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.     

BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt-beta-catenin signaling

In humans, mutations in BMPR1A, SMAD4 and PTEN are responsible for juvenile polyposis syndrome, juvenile intestinal polyposis and Cowden disease, respectively. The development of polyposis is a common feature of these diseases, suggesting that there is an association between BMP and PTEN pathways. The mechanistic link between BMP and PTEN pathways and the related etiology of juvenile polyposis is unresolved. Here we show that conditional inactivation of Bmpr1a in mice disturbs homeostasis of intestinal epithelial regeneration with an expansion of the stem and progenitor cell populations, eventually leading to intestinal polyposis resembling human juvenile polyposis syndrome. We show that BMP signaling suppresses Wnt signaling to ensure a balanced control of stem cell self-renewal. Mechanistically, PTEN, through phosphatidylinosital-3 kinase-Akt, mediates the convergence of the BMP and Wnt pathways on control of beta-catenin. Thus, BMP signaling may control the duplication of intestinal stem cells, thereby preventing crypt fission and the subsequent increase in crypt number.     

Regulation of polarized extension and planar cell polarity in the cochlea by the vertebrate PCP pathway

The mammalian auditory sensory organ, the organ of Corti, consists of sensory hair cells with uniformly oriented stereocilia on the apical surfaces and has a distinct planar cell polarity (PCP) parallel to the sensory epithelium. It is not certain how this polarity is achieved during differentiation. Here we show that the organ of Corti is formed from a thicker and shorter postmitotic primordium through unidirectional extension, characteristic of cellular intercalation known as convergent extension. Mutations in the PCP pathway interfere with this extension, resulting a shorter and wider cochlea as well as misorientation of stereocilia. Furthermore, parallel to the homologous pathway in Drosophila melanogaster, a mammalian PCP component Dishevelled2 shows PCP-dependent polarized subcellular localization across the organ of Corti. Taken together, these data suggest that there is a conserved molecular mechanism for PCP pathways in invertebrates and vertebrates and indicate that the mammalian PCP pathway might directly couple cellular intercalations to PCP establishment in the cochlea.     

Control of neurulation by the nucleosome assembly protein-1-like 2

Neurulation is a complex process of histogenesis involving the precise temporal and spatial organization of gene expression. Genes influencing neurulation include proneural genes determining primary cell fate, neurogenic genes involved in lateral inhibition pathways and genes controlling the frequency of mitotic events. This is reflected in the aetiology and genetics of human and mouse neural tube defects, which are of both multifactorial and multigenic origin. The X-linked gene Nap1l2, specifically expressed in neurons, encodes a protein that is highly similar to the nucleosome assembly (NAP) and SET proteins. We inactivated Nap1l2 in mice by gene targeting, leading to embryonic lethality from mid-gestation onwards. Surviving mutant chimaeric embryos showed extensive surface ectoderm defects as well as the presence of open neural tubes and exposed brains similar to those observed in human spina bifida and anencephaly. These defects correlated with an overproduction of neuronal precursor cells. Protein expression studies showed that the Nap1l2 protein binds to condensing chromatin during S phase and in apoptotic cells, but remained cytoplasmic during G1 phase. Nap1l2 therefore likely represents a class of tissue-specific factors interacting with chromatin to regulate neuronal cell proliferation.     

The DNA damage-dependent intra-S phase checkpoint is regulated by parallel pathways

To preserve genetic integrity, mammalian cells exposed to ionizing radiation activate the ATM kinase, which initiates a complex response-including the S-phase checkpoint pathways-to delay DNA replication. Defects in ATM or its substrates Nbs1 or Chk2 (ref. 3), the Nbs1-interacting Mre11 protein, or the Chk2-regulated Cdc25A-Cdk2 cascade all cause radio-resistant DNA synthesis (RDS). It is unknown, however, whether these proteins operate in a common signaling cascade. Here we show that experimental blockade of either the Nbs1-Mre11 function or the Chk2-triggered events leads to a partial RDS phenotype in human cells. In contrast, concomitant interference with Nbs1-Mre11 and the Chk2-Cdc25A-Cdk2 pathways entirely abolishes inhibition of DNA synthesis induced by ionizing radiation, resulting in complete RDS analogous to that caused by defective ATM. In addition, Cdk2-dependent loading of Cdc45 onto replication origins, a prerequisite for recruitment of DNA polymerase, was prevented upon irradiation of normal or Nbs1/Mre11-defective cells but not cells with defective ATM. We conclude that in response to ionizing radiation, phosphorylations of Nbs1 and Chk2 by ATM trigger two parallel branches of the DNA damage-dependent S-phase checkpoint that cooperate by inhibiting distinct steps of DNA replication.     

The SH2 tyrosine phosphatase shp2 is required for mammalian limb development

The tyrosine phosphatase Shp2 is recruited into tyrosine-kinase signalling pathways through binding of its two amino-terminal SH2 domains to specific phosphotyrosine motifs, concurrent with its re-localization and stimulation of phosphatase activity. Shp2 can potentiate signalling through the MAP-kinase pathway and is required during early mouse development for gastrulation. Chimaeric analysis can identify, by study of phenotypically normal embryos, tissues that tolerate mutant cells (and therefore do not require the mutated gene) or lack mutant cells (and presumably require the mutated gene during their developmental history). We therefore generated chimaeric mouse embryos to explore the cellular requirements for Shp2. This analysis revealed an obligatory role for Shp2 during outgrowth of the limb. Shp2 is specifically required in mesenchyme cells of the progress zone (PZ), directly beneath the distal ectoderm of the limb bud. Comparison of Ptpn11 (encoding Shp2)-mutant and Fgfr1 (encoding fibroblast growth factor receptor-1)-mutant chimaeric limbs indicated that in both cases mutant cells fail to contribute to the PZ of phenotypically normal chimaeras, leading to the hypothesis that a signal transduction pathway, initiated by Fgfr1 and acting through Shp2, is essential within PZ cells. Rather than integrating proliferative signals, Shp2 probably exerts its effects on limb development by influencing cell shape, movement or adhesion. Furthermore, the branchial arches, which also use Fgfs during bud outgrowth, similarly require Shp2. Thus, Shp2 regulates phosphotyrosine-signalling events during the complex ectodermal-mesenchymal interactions that regulate mammalian budding morphogenesis.     

