Loss of function of the mouse Sharpin gene results in Peyer's patch regression.

Peyer's patches (PP) are an important component in the immune response against intestinal pathogens. Two independent, spontaneous mutations in the mouse Sharpin gene (Sharpin(cpdm) and Sharpin(cpdm-Dem)) result in the absence of PP and disrupted splenic white pulp in adult mice, although a full complement of lymph nodes is present. Here we report that rudimentary PP begin to develop in Sharpin(cpdm) mice during embryogenesis, but lack the organizational patterns that are typical of this tissue. In the present study, small intestines examined at weekly intervals from birth to maturity showed spontaneous regression of PP in mutant mice with concurrent infiltration of granulocytes. At 5 to 6 weeks of age, only indistinct remnants of granulocytic accumulations remain. Transplantation of normal bone marrow into Sharpin(cpdm) mice at 7 days of age did not prevent regression of PP in bone marrow chimeras examined at 7 to 8 weeks of age. These findings indicate that SHARPIN expression is required for the normal development and maintenance, but not initiation, of PP.

Exclusion of the uniform tetraploid cells significantly improves specificity of the urine FISH assay.

The urine fluorescence in situ hybridization (FISH) assay (UroVysion™), with the current scoring criteria, has a higher sensitivity than routine cytopathology but a lower specificity. Among 215 urine FISH tests we performed, 45 had associated histopathology and clinical follow up. In this study, a cell with four signals for each probe was classified as a uniform tetraploid cell (UTC); a presumed reparative cell which is currently classified as an abnormal cell in the FDA approved assay. By using the existing criteria, the tests were scored as positive or negative before and after exclusion of the UTCs. Before the exclusion, 24 positive, 13 negative, seven false positive, and one false negative result were obtained with 96% sensitivity and 65% specificity. After the exclusion, the results changed to 22 positive, 19 negative, one false positive, and three false negatives resulting in a 88% sensitivity of 88% and a 95% specificity; a significant improvement in the specificity. We conclude that exclusion of the UTCs as abnormal cells would result in a more solid performance of the FISH assay.

Complex oncogenic translocations with gene amplification are initiated by specific DNA breaks in lymphocytes.

Chromosomal instability is a hallmark of many tumor types. Complex chromosomal rearrangements with associated gene amplification, known as complicons, characterize many hematologic and solid cancers. Whereas chromosomal aberrations, including complicons, are useful diagnostic and prognostic cancer markers, their molecular origins are not known. Although accumulating evidence has implicated DNA double-strand break repair in suppression of oncogenic genome instability, the genomic elements required for chromosome rearrangements, especially complex lesions, have not been elucidated. Using a mouse model of B-lineage lymphoma, characterized by complicon formation involving the immunoglobulin heavy chain (Igh) locus and the c-myc oncogene, we have now investigated the requirement for specific genomic segments as donors for complex rearrangements. We now show that specific DNA double-strand breaks, occurring within a narrow segment of Igh, are necessary to initiate complicon formation. By contrast, neither specific DNA breaks nor the powerful intronic enhancer Emu are required for complicon-independent oncogenesis. This study is the first to delineate mechanisms of complex versus simple instability and the first to identify specific chromosomal elements required for complex chromosomal aberrations. These findings will illuminate genomic cancer susceptibility and risk factors.

Homologous recombination is necessary for normal lymphocyte development.

Primary immunodeficiencies are rare but serious diseases with diverse genetic causes. Accumulating evidence suggests that defects in DNA double-strand break (DSB) repair can underlie many of these syndromes. In this context, the nonhomologous end joining pathway of DSB repair is absolutely required for lymphoid development, but possible roles for the homologous recombination (HR) pathway have remained more controversial. While recent evidence suggests that HR may indeed be important to suppress lymphoid transformation, the specific relationship of HR to normal lymphocyte development remains unclear. We have investigated roles of the X-ray cross-complementing 2 (Xrcc2) HR gene in lymphocyte development. We show that HR is critical for normal B-cell development, with Xrcc2 nullizygosity leading to p53-dependent early S-phase arrest. In the absence of p53 (encoded by Trp53), Xrcc2-null B cells can fully develop but show high rates of chromosome and chromatid fragmentation. We present a molecular model wherein Xrcc2 is important to preserve or restore replication forks during rapid clonal expansion of developing lymphocytes. Our findings demonstrate a key role for HR in lymphoid development and suggest that Xrcc2 defects could underlie some human primary immunodeficiencies.

Chromosome neighborhood composition determines translocation outcomes after exposure to high-dose radiation in primary cells.

Radiation exposure is an occupational hazard for military personnel, some health care professionals, airport security screeners, and medical patients, with some individuals at risk for acute, high-dose exposures. Therefore, the biological effects of radiation, especially the potential for chromosome damage, are major occupational and health concerns. However, the biophysical mechanisms of chromosome instability subsequent to radiation-induced DNA damage are poorly understood. It is clear that interphase chromosomes occupy discrete structural and functional subnuclear domains, termed chromosome territories (CT), which may be organized into 'neighborhoods' comprising groups of specific CTs. We directly evaluated the relationship between chromosome positioning, neighborhood composition, and translocation partner choice in primary lymphocytes, using a cell-based system in which we could induce multiple, concentrated DNA breaks via high-dose irradiation. We critically evaluated mis-rejoining profiles and tested whether breaks occurring nearby were more likely to fuse than breaks occurring at a distance. We show that CT neighborhoods comprise heterologous chromosomes, within which inter-CT distances directly relate to translocation partner choice. These findings demonstrate that interphase chromosome arrangement is a principal factor in genomic instability outcomes in primary lymphocytes, providing a structural context for understanding the biological effects of radiation exposure, and the molecular etiology of tumor-specific translocation patterns.

Novel Sxr(a) ES cell line offers hope for Y chromosome gene-targeted mice.

A mouse targeted for a Y Chromosome gene has not been reported. Because the Y Chromosome is present in only one copy, and most of its genes are critical for germ cell development, such a mouse would likely be infertile. Thus, we describe a new reproductive strategy to enable transmission of targeted Y Chromosome genes to subsequent generations. The strategy uses two segregating copies of Y Chromosome genes to mimic the autosomal condition. To achieve this, we developed a new embryonic stem cell line from the XYSxr(a) mouse, which carries a duplication of the gene-rich Y Chromosome short arm. Importantly, we demonstrate germ line transmission of the YSxr(a) chromosome and describe this significant new tool as a practical solution to enable reproduction in mice targeted for Y Chromosome genes.