mode of B cell activation and cytokine exposure. More recent work indicates that the likelihood or probability of switching increases with successive cell divisions and is largely independent of time. We have analyzed different molecular features of CSR using cell division as a reference point in an attempt to gain insight into the mechanism of division-linked switching. Our results indicated that the accessibility of Ig heavy chain constant regions targeted for CSR was established after the cells had undergone a single cell division and did not vary significantly with subsequent cell divisions. In contrast, expression of activation-induced cytidine deaminase (AID) mRNA was found to increase with successive divisions, exhibiting a striking correlation with the frequency of CSR. Levels of AID in a given division remained constant at different time points, strongly suggesting that the regulation of AID expression was division-linked and independent of time. In addition, constitutive AID expression from a transgene accelerated division-linked CSR. Thus, we propose that the division-linked increase in AID expression provides an underlying molecular explanation for division-linked CSR.

Animals↗
PubMed2005

Histone modifications associated with somatic hypermutation.

A number of modified histones, including acetylated H3 and H4 and phosphorylated H2AX (gammaH2AX), are associated with V(D)J recombination and class switch recombination (CSR). In contrast, little is known concerning the chromatin modifications associated with somatic hypermutation (SHM) in vivo. Here, we report that several modifications--including histone acetylation and H3-lysine 4 methylation--fail to demarcate an actively hypermutating immunoglobulin (Ig) locus or to correlate spatially with SHM within Ig loci. Furthermore, no obvious association between SHM and gammaH2AX could be detected. Instead, we find that the phosphorylated form of histone H2B (H2B(Ser14P)) correlates tightly with SHM and CSR. Phosphorylation of H2B within Ig variable and switch regions requires AID and may be mediated by the histone kinase Mst1. These findings indicate that SHM and CSR trigger distinct DNA damage responses and identify a novel histone modification pattern for SHM consisting of H2B(Ser14P) in the absence of gammaH2AX.

Acetylation↗
PubMed2005

B cells and osteoblast and osteoclast development.

The molecules that regulate bone cell development, particularly at the early stages of development, are only partially known. Data are accumulating that indicate a complex relationship exists between B cells and bone cell differentiation. Although the exact nature of this relationship is still evolving, it takes at least two forms. First, factors that regulate B-cell growth and development have striking effects on osteoclast and osteoblast lineage cells. Similarly, factors that regulate bone cell development influence B-cell maturation. Second, a series of transcription factors required for B-cell differentiation have been identified, and these factors function in a developmentally ordered circuit. These transcription factors have unpredicted, pronounced, and non-overlapping effects on osteoblast and/or osteoclast development. These data indicate that at least a regulatory relationship exists between B lymphopoiesis, osteoclastogenesis, and osteoblastogenesis.

Animals↗
PubMed2005

Radiosensitization of MDA-MB-231 breast tumor cells by adenovirus-mediated overexpression of a fragment of the XRCC4 protein.

Incomplete DNA repair or misrepair can contribute to the cytotoxicity of DNA double-strand breaks. Consequently, interference with double-strand break repair, by pharmacologic or genetic means, is likely to sensitize tumor cells to ionizing radiation. The current studies were designed to inhibit the nonhomologous end joining repair pathway by interfering with the function of the XRCC4/ligase IV complex. A PCR-generated fragment of the XRCC4 gene, encompassing the homodimerization and ligase IV-binding domains, was inserted into a plasmid vector (pFLAG-CMV-2) expressing the FLAG peptide and the cassette encoding FLAG-tagged XRCC4 fragment was cloned into an adenoviral vector. Both the plasmid and the corresponding adenovirus elicited robust expression of a truncated XRCC4 protein designed to compete in a dominant-negative fashion with full-length XRCC4 for binding to ligase IV. Binding of the XRCC4 fragment to ligase IV in vivo was confirmed by immunoprecipitation. Clonogenic survival assays showed that the adenovirus expressing the truncated XRCC4 protein sensitizes MDA-MB-231 breast tumor cells to ionizing radiation, presumably through interference with the functional activity of ligase IV, leading to inhibition of the final ligation step in end joining. These studies support the potential clinical utility of combining radiation therapy with agents that inhibit DNA double-strand break repair.

Adenoviridae↗
PubMed2004

Pax5-deficient mice exhibit early onset osteopenia with increased osteoclast progenitors.

Pax5 encodes BSAP, a member of the paired box domain transcription factors, whose expression is restricted to B lymphocyte lineage cells. Pax5(-/-) mice have a developmental arrest of the B cell lineage at the pro-B cell stage. We show here that Pax5(-/-) mice are severely osteopenic, missing 60% of their bone mass. The osteopenia can be accounted for by a >100% increase in the number of osteoclasts in bone measured histomorphometrically. This is not due to a lack of B cells, because other strains of B cell-deficient mice do not exhibit this phenotype. There was no difference in the number of osteoclasts produced in vitro by wild-type and Pax5(-/-) bone marrow cells. In contrast, spleen cells from Pax5(-/-) mice produce as much as five times the number of osteoclasts as control spleen cells. Culture of Pax5(-/-) spleen cells yields a population of adherent cells that grow spontaneously in culture without added growth factors for >4 wk. These cells have a monocyte phenotype, produce large numbers of osteoclasts when induced in vitro, and therefore are highly enriched in osteoclast precursors. These data demonstrate a previously unsuspected connection between B cell and osteoclast development and a key role for Pax5 in the control of osteoclast development.

Animals↗
PubMed2004

Identification of an AID-independent pathway for chromosomal translocations between the Igh switch region and Myc.

