Transcriptional read through interrupts boundary function in Drosophila

In higher eukaryotes enhancer-promoter interactions are known to be restricted by the chromatin insulators/boundaries that delimit topologically associated domains (TADs); however, there are instances in which enhancer-promoter interactions span one or more boundary elements/TADs. At present, the mechanisms that enable cross-TAD regulatory interaction are not known. In the studies reported here we have taken advantage of the well characterized Drosophila Bithorax complex (BX-C) to study one potential mechanism for controlling boundary function and TAD organization. The regulatory domains of BX-C are flanked by boundaries which function to block crosstalk with their neighboring domains and also to support long distance interactions between the regulatory domains and their target gene. As many lncRNAs have been found in BX-C, we asked whether transcriptional readthrough can impact boundary function. For this purpose, we took advantage of two BX-C boundary replacement platforms, Fab-7attP50 and F2attP, in which the Fab-7 and Fub boundaries, respectively, are deleted and replaced with an attP site. We introduced boundary elements, promoters and polyadenylation signals arranged in different combinations and then assayed for boundary function. Our results show that transcriptional readthrough can interfere with boundary activity. Since lncRNAs represent a significant fraction of Pol II transcripts in multicellular eukaryotes, it is possible that many of them may function in the regulation of TAD organization. Author SummaryRecent studies have shown that much genome in higher eukaryotes is transcribed into non-protein coding lncRNAs. It is though that lncRNAs may preform important regulatory functions, including the formation of protein complexes, organization of functional interactions between enhancers and promoters and the maintenance of open chromatin. Here we examined how transcription from promoters inserted into the Drosophila Bithorax complex can impact the boundaries that are responsible for establishing independent regulatory domains. Surprisingly, we found that even a relatively low level of transcriptional readthrough can impair boundary function. Transcription also affects the activity of enhancers located in BX-C regulatory domains. Taken together, our results raise the possibility that transcriptional readthrough may be a widely used mechanism to alter chromosome structure and regulate gene expression.