Tissue-specific regulation of chromatin insulator function mediated by an RNA-binding protein. Leah H. Matzat, Ryan K. Dale, Nellie Moshkovich, Elissa P. Lei. Laboratory of Cellular and Developmental Biology, NIDDK, Bethesda, MD.

   Chromatin insulators organize the genome into distinct transcriptional domains and contribute to cell type-specific chromatin organization. However, factors regulating tissue-specific insulator function have not yet been discovered. In this study, we identify the RNA recognition motif-containing protein, Shep, as the first known tissue-specific regulator of insulator function in any organism. Shep is a direct interactor of two individual components of the gypsy insulator complex, Su(Hw) and Mod(mdg4)2.2. Mutation of shep improves gypsy-dependent enhancer blocking in a mod(mdg4)^u1 null genetic background, indicating a role as a negative regulator of insulator activity. Furthermore, both strong loss of function or overexpression of shep results in synthetic lethality with mod(mdg4)^u1; however, synthetic lethality is not observed by overexpression of an RNA-binding point mutant. These data suggest that both shep dosage as well as RNA-binding are important for insulator activity.
   Unlike ubiquitously expressed core gypsy insulator proteins, Shep is highly expressed in the central nervous system (CNS) with lower expression in other tissues. We developed a novel, quantitative tissue-specific barrier assay to demonstrate that Shep functions as a negative regulator of insulator activity in the CNS but not in muscle tissue. Additionally, mutation of shep alters insulator complex nuclear localization in the CNS but has no effect in other tissues. Consistent with negative regulatory activity, ChIP-seq analysis of Shep in a CNS-derived cell line indicates substantial genome-wide colocalization with a single gypsy insulator component but limited overlap with intact insulator complexes. Taken together, these data reveal a novel, tissue-specific mode of regulation of a chromatin insulator.