Chromosomal translocations involving immunoglobulin heavy chain (Igh) switch regions and an oncogene such as Myc represent initiating events in the development of many B cell malignancies. These translocations are widely thought to result from aberrant class-switch recombination. To test this model, we measured translocations in mice deficient in activation-induced cytidine deaminase (AID) that lack class-switch recombination. We found that AID made no measurable contribution to the generation of initial translocations, indicating that the intrinsic fragility of the switch regions or a pathway unrelated to AID is responsible for these translocations. In contrast, the outgrowth of translocation-positive cells was dependent on AID, raising the possibility that AID is important in tumor progression, perhaps by virtue of its mutagenic properties.

Animals↗
PubMed2004

B cell-specific loss of histone 3 lysine 9 methylation in the V(H) locus depends on Pax5.

Immunoglobulin heavy chain rearrangement (V(H)-to-DJ(H)) occurs only in B cells, suggesting it is inhibited in other lineages. Here we found that in the mouse V(H) locus, methylation of lysine 9 on histone H3 (H3-K9), a mark of inactive chromatin, was present in non-B lineage cells but was absent in B cells. As others have shown that H3-K9 methylation can inhibit V(D)J recombination on engineered substrates, our data support the idea that H3-K9 methylation inhibits endogenous V(H)-to-DJ(H) recombination. We also show that Pax5, a transcription factor required for B cell commitment, is necessary and sufficient for the removal of H3-K9 methylation in the V(H) locus and provide evidence that one function of Pax5 is to remove this inhibitory modification by a mechanism of histone exchange, thus allowing B cell-specific V(H)-to-DJ(H) recombination.

Animals↗
PubMed2004

Mutational analysis of terminal deoxynucleotidyltransferase-mediated N-nucleotide addition in V(D)J recombination.

The addition of nontemplated (N) nucleotides to coding ends in V(D)J recombination is the result of the action of a unique DNA polymerase, TdT. Although N-nucleotide addition by TdT plays a critical role in the generation of a diverse repertoire of Ag receptor genes, the mechanism by which TdT acts remains unclear. We conducted a structure-function analysis of the murine TdT protein to determine the roles of individual structural motifs that have been implicated in protein-protein and protein-DNA interactions important for TdT function in vivo. This analysis demonstrates that the N-terminal portion of TdT, including the BRCA-1 C-terminal (BRCT) domain, is not required for TdT activity, although the BRCT domain clearly contributes quantitatively to N-nucleotide addition activity. The second helix-hairpin-helix domain of TdT, but not the first, is required for activity. Deletional analysis also suggested that the entire C-terminal region of TdT is necessary for N-nucleotide addition in vivo. The long isoform of TdT was found to reduce N-nucleotide addition by the short form of TdT, but did not increase nucleotide deletion from coding ends in either human or rodent nonlymphoid cells. We consider these results in light of the recently reported structure of the catalytic region of TdT.

Animals↗
PubMed2004

Up-regulation of Hlx in immature Th cells induces IFN-gamma expression.

Differentiation of Th cells from naive precursors is a dynamic process that involves multiple transcription factors acting at specific time points to regulate gene expression. In this study we show that the homeobox transcription factor Hlx is up-regulated early in Th1 cell differentiation. Mice constitutively expressing an Hlx transgene driven by a CD4 promoter showed marked reduction in the CD4+CD8+ thymocyte population. The Hlx transgenic mice generated increased numbers of Th1 cells in response to keyhole limpet hemocyanin immunization. After differentiation under Th2-polarizing conditions in vitro, the transgenic CD4 T cells expressed high levels of IFN-gamma. Intracellular cytokine staining revealed that in addition to Th2 cells, large numbers of Th0 and Th1 cells were generated from such in vitro differentiated transgenic CD4 T cells. Retrovirally overexpressed Hlx also induced the aberrant expression of IFN-gamma in normal CD4 T cells differentiated under Th2-polarizing conditions. This effect was apparent only when Hlx was introduced into the cells by retroviral infection at an early time point that led to the expression of the retrovirally transferred Hlx gene at a time comparable to that of the up-regulation of the endogenous Hlx during Th1 cell differentiation. Later infection with Hlx-expressing retrovirus showed no effect. Thus, the induction of IFN-gamma expression by Hlx depends on a permissive epigenetic state of the IFN-gamma gene locus and/or the molecular context of the immature Th cells.

Adjuvants, Immunologic↗
PubMed2004

Non-redundancy of cytidine deaminases in class switch recombination.

Class switch recombination (CSR), somatic hypermutation, and gene conversion are immunoglobulin diversification mechanisms that are strictly dependent on the activity of the activation-induced cytidine deaminase (AID). The precise role and substrate(s) of AID in these processes remain to be well defined. The closest homologue of AID is APOBEC-1, a bona fide mRNA-editing enzyme, which shares with AID the ability to deaminate cytidines within single-stranded DNA in vitro and in prokaryotic cells. To determine whether APOBEC-1 can therefore substitute for AID in activated B cells, we expressed human AID, a catalytic mutant thereof, and rat APOBEC-1 in AID-deficient murine B cells. Whereas AID rescued CSR, neither the inactive mutant nor APOBEC-1 could complement AID deficiency. This indicates that cytidine deaminase activity is necessary but not sufficient to initiate CSR, and suggests that AID is specifically targeted to its cognate substrate, the immunoglobulin genes or a distinct mRNA, by an as-yet-unknown mechanism.

APOBEC-1 Deaminase↗