Telomere dysfunction impairs DNA repair and enhances sensitivity to ionizing radiation

Telomeres are specialized nucleoprotein complexes that serve as protective caps of linear eukaryotic chromosomes. Loss of telomere function is associated with rampant genetic instability and loss of cellular viability and renewal potential. The telomere also participates in processes of chromosomal repair, as evidenced by the 'capture' or de novo synthesis of telomere repeats at double-stranded breaks and by the capacity of yeast telomeres to serve as repositories of essential components of the DNA repair machinery, particularly those involved in non-homologous end-joining (NHEJ). Here we used the telomerase-deficient mouse, null for the essential telomerase RNA gene (Terc), to assess the role of telomerase and telomere function on the cellular and organismal response to ionizing radiation. Although the loss of telomerase activity per se had no discernable impact on the response to ionizing radiation, the emergence of telomere dysfunction in late-generation Terc-/- mice imparted a radiosensitivity syndrome associated with accelerated mortality. On the cellular level, the gastrointestinal crypt stem cells and primary thymocytes showed increased rates of apoptosis, and mouse embryonic fibroblasts (MEFs) showed diminished dose-dependent clonogenic survival. The radiosensitivity of telomere dysfunctional cells correlated with delayed DNA break repair kinetics, persistent chromosomal breaks and cytogenetic profiles characterized by complex chromosomal aberrations and massive fragmentation. Our findings establish a intimate relationship between functionally intact telomeres and the genomic, cellular and organismal response to ionizing radiation.     

Construction of a mouse yeast artificial chromosome library in a recombination-deficient strain of yeast.

We have constructed a new generation yeast artificial chromosome (YAC) library from female C57BL/10 mice in a recombination-deficient strain of Saccharomyces cerevisiae carrying a mutation in the RAD52 gene. The YAC library contains 41,568 clones with an average insert size of 240 kilobases, representing a greater than threefold coverage of the mouse genome. Currently, the library can be screened by polymerase chain reaction and we have isolated positive clones at a number of loci in the mouse genome. This rad52 library should enable a long-term assessment of the effect of one of the yeast recombination pathway genes on both, genome-wide YAC clone stability and the frequency of chimaeric clones.     

Systematic screen for human disease genes in yeast

High similarity between yeast and human mitochondria allows functional genomic study of Saccharomyces cerevisiae to be used to identify human genes involved in disease. So far, 102 heritable disorders have been attributed to defects in a quarter of the known nuclear-encoded mitochondrial proteins in humans. Many mitochondrial diseases remain unexplained, however, in part because only 40-60% of the presumed 700-1,000 proteins involved in mitochondrial function and biogenesis have been identified. Here we apply a systematic functional screen using the pre-existing whole-genome pool of yeast deletion mutants to identify mitochondrial proteins. Three million measurements of strain fitness identified 466 genes whose deletions impaired mitochondrial respiration, of which 265 were new. Our approach gave higher selection than other systematic approaches, including fivefold greater selection than gene expression analysis. To apply these advantages to human disorders involving mitochondria, human orthologs were identified and linked to heritable diseases using genomic map positions.     

A double-stranded RNA binding protein required for activation of repressed messages in mammalian germ cells.

Chromatin packaging in mammalian spermatozoa requires an ordered replacement of the somatic histones by two classes of spermatid-specific basic proteins, the transition proteins and the protamines. Temporal expression of transition proteins and protamines during spermatid differentiation is under translational control, and premature translation of protamine 1 leads to precocious nuclear condensation and sterility. We have previously suggested that the double-stranded (ds) RNA binding protein Prbp (encoded by the gene Tarbp2) functions as a translational regulator during mouse spermatogenesis. Here we show that Prbp is required for proper translational activation of the mRNAs encoding the protamines. We generated mice that carry a targeted disruption of Tarbp2 and determined that they were sterile and severely oligospermic. Using immunohistological analysis, we determined that the endogenous Prm2 mRNA and a reporter mRNA carrying protamine 1 translational-control elements were translated in a mosaic pattern. We showed that failure to synthesize the protamines resulted in delayed replacement of the transition proteins and subsequent failure of spermiation. The timing of Prbp expression suggests that it may function as a chaperone in the assembly of specific translationally regulated ribonucleoprotein particles.     

