Synaptonemal complex assembly and H3K4Me3 demethylation determine DIDO3 localization in meiosis.

Synapsis of homologous chromosomes is a key meiotic event, mediated by a large proteinaceous structure termed the synaptonemal complex. Here, we describe a role in meiosis for the murine death-inducer obliterator (Dido) gene. The Dido gene codes for three proteins that recognize trimethylated histone H3 lysine 4 through their amino-terminal plant homeodomain domain. DIDO3, the largest of the three isoforms, localizes to the central region of the synaptonemal complex in germ cells. DIDO3 follows the distribution of the central region protein SYCP1 in Sycp3-/- spermatocytes, which lack the axial elements of the synaptonemal complex. This indicates that synapsis is a requirement for DIDO3 incorporation. Interestingly, DIDO3 is missing from the synaptonemal complex in Atm mutant spermatocytes, which form synapses but show persistent trimethylation of histone H3 lysine 4. In order to further address a role of epigenetic modifications in DIDO3 localization, we made a mutant of the Dido gene that produces a truncated DIDO3 protein. This truncated protein, which lacks the histone-binding domain, is incorporated in the synaptonemal complex irrespective of histone trimethylation status. DIDO3 protein truncation in Dido mutant mice causes mild meiotic defects, visible as gaps in the synaptonemal complex, but allows for normal meiotic progression. Our results indicate that histone H3 lysine 4 demethylation modulates DIDO3 localization in meiosis and suggest epigenetic regulation of the synaptonemal complex.

Dido gene expression alterations are implicated in the induction of hematological myeloid neoplasms.

The myelodysplastic/myeloproliferative diseases (MDS/MPDs) are a heterogeneous group of myeloid neoplasms that share characteristics with chronic myeloproliferative diseases and myelodysplastic syndromes. The broad spectrum of clinical manifestations makes MDS/MPDs extremely difficult to diagnose and treat, with a median survival time of 1-5 years. No single gene defect has been firmly associated with MDS/MPDs, and no animal models have been developed for these diseases. The association of deletions on chromosome 20q with myeloid malignancies suggests the presence of unidentified tumor suppressor genes in this region. Here we show that the recently identified death inducer-obliterator (Dido) gene gives rise to at least 3 polypeptides (Dido1, Dido2, and Dido3) through alternative splicing, and we map the human gene to the long arm of chromosome 20. We found that targeting of murine Dido caused a transplantable disease whose symptoms and signs suggested MDS/MPDs. Furthermore, 100% of human MDS/MPD patients analyzed showed Dido expression abnormalities, which we also found in other myeloid but not lymphoid neoplasms or in healthy donors. Our findings suggest that Dido might be one of the tumor suppressor genes at chromosome 20q and that the Dido-targeted mouse may be a suitable model for studying MDS/MPD diseases and testing new approaches to their diagnosis and treatment.

Quantitative determination of tumor cell intravasation in a real-time polymerase chain reaction-based assay.

Tumor cells acquire the ability to enter blood vessels surrounding the primary tumor, endowing them with the capacity to disseminate and become established in distant sites, originating a metastasis. Determination of the intravasation ability of tumor cells is thus important, as it can be correlated with their potential malignancy. To analyze the intravasation phenotype of human tumor cells in vivo, we performed chick embryo chorioallantoic membrane (CAM) assays. Cells were inoculated on the CAM of 9-day-old chick embryos and the membrane at the opposite side of the egg was recovered after 48 h incubation. To measure intravasation ability, we calculated the amount of human DNA in each CAM sample by real-time PCR of Alu sequences and SYBR Green 1 fluorescence detection. This analysis showed a detection limit of 1 human cell per 10(5) total cells, and we were able to distinguish between tumor cells of distinct invasive capacity. This assay has several advantages over current methods to measure intravasation ability, including the elimination of post-PCR analysis, sensitivity and easy scale-up of sample numbers.

Inhibitor of apoptosis protein from Orgyia pseudotsugata nuclear polyhedrosis virus provides a costimulatory signal required for optimal proliferation of developing thymocytes.

The inhibitors of apoptosis proteins (IAPs) constitute a family of endogenous inhibitors that control apoptosis in the cell by inhibiting caspase processing and activity. IAPs are also implicated in cell division, cell cycle regulation, and cancer. To address the role of IAPs in thymus development and homeostasis, we generated transgenic mice expressing IAP generated from the baculovirus Orgyia pseudotsugata nuclear polyhedrosis virus (OpIAP). Developing thymocytes expressing OpIAP show increased nuclear levels of NF-kappaB and reduced cytoplasmic levels of its inhibitor, IkappaBalpha. In mature thymocytes, OpIAP induces optimal activation and proliferation after TCR triggering in the absence of a costimulatory signal. OpIAP expression in immature thymocytes blocks TCR-induced apoptosis. Taken together, our data illustrate the pleiotropism of OpIAP in vivo.