Resveratrol rescues mutant polyglutamine cytotoxicity in nematode and mammalian neurons

We report that Sir2 activation through increased sir-2.1 dosage or treatment with the sirtuin activator resveratrol specifically rescued early neuronal dysfunction phenotypes induced by mutant polyglutamines in transgenic Caenorhabditis elegans. These effects are dependent on daf-16 (Forkhead). Additionally, resveratrol rescued mutant polyglutamine-specific cell death in neuronal cells derived from HdhQ111 knock-in mice. We conclude that Sir2 activation may protect against mutant polyglutamines.     

High frequency of homoplasmic mitochondrial DNA mutations in human tumors can be explained without selection

Researchers in several laboratories have reported a high frequency of homoplasmic mitochondrial DNA (mtDNA) mutations in human tumors. This observation has been interpreted to reflect a replicative advantage for mutated mtDNA copies, a growth advantage for a cell containing certain mtDNA mutations, and/or tumorigenic properties of mtDNA mutations. We consider another possibility-that the observed homoplasmy arose entirely by chance in tumor progenitor cells, without any physiological advantage or tumorigenic requirement. Through extensive computer modeling, we demonstrate that there is sufficient opportunity for a tumor progenitor cell to achieve homoplasmy through unbiased mtDNA replication and sorting during cell division. To test our model in vivo, we analyzed mtDNA homoplasmy in healthy human epithelial tissues and discovered that the model correctly predicts the considerable observed frequency of homoplasmic cells. Based on the available data on mitochondrial mutant fractions and cell division kinetics, we show that the predicted frequency of homoplasmy in tumor progenitor cells in the absence of selection is similar to the reported frequency of homoplasmic mutations in tumors. Although a role for other mechanisms is not excluded, random processes are sufficient to explain the incidence of homoplasmic mtDNA mutations in human tumors.     

Coordination of the random asynchronous replication of autosomal loci

Random monoallelic expression and asynchronous replication define an unusual class of autosomal mammalian genes. We show that every cell has randomly chosen either the maternal or paternal copy of each given autosome pair, such that alleles of these genes scattered across the chosen chromosome replicate earlier than the alleles on the homologous chromosome. Thus, chromosome-pair non-equivalence, rather than being limited to X-chromosome inactivation, is a fundamental property of mouse chromosomes.     

The Blk pathway functions as a tumor suppressor in chronic myeloid leukemia stem cells

A therapeutic strategy for treating cancer is to target and eradicate cancer stem cells (CSCs) without harming their normal stem cell counterparts. The success of this approach relies on the identification of molecular pathways that selectively regulate CSC function. Using BCR-ABL-induced chronic myeloid leukemia (CML) as a disease model for CSCs, we show that BCR-ABL downregulates the Blk gene (encoding B-lymphoid kinase) through c-Myc in leukemic stem cells (LSCs) in CML mice and that Blk functions as a tumor suppressor in LSCs but does not affect normal hematopoietic stem cells (HSCs) or hematopoiesis. Blk suppresses LSC function through a pathway involving an upstream regulator, Pax5, and a downstream effector, p27. Inhibition of this Blk pathway accelerates CML development, whereas increased activity of the Blk pathway delays CML development. Blk also suppresses the proliferation of human CML stem cells. Our results show the feasibility of selectively targeting LSCs, an approach that should be applicable to other cancers.     

Treatment-specific changes in gene expression discriminate in vivo drug response in human leukemia cells

To elucidate the genomics of cellular responses to cancer treatment, we analyzed the expression of over 9,600 human genes in acute lymphoblastic leukemia cells before and after in vivo treatment with methotrexate and mercaptopurine given alone or in combination. Based on changes in gene expression, we identified 124 genes that accurately discriminated among the four treatments. Discriminating genes included those involved in apoptosis, mismatch repair, cell cycle control and stress response. Only 14% of genes that changed when these medications were given as single agents also changed when they were given together. These data indicate that lymphoid leukemia cells of different molecular subtypes share common pathways of genomic response to the same treatment, that changes in gene expression are treatment-specific and that gene expression can illuminate differences in cellular response to drug combinations versus single agents.     

Polycomb-mediated methylation on Lys27 of histone H3 pre-marks genes for de novo methylation in cancer

Many genes associated with CpG islands undergo de novo methylation in cancer. Studies have suggested that the pattern of this modification may be partially determined by an instructive mechanism that recognizes specifically marked regions of the genome. Using chromatin immunoprecipitation analysis, here we show that genes methylated in cancer cells are specifically packaged with nucleosomes containing histone H3 trimethylated on Lys27. This chromatin mark is established on these unmethylated CpG island genes early in development and then maintained in differentiated cell types by the presence of an EZH2-containing Polycomb complex. In cancer cells, as opposed to normal cells, the presence of this complex brings about the recruitment of DNA methyl transferases, leading to de novo methylation. These results suggest that tumor-specific targeting of de novo methylation is pre-programmed by an established epigenetic system that normally has a role in marking embryonic genes for repression.