Cancer predisposition caused by elevated mitotic recombination in Bloom mice

Bloom syndrome is a disorder associated with genomic instability that causes affected people to be prone to cancer. Bloom cell lines show increased sister chromatid exchange, yet are proficient in the repair of various DNA lesions. The underlying cause of this disease are mutations in a gene encoding a RECQ DNA helicase. Using embryonic stem cell technology, we have generated viable Bloom mice that are prone to a wide variety of cancers. Cell lines from these mice show elevations in the rates of mitotic recombination. We demonstrate that the increased rate of loss of heterozygosity (LOH) resulting from mitotic recombination in vivo constitutes the underlying mechanism causing tumour susceptibility in these mice.     

Efficient Cre-loxP-induced mitotic recombination in mouse embryonic stem cells.

FLP/FRT-induced mitotic recombination provides a powerful method for creating genetic mosaics in Drosophila and for discerning the function of recessive genes in a heterozygous individual. Here we show that mitotic recombination can be reproducibly induced in mouse embryonic stem (ES) cells, by Cre/loxP technology, at frequencies ranging from 4.2 x 10(-5) (Snrpn) to 7.0 x 10(-3) (D7Mit178) for single allelic loxP sites, and to 5.0 x 10(-2) (D7Mit178) for multiple allelic lox sites, after transient Cre expression. Notably, much of the recombination occurs in G2 and is followed by X segregation, where the recombinant chromatids segregate away from each other during mitosis. It is X segregation that is useful for genetic mosaic analysis because it produces clones of homozygous mutant daughter cells from heterozygous mothers. Our studies confirm the predictions made from studies in Drosophila that suggest that X segregation will not be limited to organisms with strong mitotic pairing, because the forces (sister-chromatid cohesion) responsible for X segregation are an elemental feature of mitosis in all eukaryotes. Our studies also show that genetic mosaic analysis in mice is feasible, at least for certain chromosomal regions.     

Chfr is required for tumor suppression and Aurora A regulation

Tumorigenesis is a consequence of loss of tumor suppressors and activation of oncogenes. Expression of the mitotic checkpoint protein Chfr is lost in 20-50% of primary tumors and tumor cell lines. To explore whether downregulation of Chfr contributes directly to tumorigenesis, we generated Chfr knockout mice. Chfr-deficient mice are cancer-prone, develop spontaneous tumors and have increased skin tumor incidence after treatment with dimethylbenz(a)anthracene. Chfr deficiency leads to chromosomal instability in embryonic fibroblasts and regulates the mitotic kinase Aurora A, which is frequently upregulated in a variety of tumors. Chfr physically interacts with Aurora A and ubiquitinates Aurora A both in vitro and in vivo. Collectively, our data suggest that Chfr is a tumor suppressor and ensures chromosomal stability by controlling the expression levels of key mitotic proteins such as Aurora A.     

Trinucleotide expansion in haploid germ cells by gap repair

Huntington disease (HD) is one of eight progressive neurodegenerative disorders in which the underlying mutation is a CAG expansion encoding a polyglutamine tract. The mechanism of trinucleotide expansion is poorly understood. Expansion is mediated by misaligned pairing of repeats and the inappropriate formation of DNA secondary structure as the duplex unpairs. It has never been clear, however, whether duplex unpairing occurs during mitotic replication or during strand-break repair. In simple organisms, trinucleotide expansion arises by replication slippage on either the leading or the lagging strand, homologous recombination, gene conversion, double-strand break repair and base excision repair; it is not clear which of these mechanisms is used in mammalian cells in vivo. We have followed heritable changes in CAG length in male transgenic mice. In germ cells, expansion is limited to the post-meiotic, haploid cell and therefore cannot involve mitotic replication or recombination between a homologous chromosome or a sister chromatid. Our data support a model in which expansion in the germ cells arises by gap repair and depends on a complex containing Msh2. Expansion occurs during gap-filling synthesis when DNA loops comprising the CAG trinucleotide repeats are sealed into the DNA strand.     

Regulation of premeiotic S phase and recombination-related double-strand DNA breaks during meiosis in fission yeast.

The meiotic cell cycle is characterized by high levels of recombination induced by DNA double-strand breaks (DSBs), which appear after completion of premeiotic S phase, leading to the view that initiation of recombination depends on meiotic DNA replication. It has also been indicated that DNA replication initiation proteins may differ between the meiotic and mitotic cell cycles, giving rise to an altered S phase, which could contribute to the high level of recombination during meiosis. We have investigated these possibilities in the fission yeast Schizosaccharomyces pombe and found that core DNA replication initiation proteins used during the mitotic cell cycle, including Cdc18p (budding yeast Cdc6p), Cdc19p (Mcm2p), Cdc21p (Mcm4p) and Orp1p (Orc1p), are also required for premeiotic S phase. Reduced activity of these proteins prevents completion of DNA replication but not formation of DSBs. We conclude that recombination-related DSB formation does not depend on the completion of meiotic DNA replication and we propose two parallel developmental sequences during the meiotic cell cycle: one for premeiotic S phase and the other for initiating recombination.     