Dax1 is required for testis determination

The orphan nuclear receptor, Dax1, was originally proposed to act as an 'anti-testis' factor. We find, however, that Nr0b1 (also called Dax1 and Ahch, which encodes Dax1) is in fact required for testis differentiation.     

AID is required for germinal center-derived lymphomagenesis

Most human B cell non-Hodgkin's lymphomas (B-NHLs) derive from germinal centers (GCs), the structure in which B cells undergo somatic hypermutation (SHM) and class switch recombination (CSR) before being selected for high-affinity antibody production. The pathogenesis of B-NHL is associated with distinct genetic lesions, including chromosomal translocations and aberrant SHM, which arise from mistakes occurring during CSR and SHM. A direct link between these DNA remodeling events and GC lymphoma development, however, has not been demonstrated. Here we have crossed three mouse models of B cell lymphoma driven by oncogenes (Myc, Bcl6 and Myc/Bcl6; refs. 5,6) with mice lacking activation-induced cytidine deaminase (AID), the enzyme required for both CSR and SHM. We show that AID deficiency prevents Bcl6-dependent, GC-derived B-NHL, but has no impact on Myc-driven, pre-GC lymphomas. Accordingly, abrogation of AID is associated with the disappearance of CSR- and SHM-mediated structural alterations. These results show that AID is required for GC-derived lymphomagenesis, supporting the notion that errors in AID-mediated antigen-receptor gene modification processes are principal contributors to the pathogenesis of human B-NHL.     

The SNRPN promoter is not required for genomic imprinting of the Prader-Willi/Angelman domain in mice.

In mice and humans, the locus encoding the gene for small nuclear ribonucleoprotein N (SNRPN/Snrpn), as well as other loci in the region are subject to genomic imprinting. The SNRPN promoter is embedded in a maternally methylated CpG island, is expressed only from the paternal chromosome and lies within an imprinting center that is required for switching to and/or maintenance of the paternal epigenotype. We show here that a 0.9-kb deletion of exon 1 of mouse Snrpn did not disrupt imprinting or elicit any obvious phenotype, although it did allow the detection of previously unknown upstream exons. In contrast, a larger, overlapping 4.8-kb deletion caused a partial or mosaic imprinting defect and perinatal lethality when paternally inherited.     

Complete coverage of the Schizosaccharomyces pombe genome in yeast artificial chromosomes.

The genome of the fission yeast, Schizosaccharomyces pombe, consists of some 14 million base pairs of DNA contained in three chromosomes. On account of its excellent genetics we used it as a test system for a strategy designed to map mammalian chromosomes and genomes. Data obtained from hybridization fingerprinting established an ordered library of 1,248 yeast artificial chromosome clones with an average size of 535 kilobases. The clones fall into three contigs completely representing the three chromosomes of the organism. This work provides a high resolution physical and clone map of the genome, which has been related to available genetic and physical map information.     

Modular epistasis in yeast metabolism

Epistatic interactions, manifested in the effects of mutations on the phenotypes caused by other mutations, may help uncover the functional organization of complex biological networks. Here, we studied system-level epistatic interactions by computing growth phenotypes of all single and double knockouts of 890 metabolic genes in Saccharomyces cerevisiae, using the framework of flux balance analysis. A new scale for epistasis identified a distinctive trimodal distribution of these epistatic effects, allowing gene pairs to be classified as buffering, aggravating or noninteracting. We found that the ensuing epistatic interaction network could be organized hierarchically into function-enriched modules that interact with each other 'monochromatically' (i.e., with purely aggravating or purely buffering epistatic links). This property extends the concept of epistasis from single genes to functional units and provides a new definition of biological modularity, which emphasizes interactions between, rather than within, functional modules. Our approach can be used to infer functional gene modules from purely phenotypic epistasis measurements.     

