David Schatz - Academia.edu (original) (raw)

Papers by David Schatz

Nature Genetics, 2015

Cutaneous T cell lymphoma (CTCL) is a non-Hodgkin lymphoma of skin-homing T lymphocytes. We perfo... more Cutaneous T cell lymphoma (CTCL) is a non-Hodgkin lymphoma of skin-homing T lymphocytes. We performed exome and whole-genome DNA sequencing and RNA sequencing on purified CTCL and matched normal cells. The results implicate mutations in 17 genes in CTCL pathogenesis, including genes involved in T cell activation and apoptosis, NF-κB signaling, chromatin remodeling and DNA damage response. CTCL is distinctive in that somatic copy number variants (SCNVs) comprise 92% of all driver mutations (mean of 11.8 pathogenic SCNVs versus 1.0 somatic single-nucleotide variant per CTCL). These findings have implications for new therapeutics.

Molecular and cellular biology, 2007

The beyond 12/23 (B12/23) rule ensures inclusion of a Dbeta gene segment in the assembled T-cell ... more The beyond 12/23 (B12/23) rule ensures inclusion of a Dbeta gene segment in the assembled T-cell receptor (TCR) beta variable region exon and is manifest by a failure of direct Vbeta-to-Jbeta gene segment joining. The restriction is enforced during the DNA cleavage step of V(D)J recombination by the recombination-activating gene 1 and 2 (RAG1/2) proteins and the recombination signal sequences (RSSs) flanking the TCRbeta gene segments. Nothing is known about the step(s) at which DNA cleavage is defective or how TCRbeta locus sequences contribute to these defects. To address this, we examined the steps of DNA cleavage by the RAG proteins using TCRbeta locus V, D, and J RSS oligonucleotide substrates. The results demonstrate that the B12/23 rule is enforced through slow nicking of Jbeta substrates and to some extent through poor synapsis of Vbeta and Jbeta substrates. Nicking is controlled largely by the coding flank and, unexpectedly, the RSS spacer, while synapsis is controlled prima...

Proceedings of the National Academy of Sciences of the United States of America, Jan 23, 2015

The recombination-activating gene products, RAG1 and RAG2, initiate V(D)J recombination during ly... more The recombination-activating gene products, RAG1 and RAG2, initiate V(D)J recombination during lymphocyte development by cleaving DNA adjacent to conserved recombination signal sequences (RSSs). The reaction involves DNA binding, synapsis, and cleavage at two RSSs located on the same DNA molecule and results in the assembly of antigen receptor genes. We have developed single-molecule assays to examine RSS binding by RAG1/2 and their cofactor high-mobility group-box protein 1 (HMGB1) as they proceed through the steps of this reaction. These assays allowed us to observe in real time the individual molecular events of RAG-mediated cleavage. As a result, we are able to measure the binding statistics (dwell times) and binding energies of the initial RAG binding events and characterize synapse formation at the single-molecule level, yielding insights into the distribution of dwell times in the paired complex and the propensity for cleavage on forming the synapse. Interestingly, we find th...

Transgenic research, 2002

Telogen effluvium is a common type of hair loss. Although the morphological changes associated wi... more Telogen effluvium is a common type of hair loss. Although the morphological changes associated with telogen effluvium have been well characterized, the underlying molecular mechanisms remain unknown, and no animal models have been developed. We report here that inducible transgenic mice expressing high levels of the transcription factor, tTA (tetracycline transactivator), plus a reporter luciferase gene, show a reversible hair loss phenotype. Skin of these mice exhibits an increase in the number of hair follicles at the telogen phase, but a decreased number of follicles at the anagen phase. These changes resemble skin pathology seen in patients with telogen effluvium, which suggests that the inducible transgenic mice may be useful as a model for this disorder. Moreover, since overexpression of several other transgenes failed to cause skin pathology, the present findings also indicate types of molecular abnormalities that may cause reversible hair loss.

Molecular cell, 2000

During V(D)J recombination, the RAG1 and RAG2 proteins cooperate to catalyze a series of DNA bond... more During V(D)J recombination, the RAG1 and RAG2 proteins cooperate to catalyze a series of DNA bond breakage and strand transfer reactions. The structure, location, and number of active sites involved in RAG-mediated catalysis have as yet not been determined. Using protein secondary structure prediction algorithms, we have identified a region of RAG1 with possible structural similarities to the active site regions of transposases and retroviral integrases. Based on this information, we have identified two aspartic acid residues in RAG1 (D600 and D708) that function specifically in catalysis. The results support a model in which RAG1 contains a single, divalent metal ion binding active site structurally related to the active sites of transposases/integrases and responsible for all catalytic functions of the RAG protein complex.

