Apc modulates embryonic stem-cell differentiation by controlling the dosage of beta-catenin signaling

The Wnt signal-transduction pathway induces the nuclear translocation of membrane-bound beta-catenin (Catnb) and has a key role in cell-fate determination. Tight somatic regulation of this signal is essential, as uncontrolled nuclear accumulation of beta-catenin can cause developmental defects and tumorigenesis in the adult organism. The adenomatous polyposis coli gene (APC) is a major controller of the Wnt pathway and is essential to prevent tumorigenesis in a variety of tissues and organs. Here, we have investigated the effect of different mutations in Apc on the differentiation potential of mouse embryonic stem (ES) cells. We provide genetic and molecular evidence that the ability and sensitivity of ES cells to differentiate into the three germ layers is inhibited by increased doses of beta-catenin by specific Apc mutations. These range from a severe differentiation blockade in Apc alleles completely deficient in beta-catenin regulation to more specific neuroectodermal, dorsal mesodermal and endodermal defects in more hypomorphic alleles. Accordingly, a targeted oncogenic mutation in Catnb also affects the differentiation potential of ES cells. Expression profiling of wildtype and Apc-mutated teratomas supports the differentiation defects at the molecular level and pinpoints a large number of downstream structural and regulating genes. Chimeric experiments showed that this effect is cell-autonomous. Our results imply that constitutive activation of the Apc/beta-catenin signaling pathway results in differentiation defects in tissue homeostasis, and possibly underlies tumorigenesis in the colon and other self-renewing tissues.     

Mitotic recombination is suppressed by chromosomal divergence in hybrids of distantly related mouse strains

Mitotic recombination occurs with high frequency in humans and mice. It leads to loss of heterozygosity (LOH) at important gene loci and can cause disease. However, the genetic modulators of mitotic recombination are not well understood. As recombination depends on a high level of nucleotide sequence homology, we postulate that the frequency of somatic variants derived from mitotic recombination should be diminished in progeny from crosses between strains of mice in which nucleotide sequences have diverged. Here we report that mitotic recombination is suppressed, to various degrees in different tissues, in hybrids of distantly related mouse strains. Reintroduction of greater chromosomal homology by backcrossing restores mitotic recombination in offspring. Thus, chromosomal divergence inhibits mitotic recombination and, consequently, may act as a modifier of cancer susceptibility by limiting the rate of LOH. The suppression of mitotic recombination in some F1 hybrids in which meiotic recombination persists indicates that these processes are differentially affected by chromosomal divergence.     

Recombinational DNA double-strand breaks in mice precede synapsis

In Saccharomyces cerevisiae, meiotic recombination is initiated by Spo11-dependent double-strand breaks (DSBs), a process that precedes homologous synapsis. Here we use an antibody specific for a phosphorylated histone (gamma-H2AX, which marks the sites of DSBs) to investigate the timing, distribution and Spo11-dependence of meiotic DSBs in the mouse. We show that, as in yeast, recombination in the mouse is initiated by Spo11-dependent DSBs that form during leptotene. Loss of gamma-H2AX staining (which in irradiated somatic cells is temporally linked with DSB repair) is temporally and spatially correlated with synapsis, even when this synapsis is 'non-homologous'.     

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.     

Nras loss induces metastatic conversion of Rb1-deficient neuroendocrine thyroid tumor

Mutations in the gene encoding the retinoblastoma tumor suppressor predispose humans and mice to tumor development. Here we have assessed the effect of Nras loss on tumor development in Rb1 heterozygous mice. Loss of one or two Nras alleles is shown to significantly reduce the severity of pituitary tumors arising in Rb1(+/-) animals by enhancing their differentiation. By contrast, C-cell thyroid adenomas occurring in Rb1(+/-) mice progress to metastatic medullary carcinomas after loss of Nras. In Rb1(+/-)Nras(+/-) animals, distant medullary thyroid carcinoma metastases are associated with loss of the remaining wild-type Nras allele. Loss of Nras in Rb1-deficient C cells results in elevated Ras homolog family A (RhoA) activity, and this is causally linked to the invasiveness and metastatic behavior of these cells. These findings suggest that the loss of the proto-oncogene Nras in certain cellular contexts can promote malignant tumor progression.     

Mre11 and Ku70 interact in somatic cells, but are differentially expressed in early meiosis.

Double-strand DNA breaks (DSBs) pose a major threat to living cells, and several mechanisms for repairing these lesions have evolved. Eukaryotes can process DSBs by homologous recombination (HR) or non-homologous end joining (NHEJ). NHEJ connects DNA ends irrespective of their sequence, and it predominates in mitotic cells, particularly during G1 (ref. 3). HR requires interaction of the broken DNA molecule with an intact homologous copy, and allows restoration of the original DNA sequence. HR is active during G2 of the mitotic cycle and predominates during meiosis, when the cell creates DSBs (ref. 4), which must be repaired by HR to ensure proper chromosome segregation. How the cell controls the choice between the two repair pathways is not understood. We demonstrate here a physical interaction between mammalian Ku70, which is essential for NHEJ (ref. 5), and Mre11, which functions both in NHEJ and meiotic HR (Refs 2,6). Moreover, we show that irradiated cells deficient for Ku70 are incapable of targeting Mre11 to subnuclear foci that may represent DNA-repair complexes. Nevertheless, Ku70 and Mre11 were differentially expressed during meiosis. In the mouse testis, Mre11 and Ku70 co-localized in nuclei of somatic cells and in the XY bivalent. In early meiotic prophase, however, when meiotic recombination is most probably initiated, Mre11 was abundant, whereas Ku70 was not detectable. We propose that Ku70 acts as a switch between the two DSB repair pathways. When present, Ku70 destines DSBs for NHEJ by binding to DNA ends and attracting other factors for NHEJ, including Mre11; when absent, it allows participation of DNA ends and Mre11 in the meiotic HR pathway.     

