Identifying New Genes Involved in Centromere Establishment

The centromere is a site on the chromosome that mediates accurate cell division by serving as a platform for kinetochore assembly, and microtubule attachment during cell division. Errors in the process of chromosome segregation can contribute to genetic irregularities, such as those seen in cancer and congenital defects. Our lab uses the ectopic centromere as a tool to discover what proteins may be involved in centromere establishment, defined as the deposition of CENP-A at the locus. We use the lacO/LacI system within Drosophila S2 cells that contain a CAL1-GFP- LacI transgene and an integrated lacO array to study the ectopic centromere. The CAL1-GFP- LacI gene is controlled under the metallothionein promoter (pMT), which can be induced by using copper sulfate in the medium. This allows for expression of the gene and the tethering of CAL1-GFP-LacI protein to the lacO locus, due to the high affinity between LacI and lacO. To identify novel genes involved in ectopic centromere establishment, I performed RNAi-mediated knockdown of 12 candidate genes in CAL1-GFP-LacI Drosophila S2 cells. The cells were then analyzed using Immunofluorescence (IF) and Fluorescent In Situ Hybridization (FISH) techniques to evaluate rates of centromere establishment in comparison to the knockdown of a negative control gene, Brown. I observed that the knockdown of Iswi, Rho1, Hr96, z, and CAL1 produced a statistically significant difference in ectopic centromere formation, suggesting that these genes, and the proteins they code for, play a role in centromere establishment