LKB1 signaling in mesenchymal cells required for suppression of gastrointestinal polyposis

Germline mutations in STK11 (also known as LKB1) are found in individuals with Peutz-Jeghers syndrome (PJS) manifesting with gastrointestinal polyps that contain a prominent stromal component. Epithelia in polyps of Stk11(+/-) mice can retain a functional copy of Stk11 (refs. 2,3), and loss of heterozygosity is not an obligate feature of human polyps, raising the possibility of non-epithelial origins in tumorigenesis. Here we show that either monoallelic or biallelic loss of murine Stk11 limited to Tagln-expressing mesenchymal cells results in premature postnatal death as a result of gastrointestinal polyps indistinguishable from those in PJS. Stk11-deficient mesenchymal cells produced less TGFbeta, and defective TGFbeta signaling to epithelial cells coincided with epithelial proliferation. We also noted TGFbeta signaling defects in polyps of individuals with PJS, suggesting that the identified stromal-derived mechanism of tumor suppression is also relevant in PJS.     

Syndecan-1 is required for Wnt-1-induced mammary tumorigenesis in mice

Syndecan-1 is a cell-surface, heparan-sulphate proteoglycan (HSPG) predominantly expressed by epithelial cells. It binds specifically to many proteins, including oncoproteins. For example, it induces the assembly of a signalling complex between FGF ligands and their cognate receptors. But so far there has been no direct evidence that this proteoglycan contributes to tumorigenesis. Here we have examined the role of syndecan-1 (encoded by Sdc1) during mammary tumour formation in response to the ectopic expression of the proto-oncogene Wnt1. We crossed syndecan-1-deficient mice with transgenic mice that express Wnt1 in mammary gland (TgN(Wnt-1)1Hev; ref. 2). Ectopic Wnt-1 expression induces generalized mammary hyperplasia, followed by the development of solitary tumours (median time 22 weeks). We show that in Sdc1-/- mice, Wnt-1-induced hyperplasia in virgin mammary gland was reduced by 70%, indicating that the Wnt-1 signalling pathway was inhibited. Of the 39 tumours that developed in a test cohort of mice, only 1 evolved in the Sdc1-/- background. In addition, we show that soluble syndecan-1 ectodomain purified from mouse mammary epithelial cells stimulates the activity of a Wnt-1 homologue in a tissue culture assay. Our results provide both genetic and biochemical evidence that syndecan-1 can modulate Wnt signalling, and is critical for Wnt-1-induced tumorigenesis of the mouse mammary gland.     

Identification of a ferrireductase required for efficient transferrin-dependent iron uptake in erythroid cells

The reduction of iron is an essential step in the transferrin (Tf) cycle, which is the dominant pathway for iron uptake by red blood cell precursors. A deficiency in iron acquisition by red blood cells leads to hypochromic, microcytic anemia. Using a positional cloning strategy, we identified a gene, six-transmembrane epithelial antigen of the prostate 3 (Steap3), responsible for the iron deficiency anemia in the mouse mutant nm1054. Steap3 is expressed highly in hematopoietic tissues, colocalizes with the Tf cycle endosome and facilitates Tf-bound iron uptake. Steap3 shares homology with F(420)H(2):NADP(+) oxidoreductases found in archaea and bacteria, as well as with the yeast FRE family of metalloreductases. Overexpression of Steap3 stimulates the reduction of iron, and mice lacking Steap3 are deficient in erythroid ferrireductase activity. Taken together, these findings indicate that Steap3 is an endosomal ferrireductase required for efficient Tf-dependent iron uptake in erythroid cells.