Science (New York, N.Y.), Jan 22, 1990

The vast repertoire of immunoglobulins and T cell receptors is generated, in part, by V(D)J recom... more The vast repertoire of immunoglobulins and T cell receptors is generated, in part, by V(D)J recombination, a series of genomic rearrangements that occur specifically in developing lymphocytes. The recombination activating gene, RAG-1, which is a gene expressed exclusively in maturing lymphoid cells, was previously isolated. RAG-1 inefficiently induced V(D)J recombinase activity when transfected into fibroblasts, but cotransfection with an adjacent gene, RAG-2, has resulted in at least a 1000-fold increase in the frequency of recombination. The 2.1-kilobase RAG-2 complementary DNA encodes a putative protein of 527 amino acids whose sequence is unrelated to that of RAG-1. Like RAG-1, RAG-2 is conserved between species that carry out V(D)J recombination, and its expression pattern correlates precisely with that of V(D)J recombinase activity. In addition to being located just 8 kilobases apart, these convergently transcribed genes are unusual in that most, if not all, of their coding an...

Science (New York, N.Y.), Jan 16, 1991

The expression of the V(D)J [variable (diversity) joining elements] recombination activating gene... more The expression of the V(D)J [variable (diversity) joining elements] recombination activating genes, RAG-1 and RAG-2, has been examined during T cell development in the thymus. In situ hybridization to intact thymus and RNA blot analysis of isolated thymic subpopulations separated on the basis of T cell receptor (TCR) expression demonstrated that both TCR- and TCR+ cortical thymocytes express RAG-1 and RAG-2 messenger RNA's. Within the TCR+ population, RAG expression was observed in immature CD4+CD8+ (double positive) cells, but not in the more mature CD4+CD8- or CD4-CD8+ (single positive) subpopulations. Thus, although cortical thymocytes that bear TCR on their surface continue to express RAG-1 and RAG-2, it appears that the expression of both genes is normally terminated during subsequent thymic maturation. Since thymocyte maturation in vivo is thought to be regulated through the interaction of the TCR complex with self major histocompatibility complex (MHC) antigens, these dat...

V(D)J recombination is essential for generating a diverse array of B and T cell receptors that ca... more V(D)J recombination is essential for generating a diverse array of B and T cell receptors that can recognize and combat foreign antigens. As with any recombination event, tight control is essential to prevent the occurrence of genetic anomalies that drive cellular transformation. One important aspect of regulation is directed targeting of the RAG recombinase. Indeed, RAG accumulates at the 3 0 end of individual antigen receptor loci poised for rearrangement; however, it is not known whether focal binding is involved in regulating cleavage, and what mechanisms lead to enrichment of RAG in this region. Here, we show that monoallelic looping out of the 3 0 end of the T cell receptor a (Tcra) locus, coupled with transcription and increased chromatin/nuclear accessibility, is linked to focal RAG binding and ATM-mediated regulation of monoallelic cleavage on looped-out 3 0 regions. Our data identify higherorder loop formation as a key determinant of directed RAG targeting and the maintenance of genome stability.

Science, 2002

By creating an extremely diverse antibody repertoire, B cells protect the body against numerous i... more By creating an extremely diverse antibody repertoire, B cells protect the body against numerous infectious pathogens. Generation of this antibody repertoire depends on immunoglobulin gene modification events driven by four different molecular processes: V(D)J recombination, somatic hypermutation, class switch recombination, and gene conversion. The enzyme AID (activation-induced cytidine deaminase) is known to be involved in somatic hypermutation and class switch recombination. In their Perspective, [Fugmann and Schatz][1] explain that AID is also essential for gene conversion ([ Arakawa et al .][2]) and discuss how AID could operate in these three quite different processes. [1]: http://www.sciencemag.org/cgi/content/full/295/5558/1244 [2]: http://www.sciencemag.org/cgi/content/short/295/5558/1301

PLoS Biology, 2003

During lymphocyte development, V(D)J recombination assembles antigen receptor genes from componen... more During lymphocyte development, V(D)J recombination assembles antigen receptor genes from component V, D, and J gene segments. These gene segments are flanked by a recombination signal sequence (RSS), which serves as the binding site for the recombination machinery. The murine Jb2.6 gene segment is a recombinationally inactive pseudogene, but examination of its RSS reveals no obvious reason for its failure to recombine. Mutagenesis of the Jb2.6 RSS demonstrates that the sequences of the heptamer, nonamer, and spacer are all important. Strikingly, changes solely in the spacer sequence can result in dramatic differences in the level of recombination. The subsequent analysis of a library of more than 4,000 spacer variants revealed that spacer residues of particular functional importance are correlated with their degree of conservation. Biochemical assays indicate distinct cooperation between the spacer and heptamer/nonamer along each step of the reaction pathway. The results suggest that the spacer serves not only to ensure the appropriate distance between the heptamer and nonamer but also regulates RSS activity by providing additional RAG:RSS interaction surfaces. We conclude that while RSSs are defined by a ''digital'' requirement for absolutely conserved nucleotides, the quality of RSS function is determined in an ''analog'' manner by numerous complex interactions between the RAG proteins and the less-well conserved nucleotides in the heptamer, the nonamer, and, importantly, the spacer. Those modulatory effects are accurately predicted by a new computational algorithm for ''RSS information content.'' The interplay between such binary and multiplicative modes of interactions provides a general model for analyzing protein-DNA interactions in various biological systems.

