We have studied two related proteins that contain a repeated amino acid motif homologous to the anti-angiogenic type 1 repeats of thrombospondin-1 (TSP1). Complete sequence analysis revealed no other similarities with TSP1, but identified unique signal sequences, as well as metalloprotease and disintegrin-like domains in the NH 2 termini. We named these proteins METH-1 and METH-2 due to the novel combination of metalloprotease and thrombospondin domains. Overall amino acid sequence identity between METH-1 and METH-2 is 51.7%, yet transcript distribution revealed non-overlapping patterns of expression in tissues and cultured cell lines. To characterize these proteins functionally, we isolated full-length cDNAs, produced recombinant protein, and generated antisera to the recombinant proteins. Both METH-1 and METH-2 represent single copy genes, which encode secreted and proteolytically processed proteins. METH proteins suppressed fibroblast growth factor-2-induced vascularization in the cornea pocket assay and inhibited vascular endothelial growth factor-induced angiogenesis in the chorioallantoic membrane assay. Suppression of vessel growth in both assays was considerably greater than that mediated by either thrombospondin-1 or endostatin on a molar basis. Consistent with an endothelial specific response, METH-1 and METH-2 were shown to inhibit endothelial cell proliferation, but not fibroblast or smooth muscle growth. We propose that METH-1 and METH-2 represent a new family of proteins with metalloprotease, disintegrin, and thrombospondin domains. The distinct distribution of each gene product suggests that each has evolved distinct regulatory mechanisms that potentially allow for fine control of activity during distinct physiological and pathological states. Thrombospondin-1 (TSP1)1 is a modular protein that associates with the extracellular matrix and has the ability to inhibit angiogenesis in vivo (1, 2). Under culture conditions, TSP1 blocks capillary-like tube formation (3) and endothelial cell proliferation (4). The region responsible for the anti-angiogenic activity has been mapped to the 385-522 amino acid region of the protein, which contains three type 1 (properdin or TSP) repeats (2). Recombinant and proteolytic fragments containing these repeats exhibited angio-inhibitory activity in the rabbit corneal pocket and chorioallantoic membrane assays (2, 5, 6) and peptides derived from the second and third type 1 repeats of TSP1 inhibit endothelial cell chemotaxis and proliferation (7). Furthermore, peptides derived from the same region induce apoptosis of endothelial cells and suppress tumor growth when injected systemically (8, 9). Of the five known members of the TSP family, only TSP1 and TSP2 contain type 1 repeats, and are the only members that inhibit angiogenesis (10, 11). We hypothesized that TSP/type 1 repeats might be present in other yet unidentified proteins, in a context that enables activity as inhibitors of capillary growth. We therefore initiated a search for novel cDNAs using the anti-angio...

Terminal deletions of Drosophila chromosomes can be stably protected from end-to-end fusion despite the absence of all telomere-associated sequences. The sequence-independent protection of these telomeres suggests that recognition of chromosome ends might contribute to the epigenetic protection of telomeres. In mammals, Ataxia Telangiectasia Mutated (ATM) is activated by DNA damage and acts through an unknown, telomerase-independent mechanism to regulate telomere length and protection. We demonstrate that the Drosophila homolog of ATM is encoded by the telomere fusion (tefu) gene. In the absence of ATM, telomere fusions occur even though telomere-specific Het-A sequences are still present. High levels of spontaneous apoptosis are observed in ATM-deficient tissues, indicating that telomere dysfunction induces apoptosis in Drosophila. Suppression of this apoptosis by p53 mutations suggests that loss of ATM activates apoptosis through a DNA damage-response mechanism. Loss of ATM reduces the levels of heterochromatin protein 1 (HP1) at telomeres and suppresses telomere position effect. We propose that recognition of chromosome ends by ATM prevents telomere fusion and apoptosis by recruiting chromatin-modifying complexes to telomeres.[Keywords: Drosophila; telomere; protection; ATM; HP1; chromatin] Supplemental material is available at http://www.genesdev.org.

The CRISPR-Cas9 system is commonly employed in biomedical research; however, the precision of Cas9 is sub-optimal for gene therapy applications that involve editing a large population of cells. Variations on the standard Cas9 system have yielded improvements in the precision of targeted DNA cleavage, but often restrict the range of targetable sequences. It remains unclear whether these variants can limit lesions to a single site within the human genome over a large cohort of treated cells. Here, we demonstrate that fusing a programmable DNA-binding domain (pDBD) to Cas9 combined with the attenuation of Cas9’s inherent DNA binding affinity produces a Cas9-pDBD chimera with dramatically improved precision and increased targeting range. Because the specificity and affinity of this framework is easily tuned, Cas9-pDBDs provide a flexible system that can be tailored to achieve extremely precise genome editing at nearly any genomic locus – characteristics that are ideal for gene therapy applications.

Acute myeloid leukemia subtype M4 with eosinophilia is associated with a chromosome 16 inversion that creates a fusion gene CBFB-MYH11. We have previously shown that CBFB-MYH11 is necessary but not sufficient for leukemogenesis. Here, we report the identification of genes that specifically cooperate with CBFB-MYH11 in leukemogenesis. Neonatal injection of Cbfb-MYH11 knock-in chimeric mice with retrovirus 4070A led to the development of acute myeloid leukemia in 2-5 months. Each leukemia sample contained one or a few viral insertions, suggesting that alteration of one gene could be sufficient to synergize with Cbfb-MYH11. The chromosomal position of 67 independent retroviral insertion sites (RISs) was determined, and 90% of the RISs mapped within 10 kb of a flanking gene. In total, 54 candidate genes were identified; six of them were common insertion sites (CISs). CIS genes included members of a zinc finger transcription factors family, Plag1 and Plagl2, with eight and two independent insertions, respectively. CIS genes also included Runx2, Myb, H2-T24, and D6Mm5e. Comparison of the remaining 48 genes with single insertion sites with known leukemia-associated RISs indicated that 18 coincide with known RISs. To our knowledge, this retroviral genetic screen is the first to identify genes that cooperate with a fusion gene important for human myeloid leukemia.