RNA dependent chromatin organization in Drosophila
It has become increasingly apparent that proper control of gene expression requires complex organization of DNA at the level of chromatin. Chromatin insulators are DNA-protein complexes that influence gene expression by establishing chromatin domains subject to distinct transcriptional controls, likely through alteration of their spatial organization. Insulators enforce the strict specific and temporal expression of complex loci such as the Drosophila bithorax complex, a master regulator of body segmentation, and the vertebrate beta-globin locus, which changes in expression during erythroid development. Therefore, studying the mechanism and regulation of insulator function is essential to further understand how higher order chromatin structure influences the intricately orchestrated transcriptional programs needed for proper development and differentiation.
We seek to determine mechanisms of chromatin insulator function using the biochemically and genetically tractable model system Drosophila, which harbors the largest diversity of known chromatin insulator complexes. Several lines of evidence suggest that insulator proteins bridge distant DNA sequences dispersed throughout the genome, causing looping of the DNA and the creation of a distinct chromatin domain. Large nuclear insulator complexes termed insulator bodies are tethered stably to the nuclear matrix and may form higher order structures of chromatin loops. Interestingly, insulator body association with the nuclear scaffold can be disrupted by RNase A treatment. These findings prompted us to examine whether RNA silencing, an RNA-dependent cellular mechanism of gene regulation known to act on the level of chromatin, affects insulator activity.
Our research provides evidence for a previously unknown role for RNA silencing in gypsy insulator function as well as higher order chromatin organization. Using biochemical purification techniques, we have identified an RNA-dependent physical interaction between proteins required for proper gypsy insulator and RNA silencing function respectively. Furthermore, mutations in genes encoding RNA silencing components affect gypsy insulator activity in vivo and the formation of insulator bodies. These results suggest that RNA silencing contributes to the multimerization of insulator complexes and/or the ability of insulator bodies to interact with a nuclear scaffold. Current efforts center on identifying insulator associated RNAs using deep sequencing and characterizing novel insulator interacting proteins using genetic and biochemical approaches. Interestingly, our studies have revealed differential effects of RNA silencing between gypsy and another chromatin insulator suggesting distinct mechanisms of insulator function.
An eBriefing of the New York Academy of Sciences 
