Transcriptional regulation by Atrophin in development and neurodegeneration

Atrophin (Atro) is the only Drosophila homolog of human Atrophin-1, the DRPLA disease gene. DRPLA (Dentatorubralpallidoluysian Atrophy) is a polyglutamine disease, caused by an expansion (over 48 repeats) of a polyQ tract in Atrophin-1, and characterized by brain specific neurons degeneration, together with psychiatric and motor symptoms. Atrophins are ubiquitous transcriptional cofactors involved in neuronal development and survival. Drosophila Atrophin itself contains two polyQ tracts, Q11 and Q14, of 11 and 14 glutamines respectively. To find what is the Atro function in gene expression regulation, what are the genes whose expression is regulated by Atro, and possibly misregulated by DRPLA-modeling Atro mutations, we are coupling gene expression with chromatin profiling in flies overexpressing wild-type or polyQ-expanded Atro. We have generated transgenic flies carrying wild-type Atro and two DRPLA fly models carrying polyQ-expanded Atro forms, which show retinal neurodegeneration. Taking advantage of the TARGET (Temporal And Regional Gene Expression Targeting) system, we have induced the transgenes expression in the adult retina, performing microarrays based genome-wide expression profiling at time points preceding neurodegeneration, and we are carrying on data mining and validation. We are also generating a cell culture model based on Drosophila neurons inducibly overexpressing wt and polyQ-expanded Atro forms to verify their effects on gene expression in neuronal cells, by quantitative RT-PCR. Finally we have generated an in vivo system based on flies which inducibly express Atro fused with Dam (DNA adenine-methyltransferase), to perform genome-wide chromatin analysis by the DamID (Dam Identification) method. This technique will allow us to find out DNA-protein interaction sites by methylation profiling and to identify the Atro direct transcriptional targets.