The role of insulators in transgene transvection inDrosophila

Transvection is the phenomenon where a transcriptional enhancer activates a promoter located on the homologous chromosome. It has been amply documented inDrosophilawhere homologues are closely paired in most, if not all, somatic nuclei, but it has been known to rarely occur in mammals as well. We have taken advantage of site-directed transgenesis to insert reporter constructs into the same genetic locus inDrosophilaand have evaluated their ability to engage in transvection by testing many heterozygous combinations. We find that transvection requires the presence of an insulator element on both homologues. Homotypictrans-interactions between four different insulators can support transvection: thegypsy insulator(GI),Wari, Fab-8and1A2;GIandFab-8are more effective thanWarior1A2. We show that in the presence of insulators, transvection displays the characteristics that have been previously described: it requires homologue pairing, but can happen at any of several loci in the genome; a solitary enhancer confronted with an enhancerless reporter is sufficient to drive transcription; it is weaker than the action of the same enhancer-promoter pair incisand it is further suppressed bycis-promoter competition. Though necessary, the presence of homotypic insulators is not sufficient for transvection; their position, number and orientation matters. A single GI adjacent to both enhancer and promoter is the optimal configuration. The addition of a heterologous insulator in one homolog can positively or negatively influence transvection strength. The local landscape of enhancers and promoters is also important, indicative of complex insulator-enhancer-promoter interactions.

metaseq: a Python package for integrative genome-wide analysis reveals relationships between chromatin insulators and associated nuclear mRNA

Here we introduce metaseq, a software library written in Python, which enables loading multiple genomic data formats into standard Python data structures and allows flexible, customized manipulation and visualization of data from high-throughput sequencing studies. We demonstrate its practical use by analyzing multiple datasets related to chromatin insulators, which are DNA–protein complexes proposed to organize the genome into distinct transcriptional domains. Recent studies in Drosophila and mammals have implicated RNA in the regulation of chromatin insulator activities. Moreover, the Drosophila RNA-binding protein Shep has been shown to antagonize gypsy insulator activity in a tissue-specific manner, but the precise role of RNA in this process remains unclear. Better understanding of chromatin insulator regulation requires integration of multiple datasets, including those from chromatin-binding, RNA-binding, and gene expression experiments. We use metaseq to integrate RIP- and ChIP-seq data for Shep and the core gypsy insulator protein Su(Hw) in two different cell types, along with publicly available ChIP-chip and RNA-seq data. Based on the metaseq-enabled analysis presented here, we propose a model where Shep associates with chromatin cotranscriptionally, then is recruited to insulator complexes in trans where it plays a negative role in insulator activity.