Most carcinomas present some form of chromosome instability in combination with spindle defects. Numerical instability is likely caused by spindle aberrations, but the origin of breaks and translocations remains elusive. To determine whether one mechanism can bring about both types of instability, we studied the relationship between DNA damage and spindle defects. Although lacking apparent repair defects, primary Dido mutant cells formed micronuclei containing damaged DNA. The presence of centromeres showed that micronuclei were caused by spindle defects, and cell cycle markers showed that DNA damage was generated during mitosis. Although the micronuclei themselves persisted, the DNA damage within was repaired during S and G2 phases. DNA breaks in Dido mutant cells regularly colocalized with centromeres, which were occasionally distorted. Comparable defects were found in APC mutant cell lines, an independent system for spindle defects. On the basis of these results, we propose a model for break formation in which spindle defects lead to centromere shearing.

The t(12;22) creates an MN1–TEL fusion gene leading to acute myeloid leukemia. The fusion partner TEL (ETV6) is a member of the ETS family of transcription factors. The nature of the other fusion partner, MN1, has not been investigated in detail until now. We recently described that MN1 activates the transcription activity of the moloney sarcoma virus long terminal repeat, indicating that this protein itself may act as a transcription factor. We show here that MN1 comprises multiple transcription activating domains. A search for a bound DNA sequence revealed that MN1 has affinity for retinoic acid responsive elements. A DR5 retinoic acid responsive element was observed in the LTR. The combination of MN1 and ligand-activated retinoic acid receptor leads to a synergistic induction of expression directed by the LTR. Cotransfection of MN1 with RAC3 or p300, known coactivators of retinoic acid receptors, leads to a further synergistic induction of transcription. In addition, the effect of MN1 can be inhibited by the wild-type adenovirus ElA protein that inhibits p300 function, but not by an E1A mutant lacking the p300-binding site. GAL4-MN1-mediated transcription can be enhanced directly by RAC3 and p300. Taken together, our results indicate that MN1 is a transcription coactivator rather than a sequence-specific transcription factor, and that it may stimulate RAR/RXR-mediated transcription through interaction with p160 and p300.

The insulin-like growth factor (IGF)-I protein is a growth-promoting polypeptide that can act as an angiogenic agent in the eye. The purpose of the current study was to localize the expression of IGF-I and its receptor (IGF-IR) mRNA and IGF-IR protein in situ in the normal human eye and to examine the presence of expression in eyes with neovascular age-related macular degeneration (AMD). Formalin-fixed, paraffin-embedded slides of 4 normal control eyes and 14 eyes with choroidal neovascularization (CNV) secondary to AMD were examined. Three eyes with proliferative diabetic retinopathy were studied as the positive control. IGF-I and IGF-IR mRNA was detected by in situ hybridization with digoxigenin-labeled RNA probes. IGF-IR protein was studied by immunohistochemistry. In the normal retina, IGF-I and IGF-IR mRNA expression was found throughout the neuroretinal layers, in the retinal pigment epithelium (RPE), and in some choriocapillary and retinal capillary endothelial cells. In eyes with CNV we found IGF and IGF-IR mRNA in capillary endothelial cells, some transdifferentiated RPE, and fibroblast-like cells. IGF-IR protein was found in normal eyes in all neuroretinal layers, in the RPE, and in the choroidal vessels. In eyes with CNV, IGF-IR protein was present in the RPE monolayer, in transdifferentiated RPE, and in newly formed vessels. The colocalization of protein and receptor indicates an autocrine function of IGF-I in the normal human retina. Because IGF-I participates in ocular neovascularization, synthesis of IGF-IR and IGF-I in endothelial cells, RPE cells, and fibroblast-like cells in CNV may point toward a role for this growth factor in the pathogenesis of neovascular AMD.

Death Inducer Obliterator 3 (Dido3) is implicated in the maintenance of stem cell genomic stability and tumorigenesis. Here, we show that Dido3 regulates the expression of stemness genes in embryonic stem cells through its plant homeodomain (PHD) finger. Binding of Dido3 PHD to histone H3K4me3 is disrupted by threonine phosphorylation that triggers Dido3 translocation from chromatin to the mitotic spindle. The crystal structure of Dido3 PHD in complex with H3K4me3 reveals an atypical aromatic-cage-like binding site that contains a histidine residue. Biochemical, structural, and mutational analyses of the binding mechanism identified the determinants of specificity and affinity and explained the inability of homologous PHF3 to bind H3K4me3. Together, our findings reveal a link between the transcriptional control in embryonic development and regulation of cell division.

Fusion of the MN1 gene to TEL (ETV6) results in myeloid leukemia. The fusion protein combines the transcription activating domain of MN1 and the DNA binding domain of TEL and is thought to act as a deranged transcription factor. In addition, disruption of the large first exon of the MN1 gene is thought to inactivate MN1 function in a meningioma. To further investigate the role of MN1 in cancer, we generated Mn1 knockout mice. Mn1^+/− animals were followed for 30 months, but they had no higher incidence of tumor formation than wild-type littermates. Mn1 null mice, however, were found to die at birth or shortly thereafter as the result of a cleft palate. Investigation of newborn or embryonic day 15.5 (E15.5) to E17.5 null mice revealed that the development of several bones in the skull was abnormal. The affected bones are almost exclusively formed by intramembranous ossification. They are either completely agenic at birth (alisphenoid and squamosal bones and vomer), hypoplastic, deformed (basisphenoid, pterygoid, and presphenoid), or substantially thinner (frontal, parietal, and interparietal bones). In heterozygous mice hypoplastic membranous bones and incomplete penetrance of the cleft palate were observed. We conclude that Mn1 is an important factor in development of membranous bones.

The acquisition of massive but localized chromosome translocations, a phenomenon termed chromothripsis, has received widespread attention since its discovery over a year ago. Until recently, chromothripsis was believed to originate from a single catastrophic event, but the molecular mechanisms leading to this event are yet to be uncovered. Because a thorough interpretation of the data are missing, the phenomenon itself has wrongly acquired the status of a mechanism used to justify many kinds of complex rearrangements. Although the assumption that all translocations in chromothripsis originate from a single event has met with criticism, satisfactory explanations for the intense but localized nature of this phenomenon are still missing. Here, we show why the data used to describe massive catastrophic rearrangements are incompatible with a model comprising a single event only and propose a molecular mechanism in which a combination of known cellular pathways accounts for chromothripsis. Instead of a single traumatic event, the protection of undamaged chromosomes by telomeres can limit repetitive breakage-fusion-bridge events to a single chromosome arm. Ultimately, common properties of chromosomal instability, such as aneuploidy and centromere fission, might establish the complex genetic pattern observed in this genomic state.