Molecular Immunology, 2004

Previously, we described a tetracycline-based autoregulatory system for inducible gene expression... more Previously, we described a tetracycline-based autoregulatory system for inducible gene expression in mammalian cells and transgenic mice [Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 6522]. We have tested the ability of this system to drive functional expression in vivo of the V(D)J recombination activating genes, RAG1 and RAG2. In induced transgenic mice, transgenic RAG1 and RAG2 mRNA is observed in thymus and spleen, and expression of both transgenes on the RAG1 or RAG2 knockout backgrounds allows partial, inducible, lymphocyte reconstitution. In thymus and peripheral lymphoid organs of reconstituted animals, cells expressing CD4 and/or CD8 on their surface, also express CD3 and TCR beta chain. In these animals, V(D)J rearrangements are detected in thymus, lymph nodes, and spleen at the TRB locus, and in thymus and lymph nodes at the TRD locus. At the TRA locus, broken ends at V(D)J recombination signals are detected only in thymus, as are reciprocal signal joint products derived from deletional rearrangement. T cell reconstitution occurs in these animals whether they are induced in utero during development, or shortly after birth. A low level of B cell reconstitution is also observed. B220+IgM+ cells are observed in spleen only in induced animals, and rearrangements at IGH and IGK loci are detected in bone marrow and spleen. Broken signal ends at the IGK locus, are not detected in peripheral lymphoid organs. Inducible reconstitution of normal levels of serum immunoglobulin, including heavy chain class switch isotype variants is also observed in these animals. Further, these transgenes do not appear to interfere with lymphocyte development mediated by functionally rearranged TRB chain or IGH chain transgenes in RAG-deficient animals. These mice provide a unique system for the inducible activation of V(D)J recombination and the development of primary lymphocytes.

Journal of Experimental Medicine, 2006

It is thought that gene conversion (GCV) and somatic hypermutation (SHM) of immunoglobulin (Ig) g... more 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...

International Immunology, 2004

Class switch recombination (CSR) is the process whereby B cells alter the effector properties of ... more Class switch recombination (CSR) is the process whereby B cells alter the effector properties of their Ig molecules. Whilst much is known about the cellular regulation of this process, many of the molecular details remain elusive. Recent evidence suggests that CSR involves blunt DNA double strand breaks (dsbs), and that formation of these dsbs requires the function of the activationinduced cytidine deaminase (AID). We sought to characterize the structural properties and kinetics of induction of the DNA lesions associated with CSR. Using ligation-mediated PCR, we found that AID-dependent DNA dsbs were speci®cally induced in the Sm region of murine B cells stimulated to undergo CSR. While blunt dsbs were detected, they were only a minor species, with staggered breaks being more than an order of magnitude more abundant. In addition, these breaks could be detected at equal frequency at upstream and downstream portions of Sm, and were induced prior to expression of newly switched isotypes. Collectively, these results provide direct evidence that staggered, Sm-targeted AID-dependent dsbs are the predominant DNA lesion associated with CSR, with important implications for the mechanisms by which CSR DNA lesions are made and processed.

Immunity, 2003

V(D)J recombination occurs efficiently only between gene segments flanked by recombination signal... more V(D)J recombination occurs efficiently only between gene segments flanked by recombination signals (RSs) containing 12 and 23 base pair spacers (the 12/23 rule). A further limitation &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;beyond the 12/23 rule&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot; (B12/23) exists at the TCRbeta locus and ensures Dbeta usage. Herein, we show that extrachromosomal V(D)J recombination substrates recapitulate B12/23 restriction in nonlymphoid cells. We further demonstrate that the Vbeta coding flank, the 12-RS heptamer/nonamer, and the 23-RS spacer each can significantly influence B12/23 restriction. Finally, purified core RAG1 and RAG2 proteins (together with HMG2) also reproduce B12/23 restriction in a cell-free system. Our findings indicate that B12/23 restriction of V(D)J recombination is cemented at the level of interactions between the RAG proteins and TCRbeta RS sequences.

European Journal of Immunology, 2004

Class switch recombination (CSR), somatic hypermutation, and gene conversion are immunoglobulin d... more 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.

Current Opinion in Immunology, 2011

Activation-induced cytidine deaminase (AID) is a key enzyme for antibody-mediated immune response... more Activation-induced cytidine deaminase (AID) is a key enzyme for antibody-mediated immune responses. Antibodies are encoded by the immunoglobulin genes and AID acts as a transcription-dependent DNA mutator on these genes to improve antibody affinity and effector functions. An emerging theme in field is that many transcribed genes are potential targets of AID, presenting an obvious danger to genomic integrity. Thus there are mechanisms in place to ensure that mutagenic outcomes of AID activity are specifically restricted to the immunoglobulin loci. Cis-regulatory targeting elements mediate this effect and their mode of action is probably a combination of immunoglobulin gene specific activation of AID and a perversion of faithful DNA repair towards error-prone outcomes.