Characterization of an RNA Binding Protein Involved in Chromatin Insulation. Matthew R. King, Ryan K. Dale, Elissa P Lei. National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD.

   Chromatin insulators are DNA-protein complexes defined by the ability to prevent enhancer-promoter interaction or the spread of silent chromatin, functions termed enhancer-blocking and barrier activity, respectively. Previous work suggests that the gypsy insulator complex interacts with RNA, though none of its three core proteins contain an RNA-binding motif. To identify gypsy insulator associated transcripts, we carried out sequential RNA immunoprecipitation followed by high throughput sequencing (RIP-seq) of core gypsy proteins Centrosomal Protein 190 (CP190) and Suppressor of Hairy wing (Su(Hw)). In order to test the functional significance of their interaction, null or putative loss-of-function alleles of the genes encoding six of the most highly enriched transcripts were tested for enhancer-blocking activity. We also tested a null allele of CIP3, which encodes an RNA-binding protein known to bind one of the transcripts. Of these candidates, only CIP3 null mutants showed a negative effect on enhancer-blocking. We are currently testing the capacity of CIP3 to modulate gypsy barrier activity using a quantitative luciferase-based barrier assay. We found that CIP3 interacts with gypsy insulator proteins and at least a subset of gypsy associated transcripts. The three core gypsy proteins co-immunoprecipitate with CIP3 from embryonic nuclear extracts. Additionally, anti-CIP3 RIP of nuclear extracts followed by qRT-PCR revealed that many of the transcripts enriched in Su(Hw)/CP190 RIP-seq are also present in CIP3 RIP. Immunostaining of salivary gland polytene chromosomes of CIP3 null mutants showed that loss of CIP3 does not affect localization of gypsy insulator proteins to chromatin. Furthermore, double staining of wildtype polytenes for CIP3 and Su(Hw) shows only limited overlap of both proteins. However, CIP3 extensively colocalizes with Shep and Rm62, two RNA-binding proteins known to negatively affect gypsy insulator function. These data demonstrate a novel role for an RNA-binding protein in the regulation of gypsy insulator activity.