An initiation site for meiotic crossing-over and gene conversion in the mouse

During meiosis, the reductional segregation of homologous chromosomes at the first meiotic division requires reciprocal exchange (crossing over) between homologs. The number of crossovers is tightly regulated (one to two per homolog in mice), and their distribution in the genome is not random-recombination 'hot' and 'cold' regions can be identified. We developed a molecular assay to study these events directly in mouse germ cells. This analysis was developed with reference to the proteosome subunit beta type 9 (Psmb9, previously called Lmp2) hot-spot region identified through genetic analysis. Here we show that this hot spot is an initiation site of meiotic recombination on the basis of two observations: (i) crossover density is maximal in an interval of 210 bp and decreases on both sides of this region; (ii) a high frequency of gene conversion is found in the region of highest crossover density. We then used this strategy to carry out the first temporal analysis of meiotic recombination in mouse spermatogenesis and demonstrate that crossover events occur during the pachytene stage of meiotic prophase.     

Disruption of mouse Slx4, a regulator of structure-specific nucleases, phenocopies Fanconi anemia

The evolutionarily conserved SLX4 protein, a key regulator of nucleases, is critical for DNA damage response. SLX4 nuclease complexes mediate repair during replication and can also resolve Holliday junctions formed during homologous recombination. Here we describe the phenotype of the Btbd12 knockout mouse, the mouse ortholog of SLX4, which recapitulates many key features of the human genetic illness Fanconi anemia. Btbd12-deficient animals are born at sub-Mendelian ratios, have greatly reduced fertility, are developmentally compromised and are prone to blood cytopenias. Btbd12(-/-) cells prematurely senesce, spontaneously accumulate damaged chromosomes and are particularly sensitive to DNA crosslinking agents. Genetic complementation reveals a crucial requirement for Btbd12 (also known as Slx4) to interact with the structure-specific endonuclease Xpf-Ercc1 to promote crosslink repair. The Btbd12 knockout mouse therefore establishes a disease model for Fanconi anemia and genetically links a regulator of nuclease incision complexes to the Fanconi anemia DNA crosslink repair pathway.     

Inherited variants of MYH associated with somatic G:C-->T:A mutations in colorectal tumors

Inherited defects of base excision repair have not been associated with any human genetic disorder, although mutations of the genes mutM and mutY, which function in Escherichia coli base excision repair, lead to increased transversions of G:C to T:A. We have studied family N, which is affected with multiple colorectal adenomas and carcinoma but lacks an inherited mutation of the adenomatous polyposis coli gene (APC) that is associated with familial adenomatous polyposis. Here we show that 11 tumors from 3 affected siblings contain 18 somatic inactivating mutations of APC and that 15 of these mutations are G:C-->A transversions--a significantly greater proportion than is found in sporadic tumors or in tumors associated with familial adenomatous polyposis. Analysis of the human homolog of mutY, MYH, showed that the siblings were compound heterozygotes for the nonconservative missense variants Tyr165Cys and Gly382Asp. These mutations affect residues that are conserved in mutY of E. coli (Tyr82 and Gly253). Tyrosine 82 is located in the pseudo-helix-hairpin-helix (HhH) motif and is predicted to function in mismatch specificity. Assays of adenine glycosylase activity of the Tyr82Cys and Gly253Asp mutant proteins with 8-oxoG:A and G:A substrates show that their activity is reduced significantly. Our findings link the inherited variants in MYH to the pattern of somatic APC mutation in family N and implicate defective base excision repair in predisposition to tumors in humans.     

A role for the Rb family of proteins in controlling telomere length

The molecular mechanisms of cellular mortality have recently begun to be unraveled. In particular, it has been discovered that cells that lack telomerase are subject to telomere attrition with each round of replication, eventually leading to loss of telomere capping function at chromosome ends. Critically short telomeres and telomeres lacking telomere-binding proteins lose their functionality and are metabolized as DNA breaks, thus generating chromosomal fusions. Telomerase activity is sufficient to rescue short telomeres and confers an unlimited proliferative capacity. In addition, the tumor-suppressor pathway Cdkn2a/Rb1 has also been implicated as a barrier to immortalization. Here, we report a connection between the members of the retinoblastoma family of proteins, Rb1 (retinoblastoma 1), Rbl1 (retinoblastoma-like 1) and Rbl2 (retinoblastoma-like 2), and the mechanisms that regulate telomere length. In particular, mouse embryonic fibroblasts doubly deficient in Rbl1 and Rbl2 or triply deficient in Rbl1, Rbl2 and Rb1 have markedly elongated telomeres compared with those of wildtype or Rb1-deficient cells. This deregulation of telomere length is not associated with increased telomerase activity. Notably, the abnormally elongated telomeres in doubly or triply deficient cells retain their end-capping function, as shown by the normal frequency of chromosomal fusions. These findings demonstrate a connection between the Rb1 family and the control of telomere length in mammalian cells.     

