Incomplete replication generates somatic DNA alterations within Drosophila polytene salivary gland cells.

DNA replication remains unfinished in many Drosophila polyploid cells, which harbor disproportionately fewer copies of late-replicating chromosomal regions. By analyzing paired-end high-throughput sequence data from polytene larval salivary gland cells, we define 112 underreplicated (UR) euchromatic regions 60-480 kb in size. To determine the effects of underreplication on genome integrity, we analyzed anomalous read pairs and breakpoint reads throughout the euchromatic genome. Each UR euchromatic region contains many different deletions 10-500 kb in size, while very few deletions are present in fully replicated chromosome regions or UR zones from embryo DNA. Thus, during endocycles, stalled forks within UR regions break and undergo local repair instead of remaining stable and generating nested forks. As a result, each salivary gland cell contains hundreds of unique deletions that account for their copy number reductions. Similar UR regions and deletions were observed in ovarian DNA, suggesting that incomplete replication, fork breakage, and repair occur widely in polytene cells. UR regions are enriched in genes encoding immunoglobulin superfamily proteins and contain many neurally expressed and homeotic genes. We suggest that the extensive somatic DNA instability described here underlies position effect variegation, molds the structure of polytene chromosomes, and should be investigated for possible functions.

The molecular mechanisms of OPA1-mediated optic atrophy in Drosophila model and prospects for antioxidant treatment.

Mutations in optic atrophy 1 (OPA1), a nuclear gene encoding a mitochondrial protein, is the most common cause for autosomal dominant optic atrophy (DOA). The condition is characterized by gradual loss of vision, color vision defects, and temporal optic pallor. To understand the molecular mechanism by which OPA1 mutations cause optic atrophy and to facilitate the development of an effective therapeutic agent for optic atrophies, we analyzed phenotypes in the developing and adult Drosophila eyes produced by mutant dOpa1 (CG8479), a Drosophila ortholog of human OPA1. Heterozygous mutation of dOpa1 by a P-element or transposon insertions causes no discernable eye phenotype, whereas the homozygous mutation results in embryonic lethality. Using powerful Drosophila genetic techniques, we created eye-specific somatic clones. The somatic homozygous mutation of dOpa1 in the eyes caused rough (mispatterning) and glossy (decreased lens and pigment deposition) eye phenotypes in adult flies; this phenotype was reversible by precise excision of the inserted P-element. Furthermore, we show the rough eye phenotype is caused by the loss of hexagonal lattice cells in developing eyes, suggesting an increase in lattice cell apoptosis. In adult flies, the dOpa1 mutation caused an increase in reactive oxygen species (ROS) production as well as mitochondrial fragmentation associated with loss and damage of the cone and pigment cells. We show that superoxide dismutase 1 (SOD1), Vitamin E, and genetically overexpressed human SOD1 (hSOD1) is able to reverse the glossy eye phenotype of dOPA1 mutant large clones, further suggesting that ROS play an important role in cone and pigment cell death. Our results show dOpa1 mutations cause cell loss by two distinct pathogenic pathways. This study provides novel insights into the pathogenesis of optic atrophy and demonstrates the promise of antioxidants as therapeutic agents for this condition.

TBX3 is overexpressed in breast cancer and represses p14 ARF by interacting with histone deacetylases.

TBX3 is a transcription factor of the T-box gene family. Mutations in the TBX3 gene can cause hypoplastic or absent mammary glands. Previous studies have shown that TBX3 might be associated with breast cancer. Here, we show that TBX3 is overexpressed in malignant cells of primary breast cancer tissues by immunohistochemistry. TBX3 interacts with histone deacetylases (HDAC) 1, 2, 3, and 5. TBX3 interacts with HDAC1, 2, and 3 via two distinct binding sites. However, deletion of the repression domain (amino acids 566-624) of TBX3 completely abolishes its interaction with HDAC5. Endogenous TBX3 and HDACs interaction and colocalization are found in a breast cancer cell line by coimmunoprecipitation and immunofluorescence, respectively. The functional significance of the interaction between TBX3 and HDAC is also tested in a p14(ARF)-luciferase reporter system. Results indicate that TBX3 represses expression of p14(ARF) tumor suppressor and that a HDAC inhibitor is able to reverse the TBX3 repressive function in a dosage-dependent manner. This study suggests that TBX3 may function by recruiting HDACs to the T-box binding site in the promoter region. TBX3 repression to its targets is dependent on HDAC activity. TBX3 may serve as a biomarker for breast cancer and have significant applications in both breast cancer diagnosis and treatment.