A Robertsonian translocation suppresses a somatic recombination pathway to loss of heterozygosity

In mammals, loss of APC/Apc gatekeeper function initiates intestinal tumorigenesis. Several different mechanisms have been shown or proposed to mediate functional loss of APC/Apc: mutation in APC/Apc, non-disjunction, homologous somatic recombination and epigenetic silencing. The demonstration that, in the C57BL/6 (B6) Apc(Min/+) mouse model of inherited intestinal cancer, loss of Apc function can occur by loss of heterozygosity (LOH) through somatic recombination between homologs presents an opportunity to search for polymorphisms in the homologous somatic recombination pathway. We report that the Robertsonian translocation Rb(7.18)9Lub (Rb9) suppresses the multiplicity of intestinal adenomas in this mouse model. As the copy number of Rb9 increases, the association with the interphase nucleolus of the rDNA repeats centromeric to the Apc locus on Chromosome 18 is increasingly disrupted. Our analysis shows that homologous somatic recombination is the principal pathway for LOH in adenomas in B6 Apc(Min/+) mice. These studies provide additional evidence that neoplastic growth can initiate in the complete absence of canonical genomic instability.     

Large-scale analysis of the regulatory architecture of the mouse genome with a transposon-associated sensor

We present here a Sleeping Beauty-based transposition system that offers a simple and efficient way to investigate the regulatory architecture of mammalian chromosomes in vivo. With this system, we generated several hundred mice and embryos, each with a regulatory sensor inserted at a random genomic position. This large sampling of the genome revealed the widespread presence of long-range regulatory activities along chromosomes, forming overlapping blocks with distinct tissue-specific expression potentials. The presence of tissue-restricted regulatory activities around genes with widespread expression patterns challenges the gene-centric view of genome regulation and suggests that most genes are modulated in a tissue-specific manner. The local hopping property of Sleeping Beauty provides a dynamic approach to map these regulatory domains at high resolution and, combined with Cre-mediated recombination, allows for the determination of their functions by engineering mice with specific chromosomal rearrangements.     

Dichotomy of single-nucleotide polymorphism haplotypes in olfactory receptor genes and pseudogenes

Substantial efforts are focused on identifying single-nucleotide polymorphisms (SNPs) throughout the human genome, particularly in coding regions (cSNPs), for both linkage disequilibrium and association studies. Less attention, however, has been directed to the clarification of evolutionary processes that are responsible for the variability in nucleotide diversity among different regions of the genome. We report here the population sequence diversity of genomic segments within a 450-kb cluster of olfactory receptor (OR) genes on human chromosome 17. We found a dichotomy in the pattern of nucleotide diversity between OR pseudogenes and introns on the one hand and the closely interspersed intact genes on the other. We suggest that weak positive selection is responsible for the observed patterns of genetic variation. This is inferred from a lower ratio of polymorphism to divergence in genes compared with pseudogenes or introns, high non-synonymous substitution rates in OR genes, and a small but significant overall reduction in variability in the entire OR gene cluster compared with other genomic regions. The dichotomy among functionally different segments within a short genomic distance requires high recombination rates within this OR cluster. Our work demonstrates the impact of weak positive selection on human nucleotide diversity, and has implications for the evolution of the olfactory repertoire.     

Recombination and the Tel1 and Mec1 checkpoints differentially effect genome rearrangements driven by telomere dysfunction in yeast

In telomerase-deficient Saccharomyces cerevisiae, telomeres are maintained by recombination. Here we used a S. cerevisiae assay for characterizing gross chromosomal rearrangements (GCRs) to analyze genome instability in post-senescent telomerase-deficient cells. Telomerase-deficient tlc1 and est2 mutants did not have increased GCR rates, but their telomeres could be joined to other DNAs resulting in chromosome fusions. Inactivation of Tel1 or either the Rad51 or Rad59 recombination pathways in telomerase-deficient cells increased the GCR rate, even though telomeres were maintained. The GCRs were translocations and chromosome fusions formed by nonhomologous end joining. We observed chromosome fusions only in mutant strains expressing Rad51 and Rad55 or when Tel1 was inactivated. In contrast, inactivation of Mec1 resulted in more inversion translocations such as the isochromosomes seen in human tumors. These inversion translocations seemed to be formed by recombination after replication of broken chromosomes.     