No evidence of clonal somatic genetic alterations in cancer-associated fibroblasts from human breast and ovarian carcinomas

There is increasing evidence showing that the stromal cells surrounding cancer epithelial cells, rather than being passive bystanders, might have a role in modifying tumor outgrowth. The molecular basis of this aspect of carcinoma etiology is controversial. Some studies have reported a high frequency of genetic aberrations in carcinoma-associated fibroblasts (CAFs), whereas other studies have reported very low or zero mutation rates. Resolution of this contentious area is of critical importance in terms of understanding both the basic biology of cancer as well as the potential clinical implications of CAF somatic alterations. We undertook genome-wide copy number and loss of heterozygosity (LOH) analysis of CAFs derived from breast and ovarian carcinomas using a 500K SNP array platform. Our data show conclusively that LOH and copy number alterations are extremely rare in CAFs and cannot be the basis of the carcinoma-promoting phenotypes of breast and ovarian CAFs.     

HNPCC-like cancer predisposition in mice through simultaneous loss of Msh3 and Msh6 mismatch-repair protein functions.

Cancer predisposition in hereditary non-polyposis colon cancer (HNPCC) is caused by defects in DNA mismatch repair (MMR). Mismatch recognition is attributed to two heterodimeric protein complexes: MutSalpha (refs 2, 3, 4, 5), a dimer of MutS homologues MSH2 and MSH6; and MutSbeta (refs 2,7), a dimer of MSH2 and MSH3. These complexes have specific and redundant mismatch recognition capacity. Whereas MSH2 deficiency ablates the activity of both dimers, causing strong cancer predisposition in mice and men, loss of MSH3 or MSH6 (also known as GTBP) function causes a partial MMR defect. This may explain the rarity of MSH6 and absence of MSH3 germline mutations in HNPCC families. To test this, we have inactivated the mouse genes Msh3 (formerly Rep3 ) and Msh6 (formerly Gtmbp). Msh6-deficient mice were prone to cancer; most animals developed lymphomas or epithelial tumours originating from the skin and uterus but only rarely from the intestine. Msh3 deficiency did not cause cancer predisposition, but in an Msh6 -deficient background, loss of Msh3 accelerated intestinal tumorigenesis. Lymphomagenesis was not affected. Furthermore, mismatch-directed anti-recombination and sensitivity to methylating agents required Msh2 and Msh6, but not Msh3. Thus, loss of MMR functions specific to Msh2/Msh6 is sufficient for lymphoma development in mice, whereas predisposition to intestinal cancer requires loss of function of both Msh2/Msh6 and Msh2/Msh3.     

Insertional mutagenesis identifies multiple networks of cooperating genes driving intestinal tumorigenesis

The evolution of colorectal cancer suggests the involvement of many genes. To identify new drivers of intestinal cancer, we performed insertional mutagenesis using the Sleeping Beauty transposon system in mice carrying germline or somatic Apc mutations. By analyzing common insertion sites (CISs) isolated from 446 tumors, we identified many hundreds of candidate cancer drivers. Comparison to human data sets suggested that 234 CIS-targeted genes are also dysregulated in human colorectal cancers. In addition, we found 183 CIS-containing genes that are candidate Wnt targets and showed that 20 CISs-containing genes are newly discovered modifiers of canonical Wnt signaling. We also identified mutations associated with a subset of tumors containing an expanded number of Paneth cells, a hallmark of deregulated Wnt signaling, and genes associated with more severe dysplasia included those encoding members of the FGF signaling cascade. Some 70 genes had co-occurrence of CIS pairs, clustering into 38 sub-networks that may regulate tumor development.     

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.     

Rb regulates proliferation and rod photoreceptor development in the mouse retina

The retinoblastoma protein (Rb) regulates proliferation, cell fate specification and differentiation in the developing central nervous system (CNS), but the role of Rb in the developing mouse retina has not been studied, because Rb-deficient embryos die before the retinas are fully formed. We combined several genetic approaches to explore the role of Rb in the mouse retina. During postnatal development, Rb is expressed in proliferating retinal progenitor cells and differentiating rod photoreceptors. In the absence of Rb, progenitor cells continue to divide, and rods do not mature. To determine whether Rb functions in these processes in a cell-autonomous manner, we used a replication-incompetent retrovirus encoding Cre recombinase to inactivate the Rb1(lox) allele in individual retinal progenitor cells in vivo. Combined with data from studies of conditional inactivation of Rb1 using a combination of Cre transgenic mouse lines, these results show that Rb is required in a cell-autonomous manner for appropriate exit from the cell cycle of retinal progenitor cells and for rod development.