Population genomic analysis of outcrossing and recombination in yeast

The budding yeast Saccharomyces cerevisiae has been used by humans for millennia to make wine, beer and bread. More recently, it became a key model organism for studies of eukaryotic biology and for genomic analysis. However, relatively little is known about the natural lifestyle and population genetics of yeast. One major question is whether genetically diverse yeast strains mate and recombine in the wild. We developed a method to infer the evolutionary history of a species from genome sequences of multiple individuals and applied it to whole-genome sequence data from three strains of Saccharomyces cerevisiae and the sister species Saccharomyces paradoxus. We observed a pattern of sequence variation among yeast strains in which ancestral recombination events lead to a mosaic of segments with shared genealogy. Based on sequence divergence and the inferred median size of shared segments (approximately 2,000 bp), we estimated that although any two strains have undergone approximately 16 million cell divisions since their last common ancestor, only 314 outcrossing events have occurred during this time (roughly one every 50,000 divisions). Local correlations in polymorphism rates indicate that linkage disequilibrium in yeast should extend over kilobases. Our results provide the initial foundation for population studies of association between genotype and phenotype in S. cerevisiae.     

A genome-wide scan identifies mutations in the gene encoding phosphodiesterase 11A4 (PDE11A) in individuals with adrenocortical hyperplasia

Phosphodiesterases (PDEs) regulate cyclic nucleotide levels. Increased cyclic AMP (cAMP) signaling has been associated with PRKAR1A or GNAS mutations and leads to adrenocortical tumors and Cushing syndrome. We investigated the genetic source of Cushing syndrome in individuals with adrenocortical hyperplasia that was not caused by known defects. We performed genome-wide SNP genotyping, including the adrenocortical tumor DNA. The region with the highest probability to harbor a susceptibility gene by loss of heterozygosity (LOH) and other analyses was 2q31-2q35. We identified mutations disrupting the expression of the PDE11A isoform-4 gene (PDE11A) in three kindreds. Tumor tissues showed 2q31-2q35 LOH, decreased protein expression and high cyclic nucleotide levels and cAMP-responsive element binding protein (CREB) phosphorylation. PDE11A codes for a dual-specificity PDE that is expressed in adrenal cortex and is partially inhibited by tadalafil and other PDE inhibitors; its germline inactivation is associated with adrenocortical hyperplasia, suggesting another means by which dysregulation of cAMP signaling causes endocrine tumors.     

Complete inactivation of DNMT1 leads to mitotic catastrophe in human cancer cells

Studies have shown that DNA (cytosine-5-)-methyltransferase 1 (DNMT1) is the principal enzyme responsible for maintaining CpG methylation and is required for embryonic development and survival of somatic cells in mice. The role of DNMT1 in human cancer cells, however, remains highly controversial. Using homologous recombination, here we have generated a DNMT1 conditional allele in the human colorectal carcinoma cell line HCT116 in which several exons encoding the catalytic domain are flanked by loxP sites. Cre recombinase-mediated disruption of this allele results in hemimethylation of approximately 20% of CpG-CpG dyads in the genome, coupled with activation of the G2/M checkpoint, leading to arrest in the G2 phase of the cell cycle. Although cells gradually escape from this arrest, they show severe mitotic defects and undergo cell death either during mitosis or after arresting in a tetraploid G1 state. Our results thus show that DNMT1 is required for faithfully maintaining DNA methylation patterns in human cancer cells and is essential for their proliferation and survival.     

SGS1, the Saccharomyces cerevisiae homologue of BLM and WRN, suppresses genome instability and homeologous recombination

The Escherichia coli gene recQ was identified as a RecF recombination pathway gene. The gene SGS1, encoding the only RecQ-like DNA helicase in Saccharomyces cerevisiae, was identified by mutations that suppress the top3 slow-growth phenotype. Relatively little is known about the function of Sgs1p because single mutations in SGS1 do not generally cause strong phenotypes. Mutations in genes encoding RecQ-like DNA helicases such as the Bloom and Werner syndrome genes, BLM and WRN, have been suggested to cause increased genome instability. But the exact DNA metabolic defect that might underlie such genome instability has remained unclear. To better understand the cellular role of the RecQ-like DNA helicases, sgs1 mutations were analyzed for their effect on genome rearrangements. Mutations in SGS1 increased the rate of accumulating gross chromosomal rearrangements (GCRs), including translocations and deletions containing extended regions of imperfect homology at their breakpoints. sgs1 mutations also increased the rate of recombination between DNA sequences that had 91% sequence homology. Epistasis analysis showed that Sgs1p is redundant with DNA mismatch repair (MMR) for suppressing GCRs and for suppressing recombination between divergent DNA sequences. This suggests that defects in the suppression of rearrangements involving divergent, repeated sequences may underlie the genome instability seen in BLM and WRN patients and in cancer cases associated with defects in these genes.     

Evolution of the mammalian G protein alpha subunit multigene family.

Heterotrimeric guanine nucleotide binding proteins (G proteins) transduce extracellular signals received by transmembrane receptors to effector proteins. The multigene family of G protein alpha subunits, which interact with receptors and effectors, exhibit a high level of sequence diversity. In mammals, 15 G alpha subunit genes can be grouped by sequence and functional similarities into four classes. We have determined the murine chromosomal locations of all 15 G alpha subunit genes using an interspecific backcross derived from crosses of C57BL/6J and Mus spretus mice. These data, in combination with mapping studies in humans, have provided insight into the events responsible for generating the genetic diversity found in the mammalian alpha subunit genes and a framework for elucidating the role of the G alpha subunits in disease.     

Disruption of an imprinted gene cluster by a targeted chromosomal translocation in mice

Genomic imprinting is an epigenetic process in which the activity of a gene is determined by its parent of origin. Mechanisms governing genomic imprinting are just beginning to be understood. However, the tendency of imprinted genes to exist in chromosomal clusters suggests a sharing of regulatory elements. To better understand imprinted gene clustering, we disrupted a cluster of imprinted genes on mouse distal chromosome 7 using the Cre/loxP recombination system. In mice carrying a site-specific translocation separating Cdkn1c and Kcnq1, imprinting of the genes retained on chromosome 7, including Kcnq1, Kcnq1ot1, Ascl2, H19 and Igf2, is unaffected, demonstrating that these genes are not regulated by elements near or telomeric to Cdkn1c. In contrast, expression and imprinting of the translocated Cdkn1c, Slc22a1l and Tssc3 on chromosome 11 are affected, consistent with the hypothesis that elements regulating both expression and imprinting of these genes lie within or proximal to Kcnq1. These data support the proposal that chromosomal abnormalities, including translocations, within KCNQ1 that are associated with the human disease Beckwith-Wiedemann syndrome (BWS) may disrupt CDKN1C expression. These results underscore the importance of gene clustering for the proper regulation of imprinted genes.     

Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in beta-islet cell hyperplasia.

To ascertain the role of cyclin-dependent kinase 4 (Cdk4) in vivo, we have targeted the mouse Cdk4 locus by homologous recombination to generate two strains of mice, one that lacks Cdk4 expression and one that expresses a Cdk4 molecule with an activating mutation. Embryonic fibroblasts proliferate normally in the absence of Cdk4 but have a delayed S phase on re-entry into the cell cycle. Moreover, mice devoid of Cdk4 are viable, but small in size and infertile. These mice also develop insulin-deficient diabetes due to a reduction in beta-islet pancreatic cells. In contrast, mice expressing a mutant Cdk4 that cannot bind the cell-cycle inhibitor P16INK4a display pancreatic hyperplasia due to abnormal proliferation of beta-islet cells. These results establish Cdk4 as an essential regulator of specific cell types.     

Functions of poly(ADP-ribose) polymerase in controlling telomere length and chromosomal stability.

In most eukaryotes, poly(ADP-ribose) polymerase (PARP) recognizes DNA strand interruptions generated in vivo. DNA binding by PARP triggers primarily its own modification by the sequential addition of ADP-ribose units to form polymers; this modification, in turn, causes the release of PARP from DNA ends. Studies on the effects of the disruption of the gene encoding PARP (Adprt1, formerly Adprp) in mice have demonstrated roles for PARP in recovery from DNA damage and in suppressing recombination processes involving DNA ends. Telomeres are the natural termini of chromosomes and are, therefore, potential targets of PARP. Here, by the use of two different techniques, we show that mice lacking PARP display telomere shortening compared with wild-type mice. Telomere shortening is seen in different genetic backgrounds and in different tissues, both from embryos and adult mice. In vitro telomerase activity, however, is not altered in Adprt1-/- mouse fibroblasts. Furthermore, cytogenetic analysis of mouse embryonic fibroblasts reveals that lack of PARP is associated with severe chromosomal instability, characterized by increased frequencies of chromosome fusions and aneuploidy. The absence of PARP does not affect the presence of single-strand overhangs, naturally present at the ends of telomeres. This study therefore reveals an unanticipated role for PARP in telomere length regulation and provides insights into its functions in maintaining genomic integrity.